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161
CHAPTER 7key references
Kohl E, Steinbauer J, Landthaler M et al 201 1 Skin ageing. J Eur Acad Der -
matol Venereol 25:873–84.
An account of the key intrinsic and extrinsic factors that contribute to skin ageing.
Ladak A, Tubbs RS, Spinner RJ 2014 Mapping sensory nerve communica -
tions between peripheral nerve territories. Clin Anat 27:681–90.
Lucarz A, Brand G 2007 Current considerations about Merkel cells. Eur J
Cell Biol 86:243–51.A review of some of the controversies in Merkel cell biology, ontology and possible functions.
Miller M-C, Nanchahal J 2005 Advances in the modulation of cutaneous
wound healing and scarring. Biodrugs 19:363–81.
An overview of wound healing and scarring mechanisms, and how
recombinant growth factors and cytokines might be used therapeutically.
Pan X, Hobbs RP, Coulombe PA 2013 The expanding significance of keratin
intermediate filaments in normal and diseased epithelia. Curr Op Cell
Biol 25:47–56.A comprehensive review of the cell biology of keratins in healthy skin and in diseases such as cancer.A well-illustrated review explaining how epidermal stem cells contribute to hair follicle regeneration and also wound healing.
Ghadially R 2012 25 years of epidermal stem cell research. J Invest Dermatol
132:797–810.A concise summary of the recent progress in understanding stem cell niches in the skin and their potential clinical significance.
Haines RL, Lane EB 2012 Keratins and disease at a glance. J Cell Sci 125:
3923–8.
A compact description of keratin intermediate filament biology and diseases
associated with keratin gene mutations.
Hearing VJ 201 1 Milestones in melanocytes/melanogenesis. J Invest Derma -
tol 131:E1.
A short summary of landmarks in melanin biology with links to six other short reviews on key historical discoveries and insights germane to melanocytes in health and disease.
Hsu Y-C, Pasolli HA, Fuchs E 201 1 Dynamics between stem cells, niche and
progeny in the hair follicle. Cell 144:92–105.A detailed original study that defines the point at which stem cells in the hair follicle become irreversibly committed along a differentiation lineage. | 223 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Skin and its appendages
161.e1
CHAPTER 7
REFERENCES
Beck B, Blanpain C 2012 Mechanisms regulating epidermal stem cells.
EMBO J 31:2067–75.
A detailed review of mechanisms regulating epidermal stem cell renewal and differentiation.
Brown SJ, McLean WH 2012 One remarkable molecule: filaggrin. J Invest
Dermatol 132:751–62.
An overview of the anatomical, biochemical and clinical relevance of the
skin barrier protein filaggrin in health, atopy and allergy.
Elias PM, Gruber R, Crumrine D et al 2014 Formation and functions of
the corneocyte lipid envelope (CLE). Biochim Biophys Acta 1841:
314–18.
An update on the structure, composition and functions of the corneocyte lipid envelope with new insights from inherited and acquired disorders of lipid metabolism.
Fuchs E 2007 Scratching the surface of skin development. Nature
445:834–42.A well-illustrated review explaining how epidermal stem cells contribute to hair follicle regeneration and also wound healing.
Ghadially R 2012 25 years of epidermal stem cell research. J Invest Dermatol
132:797–810.
A concise summary of the recent progress in understanding stem cell niches
in the skin and their potential clinical significance.
Haines RL, Lane EB 2012 Keratins and disease at a glance. J Cell Sci 125:
3923–8.
A compact description of keratin intermediate filament biology and diseases associated with keratin gene mutations.
Hearing VJ 201 1 Milestones in melanocytes/melanogenesis. J Invest Derma -
tol 131:E1.A short summary of landmarks in melanin biology with links to six other short reviews on key historical discoveries and insights germane to melanocytes in health and disease.
Hsu Y-C, Pasolli HA, Fuchs E 201 1 Dynamics between stem cells, niche and
progeny in the hair follicle. Cell 144:92–105.A detailed original study that defines the point at which stem cells in the hair follicle become irreversibly committed along a differentiation lineage.Kohl E, Steinbauer J, Landthaler M et al 201 1 Skin ageing. J Eur Acad Der -
matol Venereol 25:873–84.An account of the key intrinsic and extrinsic factors that contribute to skin ageing.
Ladak A, Tubbs RS, Spinner RJ 2014 Mapping sensory nerve communica -
tions between peripheral nerve territories. Clin Anat 27:681–90.
Lucarz A, Brand G 2007 Current considerations about Merkel cells. Eur J
Cell Biol 86:243–51.A review of some of the controversies in Merkel cell biology, ontology and possible functions.
Miller M-C, Nanchahal J 2005 Advances in the modulation of cutaneous
wound healing and scarring. Biodrugs 19:363–81.An overview of wound healing and scarring mechanisms, and how recombinant growth factors and cytokines might be used therapeutically.
Pan X, Hobbs RP, Coulombe PA 2013 The expanding significance of keratin
intermediate filaments in normal and diseased epithelia. Curr Op Cell Biol 25:47–56.A comprehensive review of the cell biology of keratins in healthy skin and in diseases such as cancer.
Rozen WM, Garcia-Tutor E, Alonso-Burgos A et al 2009 The effect of anterior
abdominal wall scars on the vascular anatomy of the abdominal wall: a cadaveric and clinical study with clinical implications. Clin Anat 22:815–22.
Taylor GI, Gianoutsos MP, Morris SF 1994 The neurovascular territories of
the skin and muscles: anatomic study and clinical implications. Plast Reconstr Surg 94:1–36.
Taylor GI, Palmer JH 1987 The vascular territories (angiosomes) of the
body: experimental study and clinical applications. Br J Plast Surg 40:
1 13–41.
Wu X, Hammer JA 2014 Melanosome transfer: it is best to give and receive.
Curr Op Cell Biol 29:1–7.
Yin Z-X, Peng T-H, Ding H-M 2013 Three dimensional visualization of the
cutaneous angiosome using angiography. Clin Anat 26:282–7. | 224 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
e10SECTION 1
COMMENTARY Fluorescence microscopy in cell
biology today 1.1
(Fig. 1.1.1a). Model organisms, such as nematodes, zebrafish and fruit
flies, can be studied in this way. The newest developments in in vivo
imaging that are currently transforming the field are based on light sheets. Termed SPIM (selective plane illumination microscopy), a flat
sheet of excitation light is projected through the sample and imaged by
a perpendicularly arranged lens (Huisken et al 2004) ( Fig. 1.1.1b). This
allows high-resolution three-dimensional imaging at high speed and with minimal photo-damage to the sample.
In all the above techniques, the resolution of the final fluorescence
image is limited by the diffraction of light to be 200–300 nm; two fluo -
rescent objects separated by less than this distance would not be distin -
guishable as two separate entities. In 2008, the emerging field of
super-resolution far-field microscopy or nanoscopy was named Method
of the Year by Nature Methods, and in 2014 the Nobel Prize for Chem -
istry was awarded for the development of the techniques. By a combina -
tion of new fluorophores, optics and image analysis, the diffraction
limit was circumvented by three new methodologies. Broadly, these are
techniques based on structured illumination microscopy (SIM), in
which a grid pattern of excitation light is projected on to the sample
(Gustafsson 2000) ( Fig. 1.1.2a); stimulated emission depletion (STED)
microscopy, in which a ‘doughnut’-shaped depletion beam is used to de-excite fluorophores and narrow the excitation spot used in confocal
microscopy (Hell and Wichmann 1994, Vicidomini et al 201 1) ( Fig.
1.1.2b); or single-molecule localization techniques such as photoacti-vated localization microscopy (PALM), in which individual molecules Fluorescence microscopy is one of the most widely used tools in cell
biology today. Its major strength is that it allows the distributions of
individual, specific protein species to be mapped at submicron resolu -
tion in living cells and even whole organisms. There are several tech -
nologies that came together in the 1990s and early years of the
twenty-first century to make this possible. These are the development
of fluorescent fusion protein constructs based on genetically encoded
fluorescent proteins; advances in excitation and detector technology
allowing new levels of specificity, speed and signal-to-noise; the devel -
opment of multiphoton excitation permitting imaging deep within tissues; and new image analysis and computing techniques for the
manipulation and quantification of the resulting data. These advances
combined to make fluorescence a standard research tool, found within
most laboratories working in the biological sciences. However, a new
wave of technological development arrived in the last 5–10 years and
is only now beginning to be adopted by the biological sciences com-
munity. New fluorescent probes, optical technology and image-
processing algorithms have led to the development of super-resolution
microscopy in which fluorescence images can be obtained with resolu -
tion approaching the molecular scale. In effect, this allows the mapping of all fluorescent molecules in a sample with nanometre accuracy, a
possibility that is changing the way we think about what microscope
images mean.
Fluorescent fusion constructs allow a protein species of interest to
be genetically fused to a fluorescent protein so that it can be visualized
in a microscope (Chudakov et al 2010). Originally, this meant fusion
to the green fluorescent protein (GFP) from the Pacific jellyfish Aequoria
victoria (Tsien 1998). For the development of this technology, the 2008 Nobel Prize for Chemistry was shared between Osamu Shinomura, who
identified and purified GFP; Martin Chalfie, who created and imaged
the first fluorescent fusion (Chalfie et al 1994); and Roger Tsien, who
mutated GFP to create a whole palate of possible colours – a palate that
is now vast (Shaner et al 2005, Giepmans et al 2006). Unlike immu -
nostaining, in which fluorescently tagged antibodies to the protein of
interest are introduced into the cell, GFP is genetically encoded and is
therefore compatible with live cell imaging, so allowing the investiga -
tion of protein dynamics under physiological conditions. The newest fluorescent protein technology includes photoactivatable and colour-
switchable proteins, which are useful for tracking intracellular events
(Patterson and Lippincott-Schwartz 2002), and as timers (Subach et al
2009) and sensors of various environmental parameters, such as pH
(Tantama et al 201 1). On the hardware side, advances in laser technol -
ogy have allowed more specific excitation of the sample. Unlike mercury arc lamps, laser light is monochromatic: that is, it contains only a single
wavelength. This specificity greatly expanded the scope for multichan -
nel imaging and the visualization of several fluorescent species simul -
taneously. The latest developments in this field centre on white light
lasers and tuneable lasers for more flexible imaging (McConnell 2004).
Simultaneously, detectors have become faster and more sensitive. The
charge-coupled device (CCD) camera evolved into the electron-
multiplying CCD, which was sensitive enough to detect individual
photons and therefore image and track single molecules within cells.
Currently, compound metal-oxide semiconductor (CMOS) camera
technology is transforming microscopy with its very high frame rates
and huge fields of view. For point detectors such as those used on con -
focal and multiphoton systems, new hybrid detectors are being imple-mented, offering the Holy Grail of extremely high sensitivity and large
dynamic range.
While GFP advanced imaging into the domain of live cells, multi -
photon excitation allowed imaging in living organisms (Denk et al
1990, Helmchen and Denk 2005). Here, two low-energy photons from
a pulsed laser combine at the sample to excite the fluorophores, rather
than a single high-energy photon as is normally used. Low-energy (red) photons are scattered less by complex biological tissue, which means that high resolution can be maintained at greater imaging depths Fig. 1.1.1 Illustration of in vivo imaging by multiphoton and selective-
plane microscopy. A, In multiphoton microscopy, excitation only occurs
where the excitation photons are most dense – at the focus. Not only
does this generate intrinsic optical sectioning but also the low scattering of long-wavelength (red/infra-red) light means that focus can be deep into tissue. B, Selective-plane illumination creates a flat light sheet projected
from the side by a cylindrical lens, with fluorescence collected by a standard objective and imaged using a camera. This allows extremely high-speed three-dimensional imaging. A
BIllumination
IlluminationSample
SampleObjective
ObjectiveFluorescence
FluorescenceDylan M Owen | 225 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Fluorescence microscopy in cell biology today
e11
COMMENTAR Y 1.1
Fig. 1.1.2 The main techniques for super-resolution imaging.
A, Structured illumination microscopy (SIM) projects a pattern of
excitation light (blue lines) on to the sample (green). Multiple acquisitions
with different pattern positions and orientations allow a super-resolution
image to be reconstructed computationally. B, Stimulated emission
depletion (STED) uses a red-wavelength depletion laser beam to cancel out fluorophore excitation at the periphery of a standard confocal
excitation spot, leading to a narrower effective excitation area.
C, Single-molecule localisation microscopy (SMLM) uses special fluorophores or chemicals to image a sparse set of molecules in any one
frame exclusively, leading to individual point spread functions that can be
imaged and centroided to find the true position of the molecule. Over
many thousands of frames, the positions of all fluorophores in the sample
can be mapped. + + =+
++ =
=A
CB
Fig. 1.1.3 Example data sets acquired with SIM, STED and STORM.
A, Microtubules (green) and mitochondria (pink) imaged in U2OS cells
using SIM imaging. B, STED image of the nuclear pore protein Nup153
in the nucleus of a fixed PtK2 cell. C, STORM image of actin fibres in
Cos7 cells. A from York et al, Resolution doubling in live, multicellular
organisms via multifocal structured illumination microscopy. Nature Methods 9(7):749–754 (2012); B
from Wurm et al, Novel red fluorophores
with superior performance in STED microscopy. Optical Nanoscopy 1(1) (2012); and C
from Xu et al, Dual-objective STORM reveals three-
dimensional filament organization in the actin cytoskeleton. Nature Methods 9(2):185–188 (2012).
A
B
C
300 µm
-200 µmz
STED
ConfocalRaw data
5 µmNuclear Pore complex/ Abberior STAR635
are imaged and localized in sequence (Betzig and Chichester 1993)
(Fig. 1.1.2c).
SIM achieves resolutions of around 100 nm and is compatible with
conventional fluorophores and live cell imaging. STED typically achieves
50–100 nm in biological samples but is more challenging for live cell
imaging because of the damaging laser powers used. PALM and the related technique of direct stochastic optical reconstruction microscopy
(dSTORM) (Rust et al 2006, Heilemann et al 2008) deliver the highest
resolution of 20–30 nm, but are relatively slow and therefore mainly
used on fixed cells where new switchable fluorophores allow fluorescent molecules to be turned on stochastically. Individual molecules are then
imaged and their coordinates recorded before the fluorophores are
bleached and a new subset of molecules is activated. In this way, all
molecules in the sample are imaged in sequence, circumventing the
diffraction limit. Using PALM, the ability to acquire tables of the x, y
and z coordinates of all individual fluorescent molecules, rather than
images per se, requires new ways of thinking about the analysis and
quantification of data sets; this challenge is only just beginning. Example images acquired with these three super-resolution methods are shown
in Figure 1.1.3.
While work on new improved fluorophores, laser technology, optical
components and processing algorithms continues, more radical break -
throughs in microscopic techniques and data analysis are likely. They will focus on further enhancements to resolution, imaging speeds and
applicability to whole-organism imaging: for example, PALM is begin -
ning to be applied to live cells when the biological structure is relatively
stable (Shroff et al 2008) and the speed of STED has been greatly
improved (Chmyrov et al 2013). There is no doubt that these advances
will make fluorescence microscopy an even more valuable tool within the biological sciences.
REFERENCES
Betzig E, Chichester RJ 1993 Single molecules observed by near-field scan -
ning optical microscopy. Science 262:1422–5.
Chalfie, M, Tu Y, Euskirchen G et al 1994 Green fluorescent protein as a
marker for gene expression. Science 263:802–5.
Chmyrov A, Keller J, Grotjohann T et al 2013 Nanoscopy with more than
100,000 ‘doughnuts’ . Nat Meth 10:737–40.Chudakov DM, Matz MV, Lukyanov S et al 2010 Fluorescent proteins and
their applications in imaging living cells and tissues. Physiol Rev
90:1 103–163.
Denk W, Strickler JH, Webb WW 1990 Two-photon laser scanning fluores -
cence microscopy. Science 249:72–6. | 226 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
FluORESCENCE MICROSCO pY IN CEll b IOlOgY TOdAY
e12SECTION 1
Shroff H, Galbraith CG, Galbraith JA et al 2008 Live-cell photoactivated
localization microscopy of nanoscale adhesion dynamics. Nat Meth 5:
417–23.
Subach FV, Subach OM, Gundorov IS et al 2009 Monomeric fluorescent
timers that change color from blue to red report on cellular trafficking.
Nat Chem Biol 5:1 18–26.
Tantama M, Hung YP, Yellen G 201 1 Imaging intracellular pH in live cells
with a genetically encoded red fluorescent protein sensor. J Am Chem
Soc 133:10034–7.
Tsien RY 1998 The green fluorescent protein. Annu Rev Biochem 67:
509–44.
Vicidomini G, Moneron G, Han KY et al 201 1 Sharper low-power STED
nanoscopy by time gating. Nat Meth 8:571–3.
Wurm C, Kolmakov K, Gottfert F et al 2012 Novel red fluorophores with
superior performance in STED microscopy. Opt Nanosc 1:7.
Xu K, Babcock HP, Zhuang X 2012 Dual-objective STORM reveals three-
dimensional filament organization in the actin cytoskeleton. Nat Meth
9:185–8.
York AG, Parekh SH, Nogare DD et al 2012 Resolution doubling in live,
multicellular organisms via multifocal structured illumination micros -
copy. Nat Meth 9:749–54.Giepmans BNG, Adams SR, Ellisman MH et al 2006 The fluorescent toolbox
for assessing protein location and function. Science 312:217–24.
Gustafsson MGL 2000 Surpassing the lateral resolution limit by a factor of
two using structured illumination microscopy. J Microsc 198:82–7.
Heilemann M, van de Linde S, Schüttpelz M et al 2008 Subdiffraction-
resolution fluorescence imaging with conventional fluorescent probes.
Angew Chem Int Ed 47:6172–6.
Hell SW, Wichmann J 1994 Breaking the diffraction resolution limit
by stimulated emission: stimulated-emission-depletion fluorescence
microscopy. Opt Lett 19:780–2.
Helmchen F, Denk W 2005 Deep tissue two-photon microscopy. Nat Meth
2:932–40.
Huisken J, Swoger J, Del Bene F et al 2004 Optical sectioning deep inside
live embryos by selective plane illumination microscopy. Science 305:
1007–9.
McConnell G 2004 Confocal laser scanning fluorescence microscopy with
a visible continuum source. Opt Express 12:2844–50.
Patterson GH, Lippincott-Schwartz J 2002 A photoactivatable GFP for selec -
tive photolabeling of proteins and cells. Science 297:1873–7.
Rust MJ, Bates M, Zhuang X 2006 Sub-diffraction-limit imaging by stochastic
optical reconstruction microscopy (STORM). Nat Meth 3:793–6.
Shaner NC, Steinbach PA, Tsien RY 2005 A guide to choosing fluorescent
proteins. Nat Meth 2:905–9. | 227 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
e13
COMMENTAR Y 1.2
COMMENTARY
Stem cells in regenerative medicine 1.2
Introduction
There is a great deal of hype, hope and optimism surrounding the use
of stem cells in regenerative medicine. Historically and in recent years,
stem cell successes have attracted widespread media attention, which
has further fuelled the public perception of stem cells and what they
are capable of achieving. Although significant breakthroughs have been
made in the past decade in stem cell research and the resulting scientific
output has increased exponentially, so far, only a small proportion of
this research has been successfully translated into the clinical arena.
Scientific effort is therefore now focusing on translating this research
from ‘bench’ to ‘bedside’ and on testing the clinical efficacy and safety
of stem cell therapies through clinical trials. (For further reading, see
Dimmeler et al (2014).)
What are stem cells?
Stem cells may be defined as cells that exhibit properties of multilineage
differentiation and self-renewal (Thomson et al 1998). The term ‘mul -
tilineage differentiation’ means that cells have the potential to differen -
tiate into any of the three embryonic germ layers – ectoderm, mesoderm
or endoderm. By self-renewing, stem cells are able to generate further
stem cells, thereby propagating themselves.
Types of stem cell
Stem cells can be broadly categorized into embryonic or adult stem cells. The different types of stem cell are depicted in Figure 1.2.1. From
an immunological perspective, stem cells can also be syngeneic (from identical twins), autologous (from the same individual), allogeneic
(from a different member of the same species) or xenogeneic (from a
different species altogether). Syngeneic and autologous cells have
obvious advantages since they are unlikely to be rejected following
transplantation. The advantages and disadvantages of the different types
of stem cell are outlined in Table 1.2.1.
Embryonic stem cells (ESCs)
Embryonic stem cells are the archetypal pluripotent stem cells, derived
from the embryonic inner cell mass of the blastocyst and capable of
differentiating into any cell type (Thomson et al 1998). However,
current limitations on using ESCs include immunological rejection, safety concerns about the formation of tumours (teratomas) and ethical
dilemmas concerning the utilization of cells derived from aborted
fetuses. A recent trial of ESCs in spinal cord injury patients has been
halted due to financial constraints, although clinical trials are currently
under way in the UK and USA within the field of retinal research
(Schwartz et al 2012, Watts 201 1). Evidence suggests that human ESCs
may be generated through somatic cell nuclear transfer (cloning) tech -
niques, a challenge previously believed to be insurmountable (Tachibana
et al 2013). This has the potential of generating patient-specific
(matched) ESCs in future that will not be rejected by the patient’s immune system. Preliminary data suggest that reprogramming by
nuclear transfer may be slightly more effective than reprogramming by
transcription factors (Krupalnik and Hanna 2014, Ma et al 2014).
Amniotic fluid stem cells (AFSCs)
The amniotic fluid that surrounds the developing fetus contains a rich
stem cell population that was first discovered in 2007 (De Coppi et al
2007). Such cells are c-kit+ (CD1 17+) and fulfil the criteria of true stem
cells, in that they are pluripotent and exhibit self-renewal. Although not
in clinical trials as yet, these cells offer the prospect of correcting fetal defects either in utero or at the time of birth.Fig. 1.2.1 Different types of stem cell. Mesenchymal stem cellsEmbryonic
stem cells
Induced pluripotent stem cellsKIf4
Oct 3/4
c-Myc
Sox2Amniotic fluid
stem cells
Umbilical cord
stem cellsJonathan M Fishman, Paolo De Coppi, Martin A Birchall
Umbilical cord stem cells
Umbilical cord blood is a potential source of stem cells that may be
used to treat a variety of different diseases, including haemopoietic and
genetic diseases. Cord blood stem cells display embryonic stem cell
markers but are negative for blood cell lineage markers. The main
advantages they offer are ease of procurement with minimal risk to the
donor; ease of cryopreservation and banking for future use; and minimal
ethical concerns.
Adult stem cells
Mesenchymal stem cells (MSCs)
Mesenchymal stem cells (MSCs) are broadly multipotent stem cells that
are capable of differentiating into a variety of different tissue types,
being fairly restricted towards differentiation along the mesodermal
lineage. Their potential to differentiate into cartilage, bone or adipose tissue depends on the ability to create the appropriate microenviron-ment in which this might occur, a goal that continues to be the focus of intense study. MSCs are often derived from bone marrow but can also be enriched from a variety of other sources, including adipose tissue, synovium, skeletal muscle and placental tissues. They have the advantage of generating large numbers of cells and their application in | 228 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
STEM CEllS iN REgENERAT ivE MEdiCiNE
e14SECT iON 1
Fig. 1.2.2 Tissue-engineering an organ for transplantation.
ImplantationStem cellsScaffold
(may be natural or synthetic)
Bioreactor
Brain
Ear
Temporomandibular joint
Heart valveAorta
Pulmonary artery
Heart muscleSpinal cord within vertebral canal
Skin
Finger
KneeUrethraBladderUreterIntestinePancreasLiverChest wallTracheaJawSkull
Eye
vivo should encounter few ethical concerns when compared to the use
of ESCs, given that their utilization is comparable to bone marrow
transplantation, which is already in widespread clinical use. MSCs are
currently in numerous clinical trials for a variety of different diseases,
including the treatment of myocardial infarction, where initial results
have been promising.
induced pluripotent stem cells (iPSCs)
Induced pluripotent stem cells are a relatively novel type of stem cell. Following their discovery in 2006 (Takahashi and Yamanaka 2006),
Yamanaka was awarded the 2012 Nobel Prize in Physiology or Medi -
cine. If several critical transcription factors are introduced into a cell – Oct3/4, Sox2, Klf4, c-Myc (so-called ‘Yamanaka’ or ‘stemness’ factors),
any cell (e.g. a fibroblast) can be reverted to a pluripotent state. More
recently, it has been demonstrated that such cells may be reprogrammed
to totipotency (ability of a cell to differentiate into any cell type, includ -
ing the extra-embryonic membranes and tissues) (Abad et al 2013).
Limitations of the iPSC approach include the low efficiency of the process, safety concerns around viral transduction, immunogenicity of
the iPSCs and the risk of tumour formation. Current research is aimed
at generating iPSCs without the use of viruses, through the employment
of drug molecules (Hou et al 2013). In addition, contrary to earlier
opinion, it appears that iPSCs, being an autologous cell source, are
indeed non-immunogenic (Araki et al 2013). Phase I/II clinical trials
involving iPSCs are eagerly awaited to determine their overall efficacy and safety in disease states.
Somatic cell reprogramming – stimulus-
triggered acquisition of pluripotency (STAP): fact or fantasy?
Two papers in Nature in 2014 reported that differentiated mouse
somatic cells were able to revert to a pluripotent, or possibly even a
totipotent, phenotype after transient exposure to low pH. The repro -
gramming did not require either nuclear transfer or genetic manipula -
tion and was remarkably rapid, unlike the time taken to prepare iPSCs
(Obokata et al 2014a, Obokata et al 2014b). The reprogrammed cells
were named STAP (stimulus-triggered acquisition of pluripotency) cells.
Not surprisingly, the papers were headline news around the world. However, what seems to be too good to be true usually is just that; within weeks of publication, the methodology and the nature of the cells used in the studies were called into serious question and both papers were subsequently retracted ( Nature editorial 2014).Regenerative medicine through
tissue engineering
Tissue engineering is an interdisciplinary field that applies the princi -
ples and methods of engineering and the life sciences towards the development of biological substitutes that can restore, maintain or improve tissue function (Langer and Vacanti 1993). The main approach to tissue engineering includes using the various types of stem cell
mentioned above and seeding them, either on or within scaffolds, to create ‘off-the-shelf’ organs and tissues for transplantation ( Fig. 1.2.2).
Seeding of stem cells on to scaffolds may be undertaken either in vitro Table 1.2.1 Comparison of the different types of stem cell
Advantages Disadvantages
Embryonic
stem cellsPluripotency Fear of immunological rejection
Safety concerns – tumour (teratoma)
formation
Ethical dilemmas surrounding aborted
fetuses
Oocytes required
Amniotic fluid
stem cellsPluripotencyNon-tumorigenicCan be harvested (through
amniocentesis) and manipulated prenatally so that defect can be corrected either in utero or at the time of birthLow yield – 1% of amniotic fluid cells
are c-kit
+ (CD117+) stem cells
Further research concerning origin of
cells and characterization required
Umbilical cord
stem cellsEase of procurement with minimal
donor morbidity
Ease of cryopreservation and
banking for future use
Minimal ethical concernsLow yield of stem cells and only finite
number of cells available from donor
Problems of storage – long-term
storage may affect cell quality; cost implications; quality control issues
Mesenchymal
stem cellsMultipotentLarge numbers can be harvestedCan be enriched from a variety of
different tissues including bone marrow, adipose tissue, etc
Easily expanded in tissue culture
for tissue engineering purposes
Autologous and possess
immunomodulatory properties
Appear safe in clinical trialsMinimal ethical concernsDifferentiation dependent on an
appropriate microenvironment
Optimal mode of delivery unclearLimited long-term therapeutic potential
Induced
pluripotent stem cellsPluripotentCan be derived from any cell typeProbably non-immunogenicLow efficiency of reprogramming with
current techniques (<1%)
Safety concerns – viral vector
transduction commonly required (risks of viral infection and genetic manipulation) and risk of tumours | 229 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Stem cells in regenerative medicine
e15
COMMENTAR Y 1.2
in a bioreactor, or in vivo using the host as a ‘living’ bioreactor. Tissue
engineering has the potential to overcome existing organ shortages and
generate organs and tissues that are not rejected by the patient’s immune
system (Fishman et al 2013). The trachea was the first stem-cell-
based, tissue-engineered organ successfully transplanted into humans
(Fishman et al 201 1) ( Box 1.2.1).
Using stem cells to replace organs and
tissues through blastocyst complementation
A complementary approach to organ and tissue replacement using
interspecific blastocyst complementation has been reported recently. In
this technique, iPSCs home to developmentally deficient niches, where
they regenerate tissues of the donor cell species (Kobayashi et al 2010).
Using two transgenic pig lines, normal fluorescently-labelled pig
pluripotent stem cells have been transplanted into genetically altered
pigs lacking pancreata, resulting in the development of chimeric pigs Box 1.2.1 Clinical highlights
Several patients have now received a new trachea using tissue-
engineering techniques. The patient’s own (autologous) stem cells were harvested from the bone marrow and respiratory epithelium,
and seeded on to either decellularized (Elliott et al 2012, Macchiarini
et al 2008) or synthetic (Jungebluth et al 2011) scaffolds before
being transplanted back into the patient. Early results have been encouraging and clinical trials of full-scale partial laryngotracheal implants are anticipated in the near future.Fig. 1.2.3 Blastocyst complementation to generate human organs for transplantation. iPSCs
Injection of human iPSCs into
blastocysts genetically modified to lack specific organs
Generation of a human organin livestock animals
Organ transplantation
Patient
with fluorescent orange pancreata derived from donor cells (Matsunari
et al 2013). This is an important step towards the generation of human
organs in large animals by complementing pig embryos with human
iPSCs (Fig. 1.2.3). The clinical possibilities of using blastocyst comple -
mentation as a means of generating transplantable human organs may offer one solution to the current shortfall in organs for transplantation:
by growing human organs in livestock animals such as pigs, an unlim -
ited supply of immunologically matched human organs might be made available for transplantation. Concerns have been raised over possible
immunological rejection (targeting porcine-derived vasculature sur-
rounding the new organ), and ethical and safety issues associated with
injecting human iPSCs, capable of differentiating into any cell and/or
tissue type, into pigs (and so theoretically generating a new human–pig
hybrid species, especially if such cells home to the brain or the
germline).
Conclusion
At present, the jury is still out as to which stem cell is most effective
and safest for tissue regeneration. For this reason, all of the above stem
cells are being explored as viable options for future therapies without
relying on a single stem cell at the present time. iPSCs probably offer
most potential for the future, although it may be that different stem
cells perform differently under different conditions (e.g. some stem cells
may be better at generating one tissue than another). In addition, for
tissue engineering purposes, a stem cell is required that can generate
large numbers of cells in a relatively short period of time. Either way,
well-conducted clinical trials will be required to determine the stem
cell’s efficacy, safety and cell fate prior to its widespread use in
medicine.
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Abad M, Mosteiro L, Pantoja C et al 2013 Reprogramming in vivo pro -
duces teratomas and iPS cells with totipotency features. Nature 502:
340–5.
Araki R, Uda M, Hoki Y et al 2013 Negligible immunogenicity of terminally
differentiated cells derived from induced pluripotent or embryonic stem
cells. Nature 494:100–4.De Coppi P, Bartsch G Jr, Siddiqui M et al 2007 Isolation of amniotic stem
cell lines with potential for therapy. Nat Biotechnol 25:100–6.
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lenges in regenerative medicine. Nature Med 20:814–21. | 230 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
STEM CEllS iN REgENERAT ivE MEdiCiNE
e16SECT iON 1
Matsunari H, Nagashima H, Watanabe M et al 2013 Blastocyst complemen -
tation generates exogenic pancreas in vivo in apancreatic cloned pigs.
Proc Natl Acad Sci U S A 1 10:4557–62.
Nature editorial. 2014 STAP retracted. Nature 51 1:5–6.
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676–80.
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Schwartz SD, Hubschman JP, Heilwell G et al 2012 Embryonic stem cell
trials for macular degeneration: a preliminary report. Lancet 379:
713–20.
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derived by somatic cell nuclear transfer. Cell 153:1228–38.
Takahashi K, Yamanaka S 2006 Induction of pluripotent stem cells from
mouse embryonic and adult fibroblast cultures by defined factors. Cell
126:663–76.
Thomson JA, Itskovitz-Eldor J, Shapiro SS et al 1998 Embryonic stem cell
lines derived from human blastocysts. Science 282:1 145–7.
Watts G 201 1 Moorfields Eye Hospital is to host European trial of human
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neered tracheal replacement in a child: a 2-year follow-up study. Lancet
380:994–1000.
Fishman JM, De Coppi P, Elliott MJ et al 201 1 Airway tissue engineering.
Expert Opin Biol Ther 1 1:1623–35.
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of a decellularized skeletal muscle scaffold in a discordant xenotrans -
plantation model. Proc Natl Acad Sci U S A 1 10:14360–5.
Hou P, Li Y, Zhang X et al 2013 Pluripotent stem cells induced from mouse
somatic cells by small-molecule compounds. Science 341:651–4.
Jungebluth P, Alici E, Baiguera S et al 201 1 Tracheobronchial transplantation
with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study. Lancet 378:1997–2004.
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creas in mouse by interspecific blastocyst injection of pluripotent stem
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e17
COMMENTAR Y 1.3
COMMENTARY
Merkel cells 1.3
The epidermis, our outermost layer of skin, serves as a protective barrier
and sensory interface with the environment. In vertebrates, these func -
tions are accomplished through the work of only four conserved cell types. Keratinocytes form a water-tight barrier, melanocytes protect
against ultraviolet damage and Langerhans cells perform immune sur -
veillance. Merkel cells, which constitute less than 1% of the epidermis, are an enigmatic cell type whose function and developmental origin
have long been debated; however, recent advances show that mamma -
lian Merkel cells are epithelial derivatives that collaborate with sensory neurones to initiate touch sensation.
As early as the nineteenth century, Merkel cells were proposed to
contribute to skin’s sensory function. These cells were first described by
Friedrich Sigmund Merkel, who named them touch cells ( Tastzellen),
based on contacts with nerve terminals (Merkel 1875). Merkel cells are found in highly touch-sensitive skin areas, including glabrous (hairless)
skin of fingertips and lips, some hair follicles, and touch domes on the
trunk (Fig. 1.3.1). Most Merkel cells are innervated by a particular class
of myelinated (Aβ) sensory neurones called slowly adapting type I (SAI)
afferents (Iggo and Muir 1969). These touch receptors are unusual
because they produce biphasic discharges of action potentials that send
two types of information to the brain (see Fig. 1.3.1): dynamic responses
to moving stimuli represent an object’s spatial features (e.g. edges and curvature), whereas sustained action-potential trains signify steady pres -
sure (e.g. contact with clothing; Johnson 2001).
Merkel cells were initially proposed to serve as sensory receptor cells
based on their synapse-like contacts with nerve terminals and dense-
core vesicles filled with neurotransmitters (Halata et al 2003). Sensory
receptor cells, such as taste-bud cells or hair cells of the inner ear, trans -
duce the energy of a sensory stimulus into electrical signals, which then
trigger neurotransmitter release to excite sensory afferents. The hypo -
thesis that Merkel cells are mechanosensory receptors was challenged
by neurophysiological studies that reported touch sensitivity after
Merkel-cell loss, which suggested that Merkel cells act in a non-sensory
capacity.
Recent studies in rodent models have reconciled these views by
demonstrating that both Merkel cells and their sensory afferents are
mechanosensory cells. Three lines of evidence support this two-receptor-
site model, which was proposed more than two decades ago (Yamashita
and Ogawa 1991).
First, Merkel cells are touch-sensitive. Like inner-ear hair cells, Merkel
cells display fast mechanotransduction currents that convert cellular
displacements into electrical signals (Ikeda et al 2014, Maksimovic et al
2014, Woo et al 2014). Piezo2, a large, mechanically activated mem -
brane protein, is required for mechanotransduction currents in Merkel cells. Thus, Piezo2 is a leading candidate gene to encode sensory trans -
duction channels for touch (see Fig. 1.3.1).
Second, Merkel cells are excitatory cells. This was established with a
state-of-the-art technique called optogenetics, which uses light-gated molecules derived from Archea to activate or inhibit electrical signalling
in specific cells. Transgenic mice were engineered to selectively express
light-gated membrane proteins in Merkel cells (Maksimovic et al 2014).
When the skin is illuminated to activate Merkel cells in these mice, their
sensory afferents produce sustained volleys of action potentials that
mimic the response to static pressure (see Fig. 1.3.1). Conversely, neu-
ronal firing is inhibited when Merkel cells are optogenetically silenced. These results confirm that Merkel cells are sensory cells capable of excit -
ing afferents.
Third, Merkel cells are necessary for sustained, but not for dynamic,
firing in SAI afferents. Prototypical SAI responses are abolished in trans -
genic mouse strains that lack either Merkel cells or epidermal Piezo2
expression (Maricich et al 2009, Maksimovic et al 2014, Woo et al
2014). Instead, sustained touch produces only transient bursts of action
potentials in these mice (see Fig. 1.3.1). This finding indicates that
Merkel cells are essential for conveying information about static pres -
sure to the nervous system. Importantly, they also show that sensory afferents are independently capable of transducing dynamic touch, Fig. 1.3.1 The Merkel cell–neurite complex is a compound touch receptor.
The schematic shows the innervation of a touch dome. Merkel cells (blue)
cluster in these highly touch-sensitive spots in humans and other
mammals. Merkel cells are mechanosensory receptor cells derived from
epidermal progenitors. Adjacent Merkel cells are innervated by a
myelinated sensory afferent (yellow) that produces slowly adapting type I
(SAI) responses. This response has two phases: (1) Dynamic firing. During
a moving stimulus (blue bar), mechanotransduction channels located in the
SAI afferent open to excite a transient neuronal volley (blue action
potentials). (2) Sustained firing. Static pressure (green bar) activates the Merkel cells’ Piezo2-dependent mechanotransduction channels to excite electrical signalling. Merkel cells then signal to SAI afferent terminals to produce sustained action-potential firing (green). Although numerous neuroactive molecules localize to dense-core vesicles, the neurotransmitters and receptors (orange) that mediate excitatory signalling have not been identified. (Receptor and channel symbols © motifolio.com.)
Dermis Epidermis
Myelin sheathSAI afferent
SAI response
Dynamic firing
Sustained firingMerkel cell
Merkel-cell progenitor
Keratinocytes
Mechanotransduction
channel
Dense-core vesicles
Neurotransmitter receptor
Touch
Action
potential
trainsStatic pressureMoving stimulusEllen A Lumpkin | 232 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
MERkEl CElls
e18sECTION 1
serve additional, non-sensory functions in skin? This is particularly
important in human skin, where a substantial fraction of Merkel cells
appear to lack innervation. Are human Merkel cells derived from epi -
dermis and renewed throughout life? If so, how are these processes disrupted during ageing, which is accompanied by loss of touch recep -
tors in extremities? This condition is proposed to contribute to the decline of tactile acuity, grip strength and postural stability in the
elderly.
Mechanisms of Merkel-cell specification may also be important in a
very different human pathology – Merkel cell carcinoma (MCC). This
highly aggressive, non-melanoma skin cancer is linked to Merkel cells
based on ultrastructure, neurosecretory markers and keratin-20 immu-
noreactivity (Bhatia et al 201 1). Recent genomic profiling studies
confirm that human MCC cells express many of the same genes as
mouse Merkel cells (Haeberle et al 2004, Harms et al 2013). An impor -
tant unresolved question is whether Merkel cells or their epidermal
progenitors act as cells of origin for MCC. Immune suppression is a
probable risk factor for MCC because it is prevalent on sun-exposed
skin and in immunocompromised patients. Intriguingly, MCC has been
linked to a newly described virus, Merkel cell polyomavirus (Feng et al
2008), raising the possibility that viral infection plays a role in MCC
pathogenesis. Active areas of investigation include identifying a causal
link between MCC and polyoma viral infection, and defining mecha -
nisms that underlie the aggressiveness of MCC.
Acknowledgements
Thanks to members of the Lumpkin laboratory for helpful comments
and Ms Blair Jenkins for assistance with the figure. The author is sup-
ported by the National Institutes of Health (R01 AR051219 and R01
NS0731 19).albeit with reduced activity. Finally, touch-driven behaviours are com -
promised in rodents lacking functional Merkel cells, which indicates
that Merkel cells are important for the perception of touch (Maricich
et al 2012, Ikeda et al 2014, Woo et al 2014).
Together, these studies demonstrate that the Merkel cell–neurite
complex is a compound touch receptor with two mechanosensory cell types arranged in series (see Fig. 1.3.1). A key open question is: which
neurotransmitters convey information between Merkel cells and sensory afferents?
Transgenic mouse studies have also clarified the Merkel cell’s onto -
geny. Although embedded in a stratified epithelium, Merkel cells express
simple epithelial keratins (e.g. keratin-8, keratin-20) and produce
dozens of neurochemical markers (Moll et al 1984, Halata et al 2003,
Haeberle et al 2004). Due to this unusual assortment of epithelial and
neuronal markers, it was unclear whether Merkel cells derive from epithelial precursors or neural crest. These competing models were
tested in transgenic mice by genetically marking specific cell lineages
based on selective expression of Cre recombinase. Wnt1
Cre, which marks
all neural crest cells, failed to label Merkel cells; however, Krt14Cre, a
marker of keratinocyte-derived cells, labelled Merkel cells in all skin
areas (Morrison et al 2009, Van Keymeulen et al 2009). Moreover,
Merkel cells are replenished from epidermal progenitors in mature skin
(Van Keymeulen et al 2009, Woo et al 2010, Doucet et al 2013). Finally,
Merkel cells were abolished by conditional deletion of a developmental transcription factor, Atoh1, driven by Krt14
Cre but not Wnt1Cre (Morrison
et al 2009, Van Keymeulen et al 2009). Together, these studies provide
a starting point to define transcriptional networks that bestow a neuro -
sensory fate in epidermal cells (Bardot et al 2013, Lesko et al 2013).
Despite remarkable progress in Merkel-cell biology, important ques-
tions about human Merkel cells remain unanswered. Do Merkel cells
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Bardot ES, Valdes VJ, Zhang J et al 2013 Polycomb subunits Ezh1 and Ezh2
regulate the Merkel cell differentiation program in skin stem cells.
EMBO J 32:1990–2000.
Bhatia S, Afanasiev O, Nghiem P 201 1 Immunobiology of Merkel cell carci -
noma: implications for immunotherapy of a polyomavirus-associated cancer. Curr Oncol Rep 13:488–97.
Doucet YS, Woo SH, Ruiz ME et al 2013 The touch dome defines an epider -
mal niche specialized for mechanosensory signaling. Cell Rep 3:
1759–65.
Feng H, Shuda M, Chang Y et al 2008 Clonal integration of a polyomavirus
in human Merkel cell carcinoma. Science 319:1096–100.
Haeberle H, Fujiwara M, Chuang J et al 2004 Molecular profiling reveals
synaptic release machinery in Merkel cells. Proc Natl Acad Sci U S A 101:14503–8.
Halata Z, Grim M, Bauman KI 2003 Friedrich Sigmund Merkel and his
‘Merkel cell’, morphology, development, and physiology: review and new results. Anat Rec 271A:225–39.
Harms PW, Patel RM, Verhaegen ME et al 2013 Distinct gene expression
profiles of viral- and nonviral-associated Merkel cell carcinoma revealed by transcriptome analysis. J Invest Dermatol 133:936–45.
Iggo A, Muir AR 1969 The structure and function of a slowly adapting touch
corpuscle in hairy skin. J Physiol 200:763–96.
Ikeda R, Cha M, Ling J et al 2014 Merkel cells transduce and encode tactile
stimuli to drive A β-afferent impulses. Cell 157:664–75.
Johnson KO 2001 The roles and functions of cutaneous mechanoreceptors.
Curr Opin Neurobiol 1 1:455–61.Lesko MH, Driskell RR, Kretzschmar K et al 2013 Sox2 modulates the func -
tion of two distinct cell lineages in mouse skin. Dev Biol 382:15–26.
Maksimovic S, Nakatani M, Baba Y et al 2014 Epidermal Merkel cells are
mechanosensory cells that tune mammalian touch receptors. Nature 509:617–21.
Maricich SM, Morrison KM, Mathes EL et al 2012 Rodents rely on Merkel
cells for texture discrimination tasks. J Neurosci 32:3296–300.
Maricich SM, Wellnitz SA, Nelson AM et al 2009 Merkel cells are essential
for light-touch responses. Science 324:1580–2.
Merkel F 1875 Tastzellen und Tastkörperchen bei den Hausthieren und beim
Menschen. Archiv f mikrosk Anat 1 1:636–52.
Moll R, Moll I, Franke WW 1984 Identification of Merkel cells in human skin
by specific cytokeratin antibodies: changes of cell density and distribu -
tion in fetal and adult plantar epidermis. Differentiation 28:136–54.
Morrison KM, Miesegaes GR, Lumpkin EA et al 2009 Mammalian Merkel
cells are descended from the epidermal lineage. Dev Biol 336:76–83.
Van Keymeulen A, Mascre G, Youseff KK et al 2009 Epidermal progenitors
give rise to Merkel cells during embryonic development and adult homeostasis. J Cell Biol 187:91–100.
Woo SH, Ranade S, Weyer AD et al 2014 Piezo2 is required for Merkel-cell
mechanotransduction. Nature 509:622–6.
Woo SH, Stumpfova M, Jensen UB et al 2010 Identification of epidermal
progenitors for the Merkel cell lineage. Development 137:3965–71.
Yamashita Y, Ogawa H 1991 Slowly adapting cutaneous mechanoreceptor
afferent units associated with Merkel cells in frogs and effects of direct currents. Somatosens Motor Res 8:87–95. | 233 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
e19
COMMENTAR Y 1.4
COMMENTARY
Metaplasia 1.4
Metaplasia is the appearance, in adult life, of a patch of tissue that
normally belongs elsewhere in the body (Slack 1985, Slack 2007).
Generally, it is understood that the metaplasia originated in situ and
has not migrated from elsewhere. Therefore, metastatic tumour depos -
its, for example, are not considered to be metaplasias, and neither are tissues misplaced during embryonic development, such as thyroid resi -
dues in the thyroglossal duct.
Metaplasias are very diverse (Willis 1962). They include ectopic bone
and cartilage arising from connective tissue, squamous metaplasia of glandular epithelia, and substitution of one glandular tissue for another.
The latter group is of particular interest from a biological point of view.
These metaplastic transformations are normally between tissue types
that originated in embryonic development as neighbours in a common
cell sheet. They may remain neighbours into adult life or may become
separated. For example, intestinal metaplasia of the stomach is a well-
studied type of metaplasia in which patches of small intestine-like
epithelium are found in the stomach (Stemmermann and Hayashi
1968, Gutierrez-Gonzalez and Wright 2008). Intestinal metaplasia
found in the urinary bladder contains cell types and markers normally
found in the colon (Sung et al 2006). The bladder is not part of a
common cell sheet with the intestine in the adult, but in the embryo it is, originating from the allantoic diverticulum of the hindgut. Glandular
metaplasias are particularly common in the alimentary canal and in the
female reproductive tract (Kurita 201 1), perhaps because these are both examples of structures in which a number of different tissue types arise
from a simple epithelial tube in the embryo.
The clinical importance of metaplasias stems from the fact that they
often predispose to cancer. For example, carcinomas of the bronchus
usually arise within areas of squamous metaplasia (Auerbach et al
1961) and carcinomas of the stomach usually arise in regions of intes -
tinal metaplasia (Busuttil and Boussioutas 2009).
Metaplasias arise because the combination of gene activities that
defines the tissue type in embryonic development becomes altered in adult life. The genes in question encode a limited number of key tran -
scription factors. For example, the transcription factor p63 is essential
for the formation of squamous epithelia (Koster et al 2004) and is
always found to be expressed in patches of squamous metaplasia. In
experiments with mice, ectopic expression of the Cdx2 gene, normally
necessary for intestinal development, leads to patches of intestinal tissue
in the stomach (Silberg et al 2002, Mutoh et al 2002). Conversely, dele -
tion of Cdx2 from the intestine leads to oesophagus-like or stomach-like
patches in the intestine (Gao et al 2009, Stringer et al 2012). In tissues
that are maintained by continuous renewal from a population of stem cells, it is the stem cells whose character becomes switched in an initiat -
ing event. Once the change has occurred, it is irreversible.
A well-studied example of metaplasia is Barrett’s metaplasia,
commonly known as Barrett’s oesophagus (Falk 2002, Gilbert et al
201 1) (Fig. 1.4.1). This is characterized by the presence of columnar
SSQE
BMSSQE
BMBM
SSQEZ-lineZ-line
BM
SSQE
D EA B C
Fig. 1.4.1 Barrett’s metaplasia. A, Normal endoscopic appearance showing the junction between gastric columnar and oesophageal squamous
epithelium (Z-line). B–C, Endoscopies showing two cases of Barrett’s oesophagus. The Z-line has moved proximally and is very irregular. There are
islands of squamous tissue within the columnar region. D, Histological view, haematoxylin and eosin. E, Histological view, immunostained for CDX2
(brown). Scale bar 200 microns. Abbreviations: BM, metaplastic epithelium (Barrett’s metaplasia); SSQE, stratified squamous epithelium. Jonathan MW Slack, Leonard P Griffiths, David Tosh | 234 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
METAplAsiA
e20sECTiON 1
epithelium, the tissue also contains mucous glands connected by ducts
to the lumen, which are located in the submucosa but originate from
the original embryonic oesophageal epithelium. Barrett’s metaplasia
probably arises by transformation of the basal cells of the squamous
epithelium, or from the mucous glands, or both (Nicholson et al 2012,
Leedham et al 2008).
The predisposing cause of Barrett’s metaplasia is gastro-oesophageal
reflux, in which stomach acid, and bile from the intestine, enter the
oesophagus (Souza 2010). It is known that this environment can induce
in oesophageal cells expression of some of the key genes involved
in intestinal development, such as those encoding CDX factors and
hepatocyte nuclear factors (Eda et al 2003, Debruyne et al 2006, Piessen
et al 2007). Obesity is a well-known risk factor for reflux, partly because
of effects on intra-abdominal pressure and probably also through the
effects of cytokines released from fat (Corley et al 2007). Barrett’s meta -
plasia may progress to dysplasia and to adenocarcinoma. Progression
is associated with loss of the tumour suppressor gene CDKN2A, encod-
ing the protein p16, which is an inhibitor of cell division; and TP53,
encoding the protein p53, required for death of abnormal cells
(Spechler et al 2010, Chen et al 201 1). Studies of the clonal structure
of Barrett’s metaplasia have shown that is it complex, containing
cell populations derived from several original progenitors, with
various combinations of abnormalities arising from somatic mutation
(Leedham et al 2008).
Gastro-oesophageal reflux is normally treated with proton pump
inhibitors that reduce the levels of stomach acid. This improves associ -
ated oesophagitis but does not usually result in regression of any Bar -
rett’s metaplasia. Barrett’s metaplasia as such is not normally treated but, if it progresses to dysplasia, it may be extirpated by a variety of
endoscopic techniques (Lim and Fitzgerald 2013).epithelium in the distal oesophagus, of which some resembles intesti -
nal epithelium possessing goblet cells. Barrett’s metaplasia is important
because it is a risk factor for the development of oesophageal adeno -
carcinoma, a cancer whose incidence rose considerably in the late twen -
tieth century (Hvid-Jensen et al 201 1). In less developed countries, most
oesophageal cancers are squamous cell carcinomas, arising from the
normal squamous lining of the oesophagus but, in the Western world,
adenocarcinomas are now more common.
At least some metaplasias are monoclonal, i.e. a particular focus
consists of tissue all derived from a single cell. This is the case for intes-
tinal metaplasia of the stomach, where neighbouring metaplastic crypts
arise by fission (Gutierrez-Gonzalez et al 201 1). In Barrett’s metaplasia,
patches of crypts can also be monoclonal (Nicholson 2012), although the lesion as a whole is polyclonal. A key property of metaplastic foci
is that the new tissue is in a situation of competitive growth with the
old tissue that surrounds it. Unless the metaplastic tissue can outgrow
the surrounding tissue, then the transformation of a single stem cell
would not give rise to a macroscopic lesion. However, this aspect of
epithelial biology remains poorly understood.
The exact cell of origin of Barrett’s metaplasia is still not known
(Barbera and Fitzgerald 2010). The columnar lined epithelium of Bar -
rett’s metaplasia is in continuation with the columnar lined gastric mucosa and appears as a proximal displacement of the junction with
the stratified squamous epithelium (the Z-line). However, it is generally
thought not to be a phenomenon of cell migration alone, as animal
experiments indicate that it is possible to elicit patches of metaplasia
separated from the gastric epithelium (Gillen et al 1988, Goldstein
et al 1997). The normal oesophagus originates from an endodermal
tube lined with columnar epithelium, which becomes squamous
between about 6–7 weeks of gestation. Although lined with squamous
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Butterworths. | 235 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
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COMMENTAR Y 1.5
Electron microscopy in the
twenty-first centuryCOMMENTARY
1.5
Introduction
Biological electron microscopy (EM) is pivotal in life science research.
A measure of its ubiquity is the diversity of its capacity, e.g. three-
dimensional structural information about proteins and viruses at Å
resolution; three-dimensional reconstructions of cellular organelles and
tissues (from a few tens of nanometres to hundreds of micrometres);
and two-dimensional and topographic information and elemental
microanalysis at subcellular resolution. The capacity of EM is now
further enhanced by correlative light and electron microscopy (CLEM)
workflows, where light microscopy (LM) events are co-localized with
underlying ultrastructure. In its simplest form, LM generates a ‘Google
Earth’-like map to identify areas of interest, which are then studied at
the ultrastructural level by EM, so overcoming both the physical size
limitations imposed on samples by EM and the diffraction limits of
resolution imposed by LM (Williams and Carter 2009). CLEM can also
be combined with advanced EM sample preparation and imaging tech -
niques to provide previously unheralded levels of cytoarchitectural
insight into biological systems (McDonnald 2009, Plitzko et al 2009,
Sartori et al 2007).
However, biological EM is not without its challenges. The electron
beam is generated under vacuum at pressures and temperatures that are nominally incompatible with liquid water, yet water is the most abun -
dant cellular constituent. Moreover, carbon-based life forms also have poor contrast in the electron microscope because they are composed
mainly of light elements. In fact, carbon is so ‘transparent’ in the elec -
tron microscope that it is often employed as a film to support biological samples. To overcome these caveats, hydrated ‘live’ tissue is converted
to a ‘fixed’ state; conventional protocols employ a series of steps, includ -
ing chemical fixation, alcohol dehydration and resin infiltration. Heavy metal salts are added for positive staining of fixed and embedded speci -
mens or negative staining of whole structures that have been deposited on a support film (Glauert and Lewis 1998). More recently, cryo-fixation
has been adopted to allow the ‘solidification of a biological specimen
by cooling with the aim of minimal displacement of its components’
(Steinbrecht and Zierold 1987). By using low temperature as a physical
fixation strategy, the morphology and dimensions of the living material
are retained and soluble cellular components are not displaced, which
means that processing artefacts commonly encountered in more con -
ventional room-temperature EM techniques are either reduced or removed. Cryo-EM often allows direct observation of specimens that
have not been stained or chemically fixed.
EM in pathology
Despite the decline in diagnostic histopathology by EM from the height of its popularity in the 1980s, EM remains an important diagnostic tool
in several well-defined areas. Indeed, guidance issued by the UK’s Royal
College of Pathologists cites and supports the Association of Clinical
Pathologists Best Practice No. 160, which states that ‘Many respondents
[to a review of laboratory practice in renal pathology] expressed the
opinion that to carry out evaluation of renal biopsy specimens without
at least having the availability of electron microscopy is negligent’ (de
Haro and Furness 2012). Other specialized areas include the diagnosis
of primary ciliary dyskinesia; certain skin/connective tissue disorders,
e.g. inherited bullous lesions and Ehlers–Danlos syndrome; and spe-cialized areas of ophthalmic pathology (de Haro and Furness 2012). Diagnostic transmission electron microscopy (TEM) in the clinical setting has been largely stagnant and there has been little change in procedures or application for decades. In marked contrast, TEM in the research setting has experienced an explosion in capacity and capability, and there is considerable opportunity for twenty-first century EM to translate to, and to advance, clinical research and diagnostic pathology
(Fig. 1.5.1, Video 1.5.1).
Limitations of traditional processing
techniques and current protocols
Traditional EM processing strategies introduce artefacts (perturbations
to structure) ( Fig. 1.5.2) that affect tissue structure, e.g. by shrinkage or
swelling of the tissue under examination, the shrinkage of cellular organelles, and/or the extraction or redistribution of cellular constitu -
ents such as lipids, proteins and DNA (Boyde and Maconnachie 1979). Most chemical fixatives react with proteins and cross-link peptide
chains as part of their action (Glauert and Lewis 1998). These reactions
can be highly deleterious to epitopes, significantly compromising
immunohistochemical studies.
Cryo-fixation has two distinct advantages over chemical fixation
(Nermut and Frank 1971, Nermut 1991). It is rapid (measured in milli -
seconds), which means that the sample is preserved, hydrated and in a
‘close-to-life’ state at the point of initiating fixation, and ensures simul -
taneous immobilization of all macromolecular components (Robards and Sleytr 1995). Many protein networks are labile and prone to disrup -
tion with even a slight osmotic or temperature change; cryo-fixation minimizes these deleterious effects. Cryo-techniques allow the study of
biological samples with improved ultrastructural preservation and may
facilitate the study of dynamic processes. Rapid freezing prevents arte -
factual aggregations of proteins because tissue fixation and processing can be performed with no or minimal use of cross-linking fixatives, which means that samples retain higher antigenicity (Kellenberger
1991, Hayat 2000, Claeys et al 2004).
This level of preservation relies on vitrification (the transformation
of water from a liquid to an amorphous state without inducing the Fig. 1.5.1 A Masson trichrome stain of a renal biopsy taken for diagnostic
histopathology. The arrow highlights a glomerular capillary, fringed with
foot processes, which would be selected for investigation at higher
resolution by transmission electron microscopy (see Video 1.5.1). For
diagnostic pathology, the whole glomerulus would be examined. Key: red,
keratin; blue/green, collagen; light red/pink, cytoplasm. (Image courtesy of
M Balys.)
Roland A Fleck | 236 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
ElECTRON MiCRO sCOpY iN ThE TwENTY -fiRsT CENT uRY
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Fig. 1.5.2 An overview of electron microscopy specimen preparation workflow. Areas to the left of the dashed line are at room temperature, indicating
that samples can be handled without the requirement for specialist cryo-operating procedures. Samples to the right of the dashed line must be handled
in a manner suitable for vitreous biological samples, taking specific care to pre-cool tools before approaching or manipulating samples. Arrows denote
the direction of workflow and key stages required to process a sample from ‘living/close to life’ state to final image. Vitreous thin film (VTF) is a
technique in which a liquid sample is pipetted (4–6 µl) on to a transmission electron microscopy (TEM) grid and blotted to create a thin, even liquid film
across the surface of the grid. Ideally, the TEM grid should have a holey carbon film and, following blotting, the liquid sample will be held as a mono-
layer (approximately 12 nm thick) of particles supported within the holes. This film is then plunged rapidly into a liquid cryogen (ethane) to vitrify the
sample, generating a vitreous ice film that is amorphous (no long-range structure) and thus a desirable support phase for cryo-electron microscopy of
the encased particles (Cheng et al 2012). Other abbreviations: CEMOVIS, cryo-electron microscopy of vitreous sections; SEM, scanning electron
microscopy. (Adapted from Fleck RA 2015 Low-temperature electron microscopy. In: Wolkers W, Oldenhof H (eds), Methods in Cryopreservation and
Freeze-Drying Protocols. Methods in Molecular Biology. Berlin: Springer.)Room temperature
specimen handlingRoom temperature specimen
handling and processing
Warm/ ‘live sample’Plunge freezing
High-pressure freezingPhysically fixed vitrified specimen
Freeze fractureFixation
Processing
VisualizationFreeze fracture Freeze-substitutionCryo-ultramicrotomyVTF
Chemical fixation
Conventional room
temperature sample
preparation techniquesTokuyasu/
immunolabelling techniqueTokuyasu CEMOVIS
Ultramicrotomy
Replica
Imaging by either SEM or TEM at conventional imaging
conditions of temperature and pressureImaging by either cryo-SEM or cryo-TEM
under cryogenic conditions
nucleation of ice crystals). The nucleation of ice crystals is temperature-
and pressure-dependent. Crystallization depends on the cooling rate,
itself dependent on the thermal properties of water, the sample thick -
ness, and the heat extraction flow at the surface of the specimen. Freez -
ing is a time-dependent process. Vitrification sits at the beginning of a
workflow where the sample is either subsequently processed to a stable
room-temperature state for imaging or maintained in its vitreous state
throughout (see Fig. 1.5.2) (Fleck 2015).
There are a variety of methods for rapid freeze fixation of tissues.
However, the depth of vitrification is often limited (e.g. a few microns
for samples plunged into liquid cryogen). Of the available technolo -
gies, high-pressure freezing (HPF) is by far the most effective. First introduced by Moor and Riehle in 1968, HPF exploits the physical
benefit of high pressure (210 MPa) to reduce the cooling rate required
for the vitrification of water from several 100,000 K.s−1 to a few
1,000 K.s−1, making vitrification of relatively thick samples practicable
(Moor and Riehle 1968, Studer et al 2008). HPF machines synchro -
nize pressurization and cooling of the sample (to below the glass
transition temperature (Tg)) within 20 ms, in a highly reproducible
manner (Müller and Moor 1984, Riehle 1968, Studer et al 2001),
extending the depth of vitrification to as much as 200 µm (Studer et al
1995, Studer et al 2008).
Tissue samples prepared this way require further modification to
make them compatible with the transmission electron microscope.
They must either be converted to a room-temperature stable state (e.g. by freeze substitution into a resin), or be sectioned whilst remaining below the glass transition temperature (T
g); when this is done, images
close to the native structure of biological specimens can be achieved. However, despite its benefits, cryo-electron microscopy of vitreous sec -
tions (CEMOVIS) has not been routinely adopted, principally because of the technical difficulty of producing a ribbon of frozen sections and transferring it on to an EM grid. A double micromanipulator tool that
guides the ribbon using an electrically conductive fibre, and then posi -
tions and attaches the ribbon to an EM grid has recently been developed
(Studer et al 2014) ( Fig. 1.5.3).
Advances in instrumentation and
detector technology
EM has undergone a revolution in recent years, largely coincident
with the advent of digital charge-couple device (CCD) cameras and
increasing personal computer (PC) processing power. What was princi -
pally a two-dimensional technique is now one that readily embraces three dimensions by electron tomography (Gan and Jensen 201 1).
In this technique, a specimen is tilted through the incident beam, sequential images are acquired, and, after alignment, a weighted back-
projection is used to generate a three-dimensional tomographic volume
(see Video 1.5.1). These techniques have stimulated instrument changes.
In conventional TEM, low accelerating voltages (80–120 keV) provide
a strong beam-to-specimen interaction in order to increase contrast
from electron scattering. Cryo-TEM and tomography require higher
accelerating voltages (200–300 keV), which reduce the beam-to-
specimen interaction; the contribution of chromatic aberration to the
image is also reduced, theoretically improving resolution (Williams and
Carter 2009).
Changing the emission source has been another major technical
advance. A field emission gun (FEG) is becoming the preferred
source of the electron beam because it provides a beam of smaller diameter, higher coherence and a current density (brightness) of up
to three orders of magnitude greater than was achieved with conven -
tional thermionic emitters (e.g. tungsten or lanthanum hexaboride | 237 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Electron microscopy in the twenty-first century
e23
COMMENTAR Y 1.5
and resolution with superior detective quantum efficiency (DQE) rela -
tive to a conventional CCD camera (Milazzo et al 2010). Moreover,
direct exposure to the incident electron beam significantly improves the
signal-to-noise ratio in comparison to an SCCD (Milazzo et al 2010).
Perhaps more importantly, DDD allow images to be recorded as movies,
which means that movement caused by the electron beam can be cor -
rected computationally (Grigorieff 2013). As an example, images of ribosomes of sufficient quality to permit alignment at an accuracy that
approaches near-atomic resolution have been obtained using approxi -
mately 30,000 images (Bai et al 2013).
When combined, ZP, EFTEM and DDD offer a paradigm shift in the
capability of the transmission electron microscope and are likely to drive further advances in EM-based research. Whether in isolation or
combination, these techniques will translate from cutting edge to
routine instrumentation, heralding generic benefits to the field. Phase
contrast electron microscopy utilizing ZP has already been applied
to histochemically stained strong-phase (high-contrast heavy-metal-
stained) biological specimens (Atsuzawa et al 2009). In sections of
various thicknesses specifically stained for the Golgi apparatus, a high-contrast gain was observed even for strong-phase objects, indicating that
phase contrast electron microscopy has an application for traditional
strong-phase-contrasted biological specimens (Atsuzawa et al 2009), as
well as for low-phase cryo-fixed biological specimens (e.g. the visualiza -
tion of cyanophage Syn5 inside its host, Synechococcus, using ZP in
combination with cryo-tomography (cryo-ET) to enhance image con -
trast over conventional cryo-ET) and will permit direct identification of
subcellular components (Dai et al 2013).
Advances in the scanning
electron microscope
Cryo-FEGSEM has become well established as a research tool. The sta -
bility and emitter life of a FEG source, coupled with the stability and low keV sensitivity of a modern scanning electron microscope, is a
powerful tool. Unlike TEM, in which high accelerating voltages are used
to transmit the electrons through the specimen, SEM benefits from low
accelerating voltages that limit beam interaction with the specimen.
This is key to successful cryo-SEM but creates a challenge around gen -
erating sufficient signal for high-resolution imaging. Modern FEGSEM systems (with improved detector sensitivity and signal-to-noise ratios)
are now achieving sub-nm resolution at 1 keV, making them highly
capable tools for life science research, particularly when combined with
high-resolution coating and fracture techniques (Fleck 2015) (see Fig.
1.5.2; Fig. 1.5.4).
Large three-dimensional volumes may be generated by the assembly
of sequential two-dimensional images acquired in the scanning elec -
tron microscope. There are essentially three strategies, each with unique
strengths and weaknesses: focused ion beam (FIB), serial block face
SEM (SBFSEM) and array tomography (AT). FIB involves milling of
material in a dual-beam scanning electron microscope using a focused
beam of gallium ions to cut (mill) away layers of material. After each
cut, the surface is imaged, ultimately producing a stack of two-
dimensional images that can be aligned to generate a three-dimensional
volume. Although FIB can be combined with cryo-preparation tech -
niques, elemental microanalysis and the range of imaging modes avail -
able within the SEM, it can be challenging (problems include achieving
registry between slices, gallium contamination of the sample, and
damage to structural information caused by the milling process).
SBFSEM takes a different approach from FIB and incorporates a
microtome within the scanning electron microscope chamber (Denk
and Horstmann 2004). The microtome cuts a thin section from the
block, an image is taken, and the procedure is repeated until a three-
dimensional volume is generated ( Video 1.5.2). SBFSEM is less prone
to registration issues because the sample essentially remains locked in position below the detector. However, like FIB, this technique is destruc -
tive since, once sectioned, the sample is lost. SBFSEM uses room tem-perature processing techniques with high levels of en bloc heavy metal
staining that are required to generate a sufficiently robust back-scattered electron signal to form an image. The processing steps, and the limita -
tions of the back-scattered electron detector, mean that SBFSEM is cur -
rently incompatible with elemental microanalysis or cryo-techniques.
However, this is a nascent technology that is advancing rapidly. Novel
applications will emerge with advances in detectors, improved resins
(e.g. conductive resins) and the exploitation of charge suppression approaches in the SEM (e.g. beam deceleration).
Unlike FIB and SBFSEM, AT is not destructive and material may be
recovered and re-imaged. The technique allows samples to be prepared by any TEM preparation strategy. Material is sectioned at room tempera -
ture by ultramicrotomy; the sections are collected in order as a ribbon and may then be stained to enhance contrast, or are immunolabelled. (LaB
6)-tipped filaments) (Kersker 2001). The net results for both scan -
ning electron microscopy (SEM) and TEM have been greatly improved
signal-to-noise ratio and spatial resolution, coupled with a significant
increase in emitter life and reliability. FEG has had a major impact on
the practicality of cryo-SEM and resolution in cryo-TEM studies,
although these higher accelerating voltages carry a penalty of decreased
contrast.
Overcoming the challenges of contrast in
the transmission electron microscope
Zero-loss energy filtering is employed to improve image contrast in the
transmission electron microscope. The filter may be positioned either
in-column or post-column. Increased scattering contrast and the exclu -
sion of the blurred background (as a consequence of the interception of inelastically scattered electrons) allow specimens to be imaged closer
to focus, and increases high-resolution contrast and image resolution
(Grimm et al 1997, Grimm et al 1998).
Energy filtered transmission electron microscopy (EFTEM) imaging
to remove the contributions of inelastically scattered electrons is advan -
tageous when imaging thick biological samples. Han et al (1995) dem -
onstrated that only elastically scattered electrons contributed to the
coherent image component when imaging biological specimens of
more than 0.5 µm thick at 200 keV; optimization of the zero-loss signal
required operation at intermediate to high primary accelerating voltages
(over 200 keV). The gain due to EFTEM increases with specimen thick -
ness, with a linear dependence for light-scattering elements (Grimm
et al 1998). These results are important for the accurate recording of
images of thick biological specimens by tomography and to demon -
strate the increasing importance of high accelerating voltages in biologi-
cal TEM (Grimm et al 1998).
New technologies are overcoming previous limitations of low con-
trast in the transmission electron microscope. Zernike phase plates (ZP), which are well accepted for imaging with the light microscope,
represent a novel approach to imaging in the transmission electron
microscope. Despite technical hurdles to their use, the potential reward
in greater contrast has driven research into this technology (Fukuda
et al 2009, Glaeser 2013). An approximate five-fold increase in contrast
is possible with a π2 ZP that shifts the phase of only the scattered elec -
trons (these phase-shifted electrons interfere with the unscattered elec -
trons in the image plane), resulting in amplitude contrast rather than
phase contrast. Indirect scintillator-coupled charge-coupled-device (SCCD) cameras replaced film cameras and revolutionized EM because they provided a near-instant digital readout and obviated the physical limitation of a fixed number of image plates. Direct detection devices (DDD) avoid the fundamental inefficiencies of the electron-to-light conversion process of a scintillator and achieve unmatched sensitivity Fig. 1.5.3 Cryo-electron microscopy of vitreous sections (CEMOVIS)
performed using a cryo-ultramicrotome. The cryo-chamber (Leica EM
FC6, Leica Microsystems, Austria) is mounted on a conventional
room-temperature ultramicrotome (Leica EM UC6, Leica Microsystems,
Austria). The double manipulators (Manip, Diatome, Switzerland) are then
attached to the top of the cryo-chamber, allowing precise control of
both the vitreous ribbon and the transmission electron microscopy grid. The system is located within a glove box to maintain a low-humidity
environment around the sample that will reduce contamination during
sectioning.
| 238 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
ElECTRON MiCRO sCOpY iN ThE TwENTY -fiRsT CENT uRY
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Fig. 1.5.5 ATUMtome and tape. The ATUMtome is mounted on an PT-PC
PowerTome (RMC, USA), showing the tape being transferred from a
delivery spool (top) to receiving spool (bottom) through the boat, which
maintains a water reservoir behind the edge of the diamond knife (blue) to
float the sections and allow their capture by tape. Tape tension is
maintained throughout by a series of tensioning springs and guides. The
insert shows individual sections on tape mounted on carbon tape and
silicon wafer (to help dissipate specimen charging during imaging) ready
for viewing in the scanning electron microscope.
Fig. 1.5.4 Cryo-scanning electron microscopy (SEM) images. A, Fractured Plasmodium falciparum -infected erythrocytes. A cross-fractured infected
erythrocyte is shown; the insert depicts a high-magnification image of the p-face of the infected erythrocyte with membrane proteins visible on the
fracture plane. The scale bar represents 1 µm (main image) and 100 nm (inset). B, Cross-fractured Neisseria meningitidis showing surface membrane
protein organization and polysaccharide coat. The scale bar represents 100 nm. Images were taken with a cold-field emission gun cryo-SEM (JSM-
7401F, JEOL, Japan), equipped with a Baltec/Leica VCT100 vacuum transfer system and cryo-stage. Coating was applied after fracturing (3 nm Pt/C)
using multi-angle rotational coating (BAF060, Baltec/Leica Microsystems, Austria).
A B
Ribbons may be imaged by LM or SEM; individual sections can be
identified, recovered and imaged at the higher spatial resolution of the transmission electron microscope. Successful AT requires high-precision
x-y-z sample stages that can perform programmed, sequential (in focus)
imaging of serial sections. Arguably the most challenging aspect of the
workflow is the serial sectioning of the sample; to date, this is under -
taken almost exclusively by highly skilled operators. However, the ATUMtome (RMC, USA) employs a tape system to automate the serial
sectioning process and has recently been commercialized ( Fig. 1.5.5).
New multibeam-multidetector SEMs (e.g. 61 concurrent beams) are
being developed that will allow images of up to 1.2 billion pixels to be
acquired in 1 s (Marx 2013) and will further speed data acquisition.
Developments in thin-film technology and microfabrication are
facilitating the design of liquid cells that will permit improved imaging of samples in the liquid phase (de Jonge and Ross 201 1). In combina -
tion with recent advances in ZP, EFTEM and DDD, the new generation of liquid cells offers considerable potential for the study of complex
dynamic cellular processes.
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Nermut MV 1991 Unorthodox methods of negative staining. Micron Microsc
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Nermut MV, Frank H 1971 Fine structure of Influenza A2 (SINGAPORE) as
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sertation No. 4271: Eidgenössiche Technische Hochschule. Zurich:
Federal Institute of Technology (ETH).
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Studer D, Graber W, Al-Amoudi A et al 2001 A new approach for cryofixation
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book for Materials Science. Berlin: Springer; p. 760. | 240 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
e26SECTION 1
COMMENTARY The reaction of peripheral nerves
to injury 1.6
Main nerve trunks contain many thousands of axons. When a trunk is
stretched, but not ruptured, by dislocation of, say, the femoral or
humeral heads, or by the nearby passage of a bullet, the axons within
that nerve may respond in different ways. The degree of injury may vary
along the length of a particular nerve or transversely across its cross-
section: some axons will remain intact, some will sustain conduction
block but recover rapidly, and yet others will degenerate throughout
their entire extent distal to the injury because they have been ruptured.
A proportion recover spontaneously whilst the remainder never recover.
Seddon (1943) introduced the terms neurapraxia for conduction
block, axonotmesis for a degenerative lesion of favourable prognosis,
and neurotmesis for a degenerative lesion of unfavourable prognosis.
Sunderland (1951) expanded this classification to five degrees of
peripheral nerve injury by subdividing neurapraxia according to the
degree of connective tissue disruption, and MacKinnon and Dellon
(1988) subsequently added a sixth category. However, the fundamental distinction between neurapraxia and either axonotmesis or neurotmesis
remains the persistence of conduction in the distal segment of the nerve
in neurapraxia, and the absence of conduction in axonotmesis or neu -
rotmesis. In clinical practice, neurotmesis usually signifies transection of an entire nerve trunk, its constituent fascicles and their surrounding
investments of perineurium and epineurium; the proximal and distal
nerve stumps are frequently separated by an inter-stump gap of varying
length ( Fig. 1.6.1). The early disappearance of conduction indicates
impending or actual critical ischaemia and is probably the most impor -
tant indicator that a lesion is deepening (see below).
Neurapraxia/conduction block
Conduction block exhibits certain characteristic features: paralysis exceeds loss of sensation; proprioceptive nerves are more deeply affected
than those responsible for light touch; and sympathetic nerves to the
smooth muscles of the blood vessels and sweat glands are least affected.
There are distinct patterns of conduction block. Anoxia is important in
all of them and there are often elements of mechanical deformation,
inducing alterations of the nodal and paranodal apparatus in myeli -
nated axons, and subsequent demyelination either of whole internodes or of paranodes.Transient ischaemia Lying in one position without moving for a
long time (e.g. during coma, inebriation or anaesthesia), prolonged
sitting with the legs crossed or prolonged leaning on the elbows are all
situations in which a limb nerve may be transiently compressed against
a bone or a hard surface. The ischaemia thus induced in the compressed
nerves causes an anoxic, physiological, block of both axoplasmic trans -
port and ion channel functions along affected axons. This is seen during
operation for the exposure of limb nerves with an inflated cuff in posi -
tion: stimulation of a nerve evokes a brisk muscular response by trans -
mission through the neuromuscular junction, which then diminishes
before disappearing after about 30 minutes, whereas conduction within
the nerve itself can be detected for about another 30 minutes. On the
other hand, direct stimulation of the muscle provokes a twitch that can
be elicited for about another 2 hours; loss of this direct response signals
impending muscle death. The selective vulnerability of different popu -
lations of nerve fibres is demonstrated by the classical experiment of
applying an inflated cuff about the arm (Lewis et al 1931). The observer
experiences first a loss of superficial sensibility, then a graduated loss of muscle power. The first pain response is lost soon after superficial
sensibility fails; the delayed pain response is still detectable after
40 minutes of ischaemia. Large myelinated axons are first affected;
unmyelinated axons and autonomic fibres escape; and pilo- and vaso -
motor functions are scarcely affected. All modalities recover within a few minutes of release of the cuff; the unpleasant quality of the residual
delayed pain sensation, and the burst of painful ‘pins and needles’ after
release of the cuff, give an insight into the feelings of patients affected
by dysaesthesiae.
Slowly progressing ischaemia Conduction block caused either
by a haematoma or aneurysm, or by bleeding into compartments pro -
duces early and deep autonomic paralysis and loss of power that extends over hours or days, whilst deep pressure sense and some joint
position sense persist. These symptoms are seen in war wounds, when
nerves become compressed and strangled by scar tissue or by the cicatrix
deep to split skin grafts, and may progress over a period of weeks or
months. The recovery of function and relief of pain are rapid – at times,
dramatic – after removal of the cause in most of these cases.
Prolonged conduction block associated with focal demyel
ination Severe prolonged pressure causes local demyelination and a
conduction block that is not directly attributable to anoxia and that
may last for weeks or months (Fig. 1.6.2). Compression-induced focal
paranodal myelin deformation may be involved; experimental studies
using a cuff inflated to high pressure around a limb produced slippage
of paranodal myelin at nodes of Ranvier lying under the edges of the
cuff, i.e. where the pressure gradient between compressed and non-
compressed portions of the nerve was likely to be greatest (Ochoa et al
1972, Dyck et al 200 5). Clinically, local demyelination and conduction
Fig. 1.6.1 Axonotmesis (centre) and neurotmesis (bottom) at the moment
of injury. Note the preservation of the Schwann cell basal lamina in
axonotmesis. (Courtesy of Rolfe Birch, all rights reserved, published in Birch R, Surgical Disorders of the Peripheral Nerve. 2nd edition, 2011. Springer-Verlag, London.)
Schwann cell nucleus Basal lamina
Node of Ranvier Myelin sheath Axon
Fig. 1.6.2 The effect of mechanical compression: paranodal and
juxtaparanodal myelin slippage under the edges of an inflated pressure cuff (after Ochoa J, Fowler TJ, Gilliatt RW 1972). (Courtesy of Rolfe Birch,
all rights reserved, published in Birch R, Surgical Disorders of the Peripheral Nerve. 2nd edition, 2011. Springer-Verlag, London.)Proximal Distal Cuff
AxonRolfe Birch | 241 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
The reaction of peripheral nerves to injury
e27
COMMENTAR Y 1.6
1.6.5). (iii) A nerve displaced into a fracture or joint will usually recover
if it is extricated within a few days, but will be destroyed if a compres -
sion device has been used to stabilize the skeleton without retrieving the nerve (Fig. 1.6.6). (iv) Function may be retained for a long time in
a nerve exposed to continuing traction from a malunited fracture. (v)
Nerves may continue to work for months or years before failing when
they are exposed to the progressing fibrosis caused by radiotherapy, by overlying split skin graft or by other examples of deep scar. (vi) Pain is the cardinal symptom of the unrelieved action of a noxious agent.
Wallerian degeneration
Trauma that physically separates axons from their cell bodies produces Wallerian degeneration (WD). All affected axons in the extreme tip of block will persist until the source of the external irritation, e.g. a bony
projection, is removed (Birch and St Clair Strange 1990). Relief of pain
and recovery follow rapidly in lesions that have persisted for many
months, or even years. Mechanical deformation is the likely explanation
for the conduction block seen in cases of ‘hourglass’ constriction of
fascicles within a nerve trunk, where axons may be focally constricted
within affected fascicles ( Fig. 1.6.3).
Prolonged conduction block of war wounds The characteristic
features of classical neurapraxia (see above), caused by the nearby passage of a missile, are likely to be provoked by a momentary displace -
ment or stretching of the nerve trunks. However, a different pattern of conduction block has been recognized in recent conflicts, where the
patient is exposed, at close range, to the shock wave of an explosion
without any wound or fracture, or signs of significant injury to the soft
tissues at the level of the nerve lesion. In these cases, the smallest fibres
are most deeply affected and they may not recover. Pain is rare in both
of these variants of conduction block (Birch et al 2012).
Degenerative lesions
There are essentially two types of degenerative lesion, with very different potentials for recovery. In the first, which approximates to axonotmesis,
the tubes of Schwann cell basal lamina that surround each axon–
Schwann cell unit remain intact, which means that axons may regener -
ate in an orderly fashion across the lesion site and into the distal stump, providing that the causative agent is removed and that the length of the
distal stump (i.e. the distance from the site of injury to the end organ)
is not so long that it compromises axonal regrowth. In the second,
which approximates to neurotmesis, not only the Schwann cell basal
lamina tubes, but also the connective tissue sheaths around the fascicle
are interrupted; spontaneous axonal regeneration following this type of
injury will be imperfect and disorderly, or may not occur at all.
Deepening of a lesion in a nerve that has
not been divided
Unrelieved activity of a noxious agent on an intact nerve is signified by
pain and increasing neurological deficit, indicating that the lesion is
deepening. Nerves within a swollen ischaemic limb or tense compart -
ment become compressed and anoxic. Nerves displaced by an expand -
ing haematoma become stretched and anoxic. Nerves that have become
displaced into a fracture or joint are tethered and become subjected to
compression, stretching and anoxia. These nerves are not severed by the
initial injury; the first lesion is axonotmesis and spontaneous recovery
is probable if the cause is removed. However, if this does not happen,
the lesion continues to deepen into that of neurotmesis and spontan -
eous recovery will not occur. The speed of deepening of a lesion is
related to the cause: (i) A nerve crushed by a plate or strangled by an encircling suture may recover if the cause is removed within a few minutes (Fig. 1.6.4). (ii) A nerve that has become compressed and anoxic within a swollen ischaemic limb will recover if perfusion of the
tissues is restored within 3 hours; the likelihood of full spontaneous
recovery diminishes with the passage of every hour after that time ( Fig. Fig. 1.6.3 Hourglass ‘constriction’ of the lateral root of the median nerve:
the result of traction injury. There was full spontaneous recovery.
(Courtesy of Rolfe Birch, all rights reserved, published in Birch R, Surgical
Disorders of the Peripheral Nerve. 2nd edition, 2011. Springer-Verlag, London.)
Fig. 1.6.4 The appearance of a radial nerve after extrication from beneath
a compression plate 48 hours after the first operation. There was relief of
pain but only incomplete recovery so that subsequent flexor to extensor transfer was necessary. (Courtesy of Rolfe Birch, all rights reserved,
published in Birch R, Surgical Disorders of the Peripheral Nerve. 2nd
edition, 2011. Springer-Verlag, London.)
Fig. 1.6.5 Volkmann’s ischaemic contracture. A, The ulnar nerve exposed
during flexor muscle slide 8 weeks after supracondylar fracture. The
epineurial vessels and also the ulnar recurrent collateral vessels are occluded, and the nerve is compressed by the swollen infarcted muscle.
B
, The appearance of the hand 14 years later. (Courtesy of Rolfe Birch, all
rights reserved, published in Birch R, Surgical Disorders of the Peripheral
Nerve. 2nd edition, 2011. Springer-Verlag, London.)
A
B | 242 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
ThE REACTION Of pERIphERA l NER vES TO INjuRY
e28SECTION 1
involved in retrograde signalling to initiate expression of RAGs, particu -
larly the family of glycoprotein 130 (gp130) cytokines, see Zigmond
(201 1).) Changes in the expression of ion channels and receptors in the
neuronal cell bodies in human DRGs can be detected within a few
minutes of an injury to the nerve (Lawson 2005).
Tip of proximal stump Within a few hours of injury, the cut ends
of a transected axon seal off, forming end bulbs. The end bulb that forms at the proximal tip of axons in the proximal stump is transformed
into a growth cone from which multiple needle-like filopodia and
broader sheet-like lamellipodia grow out. The filopodia are rich in
actin, and may extend or retract within a matter of minutes. Each axon forms new branches or sprouts: collateral sprouts arise from nodes of
Ranvier at levels where the axons are still intact; terminal sprouts arise
from the tips of the surviving axons. The axon sprouts and fine cyto -
plasmic processes derived from their associated Schwann cells form clusters, regenerating units, surrounded by Schwann cell basal laminae.
Sprouts within one regenerating unit represent the regenerative effort
of one neurone and its axon. Within a few days of injury, the calibre of
axons in the proximal stumps is reduced and nerve conduction velocity
in the proximal segment falls.
Distal stump During the first 2 or 3 weeks after injury, the constitutive
tissue response within a denervated distal stump involves numerous events: some sequential, others consecutive. These include the produc-
tion of debris, as axons and their myelin sheaths are degraded; an
increase in local blood flow; activation of resident macrophages, and
recruitment and influx of exogenous macrophages; and proliferation of
fibroblasts and Schwann cells. The gliotic response produces a popula -
tion of dedifferentiated daughter Schwann cells that remain within the basal lamina tubes secreted by their parent cells, forming what are now
called Schwann tubes but which were previously known as bands of
Büngner. They behave acutely as the ‘presumed targets’ of the regrowing
axons until the actual targets (muscle or sensory end organ) have been
reinnervated: they secrete proteins that collectively facilitate axonal
regrowth, whether by providing trophic support or by establishing a
supportive growth matrix (for further reading on these interactions, see
Hall (2005), Webber et al (201 1), Arthur-Farraj et al (2012), Gordon
(2015)). There is a growing consensus that Schwann cells express dis -
tinct motor and sensory phenotypes, and that this fundamental differ -
ence affects the ability of Schwann cell tubes to selectively support regenerating neurones. For example, expression of osteopontin and
clusterin is upregulated in Schwann cells in transected peripheral
nerves: the two secreted factors appear to facilitate regeneration of
motor or sensory neurones, respectively (Wright et al 2014).
While the microenvironment of an acutely denervated distal stump
facilitates axonal regrowth because it provides a vascularized segment
of longitudinally orientated, laminin-rich basal lamina tubes filled with
axon-responsive Schwann cells, a chronically denervated distal stump
is associated with poor axonal regeneration and poor functional recov -
ery (Sulaiman and Gordon 2009). Biopsies taken during late repairs, especially when the injury has been complicated by arterial injury or
by sepsis, often show remarkably few Schwann cells lying within a
densely collagenous matrix. Myelin fragments are detectable in such
cases many months after the injury. The residual Schwann cells become
progressively less receptive to ingrowing regenerating axons with time;
experimental studies have shown that they downregulate expression of
axon-responsive receptors that are normally important in Schwann
cell–axon signalling (Li et al 1997, Li et al 1998). These findings under -
score the clinical observation that there is a relatively narrow window of opportunity when surgical intervention is most likely to produce
positive results (Lundborg 2000).
The special case of preganglionic injury
The preganglionic lesion is all too common in severe traction injuries to the brachial and lumbosacral plexuses. The roots of the spinal nerves
are torn from the spinal cord, which means that the somatic afferent
pathway is interrupted between the dorsal root ganglion and the spinal
cord (see Fig. 1.6.6). The neuronal cell bodies and their axons, investing
Schwann cells and associated basal laminae remain intact, healthy and conducting for a long time after the injury. The surviving axons include
all those with cell bodies in the dorsal root ganglion, including many
‘recurrent’ fibres in the ventral root that derived from cells in the dorsal root ganglion ( Figs 1.6.7–1.6.9). Somatic efferent fibres, being sepa -
rated from their cell bodies, degenerate; postganglionic autonomic efferent axons also degenerate, as a result of damage to their grey rami communicantes. Carlstedt (2007) reckons that about one-half of all
motor neurones in the affected spinal cord segment have disappeared
by 2 weeks after avulsion of the ventral root and he urges ‘a swift the proximal stump and throughout the distal stump degenerate, irre -
spective of their calibre or functional modality; recovery is often slow
and incomplete, and there may be considerable residual loss of func -
tion. Moreover, although research has tended to address issues associ -
ated with the molecular and cellular events that occur at the lesion site
and target tissues, it is important to remember that injury to a periph -
eral nerve that induces WD also initiates significant and often long-lasting central changes in sensory ganglia and ascending and descending
pathways within the spinal cord and brain; these changes also have a
significant impact on the likelihood of recovery. Studies of cell bodies
of human dorsal root ganglia (DRG) have revealed changes in the
expression of a wide range of receptors and a remarkable alteration in
the expression of genes regulating neuronal activity (Lawson 2005,
Rabert et al 2004, Anand et al 2008). There is an extensive literature on
WD in both central and peripheral nervous systems, and only a brief
summary of the response in the peripheral nervous system is presented
here (see Gordon (2015)).
Neuronal cell body and proximal stump The central and the
peripheral effects of WD are profound and, in neurotmesis, ultimately irreversible. Not only is the neuronal cell body separated from its con -
tinual supply of retrogradely transported neurotrophins, but also its central connections rapidly alter; many neurones thus isolated will die.
Amputation provides a model of the effect of permanent axonotomy
on the spinal cord: there is extensive loss of neurones in the dorsal root
ganglia and in the anterior horn, and a diminution of the large myeli -
nated axons in the ventral and dorsal roots (Dyck et al 1984, Suzuki
et al 1993). Neuronal death is more severe in more proximal neurot -
mesis, and less marked after axonotmesis than after neurotmesis. Neu -
rotmesis in the neonate produces a more rapid and much greater
incidence of sensory and motor neurone death than in the adult. Surviv -
ing neurones rapidly upregulate a series of genes involved in repair and neuroprotection mechanisms following injury (repair-associated genes, RAGs), which direct many of the changes seen during WD and subse-
quent recovery (e.g. Calenda et al 2012). (For a review of the molecules Fig. 1.6.6 Deepening of a lesion. A, A median nerve extricated from a
supracondylar fracture in a 9-year-old girl 3 days after injury; there was
complete recovery. B, A median nerve extricated from a supracondylar
fracture in a 13-year-old girl 8 weeks after injury; there was no recovery.
(Courtesy of Rolfe Birch, all rights reserved, published in Birch R, Surgical
Disorders of the Peripheral Nerve. 2nd edition, 2011. Springer-Verlag,
London.)
BA | 243 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
The reaction of peripheral nerves to injury
e29
COMMENTAR Y 1.6
Regeneration of end organs
Reinnervation of muscle spindles and Golgi tendon receptors is gener -
ally good after crush lesions (axonotmesis) . Regeneration after repair
of a divided nerve is much less orderly. A muscle spindle may become
reinnervated by afferents normally destined for the Golgi tendon organ;
many tendon organs remain denervated and the regenerated endings
are frequently abnormal in appearance. The reorganization of motor
units after repair causes significant alterations in the mechanical input
to an individual tendon organ.
Muscles usually exhibit weakness, impaired coordination and
reduced stamina after nerve repair. There may be selective failure of
regeneration of the largest-diameter fibres and of coactivation of the α
and γ efferents. The normal movement of joints is brought about by
smoothly coordinated and controlled activity in muscles that is pre -
cisely and delicately regulated by inhibition and facilitation of the motor neurones. The conversion of an antagonist to an agonist is the
basis of musculotendinous transfer; it is common to see patients
actively extending the knee or the ankle and toes as soon as the post -
operative splint is removed after hamstring to quadriceps transfer or anterior transfer of tibialis posterior. Reinnervated muscles usually fail
to convert after muscle transfer, irrespective of their power. Perhaps the
defective reinnervation of the deep afferent pathways from the muscle
spindles and the tendon receptors blinds muscles, which are, after all,
sensory as well as effector organs.
Cutaneous sensory receptors similarly undergo a slow degenerative
change after denervation and after 3 years they may disappear. Re -
innervation tends to reverse these changes, although the longer the
period of denervation, the less complete will be the regeneration. It is
usual to find numerous myelinated axons in the tissues bridging nerve
stumps in the human, even in failed sutures or in grafts coming to revi -
sion (Fig. 1.6.10 ) (Terenghi et al 1998); these findings encapsulate the
difference between regeneration and recovery of function.
Plasticity in the central nervous system
Nerve transfer involves connecting the proximal stump of a healthy nerve to the distal stump of one that has been injured in such a way
that direct repair is not possible.
There is considerable adaptation of the central receptor and effector
mechanisms after nerve transfer ( Fig. 1.6.1 1). Independent flexion of
the elbow without associated activity in the flexor muscles of the
forearm is usual by 24 months after transfer from the ulnar nerve to the
nerve to biceps brachii. Reinnervation of the muscle spindles and cor -
responding reorganization and remapping of the somatosensory cortex
have been confirmed after transfer of intercostal nerves to the musculo -
cutaneous nerve in adult patients with severe lesions of the brachial
plexus (Malessy et al 2003, Sai et al 1996). These central phenomena
may explain the remarkably good recovery of sensation within the hand Fig. 1.6.8 Wallerian degeneration in the ventral root of the eighth cervical
nerve, 6 weeks after avulsion from the spinal cord. A degenerate efferent
myelinated fibre (right) compared with a healthy myelinated afferent fibre
(left). Magnification ×11115. (Courtesy of Rolfe Birch, all rights reserved,
published in Birch R, Surgical Disorders of the Peripheral Nerve. 2nd
edition, 2011. Springer-Verlag, London.)
Fig. 1.6.9 Afferent and efferent fibres in a ventral root. Healthy myelinated
and unmyelinated fibres in the ventral root of the eighth cervical nerve
avulsed from the spinal cord 6 weeks previously. The myelinated efferent
fibres have undergone Wallerian degeneration and there is a notable
increase in the amount of endoneurial collagen. (Courtesy of Rolfe Birch,
all rights reserved, published in Birch R, Surgical Disorders of the
Peripheral Nerve. 2nd edition, 2011. Springer-Verlag, London.)
intervention to re-establish contact between the injured nerve cells and
the periphery with its supply of neurotrophic substances to counteract
nerve cell loss’ . After avulsion injury, human dorsal root ganglion neur -
ones show dramatic changes in the expression of genes involved in neurotransmission, trophism, cytokine function, signal transduction,
myelination, transcription regulation and apoptosis (Rabert et al 2004).
Regeneration and recovery of function
‘But the journey of the axon tip to an end organ is only the most dramatic of the phases in the process of regeneration, and its arrival is
alone no guarantee of the return of useful function. ’
Young (1942)
The cellular microenvironment of a normal peripheral nerve does not ordinarily support axonal regrowth; indeed, there appear to be ‘molecu-
lar brakes’ that prevent axonal sprouting. Somewhat counterintuitively,
it appears that their expression may be upregulated after injury. WD transforms the environment throughout the distal stump of a transected nerve to one that facilitates axonal regrowth, albeit for a relatively short period (Christie and Zochodne 2013). Manipulating the many cellular and molecular responses that occur during the injury response in the peripheral nervous system remains an as yet unachieved goal of recon -
structive surgery.Fig. 1.6.7 Intact
(afferent) myelinated
and unmyelinated fibres
in a suprascapular
nerve, 6 weeks after an
avulsion lesion of the
brachial plexus;
efferent fibres have degenerated.
Magnification ×6600.
(Courtesy of Rolfe Birch, all rights
reserved, published in
Birch R, Surgical
Disorders of the
Peripheral Nerve. 2nd
edition, 2011. Springer-
Verlag, London.) | 244 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
ThE REACTION Of pERIphERA l NER vES TO INjuRY
e30SECTION 1
whilst afferent impulses from the little and, probably, the ring fingers
entered the spinal cord through the fifth cervical nerve as shown in
Figure 1.6.12. Similar findings have been observed in four other cases
after transfer of intercostal nerves to the lateral cord ( Fig. 1.6.13). The
situation in the adult is very different. After transfer of the intercostal nerves to the lateral or medial cords, it is usual to find that stimulation
of the reinnervated skin is referred to the chest wall and only rarely to
the skin of the hand (Htut et al 2006). Concepts of the mechanisms
underlying cortical plasticity following nerve injury and the importance
of maintaining an active sensory map of the affected part in the somato -
sensory cortex during the deafferentation period are considered further
by Lundborg (2003), Rosén and Lundborg (2007), Björkman et al
(2009), Rosén et al (201 1) and Knox et al (2015) .
Laboratory and clinical studies
There are major differences between a laboratory investigation, in
which a controlled, precise and limited lesion is inflicted on a periph -
eral nerve in an anaesthetized experimental animal, and the situation faced by the clinician presented with a patient with a massive wound involving soft tissues (including blood vessels and muscles) and skeletal Fig. 1.6.10 Useless regeneration in a median nerve sutured 3 years
previously. Proximal to the suture line, the endoneurium contains thinly
remyelinated axons, some in regenerating clusters, and extensive
collagenization; there was no recovery of function. Magnification ×4300.
(Courtesy of Rolfe Birch, all rights reserved, published in Birch R, Surgical Disorders of the Peripheral Nerve. 2nd edition, 2011. Springer-Verlag,
London.)
Fig. 1.6.11 A 13-year-old boy with a right-sided lesion: avulsion of C5,
6 and 7. Repair was undertaken at 2 months as follows: accessory to
suprascapular transfer; one bundle of the ulnar nerve to the nerve to
biceps and the nerve to the medial head of triceps; medial cutaneous
nerve of the forearm to the lateral root of the median nerve. Results at
18 months show a full range of lateral rotation; abduction to 60°; power of
elbow flexion, Medical Research Council (MRC) grade 4; power of elbow
extension, MRC grade 3 +. There is a full range of active flexion and
extension without obvious co-contraction. (Courtesy of Rolfe Birch, all rights reserved, published in Birch R, Surgical Disorders of the Peripheral
Nerve. 2nd edition, 2011. Springer-Verlag, London.)
Fig. 1.6.12 The method of repair and the likely pathways for afferent
function. Abbreviations: CS, cool sensation; CW, cotton wool; JPS,
joint position sense; PP, pinprick; Vib, vibration threshold; WS, warm sensation. (Courtesy of Rolfe Birch, all rights reserved, published in Birch
R, Surgical Disorders of the Peripheral Nerve. 2nd edition, 2011.
Springer-Verlag, London.)c5
c6c7
C6, C7via
T3–T5C8
via
C5
RH Age 6y
Normal
CW , PP , VIBJPS, CS, WSlocalizationLat.SS
C5
C6
C7
C8
T1
T3
T4
T5XI
C8
T1
Fig. 1.6.13 Birth lesions of the brachial plexus. This boy had a group 4
lesion with rupture of C5 and avulsion of C6, 7, 8 and T1 at birth. At
10 weeks of age, C5 was grafted to the upper trunk and to the ventral
root of C8 and T1; accessory nerve was transferred to the ventral root
of C7; the sensory divisions of intercostal nerves T3 and T4 were
transferred to the lateral root of the median nerve. He regained a useful
grasp between thumb and index finger, and is an extremely well-adjusted
sportsman and musician. The photograph was taken at 10 years of age.
(Courtesy of Rolfe Birch, all rights reserved, published in Birch R, Surgical Disorders of the Peripheral Nerve. 2nd edition, 2011. Springer-Verlag,
London.)
after repair of severe birth lesions of the brachial plexus. Indeed, the
sensory recovery is far better than that of skeletal muscle and sympa -
thetic function. Thus, there is accurate localization to the dermatomes
of avulsed spinal nerves that had been reinnervated by intercostal nerves
transferred from remote spinal segments (Anand and Birch 2002).
Case report
Operation in a 6-month-old boy confirmed rupture of C5 with avulsion
of C6, 7, 8 and T1. The lesion was repaired by transfers of the accessory
nerve to the suprascapular nerve, of C5 to C8 and T1, and of intercostal
nerves T3, 4 and 5 to the lateral cord of the brachial plexus. At the age
of 5 years, he was able to localize cotton wool and pinprick sensation
accurately in both hands and was able to feel a pin in the affected side. Monofilament, vibration thresholds and joint position sense were iden -
tical in both hands. The thresholds for warming were 3.1°C (C5), 0.8°C
(C6), 4.1°C (C7), 3.6°C (C8) and 3.3°C (T1) on the affected side. The thresholds for cooling were 2°C (C5), 3.9°C (C6), 3.8°C (C7), 2.3°C (C8) and normal in T1. In the unaffected hand, the thresholds were less than 3.4°C for warm sensation and less than 2.2°C for cool sensa -
tion. Sweating was 70% of that of the intact palm. It seems likely that afferent impulses from the thumb, index and, probably, the middle fingers entered the spinal cord through the three intercostal nerves, | 245 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
The reaction of peripheral nerves to injury
e31
COMMENTAR Y 1.6
elements, and sometimes with other injuries that threaten life and limb.
That said, the fundamental cellular processes that underlie subsequent
regeneration are similar in the laboratory, in the injuries of civilian
practice and in the wounds of war, although in clinical practice they are
modified by the violence of the injury, by the effects of injury on associ -
ated tissues and, in particular, by ischaemia and by delay before repair. Some general conclusions may be drawn from extensive clinical and
laboratory studies.
The concept of retrograde degeneration of the axon extending to the
first internode applies only to the most benign lesion: that of experi -
mentally crushing the nerve between the tips of a jeweller’s forceps. The extent of longitudinal damage to a nerve is greater in a rupture caused
by traction than it is in ‘tidy’ transections caused by knife or glass; this
effect worsens with increasing delay before repair, is worse still when
healing has been complicated by sepsis, and worst of all in neglected
ischaemia.
Failure to repair main vessels and to ensure perfusion of tissues is
profoundly deleterious to regeneration and rules out worthwhile recov-
ery of function.
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Zigmond RE 201 1 Gp130 cytokines are positive signals triggering changes
in gene expression and axon outgrowth in peripheral neurons following
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one that facilitates regeneration to one that is less favourable. Dense
collagenization, a profusion of fibroblasts and a loss of Schwann cells
are characteristics of the chronically denervated distal stump and are
typical of late cases.
The normal architecture of a nerve is most closely restored to normal
in a well-executed, tension-free primary suture.
Regeneration through a graft falls away along its length and not
solely at the suture lines.
Delay before repair leads to increasing fibrosis and to shrinking of
the distal segment so that it becomes impossible to ensure an accurate
topographical match.
Destruction of the target tissues, of muscle and skin, limits function
even when there is strong regeneration.
Chronological age plays an important role: for example, signalling
mechanisms involved in the reinnervation of skeletal muscle may
become impaired with age (Kawabuchi et al 201 1). | 246 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
163
CHAPTER 8 Preimplantation development
involve modifications of membrane sterols or surface proteins. They
traverse the cumulus oophorus and corona radiata, then bind to specific
glycoprotein receptors on the zona pellucida, ZP3 and ZP2. Interaction
of ZP3 with the sperm head induces the acrosome reaction, in which
fusion of membranes on the sperm head releases enzymes, such as
acrosin, which help to digest the zona around the sperm head, allowing
the sperm to reach the perivitelline space. In the perivitelline space,
the spermatozoon fuses with the oocyte microvilli, possibly via two disintegrin peptides in the sperm head and integrin in the oolemma
(Figs 8.4, 8.5A).
Fusion of the sperm with the oolemma causes a weak membrane
depolarization and leads to a calcium wave, which is triggered by the
sperm at the site of fusion and crosses the egg within 5–20 seconds. The
calcium wave amplifies the local signal at the site of sperm–oocyte interaction and distributes it throughout the oocyte cytoplasm. The
increase in calcium concentration is the signal that causes the oocyte to
resume cell division, initiating the completion of meiosis II and setting
off the developmental programme that leads to embryogenesis. The
pulses of intracellular calcium that occur every few minutes for the first
few hours of development also trigger the fusion of cortical granules
with the oolemma. The cortical secretory granules release an enzyme
that hydrolyses the ZP3 receptor on the zona pellucida and so prevents
other sperm from binding and undergoing the acrosome reaction, thus
establishing the block to polyspermy. The same cortical granule secre -
tion may also modify the vitelline layer and oolemma, making them less susceptible to sperm–oocyte fusion and providing a further level of
polyspermy block.
The sperm head undergoes its protamine → histone transition as the
second polar body is extruded. The two pronuclei grow, move together
and condense in preparation for syngamy and cleavage after approxi -
mately 24 hours ( Fig. 8.5B). Nucleolar ribosomal ribonucleic acid
(rRNA), and perhaps some messenger RNA (mRNA), are synthesized in pronuclei. A succeeding series of cleavage divisions produces eight
even-sized blastomeres at approximately 55 hours, when embryonic
mRNA is transcribed.
Several examples of cells that contain male and female pronuclei,
termed ootids, have been described. Pronuclear fusion as such does not
occur; the two pronuclear envelopes disappear and the two chromo -
some groups move together to assume positions on the first cleavage spindle. No true zygote containing a membrane-bound nucleus is
formed.
The presence of the pronuclei from both parental origins is crucial
for spatial organization and the controlled growth of cells, tissues and
organs. In the mouse, embryos in which the paternal pronucleus has
been removed and replaced with a second maternal pronucleus develop
to a relatively advanced state (25 somites), but with limited develop -
ment of the trophoblast and extraembryonic tissues. In contrast, embryos in which the maternal pronucleus has been replaced by a
second paternal pronucleus develop very poorly, forming embryos of
only six to eight somites, but with extensive trophoblast. Thus it seems
that the maternal genome is relatively more important for the develop -
ment of the embryo, whereas the paternal genome is essential for the development of the extraembryonic tissues that would lead to placental
formation.
This functional inequivalence of homologous parental chromo -
somes is called parental imprinting. The process causes the expression
of particular genes to be dependent on their parental origin; some genes
are expressed only from the maternally inherited chromosome and
others from the paternally inherited chromosome. The genes involved are called imprinted genes. The requirement for both parental genomes is limited to a subset of the chromosomes. Uniparental disomy can arise through meiotic and mitotic non-disjunction events, and results in individuals who are completely disomic or who exhibit mosaicism of disomic and non-disomic cells. If imprinted genes reside on the affected chromosomes, then the uniparental disomic cells will either express a Understanding the spatial and temporal developmental processes that
take place within an embryo as it develops from a single cell into a
recognizable human is the challenge of embryology. The control of
these processes resides within the genome; fundamental questions
remain concerning the genes and interactions involved in development.
STAGING OF EMBRYOS
For the purposes of embryological study, prenatal life is divided into an embryonic period and a fetal period. The embryonic period covers
the first 8 weeks of development (weeks following ovulation and fertil -
ization resulting in pregnancy). The ages of early human embryos have previously been estimated by comparing their development with that
of monkey embryos of known postovulatory ages. Because embryos
develop at different rates and attain different final weights and sizes, a
classification of human embryos into 23 stages occurring during the
first 8 weeks after ovulation was developed most successfully by Streeter
(1942), and the task was continued by O’Rahilly and Müller (1987).
An embryo was initially staged by comparing its development with that
of other embryos. On the basis of correlating particular maternal men -
strual histories and the known developmental ages of monkey embryos,
growth tables were constructed so that the size of an embryo (specifi -
cally, the greatest length) could be used to predict its presumed age in postovulatory days (synonymous to postfertilizational days). O’Rahilly
and Müller (2000, 2010) emphasize that the stages are based on external and internal morphological criteria and are not founded on length or
age. Ultrasonic examination of embryos in vivo has necessitated the
revision of some of the ages related to stages, and embryos of stages
6–16 are now thought to be up to 3 to 5 days older than the previously
used embryological estimates (O’Rahilly and Müller 1999, 2010). Within this staging system, embryonic life commences with fertilization
at stage 1; stage 2 encompasses embryos from two cells, through com -
paction and early segregation, to the appearance of the blastocele. The developmental processes occurring during the first 10 stages of embry-
onic life are shown in Figure 8.1.
Much of our knowledge of the early developmental processes is
derived from experimental studies on amniote embryos, particularly the
chick, mouse and rat. Figure 8.2 shows the comparative timescales of
development of these species and human development up to stage 12. The size and age, in postovulatory days, of human development from
stage 10 to stage 23 is given in Figure 8.3.
Information on developmental age after stage 23 (8 weeks post
ovulation) is shown in Figure 14.3, where the developmental staging
used throughout this text is juxtaposed with the obstetric estimation of gestation that is used clinically. A critique of staging terminology and
the hazards of the concurrent use of gestational age and embryonic age
is given in Chapter 14; sizes and ages of fetuses towards the end of
gestation are illustrated in Fig. 14.9.
FERTILIZATION
The central feature of reproduction is the fusion of the two gamete
pronuclei at fertilization. In humans, the male gametes are spermato -
zoa, which are produced from puberty onwards. Female gametes are released as secondary oocytes in the second meiotic metaphase, usually
singly, in a cyclical fashion. The signal for the completion of the second meiotic division is fertilization, which stimulates the cell division cycle to resume, completing meiosis and extruding the second polar body (the second set of redundant meiotic chromosomes).
Fertilization normally occurs in the ampullary region of the uterine
tube, probably within 24 hours of ovulation. Very few spermatozoa
reach the ampulla to achieve fertilization. They must undergo capacita -
tion, a process that is still incompletely understood, and which may | 248 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Preim Plantation develo Pment
164SeCtion 2
tation genetic diagnosis (PGD). A sample (biopsy) is removed from
either the oocyte (polar bodies), the embryo itself (a blastomere) or the
blastocyst (small piece of trophectoderm), and subjected to specific
genetic testing. Oocytes and embryos can also be biopsied and screened
for aneuploidy (errors in chromosome copy numbers) in preimplanta -
tion genetic screening (PGS). Unaffected embryos can then be identified for transfer to the patient. Embryos that are surplus to immediate thera -
peutic requirements can also be cryopreserved in liquid nitrogen for later use. Propanediol or dimethylsulphoxide are commonly used as
cryoprotectants for early embryos, and, like glycerol, may be used for
blastocysts. Conception rates per cycle using ovarian stimulation, IVF
and successive transfers of fresh and cryopreserved embryos exceed
those obtained during non-assisted, or natural, conception.
PREIMPLANTATION DEVELOPMENT
Cleavage
The first divisions of the zygote are termed cleavages. They distribute the cytoplasm approximately equally among daughter blastomeres, so,
although the cell number of the preimplantation embryo increases, its
total mass actually decreases slightly ( Fig. 8.6). Cell division can be
asynchronous and daughter cells may retain a cytoplasmic link through much of the immediately subsequent cell cycle via a midbody, as a result of the delayed completion of cytokinesis. No centrioles are
present until 16–32 cells are seen, but amorphous pericentriolar mat -
erial is present and serves to organize the mitotic spindles, which are characteristically more barrel- than spindle-shaped at this time.double dose of the gene or have both copies repressed. For example,
the gene encoding the embryonal mitogen insulin-like growth factor II
is expressed from the paternally inherited chromosome, and repressed
when maternally inherited.
In vitro fertilization
In vitro fertilization (IVF) of human gametes is a successful way of
overcoming most forms of infertility ( Video 8.1). Controlled stimula -
tion of the ovaries (e.g. pituitary downregulation using gonadotrophin-releasing hormone superactive analogues, followed by stimulation
with purified follicle stimulating hormone or urinary menopausal
gonadotrophins) enables many preovulatory oocytes (often 10 or
more) to be recruited and matured, and then aspirated transvaginally
using ultrasound guidance, 34–38 hours after injection of human cho -
rionic gonadotrophin (which is given to mimic the luteinizing hormone surge). These oocytes are then incubated overnight with
motile spermatozoa in a specially formulated culture medium, in an
attempt to achieve successful in vitro fertilization. In cases of severe
male-factor infertility, in which there are insufficient normal spermato -
zoa to achieve in vitro fertilization, individual spermatozoa can be
directly injected into the oocyte in a process known as intracytoplas -
mic sperm injection (ICSI), which is as successful as routine IVF. In
cases in which there are no spermatozoa in the ejaculate, suitable material can sometimes be directly aspirated from the epididymis or surgically retrieved from the testes, and the extracted sperm are then used for intracytoplasmic injection of sperm.
It is also now possible, in some cases, to test embryos for the pres -
ence of a particular genetic or chromosomal abnormality by preimplan -
Fig . 8 .1 Developmental processes occurring during the first 10 stages of development . In the early stages, a series of binary choices determine the cell
lineages . Generally, the earliest stages are concerned with formation of the extraembryonic tissues, whereas the later stages are concerned with the
formation of embryonic tissues . Oocyte
Ootid
ZygoteTropho-
blastSyncytio-trophoblast
Cyto-trophoblast
Hypoblast
Innercellmass
EpiblastLacunae
Chorion VilliIntervillousspaces
AllantoisSecondary
yolk sacExtraemb. mesenchyme Haemopoiesis
Primary yolk
sac
Connecting stalkVisceralhypoblast
Parietalhypoblast
Extraemb.mesoblast
Amnion
Primordial germ cells
Primitive streak
Notochordal
process/plate
Embryonic
endoderm
Mesoblast
Caudal eminenceMesenchyme
Somatopleuric and
splanchnopleuric coelomic epithelium
Epithelial somites
Neuralectoderm
Neuralcrest
Ectodermal placodes
SurfaceectodermDescription of stage
FertilizationFirst cleavage
Preimplantation
Compaction
Free blastocyst
hatched from zona
Implantation
Implanted
previllus
Secondary yolk sac
Primitive streak
Notochord
Neurenteric canal
Before folding
Neural groove
Somites
Intraemb. coelom
Neural tube
Neural crestAfter folding1
2
3
4
5a
5b
5c
6a
6b
7
9
10
118
Approx.
age in days2–3 4–5 18–21 24–27 28–30
26–29 21–256
7–1216–18 1 | 249 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Preimplantation development
165
CHaPter 8
All cleavage divisions after fertilization are dependent on continuing
protein synthesis. In contrast, passage through the earliest cycles, up to
eight cells, is independent of mRNA synthesis. Thereafter, experimental
inhibition of transcription blocks further division and development,
indicating that activation of the embryonic genome is required. There
is also direct evidence for the synthesis of embryonically encoded pro -
teins at this time. As the genes of the embryo first become both active and essential, the previously functional maternally derived mRNA is
destroyed. However, protein made on these maternal templates does
persist at least during blastocyst growth. Spontaneous developmental
arrest of embryos cultured in vitro seems to occur during the cell cycle
of gene activation, but it is not caused by total failure of that activation process. Early cleavage, up to the formation of eight cells, requires
pyruvate or lactate as metabolic substrates, but thereafter more glucose
is metabolized and may be required.
The earliest time at which different types of cells can be identified
within the cleaving embryo is when 8–16 cells are present. Up to the
formation of eight cells, cells are essentially spherical, touch each other
loosely, and have no specialized intercellular junctions or significant
extracellular matrix; the cytoplasm in each cell is organized in a radially
symmetric manner around a centrally located nucleus. Once eight cells
have formed, a process of compaction occurs. Cells flatten on each other
to maximize intercellular contact, initiate the formation of gap and
focal tight junctions, and radically reorganize their cytoplasmic confor -
mation from a radially symmetric to a highly asymmetric phenotype. This latter process includes the migration of nuclei towards the centre
of the embryo, the redistribution of surface microvilli and an underly-
ing mesh of microfilaments and microtubules to the exposed surface, and the localization of endosomes beneath the apical cytoskeletal mesh. As a result of the process of compaction, the embryo forms a primitive protoepithelial cyst, which consists of eight polarized cells, in which the apices face outwards and basolateral surfaces face internally. The focal tight junctions, which align to become increasingly linear, are localized to the boundary between the apical and basolateral surfaces. Fig . 8 .2 Within developmental biology, evidence concerning the nature of developmental processes has come mainly from studies in vertebrate
embryos, most commonly amniote embryos of the chick, mouse and rat . This chart illustrates the comparative timescale of development in these
animals and in humans .
Fertilization
Incubation
Implantation
Primitive
streak
Neural
plate
Pharyngula arches
15–20 somites,
rostral neuroporeclosed
Upper
limb bud
Lowerlimb bud
Days 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Chick
Mouse
Rat
Human(days from onset
of incubation)
(days from time
of mating)
(days from time
of mating)
(days post ovulation)
Fig . 8 .3 Human developmental stages 10–23 . The greatest embryonic
length in mm (ordinate) is plotted against age in postfertilizational
weeks (abscissa), with the stages superimposed according to current
information . (Data provided by courtesy of Professor R O’Rahilly . See also
O’Rahilly and Müller (2010) .)Days post fertilization20
1530 Stage
23
22
21
20
19
18
17
16
15
14
13
1112
10
9 87625
10
5
14 21 28 35 42 49 560Size (mm) | 250 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Preim Plantation develo Pment
166SeCtion 2
elements of compaction. The entire process can function in the absence
of both mRNA and protein synthesis. Post-translational controls are
sufficient and seem to involve regulation through protein phosphoryl -
ation. Significantly, although E-cadherin is not synthesized and present on the surface of cleaving blastomeres, it first becomes phosphorylated when eight cells are visible, at the initiation of compaction.
The process of compaction is important for the generation of cell
diversity in the early embryo. As each polarized cell divides, it retains
significant elements of its polar organization, so that its daughter cells
inherit cytocortical domains, the nature of which reflects their origin and organization in the original parent cell in the eight-celled embryo. Thus, if the axis of division is aligned approximately at right angles to the axis of cell polarity, the more superficially placed daughter cell inherits all the apical cytocortex and some of the basolateral cytocortex and is polar, whereas the more centrally placed cell inherits only baso -
lateral cytocortex and is apolar. In contrast, if the axis of division is
aligned approximately along the axis of the cell polarity, two polar
daughter cells are formed. In this way, two-cell populations are formed in the 16-cell embryo that differ in phenotype (polar, apolar) and posi -
tion (superficial, deep). The number of cells in each population in any one embryo will be determined by the ratio of divisions along, and at right angles to, the axis of eight-cell polarity. The theoretical and
observed limits of the polar to apolar ratio are 16 : 0 and 8 : 8. The outer
polar cells contribute largely to the trophectoderm, whereas the inner Gap junctions form between apposed basolateral surfaces and become functional.
The process of compaction involves the cell surface and the calcium
dependent cell–cell adhesion glycoprotein, E-cadherin (also called L-CAM or uvomorulin). Neutralization of its function disturbs all three Fig . 8 .4 The fertilization pathway: a succession of steps . After a sperm binds to the zona pellucida, the acrosome reaction takes place (see detail at top) .
The outer acrosomal membrane (blue), an enzyme-rich organelle in the anterior of the sperm head, fuses at many points with the plasma membrane
surrounding the sperm head . Then those fused membranes form vesicles, which are eventually sloughed off from the head, exposing the acrosomal
enzymes (red) . The enzymes digest a path through the zona pellucida, enabling the sperm to advance . Eventually, the sperm meets and fuses with the
secondary oocyte plasma membrane and this triggers cortical and zona reactions . First, enzyme-rich cortical granules in the oocyte cytoplasm release
their contents (yellow) into the zona pellucida, starting at the point of fusion and progressing right and left . Next, in the zona reaction, the enzymes
modify the zona pellucida, transforming it into an impenetrable barrier to sperm as a guard against polyspermy (multiple fertilization) .
Neck
MidpieceHead
Perivitelline space
Zona pellucidaContinuation of reaction
Acrosome-reacted
sperm
Penetration
Calcium wave starting at sperm entry point Cortical reactionFusionZona reaction
Excluded spermInitiation of acrosome reaction
Binding
TailNucleusPlasma membrane Acrosomal contentsFusion of plasma membraneand outer acrosomal membrane Vesiculation Acrosome-reacted sperm
Inner acrosomal membraneOuter acrosomal membrane
Cortical granule
Plasma membrane
Fig . 8 .5 A, An unfertilized human secondary oocyte surrounded by the
zona pellucida; the first polar body can be seen . Spermatozoa are visible
outside the zona pellucida . B, A fertilized human ootid before fusion of
the pronuclei . Two polar bodies can be seen beneath the zona pellucida .
A B | 251 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Preimplantation development
167
CHaPter 8
apolar cells contribute almost exclusively to the inner cell mass in most
embryos.
In cleavage, the generation of cell diversity, to either trophectoderm
or inner cell mass, occurs in the 16-cell morula and precedes the forma -
tion of the blastocyst. During the 16-cell cycle, the outer polar cells continue to differentiate an epithelial phenotype, and display further
aspects of polarity and intercellular adhesion typical of epithelial cells,
while the inner apolar cells remain symmetrically organized. During
the next cell division (16–32 cells), a proportion of polar cells again
divide differentiatively, as in the previous cycle, each yielding one polar
and one apolar progeny, which enter the trophectoderm and inner cell
mass lineages, respectively. Although differentiative division at this time
is less common than at the 8- to 16-cell transition, it has the important
function of regulating an appropriate number of cells in the two tissues
of the blastocyst. Thus, if differentiative divisions were relatively infre -
quent at the 8- to 16-cell transition, they will be more frequent at the 16- to 32-cell transition, and vice versa.
After division to 32 cells, the outer polar cells complete their dif -
ferentiation into a functional epithelium, display structurally complete
zonular tight junctions and begin to form desmosomes. The nascent
trophectoderm engages in vectorial fluid transport in an apical to basal
direction to generate a cavity that expands in size during the 32- to
64-cell cycles and converts the ball of cells, the morula, to a sphere, the
blastocyst (Fig. 8.7). Once the blastocyst forms, the diversification of
the trophectoderm and inner cell mass lineages is complete, and troph -
ectoderm differentiative divisions no longer occur. In the late blastocyst, the trophectoderm is referred to as the trophoblast, which can be
divided into polar trophoblast, lying in direct contact with the inner
cell mass, and mural trophoblast, surrounding the blastocyst cavity
(Fig. 8.8).
Blastocyst
The blastocyst ‘hatches’ from its zona pellucida at 6–7 days, possibly
assisted by an enzyme similar to trypsin (see Figs 8.7C, 8.8). Tropho -
blast oozes out of a small slit; many embryos form a figure-of-eight shape bisected by the zona pellucida, especially if it has been hardened
during oocyte maturation and cleavage. Such half-hatching could result
in the formation of identical twins. Hatched blastocysts expand and
differentiation of the inner cell mass proceeds (see Fig. 8.8).
The outer cells of the blastocyst – the trophoblast or trophectoderm
– are flattened polyhedral cells, which possess ultrastructural features
typical of a transporting epithelium. The trophoblast covering the inner cell mass is the polar trophoblast and that surrounding the blastocyst cavity is the mural trophoblast. The free, unattached blastocyst is
assigned to stage 3 of development at approximately 4 days post ovula -
tion, whereas implantation (before villus development) occurs within
a period of 7–12 days post ovulation and over the next two stages of Fig . 8 .6 Successive stages of cleavage of a human ootid . A, The two-cell
stage . B, The three-cell stage . C, The five-cell stage . D, The eight-cell
stage .
A B
C D
Fig . 8 .7 Human embryos . Formation of a morula and blastocyst within
the zona pellucida and blastocyst hatching from the zona pellucida . A, A
ball of cells, the morula, with the cells undergoing compaction . B, The
blastocyst cavity is developing and the inner cell mass can be seen on
one side of the cavity . C, The blastocyst is beginning to hatch from the
zona pellucida .
B A
C
Fig . 8 .8 A human blastocyst nearly completely hatched from the zona
pellucida . The blastocyst can now expand to its full size .
development. Even at this early stage, cells of the inner cell mass are
already arranged into an upper layer (i.e. closest to the polar tropho -
blast), the epiblast, which will give rise to the embryonic cells, and a
lower layer, the hypoblast, which has an extraembryonic fate. Thus, the
dorsoventral axis of the developing embryo and a bilaminar arrange -
ment of the inner cell mass are both established at or before implanta -
tion. (The earliest primordial germ cells may also be defined at this
stage.)
Attachment to the uterine wall
On the sixth postovulatory day, the blastocyst adheres to the uterine
mucosa and the events leading to the specialized, intimate contact of trophoblast and endometrium begin. Implantation, which is the term used for this complicated process, includes the following stages: dissolu -
tion of the zona pellucida; orientation and adhesion of the blastocyst on to the endometrium; trophoblastic penetration into the endometrium; migration of the blastocyst into the endometrium; and spread and pro -
liferation of the trophoblast, which envelops and specifically disrupts | 252 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Preim Plantation develo Pment
168SeCtion 2
single ovum, or both. It is most likely to be seen in women treated with
drugs to stimulate ovulation.
The range of separation of twin embryos is reflected in the separation
of the extraembryonic membranes. The types of placentation that can
occur are shown in Figure 8.9. Monoamniotic, monochorionic placen-
tae are associated with the greatest perinatal mortality (50%), caused both by entanglement of the umbilical cords impeding the blood
supply and by various vascular shunts between the placentae, which
may divert blood from one fetus to the other. Artery–artery anastomoses
are the most common, followed by artery–vein anastomoses. If the
shunting of blood across the placentae from one twin to the other is
balanced by more than one vascular connection, development may
proceed unimpaired. However, if this is not the case, one twin may
receive blood from the other, leading to cardiac enlargement, increased
urination and polyhydramnios in the recipient, and anaemia, oligo -
hydramnios and atrophy in the donor.
Dizygotic twins have either completely separate chorionic sacs or
sacs that have fused. Such placentae are separated by four membranes, two amnia and two choria; in addition, these placentae have a ridge of
firmer tissue at the base of the dividing membranes, caused by the abut -
ting of two expanding placental tissues against each other.
FORMATION OF EXTRAEMBRYONIC TISSUES
The earliest developmental processes in mammalian embryos involve
the production of those extraembryonic structures that will support and nourish the embryo during development. Production of these layers and invades the maternal tissues (Ch. 9). The interactions between the
hatched blastocyst and the maternal endometrium are not well under -
stood. Developmentally competent embryos appear to enhance the uterine environment and promote their own implantation (Brosens
et al 2014, Macklon and Brosens 2014).
The site of implantation is normally in the endometrium of the
posterior wall of the uterus, nearer to the fundus than to the cervix; it
may be in the median plane or to one or other side, but may occur
elsewhere in the uterus, or in an extrauterine or ectopic site.
Ectopic implantation
The conceptus may be arrested at any point during its migration through the uterine tube and implant in its wall. Previous pelvic inflammation
damages the tubal epithelium and may predispose to such delay in
tubal transport. The presence of an intrauterine contraceptive device or
the use of progesterone-based oral contraceptives may also predispose
to ectopic pregnancy, probably because of alteration in the normal tubal
transport mechanisms.
Nidation of the embryo as an ectopic pregnancy most frequently
occurs in the wider ampullary portion of the uterine tube, but may also
occur in the narrow intramural part or even in the ovary itself. Most
ectopic pregnancies are anembryonic, although the continuing growth
of the trophoblast will produce a positive pregnancy test, and may cause
rupture of the uterine tube and significant intraperitoneal haemorrhage.
Ectopic pregnancies with a live embryo are the most dangerous because
they grow rapidly and may be detected only when they have eroded the
uterine tube wall and surrounding blood vessels, as early as 8 weeks of
pregnancy. Similarly, cornual ectopics (in the intramural part of the
tube) may present with catastrophic haemorrhage because there is a
substantial blood supply in the surrounding muscularis.
Ovarian or abdominal pregnancies are exceptionally rare. Although
some are presumed to have been caused by fertilization occurring in
the vicinity of the ovary (primary), most are probably caused secondar -
ily and result from an extrusion of the conceptus through the abdomi -
nal ostium of the tube.
Apart from their important clinical implications, these conditions
emphasize the fact that the conceptus can implant successfully into
tissues other than a normal progestational endometrium. Prolonged
development can occur in such sites and is usually terminated by a
mechanical or vascular accident and not by a fundamental nutritive or
endocrine insufficiency or by an immune maternal response. Abdomi -
nal implantation may occur on any organ, e.g. bowel, liver or omentum. If such a pregnancy continues, this makes removal of the placenta at
delivery or abortion hazardous as a result of haemorrhage; conse -
quently, the placenta is usually left in situ to degenerate spontaneously.
Twinning
Spontaneous twinning occurs once in about every 80 births. Monozy -
gotic twins arise from a single ovum fertilized by a single sperm. At some stage up to the establishment of the axis of the embryonic area
and the development of the primitive streak, the embryonic cells sepa-
rate into two parts, each of which gives rise to a complete embryo. The
process of hatching of the blastocyst from the zona pellucida may result
in constriction of the emerging cells and separation into two discrete
entities. There is a gradual decrease in the average thickness of the zona
pellucida with increasing maternal age, which may be causally related
to the increase in frequency of monozygotic twinning with increased
maternal age. The resultant twins have the same genotype but the
description ‘identical twins’ is best avoided, since most monozygotic
twins have differences in phenotypes. Late separation of twins from a
single conceptus may result in conjoined twins; these may be equal or
unequal, as in acardia. After twinning, monozygotic embryos enter a
period of intense catch-up growth. Despite starting out at half the size,
each twin embryo or fetus is of a size comparable to a singleton fetus
in the second trimester of pregnancy, but declines in relative size in the
last 10 weeks of pregnancy. The sex of monozygotic twins will be the
same. Monoamniotic, monochorionic, monozygotic twins are most likely to be female, as are acardiac twins. The male to female ratio for
all monozygotic twins is 0.487, and for monoamniotic, monochorionic
twins it is 0.231.
Dizygotic twins represent the most frequent form of twinning. They
result from multiple ovulations, which can be induced by gonado -
trophins or drugs commonly used in patients with infertility. Dizygotic twins may be different sexes; like-sex pairs are more common. The male to female ratio is 0.518. Multiple births greater than twinning, such as triplets or quadruplets, can arise from multiple ovulations, or from a Fig . 8 .9 Relationships of the extraembryonic membranes in different
types of twinning . A, Diamnionic, dichorionic separated; i .e . separation
of the first two blastomeres results in separate implantation sites .
B, Diamnionic, dichorionic fused; here the chorionic membranes are fused
but the fetuses occupy separate choria . C, Diamnionic, monochorionic;
reduplication of the inner cell mass can result in a single placenta and
chorionic sacs but separate amniotic cavities . D, Monoamnionic,
monochorionic; duplication of the embryonic axis results in two embryos
sharing a single placenta, chorion and amnion . E, Incomplete separation
of the embryonic axis results in conjoined twins . F, Unequal division of
the embryonic axis or unequal division of the blood supply may result in
an acardiac monster .
A
EB
C D
F | 253 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Formation of extraembryonic tissues
169
CHaPter 8mural trophoblast and, rarely, may also share gap junctions. The visceral
hypoblast cells are cuboidal; they have a uniform apical surface towards
the blastocyst cavity but irregular basal and lateral regions, with flanges
and projections underlying one another and extending into intercel -
lular spaces. There is no basal lamina subjacent to the visceral hypo -
blast, and the distance between the hypoblast cells and the epiblast basal lamina is variable.
A series of modifications of the original blastocystic cavity develops
beneath the hypoblast later than those developing above the epiblast.
While the amniotic cavity is enlarging within the sphere of epiblast
cells, the parietal hypoblast cells are proliferating and spreading along
the mural trophoblast until they extend most of the way around the
circumference of the blastocyst, converging towards the abembryonic
pole. At the same time, a space appears between the parietal hypoblast
and the mural trophoblast that limits the circumference of the hypo -
blastic cavity. A variety of terms have been applied to the parietal
hypoblast layer: extraembryonic hypoblast, extraembryonic endoderm
and exocoelomic (Heuser’s) membrane. The cavity that the layer ini -
tially surrounds is the primary yolk sac (primary umbilical vesicle), and the resultant smaller cavity lined by hypoblast is the secondary yolk sac.
It has been suggested that the secondary yolk sac forms in a variety of
ways, including cavitation of visceral hypoblast (a method similar to
formation of the amnion), rearrangement of proliferating visceral
hypoblast and folding of the parietal layer of the primary yolk sac into
the secondary yolk sac. Further development of the yolk sac is described
on page 177.
The visceral hypoblast cells are thought to be important in many
aspects of the early specification of cell lines and control of the timing
of early development. They induce the formation and position of the
primitive streak in the midline of the embryo, thus establishing the first
axis of the embryonic disc, and they influence the timing of epiblast
epithelial-to-mesenchyme transition at the primitive streak. Hypoblast
cells remain beneath the primitive streak; their experimental removal
in avian embryos causes multiple embryonic axes to form. Hypoblast
cells are also believed to be necessary for successful induction of the
head region and for the successful specification of the primordial germ
cells. With the later formation of the embryonic cell layers from the
epiblast, the visceral hypoblast appears to be sequestered into the sec -
ondary yolk sac wall by the expansion of the newly formed embryonic endoderm beneath the epiblast, although there is now evidence that
some hypoblast cells may remain in the final endoderm layer and con -
tribute to the gut, and that they may also play a role in the induction of cardiogenesis. For a review of the roles of hypoblast cells, see Stern
and Downs (2012).
After the formation of the secondary yolk sac, a diverticulum of
the visceral hypoblast, the allantois, forms towards one end of the embryonic region and extends into the local extraembryonic mesoblast.
It passes from the roof of the secondary yolk sac to the same plane
as the amnion. Further development of the allantois is described on page 178.
Extraembryonic mesoblast
By definition, extraembryonic tissues encompass all tissues that do not
contribute directly to the future body of the definitive embryo and, later,
the fetus. At stage 5, blastocysts are implanted but do not yet display
trophoblastic villi (see Fig. 8.10); they range from 7 to 12 days in age.
A feature of this stage is the first formation of extraembryonic meso -
blast, which will come to cover the amnion, secondary yolk sac and the
internal wall of the mural trophoblast, and will form the connecting
stalk of the embryo with its contained allantoenteric diverticulum. The
origin of this first mesoblastic extraembryonic layer is by no means
clear; it may arise from several sources, including the caudal region of
the epiblast, the parietal hypoblast and subhypoblastic cells. The tro -
phoblastic origin of extraembryonic mesoblast is questioned because there is always a complete basal lamina underlying the trophoblast; the
migration of cells out of an epithelium is usually associated with previ -
ous disruption of the basal lamina. Certainly, the origin of extraembry -
onic cells will change over time as new germinal populations are
established.
The first mesoblastic extraembryonic layer gives rise to the layer
known as extraembryonic mesoblast, arranged as a mesothelium with
underlying extraembryonic mesenchymal cells; this also appears to form an extracellular structure corresponding to the magma reticulare, between the mural trophoblast and the primary yolk sac in the stage 5 embryo. Later extraembryonic mesoblast populations mushroom beneath the cytotrophoblastic cells at the embryonic pole, forming
the cores of the developing villus stems, and villi and the angioblastic begins before implantation is complete. At present, it is unclear where
the extraembryonic cell lines arise. The trophoblast was considered to
be a source but evidence now points to the inner cell mass as the site
of origin. Figure 8.1 shows the sequence of development of various
tissues in the early embryo.
Epiblast and amniotic cavity
Epiblast cells are closest to the implanting face of the trophoblast and have a definite polarity; they are arranged in a radial manner with
extensive junctions near the centre of the mass of cells, supported by
supranuclear organelles. A few epiblast cells are contiguous with
cytotrophoblast cells; apart from this contact, a basal lamina surrounds
what is initially a spherical cluster of epiblast cells, and isolates them
from all other cells. Those epiblast cells adjacent to the hypoblast
become taller and more columnar than those adjacent to the tropho -
blast, and this causes the epiblast sphere to become flattened and the
centre of the sphere to be shifted towards the polar trophoblast. Amni-
otic fluid accumulates at the eccentric centre of the now lenticular
epiblast mass, which is bordered by apical junctional complexes and
microvilli. As further fluid accumulates, an amniotic cavity forms,
roofed by low cuboidal cells that possess irregular microvilli. The cells
share short apical junctional complexes and associated desmosomes,
and rest on an underlying basal lamina. The demarcation between true
amnion cells and those of the remaining definitive epiblast is clear. The
columnar epiblast cells are arranged as a pseudostratified layer with
microvilli, frequently a single cilium, clefted nuclei and large nucleoli;
the cells have a distinct, continuous basal lamina. Cell division in the
epiblast tends to occur near the apical surface, causing this region to
become more crowded than the basal region. At the margins of the
embryonic disc, the amnion cells are contiguous with the epiblast; there
is a gradation in cell size from columnar to low cuboidal within a two-
to three-cell span ( Fig. 8.10; see Fig. 9.1). Further development of the
amnion and amniotic fluid is described on page 178.
Hypoblast and yolk sac
Hypoblast is the term used to delineate the lower layer of cells of the early bilaminar disc, most commonly in avian embryos. This layer is
also termed anterior, or distal, visceral endoderm in the mouse embryo.
Just before implantation, the hypoblast consists of a layer of squamous
cells that is only slightly larger in extent than the epiblast. The cells
exhibit polarity, in that apical microvilli face the cavity of the blastocyst
and apical junctional complexes, but they lack a basal lamina. During
early implantation, the hypoblast extends beyond the edges of the
epiblast and can now be subdivided into those cells in contact with the
epiblast basal lamina, the visceral hypoblast, and those cells in contact
with the mural trophoblast, the parietal hypoblast. The parietal hypo -
blast cells are squamous; they may share adhesion junctions with the
Fig . 8 .10 The implanting conceptus at stage 5b . The embryo at this stage
is composed only of the abutting epiblast and hypoblast layers . It is
suspended within the blastocyst cavity (chorionic cavity) and surrounded
by a layer of cytotrophoblast . A large mass of syncytiotrophoblast, with
interconnecting lacunae, is penetrating the maternal endometrium . Cytotrophoblast
SyncytiotrophoblastHypoblast | 254 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Preim Plantation develo Pment
170SeCtion 2cells that will give rise to the capillaries within them and the earliest
blood cells.
Initially, the extraembryonic mesoblast connects the amnion to the
chorion over a wide area. Continued development and expansion of the extraembryonic coelom means that this attachment becomes
increasingly circumvented to a connecting stalk, which is a permanent
connection between the future caudal end of the embryonic disc and the chorion. The connecting stalk forms a pathway along which vascular anastomoses around the allantois establish communication with those of the chorion.
KEY REFERENCES
Brosens JJ, Walker MS, Teklenburg G 2014 Uterine selection of human
embryos at implantation. Sci Reports 4:3894.
Macklon NS Brosens JJ 2014 The human endometrium as a sensor of
embryo quality. Biol Reprod 91:98.
O’Rahilly R, Müller F 1987 Developmental Stages in Human Embryos.
Washington: Carnegie Institution.
O’Rahilly R, Müller F 1999 The Embryonic Human Brain. An Atlas of
Developmental Stages, 2nd ed. New York: Wiley-Liss.
O’Rahilly R, Müller F 2000 Minireview: Prenatal ages and stages – measures
and errors. Teratology 61:382–4.O’Rahilly R, Müller F 2010 Developmental stages in human embryos: revised
and new measurements. Cells Tissues Organs 192:73–84.
Stern CD, Downs KM 2012 The hypoblast (visceral endoderm): an evo-devo
perspective. Development 139:1059–69.
Streeter GL 1942 Developmental horizons in human embryos. Descriptions
of age group XI, 13 to 20 somites, and age group XII, 21 to 29 somites.
Contrib Embryol Carnegie Inst Washington 30:21 1–45.Video 8 .1 Human in vitro fertilization and early development . Bonus e-book video
| 255 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Preimplantation development
170.e1
CHaPter 8REFERENCES
Brosens JJ, Walker MS, Teklenburg G 2014 Uterine selection of human
embryos at implantation. Sci Reports 4:3894.
Macklon NS Brosens JJ 2014 The human endometrium as a sensor of
embryo quality. Biol Reprod 91:98.
O’Rahilly R, Müller F 1987 Developmental Stages in Human Embryos.
Washington: Carnegie Institution.
O’Rahilly R, Müller F 1999 The Embryonic Human Brain. An Atlas of
Developmental Stages, 2nd ed. New York: Wiley-Liss.
O’Rahilly R, Müller F 2000 Minireview: Prenatal ages and stages – measures
and errors. Teratology 61:382–4.O’Rahilly R, Müller F 2010 Developmental stages in human embryos: revised
and new measurements. Cells Tissues Organs 192:73–84.
Stern CD, Downs KM 2012 The hypoblast (visceral endoderm): an evo-devo
perspective. Development 139:1059–69.
Streeter GL 1942 Developmental horizons in human embryos. Descriptions
of age group XI, 13 to 20 somites, and age group XII, 21 to 29 somites.
Contrib Embryol Carnegie Inst Washington 30:21 1–45. | 256 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
171
CHAPTER 9 Implantation and placentation
and during leukocyte emigration from the blood into the tissues.
Flanges of syncytial trophoblast grow between the cells of the uterine
luminal epithelium towards the underlying basal lamina without
apparent damage to the maternal cell membranes or disruption of the
intercellular junctions. Instead, shared junctions, including tight junc
tions, are formed with many of the maternal uterine epithelial cells.
Implantation continues with erosion of maternal vascular endothe
lium and glandular epithelium, and phagocytosis of secretory products, until the blastocyst occupies an uneven implantation cavity in the
stroma (interstitial implantation) (Fig. 9.1). In the early postimplant
ation phase, the maternal surface is resealed by re epithelialization and
the formation of a plug, which may contain fibrin. As the blastocyst burrows more deeply into the endometrium, syncytial trophoblast
forms over the mural cytotrophoblast, but never achieves the thickness
of the syncytial trophoblast over the embryonic pole.
The syncytiotrophoblast, which expresses neither class I nor class II
major histocompatibility complex (MHC) antigens, secretes numerous
hormones; human chorionic gonadotrophin (hCG) can be detected in
maternal urine from as early as 10 days after fertilization and forms the
basis for tests for early pregnancy. This hCG prolongs the life of the
corpus luteum, which continues to secrete progesterone and oestrogens
during approximately the first 2 months of pregnancy, until these essen
tial hormones are produced by the placenta.
Menstruation ceases on successful implantation. The endometrium,
now known as the decidua in pregnancy, thickens to form a suitable
nidus for the conceptus. Decidualization of the endometrial stroma
may occur without an intrauterine pregnancy, e.g. in the presence of an
ectopic pregnancy, after prolonged treatment with progesterone, and in
the late secretory phase of a non conception cycle.
Decidual differentiation is not evident in the stroma at the earliest
stages of implantation, and it may not be until a week later that fully IMPLANTATION
Implantation involves the initial attachment of the trophoblastic wall of the blastocyst to the endometrial luminal epithelium and its decidual
response. It is now apparent that, during this process, there is an inter
active dialogue between the implanting embryo and endometrial
decidu al stromal cells. Competent preimplantation human embryos
actively enhance the uterine environment for their implantation, whereas developmentally impaired embryos induce endoplasmic retic
ulum stress responses in decidual cells that inhibit implantation
(Brosens et al 2014).
The blastocyst/trophoblast lineage gives rise to three main cell types
in the human placenta: syncytiotrophoblast cells form the epithelial
covering of the villous tree and are the main endocrine component of
the placenta; villous cytotrophoblast cells represent a germinative popu
lation that proliferates throughout pregnancy, fusing to generate syn
cytiotrophoblast; and extravillous trophoblast cells are non proliferative
and invade the maternal endometrium. The first two cell lines can be seen from stages 4 and 5 onwards. The cytotrophoblast cells that form
the mural and polar trophoblast are cuboidal and covered externally
with syncytial trophoblast (syncytiotrophoblast), a multinucleated
mass of cytoplasm that forms initially in areas near the inner cell mass
after apposition of the blastocyst to the uterine mucosa (see Fig. 8.10).
Preimplantation embryos produce matrix metalloproteinases
(MMPs) that mediate penetration of the maternal subepithelial basal
lamina by the syncytiotrophoblast. Trophoblast cells express L selectin
(usually seen as a mediator of neutrophil rolling and tethering in inflamed endothelium), and the maternal epithelium upregulates
selectin oligosaccharide based ligands. Thus, differentiating cytotro
phoblast cells appear to use processes that also occur in vasculogenesis
Fig. 9.1 The implanting conceptus at stage 6. The embryo is composed of epiblast, with the amniotic cavity above it, and hypoblast, with the secondary
yolk sac below it (see also Figs 8.10, 10.1). Both cavities are covered externally with extraembryonic mesoblast, which also lines the larger chorionic
cavity. Primary villi cover the outer aspect of the conceptus and extend into the maternal endometrium, and in places, maternal blood fills the lacunae.
Cytotrophoblast
SyncytiotrophoblastDecidua
Extraembryonic mesoblastUterine glands
Amniotic cavity
Secondary yolk sac
Lacuna
Extraembryonic coelom
(chorionic cavity)Uterine blood vessel
Amnion
Yolk sac wall
Chorion
Epithelium of
endometrial surface
Coagulum over
implanting blastocystVillous stems | 257 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Implantat Ion and placentat Ion
172SectIon 2differentiated cells are present. During decidualization, the interglan
dular tissue increases in quantity (Burton et al 2010). It contains a
substantial population of leukocytes (large granular lymphocytes,
macrophages and T cells) distributed amongst large decidual cells. The
most numerous are uterine natural killer (NK) cells, which accumulate
in the endometrium during the secretory phase of the cycle and persist
until mid pregnancy; they interact with invading extravillous tropho
blast cells expressing human leukocyte antigen G and C (HLA G,
HLAC). Decidual cells are stromal cells that contain varying amounts
of glycogen, lipid and vimentin type intermediate filaments in their
cytoplasm. They are generally rounded but their shape may vary, depending on the local packing density. They may contain one, two or
sometimes three nuclei, frequently display rows of club like cytoplas
mic protrusions enclosing granules, and are associated with a charac
teristic capsular basal lamina. Decidual cells produce a range of secretory
products, including insulin like growth factor binding protein 1 (IGF
BP1) and prolactin, which may be taken up by the trophoblast. These secretions probably play a role in the maintenance and growth of the
conceptus in the early part of postimplantational development, and can
be detected in amniotic fluid in the first trimester of pregnancy.
Extracellular matrix, growth factors and protease inhibitors pro
duced by the decidua all probably modulate the degradative activity of
the trophoblast and support placental morphogenesis and placental
accession to the maternal blood supply. Once implantation is complete,
distinctive names are applied to different regions of the decidua ( Fig.
9.2). The part covering the conceptus is the decidua capsularis; that between the conceptus and the uterine muscular wall is the decidua
basalis (where the placenta subsequently develops); and that which
lines the remainder of the body of the uterus is the decidua parietalis.
There is no evidence that their respective resident maternal cell popula
tions exhibit site specific properties.
DEVELOPMENT OF THE PLACENTA
Formation of the human placenta requires a developmental progression
that proceeds in a specific chronological order: the development of
primary, secondary and anchoring villi, and the local differentiation of
haemangioblast cells within them, occur concurrently with the modifi
cation of maternal blood vessels to ensure their patency. With the onset of the embryonic heart beat, a primitive circulation exists between the
embryo and the secondary yolk sac. An embryonic–placental circula
tion starts around week 8 of gestation; over the course of 40 weeks, the
placenta develops into a highly vascular organ.Fig. 9.2 The gravid uterus in the second month. A placental site precisely
in the uterine fundus, as indicated on the figure, is rather unusual; the
dorsal, ventral or lateral wall of the corpus uteri is more usual. The
maternal endometrium is now termed decidua; different regions are
distinguished.
Uterine
tube
Umbilical
cordConnecting
stalk
Decidua
parietalis
Chorion
Chorionic
cavity
Myometrium Plug of mucusEmbryowithinamnionDeciduacapsularisAmnioticcavityYolk sac
Vitelline
ductVillousstemsof chorionfrondosumIntervillous space
Decidua basalis
Fig. 9.3 A, The growing syncytiotrophoblast erodes decidual glands and spiral arteries, and their contents form lacunae within the syncytiotrophoblast.
Cytotrophoblastic cells extend as villi into the syncytiotrophoblast. B, Cells originating from the cytotrophoblast move into the decidua, forming interstitial
extravillous trophoblast cells, the third line of cytotrophoblastic cells. These surround the opened glands and spiral arteries in the decidua and inner
myometrium. Cells that move through the tunicae adventitia, media and interna to enter the lumen of the vessels become endovascular extravillous trophoblast. These cells remodel the walls and plug the lumen of the spiral arteries, permitting only plasma to enter the forming intervillous space. Glandular secretions in the intervillous space provide histiotrophic nutrition to the embryo.
Syncytiotrophoblast
Cytotrophoblast
Mesenchyme
AmnionLacunae
Endometrial gland
Spiral artery
DeciduaSpiral arteryEndometrial gland
Decidua
Glandular secretions in intervillous spaceEndovascular extravillous trophoblast
Plasma in intervillous spaceCytotrophoblast
Interstitial extravillous trophoblastSyncytiotrophoblast
A BAs the blastocyst implants, the syncytiotrophoblast invades the
uterine tissues, including the glands and walls of maternal blood vessels
(see Fig. 9.1; Fig. 9.3), and increases rapidly in thickness over the
embryonic pole ( Fig. 9.4). A progressively thinner layer covers the rest
of the wall towards the abembryonic pole. Microvillus lined clefts and
lacunar spaces develop in the syncytiotrophoblastic envelope (days
9–1 1 of pregnancy) and establish communications with one another.
Initially, many of these spaces contain maternal blood derived from
dilated uterine capillaries and veins, as the walls of the vessels are par
tially destroyed. As the conceptus grows, the lacunar spaces enlarge, and become confluent to form intervillous spaces.
The projections of syncytiotrophoblast into the maternal decidua are
called primary villi. They are invaded initially with cytotrophoblast and | 258 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
development of the placenta
173
cHapte R 9
Fig. 9.5 Placental development. CytotrophoblastLacunae in syncytiumDEVELOPMENT
Fetal vessels Stem villus
True villus Secondary syncytial fusion
Terminal villus Connective tissue
AmnionMaternal blood
Ingrowth of cytotrophoblast and
mesoblast bearing fetal vesselsLacunarcirculationLacunaeenlarging asintervillousspacesCytotrophoblasticcell columnSyncytiotrophoblastCotyledonary septum
Maternal vesselsin deciduaBasal plate
Intervillous
space
Chorionic
platethen with extraembryonic mesenchyme (days 13–15) to form second
ary placental villi. Capillaries develop in the mesenchymal core of the
villi during the third week post conception. The cytotrophoblast within
the villi continues to grow through the invading syncytiotrophoblast
and makes direct contact with the decidua basalis, forming anchoring
villi. Further cytotrophoblast proliferation occurs laterally so that neigh
bouring outgrowths meet to form a spherical cytotrophoblastic shell around the conceptus (Fig. 9.5; see Fig. 9.3B). Lateral projections from
the main stem villus form true villi. Elongation of growing capillaries outstrips that of the containing villi, leading to looping of vessels. This
obtrusion of both capillary loops and new sprouts results in the forma
tion of terminal villi.
As secondary villi form, single mononuclear cells become detached
from the distal (anchoring) cytotrophoblast and infiltrate the maternal decidua (Fig. 9.6; see Fig. 9.3B). These extravillous cells are the third
line of the original trophoblastic cells. Interstitial extravillous trophob
last cells invade the maternal spiral arteries from their adventitia. The
smooth muscle and internal elastic lamina are replaced with extracel
lular fibrinoid deposits (Harris 2010). Those cells that pass through the endothelial basement membrane and gain access to the vessel lumen
are termed endovascular extravillous trophoblast (see Figs 9.3B, 9.6).
These cells plug the maternal spiral arteries until the end of the first trimester; the cells also migrate antidromically, against the flow of maternal blood, along the spiral arteries as far as the inner myometrial
region (Huppertz et al 2014).
The definitive placenta is composed of a chorionic plate on its fetal
aspect and a basal plate on its maternal aspect, separated by an interven
ing intervillous space containing villous stems with branches in contact
with maternal blood (see Fig. 9.5). During the first trimester, develop
ment takes place in a low oxygen environment supported by histio
trophic nutrition from the endometrial glands, which discharge into
the intervillous space until at least 10 weeks (see Fig. 9.3A) (Burton et al
2002, Burton et al 2007, Burton and Fowden 2012). When maternal
blood bathes the surfaces of the chorion that bound the intervillous space, the human placenta is defined as haemochorial. Different grades
of fusion exist between the maternal and fetal tissues in many other
mammals (e.g. epitheliochorial, syndesmochorial, endotheliochorial).
The chorion is vascularized by the allantoic blood vessels of the body
stalk, and so the human placenta is also termed chorio allantoic
(whereas, in some mammals, a choriovitelline placenta either exists
alone or supplements the chorio allantoic variety). The human placenta
is also defined as discoidal, in contrast to other shapes in other
mammals, and deciduate because maternal tissue is shed with the pla
centa and membranes at parturition as part of the afterbirth.
Growth of the placenta
Expansion of the entire conceptus is accompanied by radial growth of the villi and, simultaneously, an integrated tangential growth and
expansion of the trophoblastic shell. Eventually, each villous stem
forms a complex that consists of a single trunk attached by its base to
the chorion, from which second and third order branches (intermedi
ate and terminal villi) arise distally. Terminal villi are specialized for
exchange between fetal and maternal circulations; each one starts as a
syncytial outgrowth and is invaded by cytotrophoblastic cells, which
then develop a core of fetal mesenchyme as the villus continues to grow.
The core is vascularized by fetal capillaries (i.e. each villus passes
through primary, secondary and tertiary grades of histological differen
tiation). The germinal cytotrophoblast continues to add cells that fuse with the overlying syncytium and so contribute to the expansion of the
haemochorial interface. Terminal villi continue to form and branch
within the confines of the definitive placenta throughout gestation,
projecting in all directions into the intervillous space (see Fig. 9.5).
From the third week until about the second month of pregnancy,
the entire chorion is covered with villous stems. They are thus continu
ous peripherally with the trophoblastic shell, which is in close apposition with both the decidua capsularis and the decidua basalis. The villi
adjacent to the decidua basalis are stouter and longer, and show a
greater profusion of terminal villi. As the conceptus continues to
expand, the decidua capsularis is progressively compressed and thinned,
the circulation through it is gradually reduced, and adjacent villi slowly
atrophy and disappear. This process starts at the abembryonic pole; by
the end of the third month, the abembryonic hemisphere of the Fig. 9.4 The conceptus
at about stage 14. The
embryonic pole shows
extensive villous
formation at the chorion
frondosum, whereas
the abembryonic pole
is smooth and
villous-free at the
chorion laeve.
(Photograph courtesy
of P Collins.) | 259 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Implantat Ion and placentat Ion
174SectIon 2
Chorionic plate
The chorionic plate is covered on its fetal aspect by amniotic epithe
lium; on the stromal side of the epithelium, a connective tissue layer
carries the main branches of the umbilical vessels (see Fig. 9.5; Fig. 9.7).
Subjacent to this are a diminishing layer of cytotrophoblast and then the inner syncytial wall of the intervillous space. The connective tissue
layer is formed by fusion between the mesenchyme covered surfaces of
amnion and chorion, and is more fibrous and less cellular than Whar
ton’s jelly (of the umbilical cord), except near the larger vessels. The umbilical vessels radiate and branch from the cord attachment, with
variations in the branching pattern, until they reach the bases of the
trunks of the villous stems; they then arborize within the intermediate
and terminal villi. There are no anastomoses between vascular trees of
adjacent stems. The two umbilical arteries are normally joined at, or
just before they enter, the chorionic plate, by some form of substantial
transverse anastomosis (Hyrtl’s anastomosis).
Basal plate
The basal plate, from fetal to maternal aspect, forms the outer wall of
the intervillous space. The trophoblast and adjacent decidua are
enmeshed in layers of fibrinoid and basement membrane like extracel
lular matrix to form a complex junctional zone. In different places, the
basal plate may contain syncytium, cytotrophoblast or fibrinoid matrix,
remnants of the cytotrophoblastic shell, and, at the site of implantation,
areas of necrotic maternal decidua (the so called Nitabuch’s stria) (see
Figs 9.5 and 9.7). Nitabuch’s stria and the decidua basalis contain
cytotrophoblast and multinucleate trophoblast giant cells derived from
the mononuclear extravillous interstitial cytotrophoblast population
that infiltrate the decidua basalis during the first 18 weeks of pregnancy.
These cells penetrate as far as the inner third of the myometrium but
can often be observed at or near the decidual–myometrial junction.
They are not found in the decidua parietalis or the adjacent myo
metrium, suggesting that the placental bed giant cell represents a dif
ferentiative end stage in the extravillous trophoblast lineage. The striae of fibrinoid are irregularly interconnected and variable in prominence. Strands pass from Nitabuch’s stria into the adjacent decidua, which contains basal remnants of the endometrial glands and large and small decidual cells scattered in a connective tissue framework that supports an extensive venous plexus.
Initially, only the central area of a placenta contains extravillous
trophoblast cells, both within and around the spiral arteries. These cells conceptus is largely denuded. Eventually, the whole chorion apposed
to the decidua capsularis is smooth and is now termed the chorion
laeve; this will later become apposed to the amnion, forming chorio
amnion (see Figs 9.2, 9.9). In contrast, the villous stems of the disc
shaped region of chorion apposed to the decidua basalis increase greatly in size and complexity, and the region is now termed the chorion
frondosum (see Fig. 9.4). The chorion frondosum and the decidua
basalis constitute the definitive placental site (see Fig. 9.2). Abnormali
ties in this process may account for the persistence of villi at abnormal
sites in the chorion laeve of the gestational sac and hence the presence
of accessory or succenturiate lobes within the membranes of the defini
tive placenta. The presence of the entire villous ring of the primitive
placenta beyond the second month post menstruation leads to the
development of placenta membranacea, a giant definitive placental
structure surrounding the whole gestational sac during the remainder
of pregnancy.
Coincidentally with the growth of the embryo and the expansion of
the amnion, the decidua capsularis is thinned and distended, and the
space between it and the decidua parietalis is gradually obliterated. By
the second month of pregnancy, the three endometrial strata recogniz
able in the premenstrual phase, i.e. compactum, spongiosum and basale, are better differentiated and easily distinguished. The glands in
the spongiosum are compressed and appear as oblique slit like fissures
lined by low cuboidal cells. By the beginning of the third month of
pregnancy, the decidua capsularis and decidua parietalis are in contact.
By the fifth month, the decidua capsularis is greatly thinned, and it
virtually disappears during the succeeding months.
Focal bleeding often occurs in the periphery of the developing pla
centa at the time of the formation of the membranes (8–12 weeks). This
complication, which is termed threatened miscarriage, is a common clinical complication of pregnancy and can lead to a complete miscar
riage if the haematoma extends to the definitive placenta (Jauniaux et al
2006).
At term, the placental diameter varies from 200 to 220 mm, the
mean placental weight is 470 g, its mean thickness is 25 mm and the
total villous surface area is 12–14 m2, providing an extensive and inti
mate interface for materno–fetal exchange; the uteroplacental circula
tion carries approximately 600 ml of maternal blood per minute. There
are no lymph vessels in the placenta and, equally, no aggregates of
lymphoid cells. Systematic evaluation and careful description of the placenta after delivery have been recommended for correlation with
later neonatal neurodevelopmental outcomes (Roescher et al 2014).Fig. 9.6 As the placenta grows, the interstitial and endovascular extravillous trophoblast cells continue to remodel the spiral arteries into large-bore,
low-resistance uteroplacental vessels. These arteries remain occluded by endovascular extravillous trophoblast until the end of the first trimester,
promoting a low oxygen environment.
Myometrium
Spiral artery
Uterine gland
Decidua
Cotyledonary septum
Glandular secretions in intervillous spaceEndovascular extravillous trophoblast
Plasma in intervillous spaceCytotrophoblast
Interstitial extravillous trophoblastSyncytiotrophoblast | 260 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
development of the placenta
175
cHapte R 9
feature occupying the peripheral margin of the placenta and commu
nicating freely with the intervillous space, has not been confirmed.
Recent anatomic and in vivo studies have shown that human placen
tation is, in fact, not truly haemochorial in early pregnancy ( Video 9.1)
(Jauniaux et al 2003). From the time of implantation, the extravillous
trophoblast not only invades the uterine tissues but also forms a con
tinuous shell at the level of the decidua. The cells of this shell anchor
the placenta to the maternal tissue and also form plugs in the tips of
the uteroplacental arteries (see Figs 9.3B, 9.6) (Burton et al 1999). The
shell and the plugs act like a labyrinthine interface that filters maternal blood, permitting a slow seepage of plasma but no true blood flow into
the intervillous space (Burton et al 2003). This creates a physiological
placental hypoxia, which may protect the developing embryo against the deleterious and teratogenic effects of oxygen free radicals. As a
result, there is a uteroplacental O
2 gradient that exerts a regulatory effect
on placental tissue development and function (Jauniaux et al 2003). In
particular, it influences cytotrophoblast proliferation and differentiation along the invasive pathway, villous vasculogenesis and the form
ation of the chorion laeve. An O
2 gradient persists within lobules of the
placenta throughout pregnancy, with the central region well oxygenated compared to the periphery, owing to the direction of maternal blood
flow (Burton and Jauniaux 201 1).
At the end of the first trimester, the endovascular extravillous tro
phoblastic plugs are progressively dislocated, allowing maternal blood
to flow progressively more freely and continuously within the intervil
lous space (see Figs 9.6, 9.7, Video 9.1 ). During the transitional phase
of 10–14 weeks’ gestation, two thirds of the primitive placenta disap
pears, the chorionic cavity is obliterated by the growth of the amniotic
sac, and maternal blood flows progressively throughout the entire pla
centa (Jauniaux et al 2003). These events bring the maternal blood
closer to the fetal tissues, facilitating nutrient and gaseous exchange
between the maternal and fetal circulations (Gutierrez Marcos et al
2012).
Structure of a placental villus
Chorionic villi are the essential structures involved in exchanges
between mother and fetus. The villous tissues separating fetal and
gradually extend laterally, reaching the periphery of the placenta around
midgestation; the extent of invasion is progressively shallower towards
the periphery. Invaded maternal vessels show a 5–10 fold dilation of
the vessel mouth and altered responsiveness to circulating vasoactive
compounds (Hung et al 2001, Burton et al 2009).
The conversion of maternal musculoelastic, spiral arteries to large
bore, low resistance uteroplacental vessels is considered key for a suc
cessful human pregnancy. Failure to achieve these changes is a feature
of common complications of pregnancy, such as early onset pre
eclampsia, miscarriage and fetal growth restriction.
From the third month onwards, the basal plate develops placental
or cotyledonary septa, which are ingrowths of the cytotrophoblast
covered with syncytium that grow towards, but do not fuse with, the
chorionic plate (see Fig. 9.5). The septa circumscribe the maternal
surface of the placenta into 15–30 lobes, often termed cotyledons. Each cotyledon surrounds a limited portion of the intervillous space associ
ated with a villous trunk from the chorionic plate. From the fourth month, these septa are supported by tissue from the decidua basalis.
Throughout the second half of pregnancy, the basal plate becomes
thinned and progressively modified: there is a relative diminution of
the decidual elements, increasing deposition of fibrinoid, and admix
ture of fetal and maternal derivatives.
Intervillous space
The intervillous space contains the main trunks of the villous stems and their arborizations into intermediate and terminal villi (see Figs 9.5,
9.7). A villous trunk and its branches may be regarded as the essential structural, functional and growth unit of the developing placenta.
At term, from the inner myometrium to the intervillous space, the
walls of most spiral arteries consist of fibrinoid matrix, within which
cytotrophoblast is embedded. This arrangement allows expansion of the arterial diameter (and so slows the rate of arterial inflow and reduces the perfusion pressure), independent of the local action of vasoconstric
tive agents. Endothelial cells, where present, are often hypertrophic. The veins that drain the blood away from the intervillous space pierce the basal plate and join tributaries of the uterine veins. The presence of a marginal venous sinus, which hitherto has been described as a constant Fig. 9.7 The arrangement of the placental tissues from the chorionic plate (fetal side) to the basal plate or decidua basalis (maternal side).
Fibrinoid deposit
CytotrophoblastCytotrophoblastic cell column
Syncytiotrophoblast
Syncytiotrophoblast
Cytotrophoblast
Fetal mesenchyme
Amniotic epitheliumAnchoring villus
Syncytial sprout
Hofbauer cellMaternal blood vessels
Fetal blood vesselsOrifices of maternal vessels
Terminal villi
Syncytial fusion
Intermediate villus
Stem villusMyometrium
Basal plate
Intervillous space
Chorionic plate | 261 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Implantat Ion and placentat Ion
176SectIon 2
plasm is more strongly basophilic than that of the cytotrophoblastic
cells and is packed with organelles consistent with its secretory pheno
type. Where the plasma membrane adjoins basal lamina, it is often infolded into the cytoplasm, whereas the surface bordering the intervil
lous space is set with numerous long microvilli, which constitute the brush border seen by light microscopy.
Glycogen is thought to be present in both layers of the trophoblast
at all stages, although it is not always possible to demonstrate its pres
ence histochemically. Lipid droplets occur in both layers and are free in the core of the villus. In the trophoblast, they are found principally
within the cytoplasm, but they also occur in the extracellular space
between cytotrophoblast and syncytium, and between the individual
cells of the cytotrophoblast. The droplets diminish in number with
advancing age and may represent fat in transit from mother to fetus,
and/or a pool of precursors for steroid synthesis. Membrane bound
granular bodies of moderate electron density occur in the cytoplasm, particularly in the syncytium, some of which are probably secretion
granules. Other membrane bound bodies, lysosomes and phagosomes,
are involved in the degradation of materials engulfed from the intervil
lous space.
Although many nuclei are dispersed within the syncytioplasm, other
are aggregated into specializations referred to as syncytial knots and
syncytial sprouts (Burton et al 2003). Knots must be distinguished from
syncytial sprouts, which are markers of trophoblast proliferation. In the immature placenta, syncytial sprouts represent the first stages in the
development of new terminal villi, which later become invaded by
cytotrophoblast and villous mesenchyme (Fogarty et al 2013). Occa
sionally, adjacent syncytial sprouts make contact and fuse to form slender syncytial bridges. The sprouts may become detached, forming
maternal syncytial emboli, which pass to the lungs. It has been com
puted that some 100,000 sprouts pass daily into the maternal circula
tion; deported trophoblast may be a mechanism by which the maternal
immune system is maintained in a state of tolerance towards paternal
antigens (Chamley et al 201 1). In the lungs, syncytial sprouts provoke
little local reaction and apparently disappear by lysis. However, they
may occasionally form foci for neoplastic growth. Syncytial sprouts are
present in the term placenta. Syncytial knots are aggregates of nuclei
with particularly condensed heterochromatin, and may represent a
sequestration phenomenon by which senescent nuclear material is removed from adjacent metabolically active areas of syncytium.
Fibrinoid deposits are frequently found on the villous surface in
areas lacking syncytiotrophoblast. They may constitute a repair mecha
nism in which the fibrinoid forms a wound surface that is subsequently
reepithelialized by trophoblast. The extracellular matrix glycoprotein
tenascin has been localized in the stroma adjacent to these sites.maternal blood are therefore of crucial functional importance. From the
chorionic plate, progressive branching occurs into the villous tree, as
stem villi give way to intermediate and terminal villi. Each villus has a
core of connective tissue containing collagen types I, III, V and VI, as
well as fibronectin. Cross banded fibres (30–35 nm) of type I collagen
often occur in bundles, whereas type III collagen is present as thinner
(10–15 nm) beaded fibres, which form a meshwork that often encases
the larger fibres. Collagens V and VI are present as 6–10 nm fibres
closely associated with collagens I and III. Laminin and collagen type
IV are present in the stroma associated with the basal laminae that
surround fetal vessels and also in the trophoblast basal lamina. Overly
ing this matrix are ensheathing cyto and syncytiotrophoblast cells
bathed by the maternal blood in the intervillous space (see Figs 9.5,
9.7; Fig. 9.8). Cohesion between the cells of the cytotrophoblast, and
also between the cytotrophoblast and the syncytium, is provided by numerous desmosomes between the apposed plasma membranes.
In earlier stages, the cytotrophoblast forms an almost continuous
layer on the basal lamina. After the fourth month, it gradually expends
itself producing syncytium, which comes to lie on the basal lamina over
an increasingly large area (56% at term), and becomes progressively
thinner. Cytotrophoblastic cells persist until term but, because the
increase in villous surface area outstrips their proliferation, they are
usually disposed singly. In the first and second trimesters, cytotropho
blastic sprouts, covered in syncytium, are present and represent a stage
in the development of new villi. Cytotrophoblast columns at the tips
of anchoring villi extend from the villous basal lamina to the maternal
decidual stroma.
The cells of the villous cytotrophoblast (Langhans cells) are pale
staining with a slight basophilia. Ultrastructurally, they have a rather
electron translucent cytoplasm and relatively few organelles. They
contain intermediate filaments, particularly in association with desmo
somes. Between the desmosomes, the membranes of adjacent cells are
separated by approximately 20 nm. Sometimes, the intercellular gap
widens to accommodate microvillous projections from the cell surfaces;
occasionally, it contains patches of fibrinoid. A smaller population of
intermediate cytotrophoblast may also be found in the chorionic villi.
This postmitotic population represents a state of partial differentiation
between the cytotrophoblast stem cell and the overlying syncytium.
The syncytiotrophoblast is the multinucleated epithelium of the
placenta and is an intensely active tissue layer, across which most trans
placental transport must occur (Ellery et al 2009). It is a selectively
permeable barrier that allows water, oxygen and other nutritive sub
stances and hormones to pass from mother to fetus, and some of the
products of excretion to pass from fetus to mother. It secretes a range of placental hormones into the maternal circulation. Syncytial cyto Fig. 9.8 A, A chorionic villus and its arterio–capillary–venous system carrying fetal blood. The artery carries deoxygenated blood and waste products
from the fetus, and the vein carries oxygenated blood and nutrients to the fetus. B–C, Transverse sections through individual chorionic villi at 10 weeks
(B) and at full term ( C). The villi would be within the intervillous space, bathed externally in maternal blood. The placental membrane, composed of fetal
tissues, separates the maternal blood from the fetal blood. A B C
Artery
Stem villusVeinArterio–capillary–
venous network
Fetal capillariesEndothelium of
fetal capillary
SyncytiotrophoblastSyncytiotrophoblast
Fibrinoid material
Nuclear aggregation or syncytial knotCytotrophoblastAnchoring villus
Hofbauer cellConnective
tissue corePlacental
membraneFetal blood
Fetal capillary
Persisting cytotrophoblast cells | 262 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Fetal membranes
177
cHapte R 9acute phase of a newly acquired maternal infection. As the maternal
circulation inside the placenta only starts to function at the end of the
first trimester, transmission of Toxoplasma to the embryos is rare during
that period, but highly damaging to the developing fetus. Plasmodium
falciparum is transferred by villous sequestration of maternal infected erythrocytes or a breakdown of the villous barrier during labour. The
presence of maternal rubella in the early months of pregnancy is of
especial importance in relation to the production of congenital anoma
lies. (For further reading about TORCH perinatal infections, caused by Toxoplasma gondii, rubella virus, CMV and herpes simplex virus (HSV),
see Stegmann and Carey (2002).)
FETAL MEMBRANES
The implanting conceptus consists initially of three cavities and their surrounding epithelia. The original blastocyst cavity, surrounded by
trophoblast, is now termed the chorionic cavity (synonymous with
extraembryonic coelom). It is a large cavity containing the much smaller
amniotic cavity and secondary yolk sac (see Fig. 9.1). The apposition
of the latter two cavities delineates the extent of the early embryo. The chorionic cavity becomes lined with extraembryonic mesoblast, which
is also reflected over the outer surface of the amnion and yolk sac. A
fourth cavity, the allantois, develops later as a caudal hypoblastic diver
ticulum that becomes embedded within the extraembryonic mesenchyme, forming the connecting stalk of the embryo. It does not have a
direct mesothelial covering.
Chorion
The chorion consists of developing trophoblast and extraembryonic
mesothelium. It varies in thickness during development, both tempor
ally and spatially. It is thickest at the implantation site throughout gesta
tion as the chorion frondosum and then the placenta, and thinner as gestation progresses over the remainder of its surface as the chorion
laeve (see Fig. 9.4). At term, the chorion consists of an inner cellular
layer containing fibroblasts and a reticular layer of fibroblasts and Hof
bauer cells, which resembles the mesenchyme of an intermediate villus.
The outer layer consists of cytotrophoblast 3–10 cells deep, resting on
a pseudo basement membrane, which extends beneath and between
the cells. Occasional obliterated villi within the trophoblast layer are
the remnants of villi present in the chorion frondosum of the first tri
mester. Although the interface between the trophoblast and decidua parietalis is uneven, no trophoblast infiltration of the decidua parietalis
occurs.
Yolk sac
As the secondary yolk sac forms, it delineates a cavity lined with par
ietal, and perhaps visceral, hypoblast, continuous with the developing
endoderm from the primitive streak (Ch. 10; see Fig. 10.10). The sec
ondary yolk sac is the first structure that can be detected ultrasono
graphically within the chorionic cavity (Jauniaux et al 1991). Its
diameter increases slightly between 6 and 10 weeks of gestation, reach
ing a maximum of 6–7 mm, after which its size decreases.
The inner cells of the yolk sac (denoted endoderm in many studies,
although this layer is restricted to the embryo itself) display a few short microvilli and are linked by juxtaluminal tight junctions (Jones and
Jauniaux 1995). Their cytoplasm contains numerous mitochondria,
whorls of rough endoplasmic reticulum, Golgi bodies and secretory
droplets, giving them the appearance of being highly active synthetic
cells. With further development, the epithelium becomes folded to
form a series of cyst like structures or tubules, only some of which com
municate with the central cavity. The cells synthesize several serum proteins in common with the fetal liver, such as α
fetoprotein (AFP),
α1antitrypsin, albumin, pre albumin and transferrin (Gulbis et al
1998). With rare exceptions, the secretion of most of these proteins is
confined to the embryonic compartments (Jauniaux and Gulbis 2000b).
The yolk sac becomes coated with extraembryonic mesenchyme,
which forms mesenchymal and mesothelial layers. A diffuse capillary
plexus develops between the mesothelial layer and the underlying sec
ondary yolk sac wall, and subsequently drains through vitelline veins to the developing liver. The mesothelial layer bears a dense covering of microvilli; the presence of numerous coated pits and pinocytotic vesi
cles gives it the appearance of an absorptive epithelium (Jones and
Jauniaux 1995, Burke et al 2013).
The secondary yolk sac plays a major role in the early embryonic
development of all mammals. In laboratory rodents, it has been The core of a villus contains small and large reticulum cells, fibro
blasts and macrophages (Hofbauer cells). Early mesenchymal cells
probably differentiate into small reticulum cells, which, in turn, produce
fibroblasts or large reticulum cells. The small reticulum cells appear to
delimit a collagen free stromal channel system through which Hofbauer
cells migrate. Mesenchymal collagen increases from a network of fine
fibres in early mesenchymal villi to a densely fibrous stroma within stem
villi in the second and third trimesters. After approximately 14 weeks,
the stromal channels found in immature intermediate villi are infilled by collagen to give the fibrous stroma characteristic of the stem villus.
Fetal placental vessels include arterioles and capillaries; lymphatic
vessels are not present. Pericytes may be found in close association with
the capillary endothelium, and from late first trimester, the vessels are
surrounded externally by a basal lamina. From the second trimester
(and a little later in terminal villi), dilated thin walled capillaries are
found immediately adjacent to the villous trophoblast; their respective basal laminae apparently fuse to produce a vasculosyncytial interface;
the distance separating the maternal and fetal circulations may be
reduced to as little as 2–3 µm. The endothelial cells are non fenestrated,
display numerous caveolae, and are linked by conspicuous junctional
complexes incorporating tight and adherens junctions.
transport across placental villi
The mechanism of transfer of substances across the placental barrier (membrane) is complex. The volume of maternal blood circulating
through the intervillous space has been assessed at 500 ml per minute.
Simple diffusion suffices to explain gaseous exchange. Transfer of ions
and other water soluble solutes is by paracellular and transcellular dif
fusion and transport; the relative importance of each of these for most
individual solutes is unknown, and the paracellular pathway is mor
phologically undefined. Glucose transfer involves facilitated diffusion, while active transport mechanisms carry calcium and at least some
amino acids. The fat soluble and water soluble vitamins are likely to
pass the placental barrier with different degrees of facility (Jauniaux and
Gulbis 2000a, Jauniaux et al 2004, Jauniaux et al 2005). The water
soluble vitamins B and C pass readily. Water is interchanged between
fetus and mother (in both directions) at approximately 3.5 litres per
hour. The transfer of substances of high molecular weight, such as
complex sugars, some lipids and hormonal and non hormonal pro
teins, varies greatly in rate and degree and is not so well understood;
energy dependent selective transport mechanisms, including receptor
mediated transcytosis, are likely to be involved.
Lipids may be transported unchanged through and between the cells
of the trophoblast to the core of the villus. The passage of maternal
antibodies (immunoglobulins) across the placental barrier confers
some degree of passive immunity on the fetus; it is widely accepted that
transfer is by micropinocytosis. Investigation of transplacental mechan
isms is complicated by the fact that the trophoblast itself is the site of
synthesis and storage of certain substances, e.g. glycogen.
The placenta is an important endocrine organ. Some steroid hor
mones, various oestrogens, βendorphins, progesterone, hCG and
human chorionic somatomammotropin (hCS), also known as placental lactogen (hPL), are synthesized and secreted by the syncytium. The tro
phoblast contains enzyme systems that are associated with the synthesis of steroid hormones, as well as enzymes that inactivate maternal hor
mones, allowing the fetus to develop and mature in a protected environ
ment; for example, the fetus is protected from 10 times higher maternal
glucocorticoid levels by the placental enzyme 1 1beta hydroxysteroid
dehydrogenase 2 (1 1beta HSD2), which converts biologically active
maternal cortisol to inactive cortisone (Murphy et al 1974).
It has been suggested that leukocytes may migrate from the maternal
blood through the placental barrier into the fetal capillaries. It has also
been shown that some fetal and maternal red blood cells may cross the
barrier. The former may have important consequences, e.g. in Rhesus
incompatibility.
The majority of drugs are small molecules and are sufficiently
lipophilic to pass the placental barrier from 7 weeks of gestation in
quantities that yield measurable concentrations in both coelomic and
amniotic fluid. Many are tolerated by the fetus but some may exert grave
teratogenic effects on the developing embryo/fetus, depending on the
gestational age and the doses used by the mother (e.g. thalidomide,
cocaine) (Ross et al 2015). A well documented association exists
between maternal alcohol ingestion and fetal abnormalities. Addiction
of the fetus can occur to substances of maternal abuse such as heroin.
A wide variety of bacteria (Listeria), protozoa ( Toxoplasma gondii,
Plasmodium falciparum) and viruses, including cytomegalovirus (CMV), human immunodeficiency virus (HIV) and rubella, are known to pass the placental barrier from mother to fetus. Toxoplasma and CMV are transferred by cellular invasion of the villous trophoblast during the | 263 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Implantat Ion and placentat Ion
178SectIon 2intra abdominal urachus; this, in turn, continues into the apex of the
bladder.
Amnion (chorio-amnion)
The original amniotic cells develop from the edges of the epiblast of
the embryonic disc, which ultimately form the interface with the skin
at the umbilical region. Between the tenth and twelfth weeks of preg
nancy, the amniotic cavity expands until it makes contact with the
chorion to form the chorio amnion, an avascular membrane that per
sists to term. The amniotic membrane extends along the connecting
stalk and forms the outer covering of the umbilical cord. After birth,
the site of this embryonic/extraembryonic junction is important because
the extraembryonic cell lines will die, causing the umbilical cord to
degenerate and detach from the body wall. In cases of anomalous
development of the ventral body wall, e.g. gastroschisis and exompha
los, the reflections of the amnion along the forming umbilical cord may be incomplete (see below).
The inner surface of the amnion consists of a simple cuboidal epi
thelium. It has a microvillous apical surface, beneath which is a cortical
web of intermediate filaments and microfilaments. There are no tight
junctional complexes between adjacent cells and cationic dyes penetrate
between the cells as far as the basal lamina. The intercellular clefts
present scattered desmosomes, but elsewhere the clefts widen and
contain interlacing microvilli. These features are consistent with selec
tive permeability properties. The epithelium synthesizes and deposits extracellular matrix into the compact layer of acellular stroma located
beneath the basal lamina, as well as the basal lamina itself.
Towards the end of gestation, increasing numbers of amniotic
cells undergo apoptosis. Apoptotic cells become detached from the
amnion and are found in the amniotic cavity at term. The highest inci
dence is in weeks 40–41, independent of the onset of labour. Apoptosis may play a role in the fragility and rupture of the fetal membranes
at term.
Human amniotic epithelial cells are thought to be pluripotent
because they arise so early from the conceptus. They can be distin
guished from the epiblast cells from day 8. Amniotic cells lack the MHC
antigen and so the amnion can be exposed to the maternal immune
system without eliciting a maternal immune response. Cultured human
amniotic epithelial cells express a range of neural and glial markers,
including glial fibrillary acidic protein, myelin basic protein, vimentin
and neurofilament proteins, suggesting that these cells may supply
neurotrophic factors to the amniotic fluid. They also appear to have a
hepatocyte gene expression profile, showing albumin production, gly
cogen storage and albumin secretion in culture. In organ culture, they
have been shown to secrete 30 fold larger amounts of albumin than in
monolayer culture, and to secrete α1antitrypsin (Takashina et al
2004). Amnion is used in the repair of corneas after trauma and as a
graft material for reconstructing vaginas in women with cloacal
abnormalities.
Prenatal changes to the chorio-amnion
At term, the surface area of the chorio amnion is 1000–1200 cm2, with
30% overlying the placenta and the remaining 70% in contact with the decidua (Myatt and Sun 2010). There is a diminution of the decidual
component of the chorion and an increase in chorionic apoptosis
towards term at the supracervical site, overlying the cervical os, which
encompasses the site of rupture (Chai et al 2013). The amnion and
choriodecidua show varying degrees of separation over the putative
rupture site; the mean rupture strength of the zone is 60% of the
remaining membranes, associated with changes in collagen organiza
tion and increase in MMP 9 (Strauss 2013). Term vaginal delivery is
associated with a three fold increase in apoptosis in the chorion close
to the rupture site, compared to fetal membranes from non labour,
elective caesarean delivery (Harirah et al 2012).
In preterm, premature rupture of membranes (PPROM), the chorion
is reported to be thinner at all sites (Fortner et al 2014). The presence
of bacteria in the chorion at term, ascending via the vagina, has been
reported without concomitant chorio amnionitis. However, chorio
amnionitis rates are higher in preterm labour and this may be a con
tributing cause (Harirah et al 2012, Fortner et al 2014). Loss of
chorio amnion intracellular cytokeratins, which become downregu
lated in infection, is thought to make the amnion vulnerable to apop
tosis, shear stress and rupture (Vanderhoeven et al 2014).
There is increasing evidence that infection and inflammation of the
chorio amnion is associated with prenatal changes to the developing
brain, especially the cortex, periventricular white matter and the devel
oping cerebral blood vessels (Harteman et al 2013, Roescher et al demonstrated as one of the initial sites of haemopoiesis. Human data
indicate that it has an absorptive role for molecules of maternal and
placental origin found in the chorionic cavity (Gulbis et al 1998), and
mediates the main movement of molecules passing from the chorionic cavity to the yolk sac and, subsequently, to the embryonic gut and
circulation.
After week 9, the cellular components of the wall of the secondary
yolk sac start to degenerate, and their function is subsumed into
exchanges at the placental chorionic plate. With embryonic develop
ment of the midgut, the connection of the yolk sac to the embryo becomes attenuated to a slender and elongated vitelline intestinal duct.
Both the yolk sac and its duct remain within the extraembryonic coelom
(chorionic cavity) throughout gestation, located between the amnion
and chorion as they fuse, near the placental attachment of the umbilical
cord.
Allantois
The allantoenteric diverticulum (see Fig. 10.10) arises early in the third
week as a solid, endodermal outgrowth from the dorsocaudal part of
the yolk sac into the mesenchyme of the connecting stalk. It soon
becomes canalized. When the hindgut is developed, the proximal
(enteric) part of the diverticulum is incorporated in its ventral wall. The
distal (allantoic) part remains as the allantoic duct and is carried ven
trally to open into the ventral aspect of the cloaca or terminal part of the hindgut ( Fig. 9.9A). The allantois is a site of angiogenesis, giving
rise to the umbilical vessels that connect to the placental circulation. The extraembryonic mesenchyme around the allantois forms the con
necting stalk, which is later incorporated into the umbilical cord.
In the fetus, the allantoic duct, which is confined to the proximal
end of the umbilical cord, elongates and thins. However, it may persist as an interrupted series of epithelial strands at term, in which case the
proximal strand is often continuous at the umbilicus with the median
Fig. 9.9 A, A Longitudinal section of a conceptus showing the cavities
associated with development. The amniotic cavity and yolk sac are both
covered with extraembryonic mesoblast and are contained within the larger chorionic cavity, which is lined with extraembryonic mesoblast. The embryo is attached to the chorion frondosum by the connecting stalk into which the allantois projects. B, A longitudinal section of a conceptus at a
later stage, showing the diminution of the chorionic cavity, expansion of the amniotic cavity, relative attenuation of the yolk sac and the structures that give rise to the umbilical cord. A
Connecting stalk
attachmentbecomes moreventrally placed
Chorionic cavity/
extraembryoniccoelom
Pericardial coelomYolk sacPlacental area (chorion frondosum)
Allantoic canal
Amniotic cavity
Chorion laeve
Yolk sac
Line of fusion
of amnion withchorion, i.e. lineof obliteratedextraembryoniccoelom
Amniotic cavityUmbilical cord
Obliterated
yolk duct
Umbilical coelom
Allantoic ductB | 264 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Umbilical cord
179
cHapte R 9of cytokines, have also been detected in amniotic fluid; the concentra
tions of most cytokines differ from those in maternal blood and reflect
the physiological status of the fetus (Tong 2013). Proteomic analysis of
human amniotic fluid at 16–18 weeks has identified more than 800
proteins and peptides (Cho et al 2007). It appears that amniotic fluid
may be acting as a pathway for the transport of signalling molecules to target cells. In animal studies, substances injected into the amniotic
cavity were detected within the embryos within 2 minutes, demonstrat
ing a rapid exchange with all fetal tissues (Tong 2013).
A number of clinical markers in amniotic fluid are used as predic
tors of fetal health, including interleukin (IL) 10, IL 6, cell free fetal
DNA and cell free fetal mRNA. Genes and proteins specific to the fetal brain, lung, kidney, blood and heart have been identified in amniotic
fluid, differential gene expression investigated in a range of trisomic
fetuses and lung maturity predicted by measurement of lecithin, sphin
gomyelin and phosphatidylglycerol (Edlow and Bianchi 2012, Ross
and Beall 2014). Bacterial species have been isolated from amniotic
fluid, and microbial invasion of the amniotic cavity is considered a
leading cause of preterm premature delivery (DiGiulio 2012). It is
thought such bacteria access the amniotic cavity via the vagina and
cervix, although many oral bacteria have also been identified, suggest
ing that a haematogenous route is also likely. The establishment of pathogenesis is influenced by microbial adhesion, biofilm formation,
and immune evasion and the maternal and fetal immune systems
(DiGiulio 2012).
UMBILICAL CORD
The formation of the connecting stalk and the early formation of the
umbilical cord are described in Chapter 10. The umbilical cord ulti
mately consists of an outer covering of flattened amniotic epithelial cells and an interior mass of mesenchyme of diverse origins (see Fig.
9.9). It contains two tubes of hypoblastic origin, the vitelline intestinal
and allantoic ducts, and their associated vitelline and allantoic (umbili
cal) blood vessels. The yolk stalk and continuing duct extend the length
of the cord, whereas the allantoic duct extends only into its proximal
part.
The mesenchymal core is derived from the somatopleuric extra
embryonic mesenchyme covering the amniotic folds, splanchnopleuric
extraembryonic mesenchyme of the yolk stalk (which carries the vitel
line vessels and clothes the yolk duct), and similar allantoic mesen
chyme of the connecting stalk (which clothes the allantoic duct and
initially carries two umbilical arteries and two umbilical veins). These
various mesenchymal compartments fuse and are gradually trans
formed into the loose connective tissue (Wharton’s jelly) that character
izes the more mature cord. The tissue consists of widely spaced elongated
fibroblasts separated by a delicate three dimensional meshwork of fine
collagen fibres, which contains a variety of hydrated glycosaminogly
cans and is particularly rich in hyaluronic acid.
The vitelline and allantoic (umbilical) vessels, which are initially
symmetrical, become modified as a result of changes in the circulation. The vitelline vessels involute, whereas most of the allantoic (umbilical)
vessels persist. The right umbilical vein disappears but the two umbilical
arteries normally remain. Occasionally, one umbilical artery may disap
pear; there is some correlation within structural anomalies, most often cardiac, in such cases. The vessels of the umbilical cord are rarely
straight, and are usually twisted into either a right or left handed cylin
drical helix. The number of turns involved ranges from a few to over 300. This conformation may be produced by unequal growth of the
vessels, or by torsional forces imposed by fetal movements. Its func
tional significance is obscure; perhaps the pulsations and contractions of the helical vessels assist the venous return to the fetus in the umbili
cal vein.
Anomalies of the fetal anterior abdominal wall, such as exomphalos
and gastroschisis, may affect the arrangement of the outer covering of amnion cells along the proximal end of the umbilical cord. Exomphalos
arises from a failure of the lateral folds along the ventral surface of the
embryo, resulting in failure of the normal embryonic regression of the
midgut from the umbilical stalk into the abdominal cavity. The abdomi
nal contents, including intestines and liver or spleen, covered by a sac
of parietal peritoneum and amnion, protrude into the base of the
umbilical cord. In gastroschisis, the insertion of the umbilical cord is intact and there is evisceration of the intestine through a small abdominal wall defect that is usually located to the right of the umbilical cord; this results in free loops of bowel in the amniotic cavity. Theories con
cerning the aetiology of this defect include abnormal involution of the right umbilical vein or disruption of the omphalomesenteric artery by ischaemia.2014). This association also extends to the neonatal period and post
natal gut maturation (Wynn and Neu 2012). Histologically confirmed
chorio amnionitis is associated with changes in cerebral blood flow
soon after birth; male infants appear to be more affected than female
(Koch et al 2014).
AMNIOTIC FLUID
The amniotic fluid, or liquor amnii, provides a buoyant medium that
supports the delicate tissues of the young embryo and allows free move
ment of the fetus up to the later stages of pregnancy. It also diminishes the risk to the fetus of injury from outside. Amniotic fluid is derived
from multiple sources throughout gestation. These include secretions
from amniotic epithelium, filtration of fluid from maternal vessels via
the parietal decidua and amniochorion, filtration from the fetal vessels
via the chorionic plate or the umbilical cord, and fetal urine and fetal
lung secretions. In early pregnancy, diffusion from intracorporeal
vessels via fetal skin provides another source. Once the gut is formed,
fetal swallowing of amniotic fluid is a normal occurrence; the fluid is
absorbed into the fetal circulation.
Amniotic fluid volume increases logarithmically during the first half
of pregnancy, from less than 10 ml at 8 weeks’ gestation to 30 ml at 22
weeks and 770 ml at 28 weeks. After 30 weeks, the volume may remain
unchanged to 36 weeks. It decreases towards the expected delivery date,
and volumes decrease sharply in post term gestation, averaging 515 ml
at 41 weeks (Beall et al 2007, Ross and Beall 2014).
Human fetal urine output from the metanephric kidney increases
from 1 10 ml/kg/day at 25 weeks to almost 200 ml/kg/day at term; this
corresponds to almost 1000 ml/day. The rate of human fetal lung secre
tion has not been measured but is presumed to be in the range of
60–100 ml/kg/day near term (Callen 2008). Animal studies indicate
that lung fluid production is about one third that of urine production,
and that half of the fluid leaving the lungs enters the amniotic fluid and
half is swallowed (Beall et al 2007, Ross and Beall 2014).
It is estimated that human fetuses swallow up to 760 ml/day of
amniotic fluid near term, although this decreases in the days before delivery. The remainder of the amniotic fluid is thought to be absorbed
via an intramembranous pathway directly across the amniotic cavity
and fetal surface of the placenta into fetal blood vessels (Beall et al
2007, Ross and Beall 2014). Intramembranous flow is thought to reach
400 ml/day at term (Callen 2008).
Amniotic fluid volume is estimated at routine antenatal ultrasound
scans, despite some concerns about its objectivity (Beattie and Rich 2007, Callen 2008, Gilbert 2012); it may be expressed as an amniotic
fluid index. A deficiency of amniotic fluid is termed oligohydramnios and absent amniotic fluid is anhydramnios. Oligohydramnios in the
second or third trimester is usually the result of urinary tract malforma
tions, e.g. bilateral renal agenesis or obstruction of the lower urinary tract, uteroplacental insufficiency or premature rupture of the mem
branes. The urachus may play a critical role in the resolution of oligo
hydramnios in lower urinary tract obstruction by acting as a fistula
between the bladder and the amniotic space. The major concern with
oligohydramnios at less than 20 weeks is the significant risk of pulmo
nary hypoplasia and neonatal death. A volume of amniotic fluid in
excess of 2 litres is generally considered to be abnormal and constitutes
polyhydramnios (see Fig. 14.5A). Maternal causes include cardiac and
renal problems and diabetes mellitus, which causes fetal hyperglycae
mia and polyuria. Fetal causes include reduced fetal swallowing due to
congenital malformations, e.g. anencephaly; upper intestinal tract
obstruction (oesophageal and duodenal atresia); compressive pulmo
nary disorders (congenital diaphragmatic hernia); and neuro muscular
impairment of swallowing.
Amniotic fluid has, for many years, been regarded as a medium that
physically supports the developing embryo and fetus. More recently, it
has also been conceptualized as part of the embryonic and early fetal
extracellular matrix, and its composition throughout pregnancy has
been investigated (Tong et al 2009). Techniques for biochemical analy
sis have revealed similarities and differences between amniotic fluid and fetal umbilical cord blood and maternal serum. Although, early in
development, amniotic fluid resembles blood plasma, it also demon
strates extremely low oxygen tension, reflecting the physiological pla
cental hypoxia noted in the first trimester (Ross and Beall 2014, Jauniaux
et al 2003). In the second and third trimesters, primary electrolytes are
similar in amniotic fluid, umbilical cord blood and maternal serum,
whereas glucose, cholesterol and some enzymes are markedly lower in amniotic fluid. Umbilical cord blood and maternal serum contain 8 and 12.5 times more protein than amniotic fluid, respectively (Tong
et al 2009). More than 100 metabolites, including cortisol and a range | 265 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Implantat Ion and placentat Ion
180SectIon 2Mature umbilical vessels, particularly the arteries, have a strong mus
cular coat that contracts readily in response to mechanical stimuli. The
outermost bundles pursue an interlacing spiral course, and when they
contract, they produce shortening of the vessel and thickening of the
media, with folding of the interna and considerable narrowing of the
lumen. This action may account for the periodic sharp constrictions of
contour, the so called valves of Hoboken, which often characterize
these vessels.
The fully developed umbilical cord is, on average, some 50 cm long
and 1–2 cm in diameter. Its length varies from 20 to 120 cm; exception
ally short or long cords are associated with fetal problems and compli
cations during labour. A long umbilical cord may prolapse through the
cervix into the vagina once the fetal membranes rupture and this may
be exacerbated by conditions that prevent the fetal head from fully
occupying the maternal pelvis, e.g. pelvic tumours (fibroids), ovarian
cysts, placenta praevia and prematurity. Compression of the cord by the
presenting part of the fetus, or an umbilical artery spasm, will lead to
fetal hypoxia and death, if untreated. The risk of perinatal death rises
as the interval from diagnosis to delivery increases. The treatment is
either funic replacement (pushing the cord back above the fetal head)
or, more commonly, immediate caesarean section, depending on factors
such as fetal viability.
The distal end of the umbilical cord usually attaches in the central
portion of the placenta, but in 0.2% of pregnancies, velamentous inser
tion is observed (i.e. into the membranes) and this may be associated Video 9.1 Ultrasound features of the maternal placental blood flow. Bonus e-book video
with vulnerability to injury and fetal haemorrhage. This is especially
important if the placenta is low lying, and may be associated with vasa
praevia, in which case fetal blood vessels run across the internal os.
Inadvertent rupture of the fetal vessels in spontaneous labour or at the
time of amniotomy (artificial rupture of membranes to induce labour)
will cause fetal haemorrhage and may prove fatal. Infection of the
umbilical cord, funisitis, is associated with chorio amnionitis and later
neurodevelopmental outcomes (Roescher et al 2014, Buhimschi and
Buhimschi 2012).
Recent studies have examined the optimal time for cutting the
umbilical cord after delivery and found that early clamping is associated with neonatal anaemia and iron deficiency. Delayed clamping, defined
as ligation of the umbilical cord 2–3 minutes after birth or when cord
pulsation stops, reduces the prevalence of iron deficiency at 4 months
of age and results in improved ferritin levels (Andersson et al 201 1,
McDonald et al 2013).
KEY REFERENCES
Brosens JJ, Salker MS, Teklenburg G et al 2014 Uterine selection of human
embryos at implantation. Sci Rep 4:3894.
Study showing that developmentally impaired human embryos elicit an endoplasmic stress response in human decidual cells and suggesting that distinct positive and negative mechanisms contribute to active selection of human embryos at implantation.
Burton GJ, Jauniaux E 201 1 Oxidative stress. Best Pract Res Clin Obstet
Gynaecol 25:287–99.A review of the evidence implicates oxidative stress in the pathophysiology of placental related complications of human pregnancy ranging from miscarriage to pre-eclampsia and premature rupture of the membranes.
Burton GJ, Jauniaux E, Watson AL 1999 Maternal arterial connections to the
placental intervillous space during the first trimester of human pregnancy: the Boyd collection revisited. Am J Obstet Gynecol 181:
718–24.Using hysterectomy specimens from the Boyd anatomy collection, this study shows that the maternal circulation to the placenta must be extremely sluggish before the eighth week of pregnancy, supporting the concept that development of the human fetoplacental unit during most of the first trimester takes place in a low-oxygen environment.
Burton GJ, Watson AL, Hempstock J et al 2002 Uterine glands provide his
tiotrophic nutrition for the human fetus during the first trimester of pregnancy. J Clin Endocrinol Metab 87:2954–9.The first study to demonstrate that the uterine glands are an important source of nutrients during organogenesis, when metabolism is essentially anaerobic. Uterine glands remain active until at least week 10 of pregnancy, and their secretions are delivered freely into the placental intervillous space, supporting the concept of histiotrophic nutrition as opposed to hemochorial nutrition.
Cho CK, Shan SJ, Winsor EJ, Diamandis EP 2007 Proteomics analysis of
human amniotic fluid. Mol Cell Proteomics 6:1406–15.This paper presents an extensive profile of the normal human amniotic fluid proteome and considers the molecular functions of amniotic fluid including the development of biomarkers for pregnancy-associated abnormalities.Gulbis B, Jauniaux E, Cotton F et al 1998 Protein and enzyme patterns in
the fluid cavities of the first trimester gestational sac: relevance to the absorptive role of secondary yolk sac. Mol Hum Reprod 4:857–62.Study demonstrating that the secondary yolk sac membrane is an important zone of transfer between the extra-embryonic and embryonic compartments and suggesting that therapeutic protocols making use of fetal somatic gene therapy could be performed by injecting transduced cells into the exocoelomic cavity.
Jauniaux E, Gulbis B 2000a Fluid compartments of the embryonic environ
ment. Hum Reprod Update 6:268–78.This paper reviews the composition, biology and role of the extraembryonic coelomic or chorionic fluid cavity and its connections with the secondary yolk sac and developing placenta during the first trimester of human
pregnancy.
Ross MG, Beall MH 2014 Amniotic fluid dynamics. In: Creasy R, Resnik R,
Iams JD et al (eds) Saunders’ Maternal Fetal Medicine: Principles and
Practice, 7th ed. Philadelphia: Elsevier, Saunders; Ch. 3, pp. 47–52.
This chapter provides an overview of the production, circulation and absorption of amniotic fluid during gestation.
Strauss JF 2013 Extracellular matrix dynamics and fetal membrane rupture.
Repro Sci 20:140–53.This paper presents the composition and biomechanical properties of the fetal membrane extracellular matrix. It considers the changes in extracellular matrix composition during normal pregnancy and term delivery and those seen in preterm premature rupture of membranes.
Tong X 2013 Amniotic fluid may act as a transporting pathway for signalling
molecules and stem cells during the embryonic development of amni
otes. J Chinese Med Ass 76:606–10.This paper considers the bi-directional substance exchange between amniotic fluid and fetal tissues, its role in transporting signaling molecules and the dynamic nature of amniotic fluid throughout pregnancy. | 266 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Implantation and placentation
180.e1
cHapte R 9REFERENCES
Andersson O, Hellström Westas L, Andersson D et al 201 1 Effect of delayed
versus early umbilical cord clamping on neonatal outcomes and iron
status at 4 months: a randomised controlled trial. BMJ 343:d7157.
Beall MH, van den Wijngaard JPHM, van Gemert MJC et al 2007 Amniotic
fluid water dynamics. Placenta 28:816–23.
Beattie RB, Rich DA 2007 Disorders of amniotic fluid, placenta and mem
branes. In: Twining P, McHugo JM, Pilling DW (eds) Textbook of Fetal
Abnormalities. London: Elsevier, Churchill Livingstone; Ch 5, pp.
75–94.
Brosens JJ, Salker MS, Teklenburg G et al 2014 Uterine selection of human
embryos at implantation. Sci Rep 4:3894.
Study showing that developmentally impaired human embryos elicit an endoplasmic stress response in human decidual cells and suggesting that distinct positive and negative mechanisms contribute to active selection of human embryos at implantation.
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181
CHAPTER 10 Cell populations at gastrulation
the region of the disc closest to the streak ‘caudal’, and the region of
the disc furthest from the streak ‘cranial’ or ‘rostral’ . With the develop
ment of the streak, the terms medial and lateral can be used. The relative
dimensions of the primitive streak and the fates of the cells that pass through it change with the developmental stage. Thus, the streak extends half way along the disc in the stage 6 embryo, reaching its greatest relative length in stage 7 and its maximum length in stage 8.
Formation of the primitive streak is induced by the underlying vis
ceral hypoblast, which remains beneath the streak even at later stages. CONCEPTUS WITH A BILAMINAR
EMBRYONIC DISC
At stage 6, the conceptus is composed of the walls of three cavities: the
large chorionic cavity is surrounded by a meshwork of trophoblast and
developing villi, and lined with extraembryonic mesoblast. The chorion,
trophoblast and extraembryonic mesoblast enclose the extraembryonic
coelom and contain the much smaller amniotic cavity and yolk sac (see
Fig. 9.1). These latter cavities abut at the embryonic bilaminar disc
where the epithelial epiblast and visceral hypoblast are approximated.
A fourth cavity, the allantois, will form as a hypoblastic diverticulum in
stage 7. The ‘bilaminar disc’ commonly referred to in embryology texts
does not yet possess the definitive layers of embryonic ectoderm and
endoderm that will give rise to embryonic structures. Only the epiblast
will give rise to the embryo; all other layers produced so far are extra
embryonic. The amnion and chorion (and surrounding mesoblast) are
part of the extraembryonic somatopleure, whereas the yolk sac, allan
tois and surrounding extraembryonic mesoblast constitute extraembry
onic splanchnopleure. At the junctional zone surrounding the margins
of the embryonic area, where the walls of the amnion and yolk sac
converge, the somatopleuric and splanchnopleuric layers of extraem
bryonic mesoblast are continuous.
The terms epiblast and hypoblast are used to make the distinction
between the earliest bilaminar disc layers and the later embryonic layers. Epiblast and hypoblast contain mixed populations of cells with
little restriction, which establish the placental structures and extraem
bryonic tissues before the production of embryonic cell lines at gastrula
tion. The older terminology depicting three germ layers that give rise to
the skin, gut lining and intervening tissues is thus incorrect for the
bilaminar and trilaminar embryonic disc. The application and retention
of this aged terminology for the early stages of embryology continue to
cause confusion and inhibit the development of more pertinent descrip
tive language to describe these early events. The earliest three cell line
ages, not layers, that give rise to the extraembryonic membranes and the embryo are trophoblast, hypoblast and epiblast; each expresses dif
ferent lineage specific genes for its establishment, mainten ance and
differentiation (Artus and Hadjantonakis 2012).
At early stage 6, the epiblast is producing extraembryonic mesen
chyme from its caudal margin. With the appearance of the primitive streak, a process is begun whereby cells of the epiblast either pass deep
to the epiblast layer to form the populations of cells within the embryo,
or remain on the dorsal aspect of the embryo to become the embryonic
ectoderm. Although human embryos do not form a ‘gastrula’ as such,
the term gastrulation is used here to denote an early period of develop
ment during which significant rearrangements, migrations and folding of the early embryo occur. The primitive streak is the site of organizer
cells analogous to those found in embryos that do undergo gastrula
tion. The appearance of the primitive streak therefore marks the begin
ning of a period when gross alterations in morphology and complex
rearrangements of cell populations occur. During this time, the epiblast
will give rise to a complex multilaminar structure with a defined cranio
caudal axis. By the end of gastrulation, cell populations from different, often widely separated, regions of the embryonic disc will become
spatially related and the embryonic shape will have been produced.
Primitive streak and node
Seen from the dorsal (epiblastic) aspect, at stage 6, the embryonic disc
appears elongated. The primitive streak is first seen in the caudal region of the embryonic disc at this stage as a collection of pluripotent cells, orientated along its long axis in the median position, conferring
the future craniocaudal axis of the embryo ( Figs 10.1–10.2). Although
the future cranial and caudal regions of the embryo are well within the boundaries of the embryonic disc, it has become the practice to term Fig. 10.1 A longitudinal section through an early conceptus. Ingression of
mesoblast is occurring at the primitive streak and the notochord is
ingressing via the primitive (Hensen’s) node. Epiblast
Primitive node
NotochordHypoblast
MesenchymeYolk sacAllantoisExtraembryonic mesoblast
Primitive streak
Amniotic
cavity
Fig. 10.2 A transverse section through the embryonic plate at the level of
the primitive streak to show the early movement of mesoblast between
the epiblast and underlying hypoblast. Primitive streak
Extraembryonic mesoblastEpiblast
Hypoblast
MesenchymeEndodermal and
notochord cells | 269 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Cell populations at gastrulation
182seCtion 2
along the long axis of the embryonic disc. By stages 9 and 10, the cells
at the lateral edge of the plate have begun to migrate laterally as free
mesenchymal cells and the plate reduces in height to two cells deep. At
stage 1 1, the migrating prechordal mesenchyme forms bilateral pre
mandibular mesenchymal condensations and is no longer a median
structure. The extent of prechordal cells remaining within the endoderm
is not clear.
Notochord
The notochord, also called chordamesoderm, the head process or
chorda, arises from epiblast cells of the medial part of the primitive
node. It passes through several stages during development. The cells of
the early notochordal process express myogenic markers transitorily as
they migrate beneath the epiblast, but later they become epithelial,
forming junctions and a basal lamina. The notochordal cells are inti
mately mixed with endodermal cells, as both cell lines ingress at the same time (see Figs 10.1–10.2; Fig. 10.4). In the stage 8 embryo, the
ingressing notochordal cells remain in the midline along the cephalo
caudal axis. They form a rostral part, which is composed of a cell mass
continuous with the prechordal mesenchyme; a mid portion, in which
cells are arranged in a tube with a central notochordal canal; and a
caudal epithelial layer of cells, the notochordal plate, which is contigu
ous with the embryonic endoderm and forms a roof to the secondary yolk sac. There is a transitory opening between the primitive node (and
amniotic cavity) and the secondary yolk sac called the neurenteric canal
(so named because its upper opening is in the future caudal floor of
the neural groove, and its lower opening is into the archenteron, which
is the primitive gut); it may still be found at stage 9, and the site of the
neurenteric canal can be recognized in stage 10 embryos. The ingression
of notochordal cells at the primitive node is matched by specification
of the overlying neural ectodermal cells, and the notochordal plate is
thus matched in length by the future neural floor plate. Both the noto
chord and the region of the floor plate of the neural tube may arise from a common progenitor cell. The early notochord is important for
the maintenance and subsequent development of the neural floor plate
and the induction of motor neurones. Removal of the notochord results
in elimination of the neural floor plate and motor neurones, and
expression of sensory cell types.
Caudal eminence
From stages 9 and 10, the region between the neurenteric canal and the cloacal membrane (see below), including the primitive streak, is termed
the caudal eminence. It consists of the caudal region of the trunk, com
posed of mesenchyme derived from the primitive streak and epiblast,
and covered with surface ectoderm. Whereas ingression of cells through
the primitive streak gives rise to the prechordal and notochordal plates, and cells rostral to the neurenteric canal (see below), the cells of the caudal eminence arise from local division of a mesenchymal popula
tion positioned caudal to the neurenteric canal. The caudal portions of the notochord, which form later in development when secondary neurulation processes begin, arise from these cell populations, sometimes termed the caudoneural hinge or junction. This tissue is thicker and Fig. 10.3 The predictive fates of the epiblast cell population at the time
the primitive streak is present. Neural ectoderm
Surface ectoderm
Mesoblast
Extraembryonic mesoblastMedial halves
of the somites
Lateral halves
of the somitesNotochord
Endoderm
Primordial germ cellsThe primitive streak may be considered to be generally homologous
with the blastopore of lower vertebrates (e.g. amphibia), with the nodal
region corresponding to the dorsal lip. Experiments clearly show the
lip of the blastopore to be a dynamic wave front on which cells are
carried into the interior to form the roof of the archenteron, a situation
analogous to ingression through the node of the prechordal plate and
endoderm. The primitive streak similarly may be considered analogous
to the coapted, or fused, lateral lips of the blastopore, and the cloacal
membrane and its immediate environs are considered analogous to the
ventral lip of the blastopore.
At the primitive streak, epiblast cells undergo a period of intense
proliferation, the rate of division being much faster than that of blasto
meres during cleavage. Streak formation is associated with the local
production of several cell layers, extensive disruption of the basal
lamina, increase in adhesive plaques and gap junctions, synthesis of
vimentin, and loss of cytokeratins by the emerging cells.
As the epiblast cells proliferate, two ridges are formed on each side
of the primitive streak, which appears to sink between them. The lower
midline portion of the streak is termed the primitive groove. The process
by which cells become part of the streak and then migrate away from
it beneath the epiblast is termed ingression.
The primitive node, or Hensen’s node, is the most rostral region of
the primitive streak. It appears as a curved ridge of cells similar in shape
to the top of an old fashioned keyhole. Cells ingressing from the ridge
pass into the primitive pit (the most rostral part of the primitive groove),
and then migrate rostrally beneath the epiblast. The primitive node has
been recorded in all stage 7 human embryos; it produces axial cell
populations, the prechordal plate, notochord, embryonic endoderm
and the medial halves of the somites. Experimental removal of the
node results in complete absence of the notochord and a failure of neurulation.
Recent studies on the dynamics of ingression at the primitive streak
have shown movement of the underlying extracellular tissues as well as
of the overlying cells. Re examination of the processes of cell movement
relative to the surrounding extracellular molecules in embryos has
found that vertical and convergent extension motion patterns, previ
ously thought to be limited to the epiblast, also occur in sub epiblastic
extracellular matrix fibrils of fibronectin (Zamir et al 2008, Szabó et al
201 1).
Position and time of ingression through the
primitive streak
Studies of cell fate have shown that epiblast cells that will pass through
the streak are randomly located within the epiblast layer before their
ingression, and that epiblast fate is determined at or before the time of
ingression through the streak, indicating that passage through the prim
itive streak is the most important factor for future differentiation.
The position and time of ingression through either streak or node
directly affect the developmental fate of cells. Passage through the streak is specified according to position, e.g. via the node, or rostral, middle
or caudal regions of the streak. Cells that ingress through the primitive
node give rise to the axial cell lines, the prechordal mesenchyme and
notochord, and to the endoderm and the medial halves of the somites.
The rostral portion of the primitive streak produces cells for the lateral
halves of the somites, whereas the middle streak produces the lateral
plate mesoblast. The adjacent caudal portion of the streak gives rise to
the primordial germ cells, which can be distinguished histologically and
histochemically, and the most caudal portion of the streak contributes
cells to the extraembryonic mesoblast until the somites are visible. A
composite of the information on the position of ingression through the
streak and node is shown in Figure 10.3. The epiblast cells that do not
pass through the streak but instead remain within the epiblast popula
tion give rise to the neural and surface ectoderm of the embryo.
Prechordal plate
The earliest cells migrating through the primitive node and streak give
rise to both the embryonic endoderm and the notochord. The pre
chordal plate is first seen at stage 7. It has been defined as a localized
thickening of the endoderm rostral to the notochordal process, although
it is seen as a highly developed mesenchymal mass in contact with the floor of the neural groove, rostral to the notochordal process, rather than as an epithelial layer. The prechordal plate is a temporary collec
tion of cells that underlies the neural plate during stage 9. It is composed of cells that are similar to, or larger and more spherical than, the ingressing endodermal cells (Müller and O’Rahilly 2003). In stage 8 embryos, the prechordal plate is up to eight cells deep and extends | 270 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Conceptus with a bilaminar embryonic disc
183
CHapter 10
Intraembryonic mesoblast (mesenchyme)
Epiblast cells ingress through the cranial and middle parts of the streak
individually, maintaining their apical epithelial contacts while elong
ating ventrally. The cells become flask shaped, with thin, attenuated
apical necks and broad basal regions. Their basal and lateral surfaces form lamellipodia and filopodia, and the apical contact is released. The
cells are now free mesoblast cells, their fibroblastic, stellate morphology
reflecting the release from the epithelial layer. Once through the streak,
the cells migrate away from it, using the basal lamina of the overlying
epiblast and extracellular matrix as a substratum. The cells contact one
another by filopodia and lamellipodia, with which they also contact
the basal lamina. Gap junctions have been observed between filopodia
and cell bodies. With the appearance of the mesoblast, spaces form
between the epiblast and visceral hypoblast that are filled with extracel
lular matrix rich in glycosaminoglycans. The migrating mesoblast has a leading edge of cells that open up the migration routes, and the follow
ing cells seem to be pulled along behind in a coordinated mass move
ment. Mesoblast formed by cells migrating through the primitive node
and rostral primitive streak will form the paraxial mesenchyme, whereas
cells migrating through the middle to caudal streak will form the lateral
plate mesenchyme (see Figs 10.1–10.2).
Embryonic ectoderm
When the ingression of cells through the primitive streak is completed, the epithelial cells remaining in the epiblast layer are termed embryonic more advanced in differentiation than the tissues derived from the early
primitive streak.
Embryonic endoderm
Before ingression, definitive embryonic endoderm cells are found in the epiblast, located at the primitive node and rostral primitive streak. In
the mouse, the endodermal cells lie beneath the epiblast mainly in the
midline, interspersed with presumptive notochordal cells, forming
the roof of the secondary yolk sac. The ingressing endoderm displaces the visceral hypoblast into the secondary yolk sac wall by a dramatic
territorial expansion that is brought about by a change in the morph
ology of the cells (see Figs 10.1–10.2, 10.4). The putative endoderm cells
are cuboidal epithelial cells within the node but they become squamous
in the endoderm layer; this could result in a four fold increase in the
surface area covered by the cells. A complete replacement of the visceral hypoblast has not yet been confirmed and there may be a mixed popu
lation of cells in the endodermal layer in the early stages. Ingression of cells through the streak and node in the human is apparent at stage 6,
and, by stage 7, a population of endoderm and notochord cells is
present beneath the epiblast (see Figs 10.1–10.2, 10.4). During stages
6–1 1, the midline roof of the secondary yolk sac becomes populated mainly by the notochordal plate, which remains in direct lateral conti
nuity with the endodermal cells. It is not until stage 1 1, after the defini
tive notochord is formed, that the endoderm cells can join across the midline. For the developmental fate of the embryonic endoderm, see Figure 12.3.Fig. 10.4 A, The unfolded embryo, showing the disposition of the intraembryonic coelom within the embryonic disc. The lines across the embryo show
the level of transverse sections through the disc shown in B–D. B–D, Transverse sections through the disc at the points indicated in A. E, A longitudinal
section through the disc.
Primitive streak AllantoisNeural tissue
Primitive streak
Connecting stalk
Cloacal membrane
Entrance to
intraembryoniccoelomA
Ectoderm
Bucco-pharyngealmembrane
Endoderm
Yolk sac
MesenchymeIntraembryonic coelom
Buccopharyngeal membraneIntraembryonic coelom
Neural tissue Primitive streak
Cloacal membrane Notochordal plateNotochordal plate Intraembryonic coelom Intraembryonic
coelomB
C DAmniotic cavity
Amniotic cavity
Connecting stalk
Allantois
Yolk sacE | 271 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Cell populations at gastrulation
184seCtion 2Mesoblast that passes in a cranial direction flanks the notochordal
plate and passes around the prechordal plate region, converging medi
ally to fuse in the midline beyond its cephalic border. This transmedian
mass, in which the heart and pericardium will develop, is initially
termed the cardiogenic mesoblast. It fuses with the junctional zone of
extraembryonic mesoblast around the extreme cephalic margin of the
embryonic area. This region will eventually form the septum transver
sum and primitive ventral mesentery of the foregut. Mesoblast passing laterally from the streak soon approaches and becomes confluent with
the extraembryonic mesoblast around the margins of the disc, i.e. at the
junctional zone where the splanchnic and somatic strata of extraembry
onic mesoblast merge. Mesoblast that streams caudally from the primi
tive streak skirts the margins of the cloacal membrane and then
converges towards the caudal midline extremity of the embryonic disc
to become continuous with the extraembryonic mesoblast of the con
necting stalk. It is unclear if the lower layer of the cloacal membrane consists of visceral hypoblast, like the more cranial primitive streak
(the hypoblast is necessary for maintaining the streak), or if it is
replaced by migrating embryonic endoderm, or if there is a region for
ingression of endoderm at the caudal end of the streak, similar to the
node cranially.
Still further caudally, the embryonic disc develops a midline diver
ticulum adjacent to the cloacal membrane. This diverticulum, the
allantois, projects into the extraembryonic connecting stalk (see Figs
9.9, 10.4; Fig. 10.6). There is little information about which cells
form the allantois, i.e. whether it is composed of visceral hypoblast,
parietal hypoblast or embryonic endoderm. The allantois later devel
ops a rich anastomotic blood supply around it, in the manner of the
yolk sac.
The generation of cells at the primitive node produces midline endo
derm, notochord and the floor plate of the future neural tube. As the
notochord grows and elongates, there is a matched growth of neural
floor plate cells until both cell lines extend to the buccopharyngeal
membrane. The epiblast lateral to the midline contains both future
surface and neural ectoderm. The latter becomes arranged between the
primitive node and buccopharyngeal membrane; cells destined to be in
the neural plate lie medially, and those destined for the neural crest lie
along the junction between the neural plate and surface ectoderm (see
Fig. 10.5). A smaller subpopulation of neuronal cells, the ectodermal
placodes, are arranged either close to the neural crest or within the
rostral limit of the neural plate itself.
FOLDING OF THE EMBRYO
In a diagrammatic representation of the trilaminar disc prior to folding and viewed from the ectodermal aspect, all of the future external surface
of the body is delimited (see Fig. 10.5). The ends of the gut tube are
specified on the ectodermal surface at the buccopharyngeal and cloacal membranes, which are regions where the ectoderm and underlying
endoderm are apposed without intervening mesoblast. In the midline
between these membranes, proliferation of the neural ectoderm matches
the underlying migration of mesoblast from the primitive streak, so
that the neural plate covers the paraxial mesenchyme on each side of the notochord (see Fig. 10.6E). As the paraxial mesenchyme segments,
the formation of the epithelial somites elevates the edges of the neural plate and initiates primary neurulation (see Fig. 10.6F; Figs 10.7–10.8).
The neural plate itself undergoes concurrent morphological changes.
The most medial cells become wedge shaped, forming the neural
groove. Further elevation of the edges of the neural groove permits fusion of the neuronal populations in the dorsal midline to form the
neural tube. The surface ectoderm forms the putative dorsal epidermis
(see Figs 10.6G, 10.7–10.8). Cells at the lateral edge of the neural plate,
termed neural crest cells, remain as a linearly arranged mesenchymal population between these two epithelia. Fusion of the neural tube
begins in the future rhombencephalic region of the embryo and pro
ceeds rostrally and caudally to about the level of somite 29. Neurulation is described further in Chapter 17. A population of neural epithelial
cells remain within the surface ectoderm; at this stage they are termed ectodermal placodes.
The representation of a person on the trilaminar disc ( Fig. 10.9)
shows, to some extent, the way in which the positions of the main body
structures are already specified in the unfolded embryo. Ectoderm lateral to the neural plate and the paraxial mesenchyme will form structures within the back. The portion of the disc between the buccopharyngeal membrane and the edge of the disc will become the ventral thoracic wall and the ventral abdominal wall cranial to the umbilicus. Further caudally, midway along the neural axis, the lateral portions of the disc will become the lateral and ventral abdominal walls of the ectoderm cells. This layer still contains a mixed population because
both surface ectoderm cells and neural ectoderm cells are present. It is
believed that these cells were originally in the cranial half of the disc
when the primitive streak first appeared, at which time the neural fated
cells were closest to the streak, and the surface ectoderm cells were most
cranial (see Fig. 10.3). The process of primary neurulation relocates
most of the neuroepithelial cells (see below).
Primordial germ cells
Although early studies on human embryos have reported primordial
germ cells, and described their development from the early endoderm
of the yolk sac and allantois, it is now clear from animal experimenta
tion that the primordial germ cells arise from epiblast ingressing at the caudal end of the primitive streak (see Fig. 10.3). It is not known
whether these cells originate from rostral regions that migrate to the streak or from local caudal regions. Extremely early segregation of the
germ cells, when the epiblast layer consists of only 10–13 cells, has been
demonstrated. It has been suggested that the primordial germ cells
remain sequestered in the extraembryonic mesenchyme at the caudal
end of the embryo until the embryonic endoderm has been produced
and gastrulation completed, and that they start to migrate along the
allantoic and hindgut endoderm as the folding of the embryo begins.
The formation of the tail fold brings the proximal portion of the allan
tois within the body, so reducing the final distance over which the cells migrate to the genital ridges. Further development of the germ cells is
described in Chapter 72.
TRILAMINAR DISC
Although the stage 8 embryo is termed a trilaminar disc, the concept
of three epithelial layers forming a trilaminar disc is incorrect; the
middle, mesoblast, layer is several cells thick with intervening extracel
lular matrix. The embryo at this stage, approximately 23 days after ovu
lation, is pear shaped, and broader cranially than caudally ( Fig. 10.5).
The upper epiblast cells are tall and form a pseudostratified columnar epithelial layer with a basal lamina, except at the primitive streak, where
the cells are ingressing to form the other layers. The more centrally
placed epiblast will give rise to neural ectoderm (neurectoderm) and
the more laterally placed epiblast will give rise to surface ectoderm. The
future neural ectoderm is seen as a neural plate that matches the length
of the notochordal plate directly beneath, being slightly wider near the
prechordal plate. The lower embryonic endoderm, a simple squamous
layer with a developing basal lamina, is not always complete at this
stage, particularly in the midline caudal to the prechordal plate, which
is still occupied by the notochordal process or plate.
The middle, mesoblast, layer is composed of free cells migrating
cranially, laterally and caudally from the primitive streak (see Fig. 10.4).
They produce extracellular matrix, which separates the epiblast and endoderm of the embryonic area and permits their passage. The streams
of mesoblast extend between the epiblast and endoderm over all of the
disc area except cranially at the buccopharyngeal membrane (where the
endoderm and ectoderm become apposed once the prechordal mesen
chyme has migrated laterally), and caudally at the cloacal membrane (a patch of thickened endoderm, similar to the buccopharyngeal mem
brane, caudal to the primitive streak). The mesoblast on each side of the notochord is termed paraxial mesenchyme.
Fig. 10.5 The extent and shape of the neural plate in an unfolded embryo. Buccopharyngeal membrane
Neural plate
Cloacal membrane | 272 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Formation of the intraembryonic coelom
185
CHapter 10
trunk. The portion of the disc beyond the cloacal membrane will form
the ventral abdominal wall caudal to the umbilicus. The circumference
of the disc, where the embryonic tissue meets the extraembryonic mem
branes, will become restricted to the connection between the ventral abdominal wall and the umbilical cord, i.e. the umbilicus.
Head folding begins at stage 9, when the fusing cranial neural plate
rises above the surface ectoderm and the portion of the disc rostral to
the buccopharyngeal membrane (which contains the cardiogenic mes
enchyme) moves to lie ventral to the developing brain (see Fig. 10.6).
The prosencephalon and buccopharyngeal membrane are now the most
rostral structures of the embryo. The previously flat region of endoderm,
which may contain cells from the prechordal plate, is now modified
into a deep tube, the primitive foregut. Tail folding can be seen in stage
10 embryos, when the entire embryo comes to rise above the level of
the yolk sac. Similar movement of the part of the disc caudal to the
cloacal membrane results in its repositioning ventral to the neural plate.
Generally, as the embryo rises above the edges of the disc, the lateral
regions of the disc are drawn ventrally and medially, contributing to
the lateral folding of the embryo.
FORMATION OF THE INTRAEMBRYONIC COELOM
At and just before stage 9 (before formation of the head fold), vesicles appear between the mesenchymal cells cranial to the buccopharyngeal
membrane and within the cranial lateral plate mesenchyme. At the
periphery of the vesicles, the mesenchymal cells develop junctional
complexes and apical polarity, and form an epithelium. The vesicles
become confluent to form a horseshoe shaped tube, the intraembryonic
coelom, which extends caudally to the level of the first somite and lat
erally into the lateral plate mesenchyme towards the extraembryonic
mesenchyme. The lateral plate mesenchyme thus develops somatopleu
ric coelomic epithelium subjacent to the ectoderm, and a splanchno
pleuric coelomic epithelium next to the embryonic endoderm (see Fig.
12.2C(iv)). At this stage, the intra and extraembryonic coeloms do not
communicate.
During development of the head fold, the morphological move
ments that organize the foregut and buccopharyngeal membrane have a similarly profound effect on the shape of the intraembryonic coelom. The midline portion of the originally flat, horseshoe shaped coelom
moves ventrally, leaving the caudal arms of the horseshoe in their origi
nal position. In this way, the midline part of the coelom, which was
originally just rostral to the buccopharyngeal membrane, comes to lie
ventral to the foregut (caudal to the buccopharyngeal membrane), and
the two lateral extensions of the coelom pass close to the lateral walls
of the foregut on each side. The caudal portions of the coelom (the two
arms of the horseshoe), which, in the unfolded disc, communicated
laterally with the extraembryonic coelom, turn 90° to lie lateral to the
gut, and communicate with the extraembryonic coelom ventrally.
Compartments of the coelom that will give rise to the body cavities
later in development can already be seen. The midline ventral portion,
caudal to the buccopharyngeal membrane, becomes the pericardial
cavity. The canals lateral to the foregut (pericardioperitoneal canals)
become the pleural cavities and the uppermost part of the peritoneal
cavity. The remaining portion of the coelom becomes the peritoneal
cavity. By stage 1 1, the intraembryonic coelom within the lateral plate
mesenchyme extends caudally to the level of the caudal wall of the yolk
sac. The intra and extraembryonic coeloms communicate widely on
each side of the midgut along the length of the embryo from the level
of the fourth somite ( Fig. 10.10).
In the early embryo, the intraembryonic coelom provides a route for
the circulation of coelomic fluid and, with the beating of the heart tube,
functions as a primitive circulation that takes nutritive fluid deep into
the embryo until it is superseded by the blood vascular system. The
coelomic channel, and the primitive circulation that passes through it,
is of paramount importance up to stage 13. Whereas the superficial
tissues of the embryo can receive nutrients via the amniotic sac and yolk
sac fluids, the deeper tissues are, until the formation of the coelom,
under conditions similar to those found in tissue culture. However,
from stage 10, exocoelomic fluid, propelled by the first contractions of
the developing heart, is brought into contact with the deeply placed mesenchyme. This early ‘circulation’ ensures that an adequate supply of nutrients reaches the rapidly increasing amount of embryonic tissue, and meets most of the requirements of the deeper mesenchymal deriva
tives. From stage 12, the endothelial system expands and fills rapidly with plasma, which passes across the locally thinned coelomic epithe
lium into the large hepatocardiac channels that project into the peri
cardioperitoneal canals at the level of the seventh somite.Neural groove
Somite
Split lateral plateNotochordF
Neural crest
Neural tube
Somite
Notochord
Intraembryonic coelomMidgutGB C
Foregut Hindgut Midgut D Neural plate Amniotic cavity
Paraxial mesenchyme
Intraembryonic coelomE
Notochordal plateYolk sacIntraembryonic
coelom
Bucco-
pharyngealmembraneYolk sacCloacal membraneAllantoisNeural tissue Amniotic cavity
Connecting stalkA
Notochord
Fig. 10.6 Head and tail (A–D) and lateral (E–G) folding of the embryo. A–D, Median sagittal
(longitudinal or axial) sections through the embryonic disc at successive stages; the relative
positions of the buccopharyngeal and cloacal membranes have been maintained; thus, the
movement of the most rostral and caudal portions of the disc can be followed. As these portions
of the disc move ventrally, the initially wide-open yolk sac becomes constricted and fore- and
hindgut divisions can be seen; the midgut is that region remaining in wide connection to the yolk
sac. E–G, Transverse sections through the midpoint of the embryonic disc at successive stages to
illustrate lateral folding that occurs as neurulation proceeds. | 273 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Cell populations at gastrulation
186seCtion 2
Fig. 10.7 Scanning electron micrographs of rat embryos at the time of
neurulation. A, Ventral view, showing the neural fold (NF), and the heart
(H), with the somatopleuric pericardial membrane and surface ectoderm
removed; the arrow indicates the entrance to the foregut via the cranial
intestinal portal. B, Dorsolateral view; the arrows indicate the extent of
rostral (to the right) and caudal (to the left) neural tube formation. (Photographs by P Collins; printed by S Cox, Electron Microscopy Unit,
Southampton General Hospital.)
NF
H
A
B
Fig. 10.8 A human embryo at stage 10, 2.1 mm long, with nine somites:
right lateral and dorsal aspects. Nearly all the yolk sac and the caudal
amnion have been excised.
First pharyngeal arch,
dorsal extremityBrain(early expansionof neural fold)
Yolk sac wall coveredin extraembryonicmesoderm
Unsegmented
paraxial
mesenchymeSomites (5 and 6)Rostral neuroporeNeural crest
Caudal neuroporeAmnion, cut edgesFig. 10.9 A representation of a person on the flat embryonic disc.
The position of the central nervous system has been matched to the
dimensions of the neural plate, and the position of the heart in the thorax
to the position of the pericardial coelom. The limbs, although represented
in this diagram, are not present on the disc at this stage. The usefulness
of this diagram lies in its illustration of the extent of the anterior body wall
both rostral to the buccopharyngeal membrane and caudal to the cloacal
membrane. The future dorsal regions of the body are found medially on
the disc, while the ventral regions of the body are situated laterally and
peripherally on the disc. After head and tail folding and lateral folding, the
peripheral edge of the disc becomes constricted as the edge of the
umbilicus.
Position of the
pericardial coelom
In spite of the importance of the coelom in defining the body cavi
ties, and of the coelomic epithelium in the production of the major
mesenchymal populations of the trunk (Streeter 1942, Langemeijer
1976, O’Rahilly and Müller 1987) (see Fig. 12.2), until recently the
overall contribution of the coelom and its epithelium to the embryo
has received relatively little attention (Carmona et al 2013). The coelom
is a single, tubular organ that may be compared to the neural tube, in
that it possesses a specialized wall that encloses a cavity. Like neural
ectoderm, proliferating coelomic epithelium is a pseudostratified
columnar epithelium with an inner germinal layer, from which cellular
progeny migrate. After the germinal phase, both epithelia ultimately
form the lining of a cavity, ependyma for the neural epithelium, and
mesothelium for the coelomic epithelium. Coelomic epithelium, like
neural epithelium, produces cells destined for different fates from dif
ferent sites and at different developmental times. Coelomic cells are like the neural epithelium, in that they have apical epithelial specializations
and tapering basal processes that are in direct contact with the underly
ing mesenchyme, without an intervening basal lamina. The possibility of the tapering processes forming directional signals for migrating
progeny, similar to the radial glia of the neural tube, has not been
examined.
EMBRYONIC CELL POPULATIONS
AT GASTRULATION
After gastrulation, the cells of the embryo contribute to two fundamen
tal types of tissue, namely: epithelial and mesenchymal. Differentiation
of specialized circulating blood cells and other cell types occurs in
sequence. Embryonic and fetal cell types are replaced later in develop
ment or after birth.
Epithelia
Epithelial populations in the embryo have many of the morphological
characteristics of differentiated epithelia, i.e. they are composed of sheets of closely packed, polarized cells, with narrow intercellular clefts containing minimal extracellular material, and a developed basal | 274 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
embryonic cell populations at gastrulation
187
CHapter 10
their early patterning are termed organizers, e.g. the primitive streak.
Later germinal epithelia give rise to system specific progenitor popula
tions, e.g. the ventricular zone of the neural tube. All epithelia other
than special germinal epithelia divide to produce embryonic growth
throughout development and may retain stem cells that will divide
throughout life.
Characteristically, epithelia clothe internal and external surfaces as
simple or compound cellular sheets that separate phases of differing
composition (e.g. the external environment and the subepithelial tissue
fluids; intravascular and extravascular fluids, and so on). Traffic of mat
erials in the intercellular clefts is limited. Traffic occurs across the cells
because their limiting membranes, which function as energy dependent
selective barriers, enhance the passage of some materials and impede the passage of others.
Mesenchyme
The terms mesoblast and mesenchyme are used in this text in a specific
manner and are not interchangeable. Previously, cells forming a popula
tion between the epiblast and hypoblast were termed mesoderm and, more recently, mesenchyme. The terms primary and secondary mesen
chyme have been used to distinguish between those cells that arise
from ingression through the primitive streak and those that arise from lamina containing specific proteins synthesized by the epithelium itself.
The cells usually show juxtaluminal lateral surface specializations, such as desmosomes, tight junctions, gap junctions, and so on, and special
izations of the apical surface, such as microvilli or cilia.
Epithelial polarity factors establish the extent of the apical, lateral
and basal domains of the individual cells forming an epithelium. Three
main control pathways have been identified in maintaining apicobasal
polarity (St Jonston and Sanson 201 1). The apical domain is specified
by the Crumbs transmembrane protein; Baz/PAR 3 specifies the posi
tion and extent of the intercellular junction; and Scribble restricts the
size of the junctional domains. Once established with an underlying
basal lamina, the basal surface displays integrins. Changes in the mor
phology of an epithelial sheet will involve relative changes in the apical,
lateral and basal domains of all cells. Planar polarity of epithelia is
driven by intercellular cortical actin and myosin. Epithelial cells are
contiguous via actomyosin contractile networks, which contact the
lateral cell membrane through E cadherin adhesion (Gorfinkiel and
Blanshard 201 1). Wnt/planar cell polarity signalling pathways are
involved in these epithelial morphogenetic movements and also in
orientated cell division which occurs in epithelial sheets (Tao et al
2009).
Embryonic epithelia differ from those in the fetus and adult. Two
distinct types can be identified. Early germinal epithelia, which give rise to epithelial or mesenchymal populations of the embryo and confer Fig. 10.10 A, An early stage in development of a human blastocyst. B, A blastocyst sectioned through the longitudinal axis of an embryo, showing the
early formation of the allantois and the connecting stalk. C, A longitudinal section of an embryo at a later stage of development; the pericardial cavity
can be seen at the most rostral part of the embryonic area. D, A longitudinal section of an embryo at a later stage, showing formation of the head
and tail folds, the expansion of the amnion and the delimitation of the umbilicus. E, A transverse section along the line a–b in D; observe that the
intraembryonic coelom communicates freely with the extraembryonic coelom. F, A longitudinal section of an embryo at a later stage, showing full
expansion of the amniotic cavity and the umbilical cord. A
a bD
EFEmbryonic pole Amniotic duct
ChorionTrophoblastConnecting stalk
Secondary yolk sacChorionic
mesoblastAllantoentericdiverticulum
Early villous stemsAmniotic cavity
Extraembryonic mesoblast
Yolk sac
Chorionic cavity/extraembryonic coelom
Connecting stalk
attachment becomes
more ventrally placed
Extraembryonic
coelomTail fold
Pericardial coelom
Head foldBuccopharyngeal
membranePlacental area (chorion frondosum)C
Amniotic cavity
Pericardial coelomEmbryonic area
Chorionic mesoblastAllantoenteric diverticulum
Villous stemConnecting stalk
Extraembryonic
coelom
Splanchnopleuric
mesoblast ofyolk sac
Secondary yolk sacAllantoic canalHindgut
Amniotic cavityMidgut
Chorion laeveSeptum transversum
Intercoelomic
communicationChorionAmnion
Intraembryonic
coelomSomite Yolk sac
Septum
transversumBuccopharyngeal
membrane
Line of fusion ofamnion with chorion,i.e. line of obliteratedextraembryonic coelomAmnioticcavityPericardialcavity
ForegutLateral body fold
Extraembryonic
coelomNeural groove
Notochord
Midgut
Yolk duct
Yolk sac wall ExtraembryonicmesoblastYolk sacAmniotic cavityForegut
Umbilical cord
Obliterated
yolk duct
Umbilical
coelom
Cloacal
membrane
Allantoic ductCloacal membraneB | 275 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Cell populations at gastrulation
188seCtion 2routes, removes the support for overlying epithelia, and disrupts the
normal branching of glandular systems.
Fibronectin deposited extracellularly along a migration pathway will
affect cells that touch it later, causing realignment of their intracellular
actin filaments and thus of their orientation; it also induces cell migra
tion. The receptors for extracellular matrix molecules such as fibronectin and laminin were originally termed integrins because they integrate
extracellular proteins and intracellular cytoskeletal elements (via α and
β subunits that span the cell membrane), allowing them to act together; the binding preference of integrins depends on their combination of
subunits and environmental conditions.
It is known that extracellular matrix is structured rather than random.
Epithelial and mesenchymal cell populations can structure the space
around them by secretion of particular matrix molecules or growth
factors, which, in turn, can organize the cells that contact them. Cell–
matrix interactions and matrix–cell interactions control the position of
migration routes and cellular ‘decisions’ to migrate or to begin to dif
ferentiate. Matrix molecules propagate developmental instructions from
cell to cell and form a far reaching four dimensional (spatial and tem
poral) mechanism of communication. Mapping the molecular path
ways and the biomechanical processes that underlie morphogenesis, the
dynamic epigenetics of early mesenchyme cells and their secreted three
dimensional extracellular matrices, and the mechanisms by which the
stiffness of the surrounding matrix affects the actions of the cells within
it and the specific deposition of fibrillary proteins creates tissue bounda
ries and zones of tension, are all the subject of ongoing research (e.g.
Davidson et al 2010, Loganathan et al 2012).
Transition between epithelial and
mesenchyme states
Transformations of cell morphology from epithelium to mesenchyme,
and vice versa, occur in specific places and times during development,
and can be seen as ways of dispersing germinal centres with increasing
restriction. The first epithelial tomesenchyme transition occurs at the
primitive streak, a germinal epithelium that confers embryonic specifi
cation on the resultant mesoblast population. The mesoblast so formed
migrates and the cells undergo mesenchyme toepithelial transitions
when they reach their final destinations. Series of small epithelial ger
minal centres, the somites, are formed, as are larger, more extensive,
germinal epithelial sheets that line the walls of the intraembryonic
coelom. The coelomic walls, especially those derived from somato
pleure and splanchnopleure, form germinal epithelia that give rise to
the major mesenchymal populations that form the viscera. The early
epithelial somites undergo further local epithelial tomesenchyme tran
sitions to form the sclerotomes, and subsequently form several germinal
epithelia in the epithelial plate of each somite. Later mesenchyme to
epithelium transitions are not associated with the formation of germi
nal epithelia; the most common involve the transition of mesenchyme
into the endothelium of the vascular system. The nephrons of the mes
onephric and metanephric systems also form from mesenchyme to
epithelial transition.neural crest ingression, respectively. Primary mesenchymal cells revert
to epithelia at their destinations. However, whereas some primary
mesen chymal cells may become epithelial within a short timeframe,
e.g. somites and lateral plate, other cells may transform later, e.g. the
epithe lium lining blood vessels. To address these terminological con
flicts, the mixed population of epiblast cells that ingress through the
primitive streak and come to lie between the epiblast and embryonic
endoderm is termed mesoblast until the cells have migrated to their
final position, at which time the populations of mesenchyme can be
identified and their fates inferred.
Mesoblastic and mesenchymal cells have no polarity. They form
junctional complexes, which are not exclusively juxtaluminal, and they
produce extracellular matrix molecules and fibres from the entire cell
surface. Mesenchymal populations are formed from a range of germinal
epithelia and by proliferation of mesenchymal cells directly, and occupy
all the regions between the various epithelial layers described above.
The term mesoderm is reserved for the coelomic epithelia that later
form mesothelia.
Mesenchymal cells support epithelia throughout the developing
body, both locally where they contribute to the basement membrane
and form the lamina propria and smooth muscle of tubes, and
generally where they differentiate into connective tissue. Specific
mesenchymal populations control the patterning of local regions of
epithelium (e.g. the zone of polarizing activity on the postaxial limb
border posterior to the apical ectodermal ridge).
Extracellular matrix
The space beneath epithelia and between mesenchyme cells is filled with extracellular matrix; both epithelial and mesenchymal cells syn
thesize extracellular matrix molecules and their receptors. Epithelial
cells produce a two dimensional basal lamina, which contains a variety
of matrix molecules, including laminin, fibronectin, type IV collagen and various proteoglycans. The particular molecules can vary during
development according to spatial and temporal patterns, resulting in
changes in the behaviour of the underlying mesenchymal cells (e.g. in
patterning of the basal regions of the skull). Mesenchymal cells produce
extracellular matrix molecules in three dimensions. Those adjacent to
an epithelial layer will connect with its basal lamina, forming a base
ment membrane that secures the epithelial layer to the underlying tissue. Cells deep within a mesenchymal population may synthesize
matrix molecules (fibrillar or granular) to separate cells locally, open
up migration routes or leave information within the matrix to act on
cell populations passing at a later time.
Molecules of the extracellular matrix are complex; they include more
than 19 individual types of collagen (some of which are capable of
being individually spliced to give more than 100 variants), proteogly
cans and glycoproteins (which come in a wide variety of forms, with and without binding proteins), and elastic fibres (see Ch. 2). Hyaluronic
acid, a glycosaminoglycan, has a vast capacity to bind water molecules and so create and structure the space between the mesenchymal cells,
thereby producing much of the overall shape of an embryo. Experimen
tal removal of hyaluronic acid prevents the formation of cell migration
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notochord and nearby median features in staged human embryos. Cells
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Streeter GL 1942 Developmental horizons in human embryos. Descriptions
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Cell populations at gastrulation
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CHapter 10REFERENCES
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Tao H, Suzuki M, Klyonarl H et al 2009 Mouse prickle 1, the homolog of
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189
CHAPTER 11 Embryonic induction and cell division
(further restrictions), they are said to be determined. Determined cells
are programmed to follow a process of development that will lead to
differentiation. The determined state is a heritable characteristic of cells
and is the final step in restriction. Once a cell has become determined,
it will progress to a differentiated phenotype if the environmental
factors are suitable.
The process of determination and differentiation within embryonic
cell populations is reflected by the ability of these populations to
produce specific proteins. Primary proteins (colloquially termed house -
keeping proteins) are considered essential for cellular metabolism, whereas proteins synthesized as cells become determined, those specific
to the state of determination, are termed secondary proteins; for
example, liver and kidney cells, but not muscle cells, produce arginase.
Fully differentiated cells produce tertiary proteins, which no other cell
line can synthesize, e.g. haemoglobin in erythrocytes. The range of
housekeeping, regulatory, and tissue-specific proteomes in adult cells is
presented in the Human Protein Atlas (www.proteinatlas.org).
As populations of cells become progressively determined, they can
be described within a hierarchy of cellular development as transiently
amplifying cells, progenitor cells, stem cells and terminally differenti -
ated cells.
Transiently amplifying cells Transiently amplifying cells undergo
proliferative cell mitosis and produce cells that are equally determined.
At some stage, and as a result of an inductive stimulus, these cells will
enter a quantal cycle that culminates in a quantal mitosis. This will
result in an increase in the restriction of their progeny, which continue
to undergo proliferative mitoses at a progressive level of determination.
The quantal mitosis corresponds to the time of binary choice when the
commitment of the progeny is different from that of the parent.
Progenitor cells Progenitor cells are already determined along a
particular pathway. They may individually follow that differentiation
pathway, or may proliferate and produce large numbers of similarly
determined progenitor cells that subsequently differentiate; neuroblasts
or myoblasts are examples of progenitor cells.
Stem cells Either individually or as a population, stem cells can both
produce determined progeny and reproduce themselves (see Commen -
tary 1.2). It is generally believed that stem cells undergo asymmetric
divisions, in which one daughter cell remains as a stem cell, while the
other proceeds along a differentiation pathway; in marked contrast,
proliferative cell division may be symmetrical, producing derived cells
with an identical determination. Human embryonic stem cells (hESCs)
are pluripotent cells that can be derived from the inner cell mass of
human blastocysts in vitro; or obtained surplus to in vitro fertilization
fertility programmes; or created from oocytes donated and fertilized for that purpose. Although not yet achieved, it is hoped that hESCs can be
coaxed down particular pathways under appropriate pharmaceutical
conditions to produce differentiated cells that will be effective in revers -
ing some degenerative diseases (e.g. dopamine-producing neurones for Parkinson’s disease, insulin-producing islet cells for diabetes), or to
replace acutely damaged tissues (e.g. motor neurones for acute spinal
cord injury, cardiomyocytes in acute myocardial infarction). Proof of
principle has been demonstrated in some animal models, and multipo -
tent haemopoietic progenitor stem cells from human umbilical cord blood are now used as an alternative treatment to bone marrow trans -
plantation for the treatment of some inherited genetic disorders (thalas -
saemia) and blood malignancies (leukaemias).
Terminally differentiated cells By virtue of their extreme special -
ization, terminally differentiated cells can no longer divide, e.g. eryth-
rocytes and neurones. Apoptosis is a particular variety of terminal differentiation in which the final outcome is the death of the individual cells or cell populations. It occurs in the developing limb, where cells
EMBRYONIC INDUCTION AND CELL DIVISION
The stages of genomic activity that unfold in the fertilized zygote from
cleavage onwards are not yet fully elucidated. An admittedly simplistic
but useful approach likens developmental processes to a series of binary
choices (see Fig. 8.1), the first being the choice between trophoblast
and inner cell mass, which specifies if the future tissue will be placenta and fetal membranes or embryo.
Bioinformatic databases use embryological ontologies wherein the
lineage of organs and tissues to be retraced to the earliest time (see
Commentary 2.1). They provide a temporal and spatial framework
against which the sequential activation of genes preceding changes in
cell and tissue morphology can be matched. They also provide a means
of viewing all of the complex partonomy, lineage data and temporal
information occurring within embryos within a specific timeframe and
of linking these to adult anatomical tissue types. As our knowledge of
the complexity of early developmental events increases, ontological
databases will assist the correlation of embryological information:
current limitations are discussed in Bard (2012).
Our knowledge of the early events that occur in a range of mam -
malian species and our interpretation of the significance of particular
sequences of gene expression is limited. The upregulation of early genes
appears to reflect the time period of implantation. In the mouse,
implantation occurs one day after blastocyst formation, thus genes
relevant to implantation are expressed early, whereas in cattle, implan -
tation occurs after 14 days, by which time gastrulation has occurred and neurulation has been initiated (Oron 2012).
Within the developing blastocyst, the position of cells relative to
each other leads to epigenetic changes. The acquisition of cell polarity
is fundamental, because this specifies apical and basolateral domains,
cell surface specializations, the position and nature of cell:cell junctions
and the concomitant positioning of intracellular organelles and
cytoskeletal elements, which in turn leads to the location of future
deposition of extracellular proteins. The two cell phenotypes, epithelial
and non-epithelial cells, are specified during cleavage and early compac -
tion within the zona pellucida. All relevant genes required to maintain these cell shapes and behaviours are upregulated at these very early
stages. During the formation of trophoblast, hypoblast (primitive endo -
derm) and epiblast, the cells respond locally to their environment, specifically to other cells that are positioned lateral and basal to them.
The genes operating at these early times in the mouse embryo are given
in Takaoka and Hamada (2012).
Cell populations within the embryo interact to provide the develop -
mental integration and fine control necessary to achieve tissue-specific
morphogenesis. In the early embryo, such interactions may occur only
if particular regions of the embryo are present, e.g. signalling centres or
organizers. As the embryo matures, some interactions tend to occur
between adjacent cell populations, e.g. epithelium and mesenchyme,
and later between adjacent differentiating tissues, e.g. between nerves
and muscle, or between muscle and skeletal elements. The interactions
between adjacent epithelia and underlying connective tissue continue
throughout embryonic and fetal life and extend into postnatal life. In
the adult, these interactions also permit the metaplastic changes that
tissues can undergo in response to local environmental conditions.
Tissue interactions result in changes or reorganization of one or
both tissues, which would not have occurred in the absence of the
tissue interactions. The process of tissue interaction is also called induc -
tion, i.e. one tissue is said to induce another. The ability of a tissue to
respond to inductive signals is called competence, and denotes the ability of a cell population to develop in response to the environments present in the embryo at that particular stage. After a cell population has been induced to develop along a certain pathway, it will lose com -
petence and become restricted. Once restricted, cells are set on a par -
ticular pathway of development; after a number of binary choices
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Embryonic induction and c Ell division
190sEction 2mesenchyme, which results in the morphogenesis of mammary
gland-like structures; chickens do not normally develop mammary
glands.
Tissue interactions continue into adult life and are probably respon -
sible for maintaining the functional heterogeneity of adult tissues and
organs. This is exemplified by the complex tissue heterogeneity, with
sharply compartmentalized boundaries, that occurs in the oral cavity.
The junctions between the mucosa of the vestibule and the lip, and
between the vermilion border and the facial skin, are distinct bound -
aries of specific epithelial and mesenchymal differentiation, and are almost certainly maintained by continuing epithelial–mesenchymal
interactions in adult life. Perturbation of these interactions throughout
the body may underlie a wide variety of adult diseases, including sus -
ceptibility to cancer and proliferative disorders.
Signalling between embryonic cells
and tissues
Cellular interactions may be signalled by four principal mechanisms:
direct cell–cell contact; cell adhesion molecules and their receptors;
extracellular matrix molecules and their receptors; and growth factors
and their receptors. Many of these mechanisms interact, and it is likely
that combinations of them are involved in development. Figure 1 1.1
illustrates some ways by which mesenchymal cells could signal to epi -
thelial cells. An additional set of identical mechanisms could operate
for epithelial–mesenchymal cell signalling. Clearly, the complexity of
these mechanisms will increase in reciprocal interactions; moreover, a
single molecule may have different effects on epithelial and mesen -
chymal cells.
Direct cell–cell contact permits the construction of gap junctions,
which are important for communication and the transfer of informa -
tion between cells. The transient production of gap junctions is seen as epithelial somites are formed, between neuroepithelial cells within
rhombomeres, and in the tunica media of the outflow tract of the heart. die along the pre- and postaxial limits of the apical ectodermal ridge,
limiting its extent, and also between the digits, permitting their
separation.
Tissue interactions
There are two types of cell and tissue interaction, namely: permissive
and instructive.
In a permissive interaction, a signal from an apposing tissue is neces -
sary for the successful self-differentiation of the responding tissue. This
means that a particular cell population (or the matrix molecules
secreted by the cells that it contains) will maintain mitotic activity in
an adjacent cell population. Since a variety of different cell populations
may permit a specific cell population to undergo mitosis and cell dif -
ferentiation, no specific instruction or signal that may limit the devel -
opmental options of the responding tissue is involved; this signal
therefore does not influence the developmental pathway selected and
there is no restriction. The responding tissue has the intrinsic capacity
to develop, and only needs appropriate environmental conditions in
order to express this capacity. Permissive interactions often occur later
in development, when a tissue whose fate has already been determined
is maintained and stabilized by another.
An instructive (directive) interaction (induction) changes the cell
type of the responding tissue, so that the cell population becomes
restricted. Wessells (1977) proposed four general principles in most
instructive interactions: in the presence of tissue A, responding tissue B develops in a certain way; in the absence of tissue A, responding tissue
B does not develop in that way; in the absence of tissue A, but in the
presence of tissue C, tissue B does not develop in that way; and in the
presence of tissue A, a tissue D, which would normally develop differ-
ently, is changed to develop like tissue B.
These principles are exemplified during induction of the lens vesicle
by the optic cup. An example of last-mentioned principle is the experi -
mental association of chicken flank ectoderm with mouse mammary
Epithelium Basal lamina Mesenchyme cell
1 1 Direct cell–cell contact by gap junctions.
3 A soluble factor (growth factor) reacting with a receptor for that factor on the epithelial cells.
4 Extracellular matrix molecule secreted by the mesenchyme cells interacting with a receptor on the epithelial cell.
5 A soluble factor (growth factor) secreted by a mesenchymal cell having a biphasic action interacting (i) with a receptor on an
epithelial cell, causing it to express a specific extracellular matrix molecule receptor; (ii) with a receptor on a mesenchyme cell,
causing it to secrete a specific extracellular matrix molecule which then interacts with the induced epithelial receptor.
6 A soluble factor (growth factor) secreted by a mesenchyme cell interacting with a receptor on an epithelial cell, causing it to
express a receptor or secrete a factor, which interacts with another factor synthesized, or receptor expressed, by another
mesenchyme cell.
7 A soluble factor secreted by a mesenchyme cell interacting with a receptor on an epithelial cell, causing it to synthesize an
extracellular matrix molecule (or a receptor for such a molecule) which then interacts with a specific receptor for that molecule
on another mesenchyme cell.
8 A soluble factor secreted from a mesenchyme cell interacting with a receptor on an epithelial cell, causing it to synthesize a
molecule which stabilizes or enhances the interaction between a mesenchymal-derived factor and its epithelial receptor. 9 A soluble factor secreted by a mesenchyme cell interacting with a receptor on an epithelial cell, causing the inhibition of
synthesis/assembly of a factor or receptor.
10 A soluble factor secreted by a mesenchyme cell binding to the extracellular matrix of the basal lamina, where it remains active
and subsequently interacts with a receptor on an epithelial cell which appears at a later developmental time. 2 Cell–cell contact by cell adhesion molecules.2
3
4
5
6
7
8
9
10
Fig. 11.1 The many ways by which mesenchyme cells could signal to epithelial cells. Precisely the same mechanisms can operate in reverse, i.e.
epithelium to mesenchyme. | 279 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
morphogenesis and pattern formation
191
cHaPtEr 11at the tip of the acinus is therefore split into two, and two branches
develop from this point.
Pattern formation concerns the processes whereby the individual
members of a mass of cells, initially apparently homogeneous, follow
a number of different avenues of differentiation that are precisely
related to each other in an orderly manner in space and time. The pat -
terns embraced by the term apply not only to regions of regular geo -
metric order, e.g. the crystalline lens, but also to asymmetric structures
such as the limb. For such a process to occur, individual cells must be
informed of their position within the embryo, and utilize that informa -
tion for appropriate differentiation. Patterning of regions is seen in: the
Fig. 11.2 In addition to the mechanisms described in Figure 11.1, cells
may also communicate by the reception, production and secretion of
growth factors. A typical embryonic mesenchyme cell may receive and
produce growth factors in this way.
1 Endocrine Delivery of growth factor by the blood stream from a distant biosynthetic site to the
target mesenchyme cell.
2 Autocrine Synthesis of growth factor by the cell, its secretion, binding and activation of a surface
receptor elsewhere on its own surface. 3 Paracrine Synthesis of growth factor by the cell, its secretion and diffusion to an adjacent cell
(or group of cells) where it binds to and activates a cell surface receptor. 4 Juxtacrine Synthesis of growth factor by the cell. The growth factor remains on the cell surface
and binds to and activates a receptor on an immediately adjacent cell. 5 Intracrine Synthesis of growth factor within the cytoplasm of the cell. The growth factor moves to
the nucleus and binds and activates its own nuclear receptors. 6 Matricrine Synthesis and export of growth factor from the cell. The growth factor binds to the
extracellular matrix where it remains active and subsequently binds to and activates a receptor for
that growth factor on the same or a different cell.Extracellular matrix Blood vessel Mesenchyme cell
43
5
61
2
Fig. 11.3 Branching of a tubular duct may occur as a result of an interaction between the proliferating epithelium of the duct and its surrounding
mesenchyme and extracellular matrix. A, Mesenchymal cells initiate cleft formation by producing collagen III fibrils locally within the development clefts
and hyaluronidase over other parts of the epithelium. Collagen III prevents local degradation of the epithelial basal lamina by hyaluronidase and slows
the rate of mitosis of the overlying epithelial cells. B, In regions where no collagen III is produced, hyaluronidase breaks down the epithelial basal lamina
and locally increases epithelial mitoses, forming an expanded acinus (see arrows) . (Adapted from Gilbert SF 1991 Developmental Biology. Sunderland,
MA: Sinauer Associates.)Narrow cleft
Mesenchyme cellsCollagen fibrils
HyaluronidaseCollagen
Epithelial cells
Collagen fibrilsHyaluronidase produced
by mesenchyme cellsA BEndogenous electrical fields are also believed to have a role in cell–cell
communication. Such fields have been demonstrated in a range of
amphibian embryos, and in vertebrate embryos during primitive streak
ingression. Neuroepithelial cells are electrically coupled, regardless of
their position relative to inter-rhombomeric boundaries.
The spatial and temporal distribution of a variety of cell adhesion
molecules has been localized in the early embryo. The appearance of
these molecules correlates with a variety of morphogenetic events that
involve cell aggregation or disaggregation; e.g. an early response of
groups of cells to embryonic inductive influences is the expression of
cadherins, calcium-dependent adhesion molecules typically found in
epithelial populations. Other molecules found in the extracellular
matrix, e.g. fibronectin and laminin, inter alia, can modulate cell adhe -
sion by their degree of glycosylation. Self-assembly or cross-linking by matrix molecules may affect cell adhesiveness by increasing the avail -
ability of binding sites or by obscuring them.
Extracellular matrix molecules include localized molecules of the
basal lamina, e.g. laminin, fibronectin, and much larger complex asso -
ciations of collagen, glycosaminoglycans, proteoglycans and glycopro -
teins between the mesenchyme cells. Mutations of the genes that code for extracellular matrix molecules give rise to a number of congenital
disorders, e.g. mutations in type I collagen produce osteogenesis imper -
fecta; mutations in type II collagen produce disorders of cartilage; and mutations in fibrillin are associated with Marfan’s syndrome.
Growth factors are distinguished from extracellular matrix mol -
ecules. They can be delivered to, and act on, cells in a variety of ways, namely: endocrine, autocrine, paracrine, intracrine, juxtacrine and
matricrine (Fig. 1 1.2). Many growth factors are secreted in a latent form,
e.g. associated with a propeptide (latency-associated peptide) in the
case of transforming growth factor beta, or attached to a binding
protein, in the case of insulin-like growth factors.
MORPHOGENESIS AND PATTERN FORMATION
Morphogenesis may be described as the assumption of form by the
whole, or part, of a developing embryo. As a term, it is used to denote
the movement of cell populations and the changing shape of an
embryo, particularly during early development.
The most obvious examples of morphogenesis are the large migra -
tions that occur during gastrulation; local examples include branching
morphogenesis, which occurs in the developing lungs and kidneys, for
example, and in most glandular organs. The development of branches
from a tubular duct occurs over a period of time. An interaction between
the proliferating epithelium of the duct and its surrounding mesen -
chyme and extracellular matrix results in a series of clefts that produce a characteristic branching pattern (Fig. 1 1.3). During tubular and acinar
development, hyaluronidase secreted by the underlying mesenchymal
cells breaks down the basal lamina produced by the epithelial cells; this
increases epithelial mitoses locally and results in an expanding acinus.
Cleft formation is initiated by the mesenchyme, which produces col-
lagen III fibrils within putative clefts. (If the collagen is removed, no
clefts develop, whereas if excess collagen is not removed, supernumer -
ary clefts appear.) The collagen acts to protect the basal lamina from the effects of the hyaluronidase, which means that the overlying epithe -
lia have a locally reduced rate of mitosis. The region of rapid mitoses | 280 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Embryonic induction and c Ell division
192sEction 2progress zone and zone of polarizing activity within the limbs; the fates
of the medial and lateral, and later the cranial and caudal, halves of the
somites; and the neural crest mesenchyme within the pharyngeal
arches. For details of patterning in vertebrate development, see Tickle
(2003).
Genes in development
Two related themes have emerged from experimental studies of devel -
opment: first, that the control of embryonic morphology has been
highly conserved in evolution between vertebrates and invertebrates;
and second, that this control involves families of genes coding for
proteins that act as transcriptional regulators.
Homeobox genes are believed to be responsible, at least in part, for
the evolutionary origin of the embryonic body plan (Robert 2001).
Experimental study of transgenic animals in which the homeobox genes
have been knocked out provide some evidence of their function;
however, because developmental processes permit significant recovery
from insult, some of the outcomes cannot be directly interpreted as
demonstrating the effect of such gene loss.
Other gene families required for normal development include the
T-box family, Helix-Loop-Helix transcription factors and Sox genes; the
signalling factors, transforming growth factor beta (TGF- β), bone mor-
phogenetic proteins (BMPs), fibroblast growth factor family (FGF), the Wnt family and hedgehog signalling molecules. A range of cell receptor molecules are also required. For details on individual members within
this range of factors, see Carlson (2014).
Experimental approaches to embryology
One of the most exciting techniques to provide information on cell
movements and fates during development is the use of chimeric
embryos. Small portions of an embryo are excised and replaced with
similar portions of an embryo from a different species at the same stage,
and the resulting development is then studied. This technique has been
particularly effective using chick and quail embryos because the nucle -
olus is especially prominent in all quail cells, whereas it is not promi -
nent in chick cells, which means that quail cells may be easily identified within a chick embryo after chimeric transplantation (Le Douarin
1969). The technique has also confirmed the reciprocity of tissue inter -
action between the embryonic species, a phenomenon that had previ -
ously been illustrated, for a limited period, in co-cultures of embryonic
avian and mammalian tissues. Somite development and vertebral for -
mation have been studied in mouse–chick chimeras (Fontaine-Pérus 2000). The production, in vitro, of human–animal chimeric cell lines is
providing new ways of studying cellular pathways, as is the introduction of human artificial chromosome vectors into animal cells to study their
interaction.
KEY REFERENCES
Bard J 2012 A new ontology (structured hierarchy) of human developmental
anatomy for the first 7 weeks (Carnegie stages 1–20). J Anat 221:
406–16.
Carlson BM 2014 Human Embryology and Developmental Biology, 5th ed.
Philadelphia: Elsevier, Saunders; Ch. 4.
Fontaine-Pérus J 2000 Mouse–chick chimera: an experimental system for
study of somite development. Curr Top Dev Biol 48:269–300.
Le Douarin NM 1969 Particularités du noyau interphasique chez la caille
japonaise (Coturnix coturnix japonica). Utilisation de ces particularités
comme ‘marquage biologique’ dans les recherches sur les interactions tissulaires et les migrations cellulaires au cours de l’ontogenèse. Bull Biol Fr Belg 103:435–52.Oron E, Ivanova N 2012 Cell fate regulation in early mammalian develop -
ment. Phys Biol 9:045002.
Robert JS 2001 Interpreting the homeobox: metaphors of gene action and
activation in development and evolution. Evol Dev 3:287–95.Takaoka K, Hamada H 2012 Cell fate decisions and axis determination in the early mouse embryo. Development 139:3–14.
Tickle C 2003 Patterning in Vertebrate Development. Oxford: Oxford Uni -
versity Press.
Wessells NK 1977 Tissue Interaction and Development. Menlo Park, CA:
Benjamin. | 281 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Embryonic induction and cell division
192.e1
cHaPtEr 11REFERENCES
Bard J 2012 A new ontology (structured hierarchy) of human developmen -
tal anatomy for the first 7 weeks (Carnegie stages 1–20). J Anat 221:
406–16.
Carlson BM 2014 Human Embryology and Developmental Biology, 5th ed.
Philadelphia: Elsevier, Saunders; Ch. 4.
Fontaine-Pérus J 2000 Mouse–chick chimera: an experimental system for
study of somite development. Curr Top Dev Biol 48:269–300.
Le Douarin NM 1969 Particularités du noyau interphasique chez la caille
japonaise (Coturnix coturnix japonica). Utilisation de ces particularités
comme ‘marquage biologique’ dans les recherches sur les interactions tissulaires et les migrations cellulaires au cours de l’ontogenèse. Bull Biol Fr Belg 103:435–52.Oron E, Ivanova N 2012 Cell fate regulation in early mammalian develop -
ment. Phys Biol 9:045002.
Robert JS 2001 Interpreting the homeobox: metaphors of gene action and
activation in development and evolution. Evol Dev 3:287–95.
Takaoka K, Hamada H 2012 Cell fate decisions and axis determination in
the early mouse embryo. Development 139:3–14.
Tickle C 2003 Patterning in Vertebrate Development. Oxford: Oxford Uni -
versity Press.
Wessells NK 1977 Tissue Interaction and Development. Menlo Park, CA:
Benjamin. | 282 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
193
Cell populations at the start
of organogenesisCHAPTER 12
contains the transmedian pericardial cavity, which communicates
dorsocaudally with right and left pericardioperitoneal canals. These
pass dorsally to the transverse septum mesenchyme and open caudally
into the extraembryonic coelom on each side of the midgut. The intra
embryonic mesenchyme has begun to differentiate and the paraxial
mesenchyme is being segmented into somites. Neural groove closure is
progressing caudally, so that a neural tube is forming between the newly
segmenting somites. Rostrally, the early brain regions, which have not
yet fused, can be discerned. The neuroepithelium is separated from the
dorsal aspect of the gut by the notochord. The earliest blood vessels
have appeared and a primitive tubular heart occupies the pericardium.
The chorionic circulation is soon to be established, after which the
embryo rapidly becomes completely dependent on the maternal blood
stream for its requirements. The embryo is connected to the developing
placenta by a mesenchymal connecting stalk in which the umbilical
vessels develop, and which also contains the allantois, a hindgut diver
ticulum. The lateral body walls are still widely separated. The embryo has contact with three different vesicles: the amnion, which is in contact
with the surface ectoderm; the yolk sac, which is in contact with the
endoderm; and the chorionic cavity, containing the extraembryonic
coelom, which is in contact with the intraembryonic coelomic lining
(see Fig. 10.10).
The early body plan of the embryo is segmented. The boundaries
between the segments are maintained by the differential expression of
genes and proteins that restrict cell migration in these regions. Organo
genetic processes either retain the segmental plan, e.g. spinal nerves, or replace it locally, e.g. the modifications of somatic intersegmental
vessels by the development of longitudinal anastomoses. Abnormalities
may result from improper specification of segments along the cephalo
caudal axis and may fail to produce the appropriately modified segmen
tal plan.
The degree to which vertebrate embryos are developmentally con
strained at this period of development is controversial. Comparative
studies on the timing at which specific embryonic structures appear,
heterochrony, have shown that other embryonic species do not follow
the same developmental sequence as humans (Richardson and Keuck
2002). Although some developmental mechanisms are highly con
served, e.g. the homeobox gene codes, others may have been dissociated and modified in different vertebrate species during evolution.
Organogenesis, the further development of body regions and organs
that is described elsewhere in this book, starts from about stage 10
(approximately 28 days). Although it is both conventional and conven
ient to consider the further development of each body system on an individual basis, not only do all systems develop simultaneously, but
also they interact and modify each other as they develop. This necessary
interdependence is supported by the evidence of experimental embry
ology and reinforced by the phenomena of growth anomalies, which
cut across the artificial boundaries of systems in most instances. For
these reasons, it is recommended that the development of an individual
system or body region should be studied in relation to others, especially
those most closely associated with it, whether spatiotemporally or
causally.
EMBRYONIC CELL POPULATIONS AT THE START
OF ORGANOGENESIS
The developmental processes operating in the embryo between stages
5 and 9 enabled the construction of the bi and trilaminar embryonic
disc, the intraembryonic coelom and new proliferative epithelia. From
the end of stage 10, a range of local epithelial and mesenchymal popula
tions now interact to produce viscera and appendages. The inductive influences on these tissues and their repertoire of responses are very different from those seen at the onset of gastrulation. The range of tissues present at the start of organogenesis, when the body plan is clear, SPECIFICATION OF THE BODY AXES AND THE
BODY PLAN
Embryos may be thought of as being constructed with three orthogonal
spatial axes (cephalocaudal, dorsoventral and laterolateral), plus a tem
poral axis. In mammalian embryos, axes cannot be specified at very early stages; embryonic axes can be defined only after the early extra
embryonic structures have been formed and the inner cell mass can be
seen. The position of the future epiblast can be predicted in human
embryos when the hollow blastocyst has formed. The inner cell mass
becomes (seemingly) randomly located on the inside of the trophecto
derm and forms a population of epiblast cells subjacent to the trophoblast. This region implants first. It is not known whether the
trophectoderm in contact with the inner cell mass initiates implanta
tion, so that the future dorsal surface of the embryo is closest to the disrupted maternal vessels at the implantation site, or whether the inner
cell mass can travel around the inside of the trophoblast to gain a posi
tion subjacent to the implantation site once implantation has started.
Axes may be conferred on the whole embryonic disc, which is ini
tially flat and mainly two dimensional. However, their subsequent ori
entation in the folded three dimensional embryo will be completely
different. The dorsal structures of the folded embryo form from a cir
cumscribed central ellipse of the early flat embryonic disc (see Fig.
10.5). Lateral and ventral structures form from the remainder of the disc, and the peripheral edge of the disc eventually becomes constricted
at the umbilicus (see Figs 10.9–10.10). Although the appearance of part
of the epiblast is taken to specify the dorsal surface of the embryo, the inner layer, i.e. the hypoblast, is not, by default, a ventral embryonic
structure.
The primary, cephalocaudal, axis is conferred by the appearance of
the primitive streak in the bilaminar disc. The primitive streak patterns
cells during ingression, and so also specifies the dorsoventral axis,
which becomes apparent after embryonic folding. The position of
ingression through the streak confers axial, medial or lateral character
istics on the forming mesenchyme cells. The axial and medial popula
tions remain as dorsal structures in the folded embryo, and the surface ectoderm above them will exhibit dorsal characteristics. The lateral plate
mesenchyme will assume lateral and ventral positions after embryonic
folding, and the surface ectoderm above this population will gain
ventral characteristics.
The third and last spatial axis is the bilateral, or laterolateral, axis,
which appears as a consequence of the development of the former two
axes. Initially, the right and left halves of the embryonic body are bilat
erally symmetric. Lateral projections, the upper and lower limbs, develop in two places on each side of the body wall (somatopleure).
With the last axis established, the temporal modification of the
original embryonic axes can be seen. The segmental arrangement of the
cephalocaudal axis is very obvious in the early embryo and is retained
in many structures in adult life. Similarly, dorsal embryonic structures
remain dorsal and undergo relatively little change. However, structures
that were originally midline and ventral, especially those derived from
splanchnopleuric mesenchyme, e.g. the cardiovascular system and the
gut, are subject to extensive shifts, and change from a bilaterally sym
metric arrangement to an entire body that is now chiral, i.e. has distinct left and right sides.
The development of all the body organs and systems, organogenesis,
begins after the dramatic events of gastrulation, when the embryo has
attained a recognizable body plan. In human embryos, this corresponds to the end of stage 10 ( Fig. 12.1). The head and tail folds are well
formed, with enclosure of the foregut and hindgut (proenteron and metenteron), although the midgut (mesenteron) is only partly con
stricted from the yolk sac. The forebrain projection dominates the cranial end of the embryo, and the buccopharyngeal membrane and cardiac prominence are caudal and ventral to it. The cardiac prominence | 283 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Cell populations at the start of organogenesis
194seCtion 2
then passes over the occipital and cervicothoracic parts of the embryo,
superficial to the four occipital somites and later to the occipitocervical
junction. Further caudally, it is associated with the upper limb field,
where it will give rise to the apical ectodermal ridge. O’Rahilly and
Müller (1985) have called the portion of the ring between the upper and lower limbs the intermembral part. It overlies the underlying
intraembryonic coelom, and later (between stages 12 and 13), the
mesonephric duct and ridge. In stages 14 and 15, this portion of the
ectodermal ring gives rise to the mammary line. Caudal to the lower
limb field, in the unfolded embryo, the ring passes distal to the cloacal
membrane. In the folded embryo, this region becomes superior to the
cloacal membrane and corresponds to the ectoderm associated with the
external genitalia, particularly the genital tubercle and urogenital swell
ings, where it influences the internal and external descent of the testis
and the formation of the inguinal canal.
Neural ectoderm
The neuroepithelium at the time of primary neurulation is pseudos
tratified. It has a midline hinge region, which, with concomitant
wedging of the cells in the lateral wall of the neural groove, promotes
neural tube formation. The processes of the neuroepithelium abut on
to internal and external limiting membranes. This epithelium prolifer
ates to form all the cell lines of the central nervous system and, via the production of neural crest, all the cell lines of the peripheral nervous
system.
Notochord
During stage 10, the notochordal plate undergoes a process that is
similar to, but a mirror image of, neurulation, and forms an epithelial
tube from caudal to rostral, ending with the pharynx. The notochordal
plate forms a deep groove, the vertical edges of the groove move medi
ally and touch, and then the endodermal epithelium from each side fuses ventral to the notochord. The cells swell and develop an internal
pressure (turgor) that confers rigidity on the notochord. The notochord
is surrounded by a basal lamina, which is initially referred to as a peri
notochordal sheath; this term is subsequently applied to mesenchymal populations that surround the notochord. After stage 1 1, the tubular notochord is in contact with the neural tube dorsally and the endoderm ventrally. It is not a proliferative epithelium, but it has inductive effects on the overlying neural tube and the adjacent somites, and later pro
vides a focus for sclerotomal migration.is given below and shown in Figures 12.1 and 12.2. For a summary of
the fates of the embryonic cell populations, see Figure 12.3 .
Epithelial populations in the embryo
Surface ectoderm
During embryogenesis, the surface ectoderm shows regional differences
in thickness. Ectoderm over the dorsal region of the head and trunk is
thin, as is the pericardial covering; this has been interpreted as a con
sequence of the expansion of this epithelium over structures that are enlarging rapidly as development proceeds. After the surface ectoderm
has completed a number of early interactions it forms the periderm,
which remains throughout fetal life and differentiates into epidermis.
Ectodermal ring and ectodermal placodes
The ectoderm on the head and lateral borders of the embryo shows a
zone of epithelial thickening, the ectodermal ring, which can be dis
cerned from stage 10 and is completed by stage 12. Rostrally, it contains populations of neuroectoderm that remain in the ectoderm after
primary neurulation and are termed ectodermal placodes; these pla
codes may be considered to be neuroepithelial cells that remain within the surface ectoderm until central nervous system development has
progressed sufficiently for their inclusion into sensory epithelia and
cranial nerve ganglia. The neuronal placodes may invaginate in toto to
form a vesicle, or remain as a neuronal layer, or contribute individually to neuronal structures with cells of other origins. The midline ectoder
mal thickening, the adenohypophysial placode, invaginates as Rathke’s pouch and forms a vesicle immediately rostral to the buccopharyngeal
membrane. The ectodermal ring then passes bilaterally to encompass
the olfactory and optic placodes, which give rise to the olfactory sensory
epithelium and the lens of the eye, respectively. It then overlays the
pharyngeal arches, where it gives rise to epibranchial placodes; these
remove themselves individually from the ectoderm at stage 10–1 1 and
become associated with the neural crest cells within the cranial sensory ganglia supplying the arches. It also forms specializations of the ecto
derm on the frontonasal, maxillary and mandibular processes, which give rise to the tooth buds and the outer coating of the teeth. The paired otic placodes overlying the rhombencephalon at the lateral portion of the second pharyngeal arch invaginate to form the otic vesicles, which give rise to the membranous labyrinth of the ear. The ectodermal ring Fig. 12.1 A, The embryo at stage 11, showing the position of the intraembryonic coelom (contained by the walls coloured blue). B, The three major
epithelial populations within a stage 11 embryo, viewed from a ventrolateral position. The neural tube lies dorsal to the gut. Ventrally, the intraembryonic
coelom crosses the midline at the level of the foregut and hindgut, but is lateral to the midgut and a portion of the foregut.
Pericardial cavityPericardial cavity
Amnion
Yolk sac wall Entrance to pericardioperitoneal canal
Yolk sac wall
Peritoneal cavityPharynx
Neural tube
Midgut
HindgutPericardioperitoneal canal
ForegutNeural tube
Somite
Left umbilical vein
Umbilical cordB A | 284 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
embryonic cell populations at the start of organogenesis
195
Chapter 12
Somites
Somites are discrete epithelial spheres formed by the transformation of
paraxial mesoblast cells to epithelium once their migration is complete.
Specific regions of the somitic epithelium form local proliferative
centres and, like neural and coelomic epithelium, it produces cells
destined for different fates, at different developmental times. Somites
can be seen on each side of the fusing neural tube in the human embryo
from stage 9. Development proceeds in a craniocaudal direction. The
original epithelial somites form the base of the skull, and the vertebral
column and ribs; the dorsolateral portion, the dermomyotome, gives
rise to the majority of the skeletal muscle of the body, including that
in the limbs.
The relative dispositions of the neural, endodermal and coelomic
epithelia are shown in Figure 12.1.
Mesenchymal populations in the embryo
In the stage 10 embryo, the major mesenchymal populations are in
place. Mesoblast is still being generated at the primitive streak and moving into the presomitic mesenchymal population adjacent to the notochord. Some mesoblast is also contributing to the lateral regions Endoderm
The craniocaudal progression of development means that the endo
derm of the early stomodeum develops ahead of other portions of the
endodermal epithelium. The development of the pharyngeal arches and
pouches (Ch. 36) is closely associated with the development of the
neural ectoderm and proliferation of the neural crest. The respiratory
diverticulum arises slightly later, when the postpharyngeal gut may
also be distinguished. The endoderm gives rise to the epithelial lining of the respiratory and gastrointestinal tracts, the biliary system, and the
bladder and urethra.
Coelomic epithelium
The coelomic epithelium lines the intraembryonic coelom, which is
subdivided into a midline pericardial cavity, two bilateral pericardio
peritoneal canals, and the initially bilateral peritoneal cavities; the
latter are continuous with the extraembryonic coelom. The coelomic epithelium is a germinal epithelium. It produces the myocardium
and connective tissue populations for the viscera, and also gives rise to the supporting cells for the germ cells, the epithelial lining of the uro
genital tracts, and the mesothelial lining of the pericardial, pleural and peritoneal cavities.Fig. 12.2 A, Mesoblast populations within the early embryonic disc. B, A stage 11 embryo, showing the position of the intraembryonic coelom
(contained by the walls coloured blue) and the positions of the sections (i)–(iv) shown in C. C, Transverse sections, arranged cranial to caudal, from a
stage 11 embryo; the populations of mesenchyme and the sites of mesenchymal proliferation are indicated. Unsegmented paraxial
mesenchymeA
C
(i)(i)
(ii)(ii)
(iii)
(iii)
(iv)
(iv)Lateral plate mesenchymeBuccopharyngeal membrane
Cloacal membrane
Pericardial
cavityNeural tube
Amnion
Yolk sac wall
Entrance to
pericardio-
peritoneal canalPrechordal mesenchyme
Notochord
Neural crest mesenchyme
Aorta
Endocardium
Developing myocardium
Notochordal plate
Aorta
Gut
Pericardioperitoneal canal
Umbilical cordSomite
MidgutLeft
umbilical veinPharynx
Pericardio-
peritoneal
canalUnsegmented paraxial mesenchyme
Proliferative coelomic epithelium
Pericardial cavity
Neural crest mesenchyme
Epithelial somite
Aorta
Proliferative
splanchnopleuric
epitheliumNeural crest mesenchyme
Notochordal plate
Pericardioperitoneal canal
Gut
Intraembryonic coelom
(peritoneal cavity)GutProliferative somatopleuriccoelomic epitheliumNotochordal plate
AortaParaxial mesenchyme
Proliferative somatopleuric
coelomic epithelium
Endothelium of sinus venosus
Septum transversum
mesenchymeGut
Neural crest mesenchyme
Epithelial somite
Region of intermediate
mesenchyme
Proliferative splanchnopleuric
coelomic epitheliumB | 285 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Cell populations at the start of organogenesis
196seCtion 2
Fig. 12.3 Structures that will be derived from specific epithelial and mesenchymal populations in the early embryo. Abbreviation: CNS, central nervous
system. Endoderm epithelium
Primitive gut
Foregut – recesses, diverticula and glands of the
pharynx.
General mucous glandular and duct-lining cells and
the main follicular cells of the thyroid.
Epithelium of pharyngeal pouches (tonsil, middle
ear cavity, thymus, parathyroids 3 and 4, C cells of thyroid), adenoids, epithelial lining of the auditorytube, tympanic cavity, tympanic antrum, internallamina of the tympanic membrane.
Respiratory tract – epithelial lining, secretory and
duct-lining cells of the trachea, bronchi, bronchiolesand alveolar sacs.
Epithelial lining, secretory and duct-lining cells of
the oesophagus, stomach and duodenum.
Hepatocytes of liver, biliary tract, exocrine and
endocrine cells of the pancreas.
Midgut – epithelial lining, glandular and duct-lining
cells of the duodenum, jejunum, appendix, caecum,part of transverse colon.
Hindgut – epithelial lining, glandular and
duct-lining cells of part of the transverse,descending and sigmoid colon, rectum, upper partof anal canal.
Allantois – urinary bladder, vagina, urethra,
secretory cells of the prostate and urethral glands.Coelomic wall epithelium Mesenchyme
Surface ectoderm epithelium Neural plate epithelium Neural crestWalls of intraembryonic coelom
Primitive pericardium – myocardium, parietal
pericardium.
Pericardioperitoneal canals – visceral, parietal
and mediastinal pleura, pleuroperitonealmembranes contributing to diaphragm.
Splanchnopleuric epithelium – visceral peritoneum
of stomach, peritoneum of lesser and greateromenta, falciform ligament, lienorenal andgastrosplenic ligaments.
Somatopleuric epithelium – parietal peritoneum.Primitive peritoneal cavity
Splanchnopleuric epithelium – visceral peritonealcovering of mid- and hindgut, the mesentery,transverse and sigmoid mesocolon.
Pronephros, epithelial lining of mesonephric ducts,
vas deferens, epididymis, seminal vesicles,ejaculatory duct, ureters, vesical trigone.
Müllerian ducts, epithelial lining of uterine tubes,
body and cervix of uterus, vagina, broad ligament ofuterus.
Germinal epithelium of gonad (note the germ cells
are not included on this chart because of their earlysequestration into the extraembryonic tissues).
Germinal epithelium forming cortex of suprarenal
gland.
Somatopleuric epithelium – parietal peritoneum,
tunica vaginalis of testis.Paraxial mesenchyme(somites and somitomeres)Sclerotome – vertebrae and portions of theneurocranium, axial skeleton.Myotome – all voluntary muscles of the head, trunkand limbs.Dermatome – dermis of skin over dorsal regions.
Intermediate mesenchyme – connective tissue of
gonads, mesonephric and metanephric nephrons,smooth muscle and connective tissues of thereproductive tracts.Septum transversum – epicardium, fibrouspericardium, portion of diaphragm, oesophagealmesentery, sinusoids of liver, tissue within lesseromentum and falciform ligament.
Lateral plate mesenchyme
Splanchnopleuric layer – smooth muscle andconnective tissues of respiratory tract andassociated glands.
Smooth muscle and connective tissues of intestinal
tract, associated glands and abdominal mesenteries.
Smooth muscle and connective tissue of blood
vessels (also see below).
Somatopleuric layer – appendicular skeleton,
connective tissue of limbs and trunk, includingcartilage, ligaments and tendons.
Dermis of ventral body wall and limbs.
Mesenchyme of external genitalia.
Angiogenic mesenchyme
Endocardium of heart, endothelium of blood andlymphatic vessels, vessels of choroid plexus,sinusoids of liver and spleen, circulating blood cells,microglia, tissue macrophages.
Ectodermal placodesAdenohypophysis.
Sensory neurones of the cranial ganglia V, VII, VIII, IX, X.
Olfactory receptor cells and olfactory epithelium.Epithelial walls of the membranous labyrinth, the
cochlear organ of Corti.
Lens of the eye.
Enamel organs of the teeth.Cranial structures
Secretory and duct-lining cells of the lacrimal,nasal, labial, palatine, oral and salivary glands.
Epithelia of the cornea and conjunctiva.Epithelial lining of the external acoustic meatus and
external epithelium of the tympanic membrane.
Epithelial lining of the lacrimal canaliculi and
nasolacrimal duct.
Epithelial lining of the paranasal sinuses, lips,
cheeks, gums and palate.
Epidermal structures
Most of the cutaneous epidermal cells, thesecretory, duct-lining and myoepithelial cells of thesweat, sebaceous and mammary glands.
Hair and nails.
Proctodeal epithelium and epithelium of the
terminal male urethra.CNS – Brain and spinal cordNeurohypophysis.
Prosencephalon (telencephalon and diencephalon)
– cerebral hemispheres, basal nuclei.
Mesencephalon – cerebral peduncles, tectum,
tegmentum.
Rhombencephalon (metencephalon and
myelencephalon) – cerebellum, pons, medullaoblongata.
Spinal cord.All cranial and spinal motor nerves.All CNS neurones, including preganglionic efferent
neurones, with somata within the CNS.
Astrocytes and oligodendrocytes.Ependyma lining the cerebral ventricles, aqueduct
and central canal of brain and spinal cord,tanycytes, cells covering the choroid plexuses,circumventricular cells.
Retina and optic nerve (II), epithelium of the iris,
ciliary body and processes.Neural derivativesSensory neurones of the cranial ganglia V, VII, VIII, IX, X.
Sensory neurones of the spinal dorsal root ganglia and
their peripheral sensory receptors.
Satellite cells in all sensory ganglia.Sympathetic ganglia and plexuses: neurones and
satellite cells.
Parasympathetic ganglia and plexuses: neurones and
satellite cells.
Enteric plexuses: neurones and glial cells.Schwann cells of all the peripheral nerves.Medulla of the suprarenal gland. Chromaffin cells.
Carotid body type I cells (and type II, satellite type cells).Calcitonin-producing cells (C cells).
Melanocytes.
Mesenchymal derivatives in the head
Frontal, parietal, squamous temporal, nasal, vomer,palatine bones, maxillae and mandible.
Meninges.
Choroid and sclera of eye.
Connective tissue of lacrimal, nasal, labial, palatine,
oral and salivary glands.
Dentine of teeth.
Connective tissues of head, including cartilage,
ligaments and tendons.
Connective tissues of thyroid gland and of the
pharyngeal pouches, i.e. parathyroid glands, thymus.
Tunica media of the outflow tract of the heart and the
great vessels. | 286 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
embryonic cell populations at the start of organogenesis
197
Chapter 12the primitive streak, none of the cells that arise from the neural crest
become arranged as epithelia. The development and fate of head and
trunk neural crest cells are very different and therefore they will be
considered separately.
head neural crest
Head neural crest migrates before the neural tube closes. Two popula
tions of crest cells develop. Some retain a neuronal lineage and con
tribute to the somatic sensory and parasympathetic ganglia in the head and neck. Others produce extensive mesenchymal populations; the
crest cell population arising from the head is larger than that found at
any trunk level. Each brain region has its own crest population that
migrates dorsolaterally around the sides of the neural tube to reach the
ventral side of the head. Crest cells surround the prosencephalic and
optic vesicles, and occupy each of the pharyngeal arches (Ch. 36). They
provide mesenchyme cells that will produce the connective tissue in
parts of the neuro and viscerocrania. All cartilage, bone, ligament,
tendon, dermal components and glandular stroma in the head are derived from the head neural crest. Head neural crest also contributes
to the tunica media of the aortic arch arteries.
trunk neural crest
Unlike its counterpart in the head, trunk neural crest is formed as the
neural tube closes craniocaudally, which means that various stages of
crest development can be found in the more caudal regions of an
embryo. As the neural tube begins to fuse dorsally in the midline, the
neural crest cells lose their epithelial characteristics and junctional con
nections, and form a band of loosely arranged mesenchyme cells imme
diately dorsal to the neural tube and beneath the ectoderm. Initially,
most of the crest cells lie with their long axes perpendicular to the long
axis of the neural tube. Later, the cell population expands laterally and
around the neural tube as a sheet. Trunk neural crest cells move from
their position dorsal to the neural tube via three routes (see Fig. 17.1 1):
dorsolaterally, to form dorsal root ganglia throughout the trunk; ven
trally, to form sympathetic ganglia, enteric nerves and the suprarenal
medulla; and rostrocaudally, along the aorta, to form the pre aortic
ganglia. In a second migration route, crest cells pass dorsolaterally
between the ectoderm and the epithelial plate of the somite into the
somatopleure, where they eventually form melanocytes in the skin.
Lateral plate
Lateral plate is the term for the early unsegmented mesoblast popula
tion lateral to the paraxial mesenchyme. Mesoblastic cells, which arise
from the middle of the primitive streak (primary mesenchyme), migrate
cranially, laterally and caudally to reach their destinations, where they
revert to epithelium and form a continuous layer that adheres to the
ectoderm dorsally and the endoderm ventrally. The epithelium faces a
new intraembryonic cavity, the intraembryonic coelom, which becomes
confluent with the extraembryonic coelom and provides a route for the
circulation of coelomic fluid through the embryo. Once formed, the
intraembryonic coelomic wall becomes a proliferative epithelium that
produces new populations of mesenchymal cells. The mesenchymal
population subjacent to the ectoderm is termed somatopleuric mesen
chyme and is produced by the somatopleuric coelomic epithelium. The mesenchymal population surrounding the endoderm is termed splanch
nopleuric mesenchyme and is produced by the splanchnopleuric coelomic epithelium (see Fig. 12.2).
It is important to note that these terms are relevant only caudal to
the third pharyngeal arch. Rostral to this, there is a sparse mesenchymal
population between the pharynx and the surface ectoderm prior to
migration of the head neural crest, and there are no landmarks with
which to demarcate lateral from paraxial mesenchyme. This unsplit
lateral plate is believed to contribute to the cricoid and arytenoid car
tilages, the tracheal rings and the associated connective tissue.
somatopleuric mesenchyme
Somatopleuric mesenchyme produces a mixed population of connec
tive tissues and has a significant organizing effect at the level of the
developing limbs. The pattern of limb development is controlled by
information contained in the somatopleuric mesenchyme. Regions of
the limb are specified by interaction between the surface ectoderm
(apical ectodermal ridge) and underlying somatopleuric mesenchyme;
together, these tissues form the progress zone of the limb. The somatopleuric mesenchyme in the limb bud also specifies the postaxial border of the developing limb. Somatopleuric mesenchyme gives rise to the connective tissue elements of the appendicular skeleton, including the pectoral and pelvic girdles and the bones and cartilage of the limbs, and their associated ligaments and tendons. It also gives rise to the dermis of the skin of the ventral and lateral body walls and of the limbs.of the embryo. The different mesenchymal populations within the
embryo from stage 10 onwards are described below. The relative
dispositions of the early mesenchymal populations are shown in
Figure 12.2.
Axial mesenchyme
The first epiblast cells to ingress through the primitive streak form the
endoderm and notochord, and initially occupy a midline position. The
earliest population of endodermal cells rostral to the notochordal plate
is termed the prechordal plate. The notochordal cells remain medially
and the endodermal cells subsequently flatten and spread laterally. The
population of cells that remain mesenchymal in contact with the floor
of the neural groove, just rostral to the notochordal plate, is termed
prechordal mesenchyme (Fig. 12.4). These axial mesenchyme cells are
tightly packed, unlike the more lateral paraxial cells but, unlike the
notochord, they are not contained in an extracellular sheath. They are
displaced laterally at the time of head flexion and form bilateral pre
mandibular mesenchymal condensations. They become associated with the local paraxial mesenchyme. Orthotopic grafting has demonstrated
that these cells leave the edges of the prechordal mesenchyme and
migrate laterally into the periocular mesenchyme, where they give rise
to all of the extrinsic ocular muscles.
Paraxial mesenchyme
Paraxial mesoblast is a transient structure that forms somites at its cranial end, whilst unsegmented mesoblast is added caudally by the
primitive streak. Epiblast cells that migrate through the primitive node
and rostral primitive streak during gastrulation form mesoblast cells
that migrate to a position lateral to the notochord and beneath the
developing neural plate. Cells that ingress through the primitive node
form the medial part of this paraxial mesoblast, and cells that ingress
through the rostral streak form the lateral part (see Fig. 10.3). The
paraxial mesoblast extends cranially from the primitive streak to the prechordal plate, which is immediately rostral to the notochord. Before
somite formation, this mesenchymal tissue is also termed presomitic
or unsegmented mesenchyme in mammals (analogous to the segmen
tal plate in birds). Paraxial mesoblast rostral to the otic vesicle was previously believed not to segment.
Caudal to the otic vesicle, the paraxial mesoblast on each side of the
rhombencephalon segments into somites as the neural folds elevate
and neurulation begins; somites are therefore post otic. During somito
genesis, mesoblastic cells show changes in shape and in cell–cell adhe
sion, and become organized into epithelial somites; this process begins
at the eighth somitomere, just caudal to the midpoint of the noto
chordal plate. Somite one is also termed the first occipital somite. The post otic paraxial cell population is termed paraxial mesoderm in con
temporary literature.
Skeletal muscle throughout the body is derived from paraxial
mesoblast derived somatic epithelium, which proliferates to form
myoblastic populations that migrate to the head, body and limbs.
Septum transversum
Early mesenchyme that invaginates through the middle part of the
primitive streak comes to lie rostral to the buccopharyngeal membrane,
where the cells form the epithelial wall of the pericardial coelom. As
this epithelium proliferates, the visceral pericardial wall gives rise to
myocardium. The parietal pericardial wall forms mesenchyme, initially
termed precardiac, or cardiac, mesenchyme, which is able to induce
proliferation of hepatic endodermal epithelium. With further prolifera
tion, the precardiac mesenchyme forms a ventral mass caudal to the heart, the septum transversum, separating the foregut endoderm from
the pericardial coelom (see Fig. 12.2). By stage 1 1, the septum transver
sum extends dorsally on each side of the developing gut and becomes continuous with the mesenchymal populations that proliferate from
the walls of the pericardioperitoneal canals. Cells of the septum trans
versum give rise to the sinusoids of the liver, the central portions of the diaphragm and the epicardium.
Neural crest
The neural crest is unique: it gives rise to neural populations in the
head and trunk, and also provides an extensive mesenchymal popula
tion in the head with attributes similar, in terms of patterning, to
somatopleuric mesenchyme. Neural crest cells arise from cells that lie initially at the outermost edges of the neural plate, between the pre
sumptive epidermis and the neural tube, and are committed to a neural crest lineage before the neural plate begins to fold. After neurulation, neural crest cells form a transient axial population and then disperse, in some cases migrating over considerable distances, to a variety of dif
ferent developmental fates. Unlike mesoblast, which is produced from | 287 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Cell populations at the start of organogenesis
198seCtion 2
Fig. 12.4 The organization of the head and pharynx in an embryo at about stage 14. The individual tissue components have been separated but are
aligned in register through the numbered zones.
PATTERNS OF GENE EXPRESSIONHOX-b5
HOX-b4HOX-b3HOX-b2
HOX-a1
CRABP
RAR-β
Wnt-1Engrailed-2
HOX-b1
Krox 20
Wnt-2
AXIAL STRUCTURES
MIGRATION OF NEURAL CREST MESENCHYME
PARAXIAL MESENCHYME
BRAIN AND CRANIAL MOTOR NERVES
CRANIAL SENSORY AND PARASYMPATHETIC GANGLIA
SUGGESTED ORIGIN OF STRIATED MUSCLES
FROM PARAXIAL MESENCHYME
PHARYNGEAL ARCH ARTERIES
ENDODERMAL PHARYNGEAL POUCHES4th 3rd 2nd 1st
4th 3rd 2nd 1st1 2
34
5 12 3 4 5 6 7
12 3 4 5 6 7
1234 5 1 234
1 2 3 41 2 3 456 7XII
X IX VII VVI
IIIIVPharyngeal arches:Rhombomeres
765432 1
SomitesSomitomeres
Proximal VII (root)
OticNeural crest shown in green and
ectodermal placodes in grey
Neural crest shown in
green around great vessels
Neural crest shown in green
around pharyngeal pouchesRhombencephalon
Otic vesicle
Otic vesicle
Proximal X (jugular)
Proximal IX (superior)
VIII (vestibulocochlear)
Distal X (nodose)
Distal IX (petrosal)
3rd arch
Laryngeal
Glossal2nd arch (hyoid)
1st arch (mandibular)
Descending aorta
Ductus arteriosus
Pulmonary artery
Ascending aorta
Inferior parathyroid
Superior parathyroid
Oesophagus
Lung budUltimobranchial bodySpinal cord
Notochord
Optic cup
MandibularMaxillary
Frontonasal
Ciliary
Prechordal mesenchyme migrates
to form extraocular musclesV (trigeminal)
PterygopalatineDistal VII (geniculate)Submandibular
Lateral rectus
Inferior rectus
EyeSuperior rectus
Superior oblique
Medial rectus
Internal carotid artery
Tubotympanic recessPharyngeal arch arteries
Pharyngeal tonsilThyroid
ThymusInferior obliqueProsencephalonPrechordal mesenchymeMesencephalon | 288 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
199
Chapter 12Key references
endodermal tissues are necessary for endothelial differentiation, par
ticularly the early foregut. Angioblastic mesenchyme forms early in
the third week of development from extraembryonic mesenchyme in
the splanchno pleure of the yolk sac, in the body stalk (containing the
allantois), and in the somatopleure of the chorion. The peripheral cells
flatten as a vascular endothelium, whereas the central cells transform
into primitive red blood corpuscles. Later, contiguous islands merge,
forming a continuous network of fine vessels. Intraembryonic blood
vessels are first seen at the endoderm–mesenchyme interface within the
lateral splanchnic mesenchyme at the caudolateral margins of the
cranial intestinal portal. Angioblastic competence has been demon
strated within the ventral (splanchnopleuric) mesenchymes with which
the endoderm interacts. However, the notochord and prechordal plate
do not contain angiogenic cells. Similarly, ectodermal tissues do not
appear to give rise to angiogenic cells. Somites, derived from paraxial
mesenchyme, have been shown to be a source of angioblasts that either
differentiate with the somite derivatives, or migrate to the neural tube,
ventrolateral body wall, limb buds, mesonephros and the dorsal part
of the aorta.
The earliest angiogenic mesenchymal cells form blood vessels by
vasculogenesis, a process in which new vessels (e.g. endothelial heart
tubes, dorsal aortae, umbilical and early vitelline vessels) develop in situ
(Ch. 13). Later vessels develop by angiogenesis, sprouting and branch
ing from the endothelium of pre existing vessels; this process is the
means by which most other vessels develop. The ultimate pattern of
vessels is controlled by the surrounding, non angiogenic mesenchyme;
vessels become morphologically specific for the organ in which they
develop, and also immunologically specific, expressing organ specific
proteins.splanchnopleuric mesenchyme
Splanchnopleuric mesenchyme surrounds the developing gut and res
piratory tubes, contributing connective tissue cells to the lamina propria
and submucosa, and smooth muscle cells to the muscularis mucosae
and muscularis externa. It plays a patterning role in endodermal devel
opment, specifying the region and villus type in the gut, and the branch
ing pattern in the respiratory tract.
Intermediate mesenchyme
Intermediate mesenchyme is a loose collection of cells found between the somites and the lateral plate (see Fig. 12.2). Its development is
closely related to the progress of differentiation of the somites and the proliferating coelomic epithelium from which it is derived. Intermedi
ate mesenchyme is not present before somitogenesis or the formation of the eighth somite. In embryos with 8–10 somites, it is present lateral
to the sixth somite but does not extend cranially. The mesenchyme cells
are arranged as layers, one continuous with the dorsal side of the par
axial mesenchyme and the somatopleure, the other with the ventral side
of the paraxial mesenchyme and the splanchnopleure.
As development proceeds, the intermediate mesenchyme forms a
loosely packed dorsolateral cord of cells, which lengthens at the caudal
end and ultimately joins the cloaca. It gives rise to the nephric system,
gonads and reproductive ducts.
Angioblastic mesenchyme
Mesenchymal cells, which give rise to the cellular elements of the blood, the endothelium and the mesenchymal layers of the tunica
externa and adventitia of blood and lymphatic vessels, arise from
extraembryonic and intraembryonic sources. Evidence suggests that
KEY REFERENCES
O’Rahilly R, Müller F 1985 The origin of the ectodermal ring in staged
human embryos of the first 5 weeks. Acta Anat 122:145–57.Richardson MK, Keuck G 2002 Haeckel’s ABC of evolution and develop
ment. Biol Rev Camb Philos Soc 77:495–528. | 289 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Cell populations at the start of organogenesis
199.e1
Chapter 12REFERENCES
O’Rahilly R, Müller F 1985 The origin of the ectodermal ring in staged
human embryos of the first 5 weeks. Acta Anat 122:145–57.Richardson MK, Keuck G 2002 Haeckel’s ABC of evolution and develop
ment. Biol Rev Camb Philos Soc 77:495–528. | 290 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
200CHAPTER 13 Early embryonic circulation
guidance, where the distal tip cell extends supported by proximal stalk
cells that may divide; sprout elongation in response to cues from the
extracellular matrix; lumen formation, which is initially by the conflu -
ence of intracellular vacuoles, followed by establishment of apical basal polarity of the cells of the endothelial sprout; and fusion of the tip cell
to another sprout tip with confluence of the two lumina established
(Chappell et al 2012, Kuijper et al 2007). The developmental processes
of angiogenesis are similar to those seen in both neoplasia and acute inflammation in adult tissue. The scientific literature on angiogenesis
is extensive and the process will not be considered in detail here; the
interested reader is directed to Senger and Davis (201 1), Beets et al
(2013), Herbert and Stainier (201 1) and Eichmann and Pardanaud
(2014).
Studies have considered the development of capillary beds within
the surrounding mesenchymal tissues destined to become laminae pro -
priae, but few have looked at how capillary beds form within tissues
undergoing morphogenetic movements. Czirok et al (201 1) discussed
the temporal imperative for free-living embryos to develop rapidly in
contrast to amniote embryos, which do not have to fend for themselves.
They concluded that amniote vascular patterns of development, prior
to the establishment of a circulation, are not predetermined by a
network of patterning genes and cell signalling pathways, but rather that
the tissue movements during organogenesis and the realignment and
tension that develop within extracellular matrix fibres may guide cell
migration and the position of early vessels.
Extracellular matrix plays a critical role in angiogenesis and vessel
stabilization in the embryo and in the adult; differences in the composi -
tion and abundance of specific extracellular matrix components between The early embryonic circulation is symmetrical ( Fig. 13.1). It is modi -
fied throughout development to produce a functioning fetal circulation
that is connected to the placenta, and changes rapidly at birth to accom -
modate disconnection from the placenta and the start of gaseous exchange in the lungs. Major restructuring of early vessels occurs as
the embryo grows; anastomoses form and then disappear, capillaries fuse and give rise to arteries or veins, and the direction of blood flow
may reverse several times before the final arrangement of vessels is
completed.
ANGIOGENESIS
The earliest circulatory components develop by vasculogenesis in the extraembryonic tissues. The endothelial heart tubes, dorsal aortae,
umbilical and early vitelline vessels arise by vasculogenesis within the
embryo. Further vessel development occurs by a process of angiogenesis
in which angioblasts, arising in splanchnic and somitic tissues, add
endothelial sprouts and branches to earlier vessels. None of the main
vessels of the adult arises as a single trunk in the embryo. A capillary
network is first laid down along the course of each vessel; the larger
arteries and veins are defined by selection and enlargement of definite
paths in this network. Lymphatic vessels develop after the main arteries
and veins are formed; they arise initially by angiogenesis from the car -
dinal veins and subsequently by proliferation of lymphangioblasts to form lymphatic capillaries.
Five stages have been described during angiogenesis: endothelial tip
cell specification and sprout initiation; sprout elongation and local
Fig. 13.1 The early, symmetrical blood vascular system. A, Ventrolateral view of the endothelial profile of the heart, the first aortic arch arteries and the
dorsal aorta shown in relation to the major epithelial populations. B, Ventrolateral view of the main venous channels shown in relation to the major
epithelial populations. C, Left lateral view of the blood vascular system of a stage 11 human embryo; only the endothelial lining of the heart tube is
shown. At this stage, the arteries and veins are in the process of development and no true circulation is yet established.
Vitelline plexus
Yolk stalk (cut)
Umbilical arteriesOtic placodeRudiment of second
aortic archFirst aortic arch
Primitive head vein
Right atrium
Rudiment of common
cardinal vein
Rudiments of
postcardinal vein
Dorsal aortaVentricleAortic sac A B C
Left umbilical arteryDorsal aortaVentricleAtriumLeft first aortic arch Left precardinal vein
Left postcardinal veinLeft common cardinal vein
Sinus venosus
Septum transversumPericardial cavity
Left umbilical vein | 291 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Angiogenesis
201
CHAPTER 13
As the heart muscle thickens, compacts and strengthens, the cardiac
orifices become both relatively and absolutely reduced in size, the valves
increase their efficiency, and the large arteries acquire their muscular
walls and undergo a relative reduction in size. From this time onwards,
the embryo is dependent for its nourishment on the expanding capillary
beds, and the function of the larger arteries becomes restricted to that
of controllable distribution channels to keep the embryonic tissues
constantly and appropriately supplied.
The heart starts to beat early, before the development of the conduc -
tion system, and a circulation is established before a competent valvular
mechanism has formed. Cardiac output increases in proportion with
the weight of the embryo and cardiac rate increases with development.
However, most of the increase in cardiac output results from a geomet -
ric increase in stroke volume. When dorsal aortic blood flow is matched to embryonic weight, blood flow remains constant over a more than
150-fold change in mass of the embryo.
After head folding, the embryo has bilateral primitive aortae, each
consisting of ventral and dorsal parts that are continuous through the
first embryonic aortic arches (Ch. 36). The ventral aortae are fused and
form a dilated aortic sac. The dorsal aortae run caudally, one on each
side of the notochord. In the fourth week, they fuse from about the
level of the fourth thoracic to that of the fourth lumbar segment to
form a single definitive descending aorta (see Fig. 13.1A,C; Fig. 13.2B).
In general, more mature endothelial channels are seen in the rostral, more advanced regions of the embryo whereas, more caudally, a
changing capillary plexus constantly remodels until it becomes conflu -
ent with the vascular channels of the connecting stalk. The dorsal continuation of the primitive dorsal aortae directs blood into an anas -
tomosing network around the allantois, which will form the umbilical arteries. Blood is channelled back to the developing heart from the
allantois via umbilical veins, from anastomoses in the primitive yolk
sac via the vitelline veins, and from the body via pre- and postcardinal
veins that join to form the common cardinal veins (see Figs 13.1B,C,
13.2A).
Vascular anomalies Localized defects of vascular development,
characterized by focal increases in the number of vessels that are abnor -
mally tortuous and enlarged, have been shown to have genetic causes
and mapped to the predisposing gene and chromosomal locus (Boon
et al 201 1). It has been suggested that a range of capillary malforma -
tions, venous malformations, telangiectasia and lymphatic malforma -
tions, including lymphoedema, is associated with factors identified in the early formation of blood and lymphatic vessels (Brouillard and
Vikkula 2007, Boon et al 201 1).the embryo and adult mean that there are likely to be significant dif -
ferences in the process at different ages. Fibronectin is required for
normal vascular development; its deposition is related to interactions
between endothelial cells and pericytes, and local tensional forces that
occur between blood vessel basement membrane and local extracellular
matrix assembly (Davis and Senger 2005; Senger and Davis 201 1). Early
blood vessels are initially surrounded by a fibronectin-rich matrix that is later incorporated into the endothelial basal lamina along with
laminin, a particularly early constituent. Several layers of fibronectin-
expressing cells are seen around larger vessels, such as the dorsal aortae.
The endothelium does not synthesize a basal lamina in those regions
where remodelling and angiogenesis are active, and the mesenchyme
around such endothelium does not express α-actin or laminin until
branching has stopped and differentiation of the tunica media begins (after a stable vascular pattern has formed). It is not known how dif -
ferentiation of pericytes and smooth muscle cells is induced; the major -
ity of arteries accumulate medial smooth muscle from the surrounding
mesenchyme.
Although studies have elucidated the genes involved in vasculogen -
esis and angiogenesis, the drivers that lead to development of asym -
metric vessels from an early symmetrical pattern and the specification of the position of particular arteries, veins and lymphatic vessels are still
not clear. In early development, the arteries of the embryo are dispro -
portionately large and their walls consist of little more than a single layer of endothelium. The cardiac orifices are also relatively large and
the force of the cardiac contraction is weak; consequently, the circula -
tion is sluggish, despite the rapid rate of contraction of the developing heart. However, the tissues are able to draw nourishment not only from
the capillaries but also from the large arteries and the intraembryonic
coelomic fluid.
It has been suggested that the rapidly expanding cardiovascular
system is filled with plasma by the movement of fluid from the intra -
embryonic coelom to the veins. In general, the wall of the intraembry -
onic coelom is composed of proliferating cells that produce the
splanchnopleuric and somatopleuric mesenchymal populations. How -
ever, the walls of a portion of the pericardioperitoneal canals are thin -
ner, and possibly more permeable to coelomic fluid, at the time when
the canals surround the hepatocardiac channels (veins). The latter
lie between the hepatic plexus and the sinus venosus; the hepatocar -
diac channel on the right side is more developed and on the left it is more plexiform, with only a transitory connection to the sinus veno -
sus. The differentiation of this specific coelomic region occurs just in advance of the expansion and filling of the right and left atria, at about stage 12.Fig. 13.2 Profile reconstructions of the blood vascular system of a stage 13 human embryo. The early circulation is now asymmetrical; the venous
vessels are enlarging on the right and diminishing on the left. A, Seen from the right side, showing the main venous channels to the heart. B, Seen from
the left side, showing the aortic arch arteries, the main vessels arising from the dorsal aorta and umbilical arteries. Note that only the endothelial lining of
the heart chambers is shown and, because the muscular wall has been omitted, the pericardial cavity appears much larger than the contained heart.
Otocyst
Second aortic arch
Left umbilical arteryThird aortic arch Internal carotid artery
Truncus arteriosus
Right atrium
Left atrium
Left ventricle
Right umbilical artery
Caudal
plexusDeveloping liver sinusoids
in septum transversumAorta
Lung bud
Primary plexus
to intestineLeft common
cardinal vein
Left
umbilical vein
Left
vitelline vein
Vitelline arteryPrimary head vein
Internal carotid artery
Optic
vesicle
Truncus
arteriosus
Right ventricle
Left ventricle
Right sinus hornRight vitelline veinRight atrium
Precardinal veinThird aortic archSecond aortic arch
Somite IV
Right common
cardinal vein
Aorta
Hepatocardiac
channel
Upper limb bud
Postcardinal vein
Right umbilical vein
0.2
cm 0.2 cmA
B
Hindgut | 292 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
EARly EmbRyoni C CiRCulATion
202SECT ion 2renal artery arises from the most caudal. Additional renal arteries are
frequently present and may be regarded as branches of persistent lateral
splanchnic arteries.
Ventral splanchnic arteries
The ventral splanchnic arteries are originally paired vessels distributed to the capillary plexus in the wall of the yolk sac. After fusion of the
dorsal aortae, they merge as unpaired trunks that are distributed to the
increasingly defined and lengthening primitive digestive tube. Longitu -
dinal anastomotic channels connect these branches along the dorsal and ventral aspects of the tube, forming dorsal and ventral splanchnic
anastomoses (see Fig. 13.3). These vessels obviate the need for so many
‘subdiaphragmatic’ ventral splanchnic arteries, and these are reduced to three: the coeliac trunk and the superior and inferior mesenteric arter -
ies. As the viscera supplied descend into the abdomen, their origins migrate caudally by differential growth: the origin of the coeliac artery
is transferred from the level of the seventh cervical segment to the level
of the twelfth thoracic; the superior mesenteric from the second thoracic
to the first lumbar segment; and the inferior mesenteric from the twelfth
thoracic to the third lumbar segment. However, above the diaphragm,
a variable number of ventral splanchnic arteries persist, usually four or
five, and supply the thoracic oesophagus. The dorsal splanchnic anas -
tomosis persists in the gastroepiploic, pancreaticoduodenal and primary
branches of the colic arteries, whereas the ventral splanchnic anasto -
mosis forms the right and left gastric and the hepatic arteries.
EMBRYONIC VEINS
The early embryonic veins are often segregated into two groups, visceral and somatic, for convenience and apparent simplicity. The visceral
group contains the derivatives of the vitelline and umbilical veins, and
the somatic group includes all remaining veins. It should be noted that,
with time, embryonic veins change the principal tissues they drain. They
may receive radicles from obviously parietal tissues, which become
confluent with drainage channels that are clearly visceral, and so form
a compound vessel. The arrangement of the early embryonic veins is
initially symmetrical. The primitive tubular symmetric heart receives its
venous return through the right and left sinual horns, which are initially
embedded in the mesenchyme of the septum transversum. Each horn
receives, most medially, the termination of the principal vitelline vein;
more laterally, the umbilical vein; and, most laterally, having encircled
the pleuroperitoneal canal, the common cardinal vein. This symmetric
pattern changes as the heart and gut develop and the cardiac return is
diverted to the right side of the heart.
Vitelline veins
The vitelline veins drain capillary plexuses that develop in the splanch -
nopleuric mesenchyme of the secondary yolk sac. With head, tail and
lateral fold formation, the upper recesses of the yolk sac are enclosed
within the embryo as the splanchnopleuric gut tube, which extends
from the stomodeal buccopharyngeal membrane to the proctodeal
cloacal membrane. Derivatives from all these levels possess a venous
drainage that is originally vitelline.
Early umbilical veins
The umbilical veins form by the convergence of venules that drain the
splanchnopleure of the extraembryonic allantois. The human endoder-
mal allantois is very small; it projects into the embryonic end of the
connecting stalk, which is therefore regarded as precociously formed
allantoic mesenchyme, whereas the umbilical vessels are considered to
be allantoic. The peripheral venules drain the mesenchymal cores of the
chorionic villous stems and terminal villi (extraembryonic somatopleu -
ric structures). These are the radicles of the vena umbilicalis impar (usually single), which traverses the compacting mixed mesenchyme of
the umbilical cord to reach the caudal rim of the umbilicus. Here, the
single cordal vein divides into primitive right and left umbilical veins.
Each curves rostrally in the somatopleuric lateral border of the umbili -
cus, i.e. where intraembryonic and extraembryonic or amniotic somato -
pleure are continuous, lying lateral to the communication between
both the intraembryonic and the extraembryonic coeloms. Rostrolateral to the umbilicus, the two umbilical veins reach, enter and traverse the junctional mesenchyme of the septum transversum and connect with septal capillary plexuses. They then continue, entering their correspond -
ing cardiac sinual horns lateral to the terminations of the vitelline veins. EMBRYONIC ARTERIES
Initially, the dorsal aortae are the only longitudinal vessels present.
Their branches all run at right angles to the long axis of the embryo.
Later, these transverse arteries become connected by longitudinal anas-
tomosing channels that persist in part, forming arteries such as the
vertebral, internal thoracic, superior and inferior epigastric, and gastro -
epiploic. Each primitive dorsal aorta gives off somatic arteries (interseg -
mental branches to the body wall), a caudal continuation that passes
into the body stalk (the umbilical arteries), lateral splanchnic arteries
(paired segmental branches to the mesonephric ridge), and ventral
splanchnic arteries (paired segmental branches to the digestive tube).
Somatic arteries
The somatic arteries are intersegmental in position. They persist, almost
unchanged, in the thoracic and lumbar regions, as the posterior inter -
costal, subcostal and lumbar arteries. Each gives off a dorsal ramus, which passes backwards in the intersegmental interval and divides into
medial and lateral branches to supply the muscles and superficial
tissues of the back ( Fig. 13.3). The dorsal ramus also gives off a spinal
branch, which enters the vertebral canal and divides into a series of branches that supply the walls and joints of the osteoligamentous canal,
and neural branches to the spinal cord and spinal nerve roots. After
giving off its dorsal ramus, each intersegmental artery runs ventrally in
the body wall, gives off a lateral branch and terminates in muscular and
cutaneous rami.
Early umbilical arteries
Initially, the umbilical arteries are the direct caudal continuation of the primitive dorsal aortae. They are present in the body stalk before any
vitelline or visceral branches emerge, indicating the dominance of the
allantoic over the vitelline circulation in the human embryo. (On a
comparative basis, the umbilical vessels are chorio-allantoic and there -
fore ‘somatovisceral’ .) After the dorsal aortae fuse, the umbilical arteries arise from their ventrolateral aspects and pass medial to the primary
excretory duct to the umbilicus. Later, the proximal part of each umbili -
cal artery is joined by a new vessel that leaves the aorta at its termination and passes lateral to the primary excretory duct. This, possibly the fifth
lumbar intersegmental artery, constitutes the dorsal root of the umbili -
cal artery (the original stem, the ventral root). The dorsal root gives off the axial artery of the lower limb, branches to the pelvic viscera and,
more proximally, the external iliac artery. The ventral root disappears
entirely, and the umbilical artery now arises from that part of its dorsal
root distal to the external iliac artery, i.e. the internal iliac artery.
Lateral splanchnic arteries
The lateral splanchnic arteries supply, on each side, the mesonephros,
metanephros, testis or ovary, and the suprarenal gland. All these struc -
tures develop, in whole or in part, from the intermediate mesenchyme, later termed the aorta-gonad-mesonephros region. One testicular or ovarian artery and three suprarenal arteries persist on each side. The phrenic artery branches from the most cranial suprarenal artery, and the Fig. 13.3 The segmental and intersegmental arteries. The small red
dilations indicate the positions of the longitudinal anastomoses. Anterior branchLateral
branchVentral
branch of
somatic
artery
Lateral
splanchnic
arteryIntersegmental somatic artery
Dorsal branch of
somatic arterySpinal branch
Dorsal aorta
Kidney
Ventral
splanchnicartery
Gut | 293 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Embryonic veins
203
CHAPTER 13
mented by a range of bilateral longitudinal channels that anastomose
with the posterior cardinal system and with each other. These channels
are the subcardinal, supracardinal, azygos line, subcentral and precostal
veins (Fig. 13.4).
Subcardinal veins
Subcardinal veins form in the ventromedial parts of the mesonephric ridges and become connected to the postcardinal veins by a number of
vessels traversing the medial part of the ridges. The subcardinal veins
assume the drainage of the mesonephros and intercommunicate by a
pre-aortic anastomotic plexus, which later constitutes the part of the
left renal vein that crosses anterior to the abdominal aorta.
Supracardinal veins
Supracardinal veins form dorsolateral to the aorta and lateral to the
sympathetic trunk and take over the intersegmental venous drainage from the posterior cardinal vein. The supracardinal veins are also referred to as the thoracolumbar line or lateral sympathetic line veins.
Azygos line veins
Azygos line veins form dorsolateral to the aorta and medial to the sympathetic trunk. These channels, also referred to as the medial This early symmetric disposition of the vitelline veins and anastomoses,
umbilical and common cardinal veins, and the locus of the hepatic
primordial complex are summarized in Figures 13.1B and 13.2A. For
further development of the vitelline and umbilical veins, see Chapter
60 (Figs 60.8–60.10).
Cardinal veins and somatic
venous complexes
The initial venous channels in the early embryo have traditionally been termed cardinal because of their importance at this stage. The cardinal
venous complexes are first represented by two large vessels on each side,
the precardinal portion being rostral and the postcardinal being caudal
to the heart. The two veins on each side unite to form a short common
cardinal vein, which passes ventrally, lateral to the pleuropericardial canal, to open into the corresponding horn of the sinus venosus (see Figs 13.1B and 13.2A; Fig. 13.4B). The precardinal veins undergo
remodelling as the head develops. The postcardinal veins, which, in the early embryo, drain the body wall, are insufficient channels for venous return from the developing mesonephros and gonads and for the growing body wall. As the embryo increases in size, they are supple -Fig. 13.4 Somatic venous development. A, A schematic section through the embryonic trunk. Principal longitudinal veins are colour-coded.
Interconnections and intersegmental veins are uncoloured. B, The development of the principal somatic veins, from the early symmetric state, through
states of increasing asymmetry, to the definitive arrangement. Abbreviations: IVC, inferior vena cava.
Precardinal veins Internal
jugular veins
Subcardinal veins
PostcardinalveinPostcardinal veinHepatocardiac veinsSinus venosus
Superior
vena cava
Azygos veinCommon cardinal vein
Superior
intercostal vein
Oblique vein
and ligamentof left atriumBrachiocephalic veins
Obliqueinterprecardinalanastomosis
Azygos line vein
Supracardinal veinIntersubcardinal
anastomosis
(renal collar)
Interpostcardinal
anastomosisHemiazygos vein
Common iliac veinsIVC hepatic
segment
IVC subhepatic
segment
IVC subcardinal
segment
Left suprarenal vein
Left renal veinLeft gonadal vein
IVC supracardinal
segmentA
(i) B
(ii)(iii)
(iv)Postcardinal vein
Supracardinal vein (thoracolumbar line vein)
Subcardinal vein
Early symmetrical
disposition of veinsProgressive asymmetry; right-sided dominance;
some channels enlarge; others retrogressMaturation and tributaries of superior vena
cava; segments of definitive inferior vena cavaAzygos line vein (medial sympathetic line vein)Subcentral vein
Hepatic segment of inferior vena cava
(and right vitelline vein)
Subhepatic segment of IVC | 294 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
EARly EmbRyoni C CiRCulATion
204SECT ion 2
mechanotransduction with lymphatic endothelial cells elongating as a
consequence of high interstitial fluid volume (Planas-Paz et al 2012).
For more details on the morphogenesis of lymphatic vessels, see Tatin
and Makinen (2014).
The formation of jugular lymph sacs and their normal remodelling
as the lymphatic system develops are of particular interest because delay or disruption is noted in aneuploidic fetuses. Increased accumulation
of tissue fluid between the skin and soft tissues, posterior to the forming
cervical spine – termed nuchal translucency – can be identified on
ultrasound examination between 10 and 14 weeks’ gestation. Approxi -
mately 75% of trisomy 21 fetuses show an increased distance of nuchal translucency at this time (see Fig. 14.4). In the majority of cases, early
nuchal fluid accumulation resolves for normal and chromosomally abnormal fetuses between 14 and 20 weeks, and is thought to reflect
increased lymphatic system development, the onset of urine production
by the metanephric kidneys and a decrease in placental resistance
(Nafziger and Vilensky 2014).
In the fetus, lymphatic flow rates appear to be higher than in the
neonate, which, in turn, are higher than in the adult; overall fetal lymph
flow is thought to be five times greater than in adults (Bellini et al
2006). The production of pulmonary fluid secreted into the amniotic
cavity is related to the lymphatic drainage of the lungs. In the neonate,
pulmonary ventilation is important in regulating lymphatic flow in the
lungs. The process of parturition causes significant changes in the dis-
tribution of body water in the neonate, including movement of blood
from the placenta to the fetus and a temporary shift of fluid from the
intravascular compartment to the interstitial compartment; restoration
of fluid balance between intravascular, lymphatic and interstitial com -
partments occurs concomitant with an increase in postnatal oxygena -
tion (Bellini et al 2006).
LYMPH NODES AND LYMPHOID TISSUES
Lymph vessels can be seen in the embryo in the cervical region from stage 16. Lymph nodes, which provide regional proliferative foci for
lymphocytes, have been identified from week 9. Early lymph sacs
become infiltrated by lymphoid cells, and the outer portion of each sac
becomes the subcapsular sinus of the lymph node. Morphological dif -
ferentiation of medullary and cortical compartments has not been
observed until the end of week 10 (Tonar et al 2001). At the same time
as these early lymph nodes are developing, the nasopharyngeal wall is
infiltrated by lymphoid cells that are believed to herald the early devel -
opment of the tubal and pharyngeal tonsils.
In the neonate, a considerable proportion of the total amount of
lymphoid tissue is localized in lymph nodes; the subsequent increase
in the amount of lymphoid tissues that occurs during childhood reflects the growth of these nodes. Definitive follicles with germinal centres are formed during the first postnatal year. The pharyngeal tonsil reaches its maximal development at 6 years and its subsequent involution is com-pleted by puberty. Details of the development of gut-associated lym -
phoid tissue are given in Chapter 60.sympathetic line veins, gradually take over the intersegmental venous
drainage from the supracardinal veins. The intersegmental veins now
reach their longitudinal channel by passing medial to the autonomic
trunk, a relationship that the lumbar and intercostal veins subsequently
maintain. Cranially, the azygos lines join the persistent cranial ends of
the posterior cardinal veins.
Subcentral veins
Subcentral veins form directly dorsal to the aorta in the interval between
the origins of the paired intersegmental arteries. These veins communi -
cate freely with each other and with the azygos line veins; these con -
nections ultimately form the retro-aortic parts of the left lumbar veins
and of the hemiazygos veins.
Precostal or lumbocostal venous line
A precostal or lumbocostal venous line, anterior to the vertebrocostal
element and posterior to the supracardinal, is recognized by some
authorities. A possible derivative is the ascending lumbar vein.
Further development of the somatic veins
The supracardinal veins lie lateral to the aorta and the sympathetic trunks, which therefore intervene between them and the azygos lines
(see Fig. 13.4). They communicate caudally with the iliac veins and
cranially with the subcardinal veins in the neighbourhood of the pre-aortic intersubcardinal anastomosis. The supracardinal veins also com -
municate freely with each other through the medium of the azygos lines and the subcentral veins. The most cranial of these connections, together
with the supracardinal–subcardinal and the intersubcardinal anasto-
moses, complete a venous ring around the aorta below the origin of the
superior mesenteric artery, termed the ‘renal collar’ .
The ultimate arrangement of these embryonic abdominal and thor -
acic longitudinal cardinal veins may be summarized as follows. The terminal part of the left postcardinal vein forms the distal part of the
left superior intercostal vein. On the right side, its cranial end persists
as the terminal part of the azygos vein. The caudal part of the subcar -
dinal vein is, in part, incorporated in the testicular or ovarian vein and partly disappears. The cranial end of the right subcardinal vein is incor -
porated into the inferior vena cava and also forms the right suprarenal vein. The left subcardinal vein, cranial to the intersubcardinal anasto -
mosis, is incorporated into the left suprarenal vein. The renal and tes -
ticular or ovarian veins on both sides join the supracardinal–subcardinal
anastomosis. On the left side, this is connected directly to the part of
the inferior vena cava that is of subcardinal status via an intersubcardi -
nal anastomosis. The right supracardinal vein forms much of the post -
renal (caudal) segment of the inferior vena cava. The left supracardinal
vein disappears entirely. The right azygos line persists in its thoracic part
to form all but the terminal part of the azygos vein. Its lumbar part can
usually be identified as a small vessel that leaves the vena azygos on the
body of the twelfth thoracic vertebra and descends on the vertebral
column, deep to the right crus of the diaphragm, to join the posterior
aspect of the inferior vena cava at the upper end of its postrenal segment.
The left azygos line forms the hemiazygos veins. The subcentral veins
give rise to the retro-aortic parts of the left lumbar veins and of the
hemiazygos veins (see Fig. 13.4).
LYMPHATIC VESSELS
The earliest lymphatic vessels arise from budding of lymphatic endothe -
lial cells from the cardinal veins to form lymph sacs (Eichmann et al
2005). Six early lymph sacs can be identified; two are paired (the jugular and the posterior lymph sacs) and two are unpaired (the retroperitoneal
sac and the cisterna chyli). In lower mammals, an additional pair (sub -
clavian) is present, but in the human embryo, these are merely exten -
sions of the jugular sacs.
The jugular lymph sac is the first to appear, at the junction of the
subclavian vein with the precardinal vein, with later prolongations
along the internal and external jugular veins. The posterior lymph sac
encircles the left common iliac vein. The retroperitoneal sac appears in
the root of the mesentery near the suprarenal glands. The cisterna chyli
appears opposite the third and fourth lumbar vertebrae ( Fig. 13.5). The
lymph vessels bud out from the lymph sacs along lines that correspond
more or less closely with the course of embryonic blood vessels (most commonly, veins); many also arise de novo in the mesenchyme and
establish connections with existing vessels. In the body wall and the wall of the intestine, the deeper plexuses are the first to be developed; the vessels in the superficial layers are gradually formed by continued growth. Lymph vessel expansion appears to be influenced by local
Fig. 13.5 Relative positions of the primary lymph sacs (as originally
determined by Sabin (1912) ). Internal jugular vein
External jugular vein
Left common cardinal vein
Left postcardinal vein
Left suprarenal vein
Left renal veinRetroperitoneal lymph sacLeft brachiocephalic vein
Left suprarenal vein
Left renal veinRetroperitoneal lymph sac
Posterior lymph sacCisterna chyliPostrenal part of
inferior vena cavaPrerenal part of
inferior vena cavaSuperior vena cavaRight brachiocephalic veinJugular lymph sac | 295 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Early embryonic circulation
204.e1
CHAPTER 13Lymphatic endothelial cells in lymphatic capillaries display a dis -
continuous basal lamina and basement membrane. They are held by
anchoring filaments to the surrounding extracellular matrix. Their dif -
ferentiation in the mouse starts with expression of Sox-18 and Prox-1, a Prospero homeobox transcription factor, in a subset of cardinal veins
(Park et al 201 1, Zhou et al 2010). Prox-1 is the most specific and func -
tional lymphatic marker: its disruption leads to failure of lymphatic vessel development. Abnormal or absent lymphatic development is also
seen with lack of vascular endothelial growth factor 3 (VEGFR3) and
lack of angiopoietin 1, respectively (Yamashita 2007). Abnormalities of
development and of remodelling of primary lymphatic vessels also
occur if Akt, a serine/theonine protein kinase, is absent, possibly due
to insufficient recruitment of smooth muscle cells to larger lymphatic
vessels (Zhou et al 2010). | 296 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
EARly EmbRyoni C CiRCulATion
204.e2
SECT ion 2REFERENCES
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notch and BMP shaping waves. Trends Genet 29:140–9.
Bellini C, Boccardo F, Bonioli E et al 2006 Lymphodynamics in the fetus
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Boon LM, Ballieux F, Vikkula M 201 1 Pathogenesis of vascular anomalies.
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Brouillard P, Vikkula M 2007 Genetic causes of vascular malformations.
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cular development. In: Feige JJ, Pagès G, Soncin F (eds) Molecular
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Eichmann A, Yuan Li, Moyon D et al 2005 Vascular development: from
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weeks gestation in foetuses with Trisomy 21: an incredible medical
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Planas-Paz L, Strilić B, Goedecke A et al 2012 Mechanoinduction of lymph
vessel expansion. EMBO J 31:788–804.
Sabin FR 1912 On the origin of the abdominal lymphatics in mammals
from the vena cava and the renal glands. Anat Rec 6: 335–42.
Senger DR, Davis GE 201 1 Angiogenesis. Cold Spring Harb Perspect Biol
3:1–19.
Tatin F, Makinen T 2014 Lymphatic vascular morphogenesis. In: Feige JJ,
Pagès G, Soncin F (eds) Molecular Mechanisms of Angiogenesis. From
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pp. 25–44.
Tonar Z, Kocova J, Liska V et al 2001 Early development of the jugular lym -
phatics. Sb Lek 102:217–25.
Yamashita JK 2007 Differentiation of arterial, venous and lymphatic
endothelial cells from vascular progenitors. Trends Cardiovasc Med 17:59–63.
Zhou F, Chang Z, Zhang L et al 2010 Akt/protein kinase B is required for
lymphatic network formation, remodelling and valve development. Am J Pathol 177:2124–33. | 297 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
205
CHAPTER 14 Pre- and postnatal development
related to early embryonic stages, and once the number of somites is
too great to count with accuracy, the degree of development of the
pharyngeal arches is often used. External staging becomes more obvious
when the limb buds appear. The upper limb bud is clearly visible at
stage 13 and, by stage 16, the acquisition of a distal paddle on the upper
limb bud is characteristic. At stage 18, the lower limb bud now has a
distal paddle, whereas the upper limb bud has digit rays that are begin -
ning to separate. By stage 23, the embryo has a head that is almost erect and rounded, and eyelids are beginning to form. The limbs look far
more in proportion and fingers and toes are separate. At this stage, the
external genitalia are well developed, although they may not be suffi -
ciently developed for the accurate determination of the sex.
Historically, the onset of bone marrow formation in the humerus
was used by Streeter to indicate the end of the embryonic and the begin -
ning of the fetal period of prenatal life. The fetal period occupies the
remainder of intrauterine life; growth is accentuated, although differ -
entiative processes continue up to and beyond birth. Overall, the fetus
increases in length from 30 mm to 500 mm, and increases in weight
from 2–3 g to more than 3000 g.
Fetal staging
Currently, there is no satisfactory system of morphological staging of the fetal period of development, and the terminology used to describe
this time period reflects this confusion. The terms ‘gestation’, ‘gesta -
tional age’ and ‘gestational weeks’ are considered ambiguous by O’Rahilly and Müller (2000), who recommend that they should be
avoided. However, they are widely used colloquially within obstetric
practice. Staging of fetal development and growth is based on an esti -
mate of the duration of a pregnancy. Whereas development of a human
from fertilization to full term averages 266 days, or 9.5 lunar months
(28 day units), the start of pregnancy is traditionally determined clini -
cally by counting days from the last menstrual period; estimated in this
manner, pregnancy averages 280 days, or 10 lunar months (40 weeks).
Figure 14.3 shows the embryonic timescale used in all descriptions of
embryonic development and the obstetric timescale used to gauge the
stage of pregnancy.
The predicted date of full term and delivery is revised after routine
ultrasound examination of the fetus. Early ultrasound estimation of
gestation increases the rate of reported preterm delivery (delivery at
<37 weeks) compared with estimation based on the date of the last
menstrual period (Yang et al 2002), possibly because delayed ovulation
is more frequent than early ovulation; the predicted age of a fetus esti-
mated from the date of the last menstrual period may differ by more
than 2 weeks from estimates of postfertilization days.
Obstetric staging
In obstetric practice, the duration of the period of gestation is regarded
as 9 calendar months, which is approximately 270 days. The period of
pregnancy is divided into thirds, termed trimesters. The first and second
trimesters each cover a period of 12 weeks, and the third trimester covers
the period from 24 weeks to delivery. Although the expected date of
delivery is computed at 40 weeks of pregnancy, the term of the preg -
nancy, i.e. its completion resulting in delivery, is considered normal
between 37 and 42 weeks. Neonates delivered before 37 weeks are called
preterm (or premature); those delivered after 42 weeks are post-term.
The period from the end of week 24 and up to 7 days after birth is
termed the perinatal period. Fetuses that are delivered and die before
24 weeks are considered to be miscarriages of pregnancy, although tech -
nological advances in neonatal care can now assist the delivery and
support of infants younger than 24 weeks. Infants born after 24 weeks
of pregnancy who subsequently die are classed as stillborn and contrib-
ute to the statistics of perinatal mortality. Studies that discuss fetal PRENATAL STAGES
The absolute size of an embryo or fetus does not afford a reliable indi -
cation of either its chronological age or the stage of structural organiza -
tion, even though graphs based on large numbers of observations have
been constructed to provide averages. All such data suffer from the dif -
ficulty of timing the moment of conception in humans. It has long been customary to compute the age, whether in a normal birth or an
abortion, from the first day of the last menstrual period of the mother
but, as ovulation usually occurs near the fourteenth day of a menstrual
cycle, this ‘menstrual age’ is an overestimate of about 2 weeks. Where a
single coitus can be held to be responsible for conception, a ‘coital age’ can be established, and the ‘fertilization age’ cannot be much less than
this because of the limited viability of both gametes. It is usually held
that the difference may be several days, which is a highly significant
interval in the earlier stages of embryonic development. Even if the
time of ovulation and coitus were known in instances of spontaneous
abortion, not only would some uncertainty still persist with regard to
the time of fertilization, but also there would remain an indefinable
period between the cessation of development and the actual recovery
of the conceptus.
To overcome these difficulties, early embryos have been graded or
classified into developmental stages or ‘horizons’, on the basis of both
internal and external features. The study of the Carnegie collection of
embryos by Streeter (1942, 1945, 1948), and the continuation of this
work by O’Rahilly and Müller (1987), provided, and continues to
provide, a sound foundation for embryonic study and a means of com -
paring stages of human development with those of the animals rou -
tinely used for experimental study, namely: the chick, mouse and rat.
Recent use of ultrasound for the examination of human embryos and
fetuses in utero has confirmed much of the staging data.
The development of a human from fertilization to birth is divided
into two periods: embryonic and fetal. The embryonic period has been
defined by Streeter as 8 weeks post fertilization, or 56 days. This time -
scale is divided into 23 Carnegie stages, a term introduced by O’Rahilly
and Müller (1987) to replace developmental ‘horizons’ . The designation
of stage is based on external and internal morphological criteria and
not on length or age.
Embryonic staging
Embryonic stages 1–10 are shown in detail in Figure 8.1. Estimations
of embryonic length may be 1–5 mm less than equivalent in vivo esti-
mates, reflecting the shrinkage caused by the fixation procedures that
are inevitably used in embryological studies. O’Rahilly and Müller
(2000, 2010) have revised some of the ages that were previously assigned to early embryonic stages, pointing out that interembryonic variation
may be greater than had been thought and that, consequently, some
ages may have been underestimated. They note that, as a guide, the age
of an embryo can reasonably be estimated from the embryonic length
within the range 3–30 mm, by adding 29 to the length. Correlating the
age of any stage of development to an approximate day may be unreli -
able, and a generalization using the number of weeks of development
might now be more appropriate.
The stages of development encompass all aspects of internal and
external morphogenetic change that occur within the embryo within
the duration of the stage. They are used to convey a snapshot of the
status of the development of all body systems within a particular time -
frame. Figure 14.1 shows the external appearance of embryos from
stage 6 to stage 23, with details of their size and age in days. The correl -
ation of external appearance of the embryo with internal development is shown in Figure 14.2.
Obvious external features provide some guidance to the changes
occurring within embryos during successive stages. Somite number is | 298 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Pre- and Postnatal develo Pment
206seCtIon 2
defects, lung immaturity, renal agenesis, polyhydramnios and defects of
the anterior abdominal wall (gastroschisis and exomphalos). Estima-
tions are made of the following: biparietal skull diameter, taken through
a plane of section that traverses the third ventricle and thalami; head
circumference through a plane that traverses the third ventricle and
thalami plus the cavum septi pellucidi anteriorly and the tentorial
hiatus posteriorly; abdominal circumference through a plane where the
transverse diameter of the liver is greatest, the appearance of the lower
ribs is symmetrical and the junction of the left and right portal veins is
identified; and femoral length, which measures the ossified portions of
the diaphysis and metaphysis ( Fig. 14.4).
Between 15 and 28 weeks, the biparietal diameter is the most accu -
rate index of fetal menstrual age and the expected date of delivery. Other measurements that may also be taken include transverse cerebellar
diameter and foot length. The amount and type of fetal movement,
breathing movements and visceral functions, exemplified by bladder
emptying, peristaltic action and colonic echogenicity, are noted. Three-
and four-dimensional ultrasound scans are now routinely constructed
(Video 14.1).
Gender can be identified later in development but this information
is not always routinely passed on to parents.
Constructions of ultrasound biometry charts for fetal aging now take
into account the ethnic population under consideration; it is recom -
mended that locally developed charts specific to the population should be used. The use of these charts means that factors that may influence
fetal biometry, including maternal age and nutritional status, maternal
height, weight, parity and smoking habits, are noted, facilitating accu -
rate prediction of small-for-date and growth-retarded fetuses. The World
Health Organization (WHO) has published a protocol for the collec-
tion of data for a multicentre study of fetal growth (Merialdi et al 2014).
INTERGROWTH-21st, an international, multicentre project, also aims
to standardize protocols for collecting anthropometric measurements and constructing charts describing optimal fetal and preterm postnatal
growth (Sarris et al 2013, Uauy et al 2013).
An outcome of routine ultrasound examination of embryos and
fetuses for anomalies may be a change to the perinatal management,
development and the gestational age of neonates, particularly those
born before 40 weeks’ gestation, use the obstetric estimated stages and
age (menstrual age), unless they specifically correct for this. If a fetal
ageing system is used, it is important to remember that the age of the
fetus may be 2 weeks more than a comparable fetus that has been aged
from postovulatory days.
Ultrasound staging
The difficulty of correlating the appearance of a chorionic sac, embryo or fetus on an ultrasound scan with age during the first trimester is
related to the specificity of reporting the age. An age reported as within
a week of pregnancy, e.g. week 12, will cover a period of 6 days (12 weeks
0 days up to 12 weeks 6 days). Therefore, it is recommended that sono -
graphic estimation of age should be given as menstrual weeks and days
(i.e. 12 weeks indicates 12 weeks 0 days) (Galan et al 2008).
First trimester scan An early ultrasound scan will detect implanta-
tion and viability of the embryo once a heart beat is detected, confirm multiple pregnancy and estimate the date of delivery. Cardiac activity
can be identified by the sixth menstrual week (Galan et al 2008). Crown–
rump length measurement is the most accurate predictor of menstrual
age of the embryo during the first trimester (Galan et al 2008). Nuchal
translucency is measured between 10 and 14 weeks to diagnose trisomy
21. The development of three-dimensional ultrasound scanning has
enabled early detection of many anomalies previously diagnosed in the
second trimester, including anencephaly, hydrocephalus and encepha -
locele (Pretorius et al 2014). Normograms of fetal spine growth have
been constructed for a Taiwan population, which show mean spine
length increasing linearly between 1 1 and 14 weeks (Cheng et al 2010).
The study also noted a tendency for spinal extension as early as 1 1 weeks.
Second trimester scan Routine scanning at 18–20 weeks (obstetric
staging) is used to confirm the delivery date, and assess not only the
position of the placenta but also the presence of fetal anomalies that would require special intervention following delivery, such as cardiac Fig. 14.1 The external appearance and size of embryos between stages 6 and 23. Early in development, external features are used to describe the
stage, e.g. somites, pharyngeal arches or limb buds. (Adapted with permission from Rodeck CH, Whittle MJ 1999 Fetal Medicine. London: Churchill
Livingstone.)Size (mm) 0.4 1.5–36 10
Approximate age
(days)16–18 26–294–613
30–338–1116
35–4013–1718
41–4521–2320
46–5028–3023
53–58Embryonic stageStage 18 Stage 16 Stage 13Eyelids Pinna Mandibular
processMaxillaryprocess
Opticvesicle
Otic vesicleFirst pharyngeal arch Neurulation
Somites Cerebralhemisphere
Connectingstalk Connectingstalk
ConnectingstalkYolksac
Reflection of amnion
Epiblast population
Primitive node
Primitive streakConnecting stalkPericardial
cavityand heart
Lower limbbudUpper limbbudHand
Umbilicalcord
Stage 10
Stage 6 | 299 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Prenatal stages
207
CHaPter 14
Fig. 14.3 The two timescales used to depict human development. Embryonic development, in the upper scale, is counted from fertilization (or from
ovulation, i.e. in postovulatory days; see O’Rahilly and Müller (1987)). Throughout this book, times given for development are based on this scale. The
clinical estimation of pregnancy is counted from the last menstrual period and is shown on the lower scale; throughout this book, fetal ages relating to
neonatal anatomy and growth will have been derived from the lower scale. Note that there is a 2-week discrepancy between these scales. The perinatal period is very long because it includes all preterm deliveries. Embryonic
stagesImplantation period Early neonatal period (birth–7 days)Late neonatal period (7–28 days)
Fetal period Perinatal period
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
1
Implantation
Last menstruation Estimated date of deliveryFertilization2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 41
1 2 3 4 5 6 7 8 937 38 39 40
10Age of embryo (weeks)
Age of embryo (months)
Pregnancy (weeks)
Pregnancy (lunar months)
First trimester Second trimester Third trimester (of pregnancy)Fig. 14.2 A timetable of development of the body systems. The development of individual systems can be seen progressing from left to right. Embryonic
stages and weeks of development are shown. Embryonic stages are associated with external and internal morphological features rather than embryonic
length. To identify the systems and organs at risk at any time of development, follow a vertical progression from top to bottom. Embryonic stages
Weeks post ovulation
External appearance
Nervous
Respiratory
Gastrointestinal
Urinary
Reproductive
Cardiovascular
MusculoskeletalHead and tail folding
Neurulation
Fore-, mid-,
hindgutMidgut loop returns to abdomenPharyngeal pouches dorsal and ventral
PancreasThyroid
Liver Midgut loop rotating
Urorectal septum Rotation of stomachTrachea
Lung buds Further division of bronchi
Primary bronchi
Mesonephros
Mesonephric duct
Ureteric bud
Germ cells in allantois wall
Primitive vascular systemSeptum primum
Somite period Cartilaginous
part of skullMembranous part of skull
Forelimb bud Forelimb digit rays
Hindlimb bud20 days .................................. 30 daysSeptation of ventricles
Heart beats Spleen Septum secundum
Heart tubeMüllerian ducts Uterus and uterine tubes Testis at inguinal canal
Indifferent gonad Testis differentiating Vagina Prostate
External genitalia indifferent External genitalia differentiatingMetanephric nephrons Kidneys ascend
Major calyces Minor calycesAnterior lobe pituitaryPosterior lobe pituitaryFirst neural crest cellsOtic vesicle
Optic cup Membranous labyrinthPharyngeal arches Palate External earUpper lip Digits on hand Eyelids fuse
1 2 3 4 5 6 7 8 9 10 11 127 6 8 9 10 11 12 13 14 1516 23 17 22 21 18 19 20 | 300 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Pre- and Postnatal develo Pment
208seCtIon 2
Fig. 14.4 Ultrasound planes examined in a routine second-trimester antenatal scan (left) and measurements taken to predict the estimated date of
delivery (right). The top three images are in the sagittal plane ( A). The other planes are transverse, apart from J, which is longitudinal. Abbreviations:
Ao, aorta; DA, ductus arteriosus; DV, ductus venosus; LA, left atrium; LPV, left portal vein; LV, left ventricle; PA, pulmonary artery; RA, right atrium;
RPV, right portal vein; RV, right ventricle; SVC, superior vena cava.
Nuchal translucency (A)
Transventricular (B)
Heart - three-vessel view (E)
Cord insertion (H) Bladder (I)Heart - four-chamber view (F)Transcerebellar (D)A
B
C
D
E
F
G
H
I
JLongitudinal spine (A)
Aortic arch (A)
Transthalamic – head circumference (C)Transthalamic – biparietal diameter (C)
Abdominal circumference (G)
Femur length (J)Ultrasound planes examined in 20-week scan
20-week scan biometry for
delivery date estimation
Posterior
ventricle
Cerebellum
BladderCisternamagna
MidlineMidline
Thalami
Midline
ThalamiMidline
CordinsertionCordinsertionStomachRPVLPV
DV
BowelRA
LALVRVPA
DA
Spine
Spine
Spine
SpineAo
SVC
Spine | 301 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Growth in utero
209
CHaPter 14still disproportionately large. (Although the rates of growth of the trunk
and lower limbs increase during the remainder of intrauterine life, the
disproportion is present after birth and, to a diminishing degree, is
retained throughout childhood and on into the years of puberty.) A
covering of primary hair, lanugo, appears. Towards the end of this
period, sebaceous glands become active; the sebum that is secreted
blends with desquamated epidermal cells to form a cheesy covering
over the skin, the vernix caseosa, which is usually considered to protect
the skin from maceration by the amniotic fluid. At about this time, the
mother becomes conscious of fetal movement, formerly termed
‘quickening’ .
In the sixth month, the lanugo darkens, the vernix caseosa is more
abundant and the skin becomes markedly wrinkled. The eyelids and
eyebrows are now well developed. During the seventh month, the hair
of the scalp is lengthening and the eyebrow hairs and the eyelashes are
well developed. The eyelids themselves separate and the pupillary mem -
brane disappears. The body becomes more plump and rounded in contour and the skin loses its wrinkled appearance as a result of the
increased deposition of subcutaneous fat. Fetal length has increased to
approximately 350 mm and weight to about 1.5 kg. Towards the end
of this month, the fetus is viable; if born prematurely, it may be able
to survive without the technological assistance found in neonatal inten -
sive care units and its postnatal development can proceed normally.
Throughout the remaining lunar months of normal gestation, the
covering of vernix caseosa is prominent. There is a progressive loss of lanugo, except for the hairs on the eyelids, eyebrows and scalp. The
bodily shape becomes more infantile but, despite some acceleration in
their growth, the legs have not quite equalled the arms in length pro -
portionately, even at the time of birth. The thorax broadens relative to the head, and the infra-umbilical abdominal wall shows a relative
increase in area, so that the umbilicus gradually becomes more centrally
situated. Average lengths and weights for the eighth, ninth and tenth
months are 40, 45 and 50 cm and 2, 2.5 and 3–3.5 kg, respectively. The
rate of fetal growth slows from 36 to 40 weeks in response to the physi -
cal limitation imposed by the maternal uterus. Birth weight thus reflects
the maternal environment more than the genotype of the child. This
slowing of the growth rate enables a genetically larger child developing
within a small mother to be delivered successfully. After birth, the
growth rate of the neonate increases; the rate of weight gain reaches a
peak some 2 months postnatally.
Just before birth, the lanugo almost disappears and the umbilicus is
central. The testes, which begin to descend with the processus vaginalis
of peritoneum during the seventh month and are approaching the
scrotum in the ninth month, are usually scrotal in position. The ovaries
are not yet in their final position at birth; although they have attained
their final relationship to the uterine folds, they are still above the level
of the pelvic brim.
Fetal surgery
The use of routine ultrasound examination and MRI of fetuses has led
to the development of a number of prenatal surgical interventions: e.g.
to correct placental or membrane anomalies resulting in twin-to-twin
transfusion and the production of amniotic bands (Deprest et al 2014);
or to offer improved outcomes for meningomyelocele on the basis that
the neural tissue may become secondarily damaged due to exposure to
amniotic fluid and mechanical traumatic injury during gestation
(Adzick 2013, Danzer and Johnson 2014, Cohen et al 2014).
GROWTH IN UTERO
Alterations in the availability of nutrients to the fetus at particular stages of pregnancy elicit adaptive responses by the fetus. These may ensure
fetal coping but may also result in pathology in adult life; the nutri -
tional status of pregnant females is therefore of fundamental impor -
tance for the health of the next generation. Under- or overnutrition
during fetal life may lead to metabolic changes in childhood, adoles -
cence and adulthood, and pass to successive generations. Poor nutrition at critical stages of fetal life permanently alters the normal developmen -
tal pattern of a range of organs and tissues, e.g. the endocrine pancreas,
liver and blood vessels, resulting in their pathological responses to
certain conditions in later adult life (Barker et al 1993, Catalano and
Hauguel-De Mouzon 201 1). Maternal hyperglycaemia leads to changes in fetal metabolism and pathology in the child and adult (Pedersen 1952).
An increase in placental size occurs in pregnancy as an adaptive
response to both high altitude and mild undernutrition, particularly
i.e. to the time, method and place of delivery of the fetus, or the parents
may choose to terminate the pregnancy to minimize concerns about
fetal and neonatal suffering and long-term disability. Termination may
be chosen for severe, untreatable inherited metabolic disorders (e.g.
Tay–Sachs disease), severe chromosomal anomalies (e.g. trisomy 13),
lethal bone dysplasias, lethal anomalies such as anencephaly and other
extreme neurological defects, and bilateral renal agenesis (Cass 201 1).
Improvements in prenatal screening and diagnosis have led to an
overall increase in the prevalence of reported birth defects and overall
lower perinatal mortality rates, reflecting increased early terminations
of pregnancy (Cass 201 1).
Magnetic resonance imaging
Advances in prenatal ultrafast magnetic resonance imaging (MRI) now
provide further ways to detect fetal anomalies, capturing particularly
clear images (Sepulveda et al 2012) ( Fig. 14.5). MRI is considered a
useful adjunct to ultrasound imaging at 20–22 weeks because it enables
better management planning for known or suspected anomalies (Reddy
et al 2014).
Fetal development
Although accurate morphological stages are not available for the fetal
period, the developmental progression is broadly clear. During the
fourth and fifth months, the fetus has a head and upper limbs that are
Fig. 14.5 MRI scans of fetal anomalies. A, Polyhydramnios may arise as
a result of oesophageal compression, impaired swallowing and lack of
absorption of amniotic fluid from the gut. B, Gastroschisis with loops of
bowel in the extracellular coelom (chorionic cavity) and normal insertion
of the umbilical cord. (With permission from Sepulveda W, Ximenes R,
Wong AE, et al 2012 Fetal magnetic resonance imaging and three-
dimensional ultrasound in clinical practice: Applications in prenatal diagnosis. Best Pract Res Clin Obstet Gynaecol 26:593–624.)
A
Bowel
Umbilical cord
B | 302 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Pre- and postnatal development
209.e1
CHaPter 14Other conditions have also been treated prenatally with limited
improvement in mortality. Thus, congenital diaphragmatic hernia may
be treated by percutaneous fetoscopic endoluminal tracheal occlusion
from 25 to 33 weeks’ gestation to prevent loss of lung fluid and enhance
lung growth (Deprest et al 2004). Fetal airway patency has also been
preserved during birth through perinatal ex utero intrapartum (EXIT)
treatment, where a portion of the fetus is delivered through a hysterec -
tomy incision for surgery while the fetus remains attached to the utero -
placental circulation. After the airway procedure is completed, the fetus
is completely delivered (Deprest et al 2014). | 303 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Pre- and Postnatal develo Pment
210seCtIon 2the rapid maturation of some systems and the compensatory growth of
others (in terms of responses to the effect of gravity or enteral feeding
or exposure to microorganisms).
Details of the relative positions of the viscera and the skeleton in a
full-term neonate are shown in Figures 14.6–14.8. The newborn infant
is not a miniature adult, and extremely preterm infants are not the same as full-term infants. There are immense differences in the relations of
some structures between the full-term neonate, child and adult, and
there are also major differences between the 20-week-gestation fetus
and the 40-week fetus, just before birth. The study of fetal anatomy at
20, 25, 30 and 35 weeks is vital for the investigative and life-saving
procedures carried out on preterm infants today.
Neonatal measurements and period
of time in utero
The tenth to ninetieth centile ranges for length of full-term neonates
are 48–53 cm ( Fig. 14.9A). Length of the newborn is measured from
crown to heel. In utero, length has been estimated either from crown–
rump length, i.e. the greatest distance between the vertex of the skull and the ischial tuberosities, with the fetus in the natural curved posi -
tion, or from the greatest length exclusive of the lower limbs. Greatest length is independent of fixed points and thus much simpler to measure;
it is generally taken to be the sitting height in postnatal life, and is the
measurement recommended by O’Rahilly and Müller (2000) as the
standard in ultrasound examination.
At birth, weight reflects the maternal environment, the number of
conceptuses, the sex of the baby and the parity of the mother. Generally, full-term female babies are lighter than full-term males, twins are lighter
than singletons, and later children tend to be heavier than the first-born.
The tenth to ninetieth centile ranges for the weight of a full-term infant
at parturition are 2700–3800 g (Fig. 14.9B ), the average being 3400 g;
75–80% of this weight is body water and a further 15–28% is composed of adipose tissue.
Birth weight is noted against charts appropriate for ethnicity and
categorized as low, normal and high. Low birth weight has been defined
as less than 2500 g, very low birth weight as less than 1500 g, and
extremely low birth weight as less than 1000 g. Infants may weigh less
than 2500 g but not be premature by gestational age. Measurement of
the range of weights that fetuses may attain before birth has led to the production of weight charts, which allow babies to be described accord -
ing to how appropriate their birth weight is for their gestational age, e.g. small for gestational age, appropriate for gestational age or large for
gestational age ( Fig. 14.10). Small-for-gestational-age infants, also
termed ‘small-for-dates’, are often the outcome of intrauterine growth retardation.
For both premature and growth-retarded infants, an assessment of
gestational age, which correlates closely with the stage of maturity, is
desirable. Gestational age at birth is predicted by its proximity to the
estimated date of delivery and the results of ultrasonographic examin -
ations during pregnancy. It is currently assessed in the neonate by evalu -
ation of a number of external physical and neuromuscular signs. Scoring
of these signs results in a cumulative score of maturity that is usually
within ±2 weeks of the true age of the infant. The scoring scheme has
been devised and improved over many years.
Estimation of large-for-dates infants is based on assessment of fetal
weight through ultrasound evaluation and some biometrical indices. Assessment of anterior abdominal wall width is thought to predict
large-for-gestational-age babies (Walsh and McAuliffe 2012). Fetal mac -
rosomia is defined as an absolute birth weight greater than 4000 g,
4500 g or 5000 g, or as a customized birth weight centile greater than
the ninetieth, ninety-fifth or ninety-seventh centile for the infant’s gesta -
tion age (Walsh and McAuliffe 2012, Schwartz et al 2014). The precise
definition may not necessarily be helpful, as some at-risk infants, not
identified as large for dates from growth curve charts, might go unrec -
ognized (Larma and Landon 201 1). There is a correlation between macrosomia and short maternal stature; macrosomic fetuses are at risk
of shoulder dystocia and brachial plexus injuries during vaginal
delivery.
GROWTH IN INFANCY AND CHILDHOOD
After birth, there is a general decrease in the total body water but a rela -
tive increase in intracellular fluid. Normally, the newborn loses about
10% of its birth weight by 3–4 days postnatally because of loss of excess
extracellular fluid. By 1 year, total body water makes up 60% of the body
weight.during mid-pregnancy. However, although a larger placenta may be better able to deliver the full nutritional requirements of the fetus, the
perfusion of a larger placenta is not without problems. It may produce
changes in fetal blood flow and placental enzymes, and in the normal
structure of the fetal vessel wall or of its responses to circulating
trophins, e.g. catecholamines or angiotensin II, which will continue
into adult life. Undernutrition in later pregnancy does not produce the
same sequelae and placental enlargement does not occur. However, fetal
growth slows and fetal wasting may occur as oxygen, glucose and amino
acids are redistributed to the placenta to maintain its function.
Transition to extrauterine life
The type of birth confers developmental outcomes on the neonate that affect its subsequent growth. Caesarean section for preterm delivery
appears not to be associated with improved neonatal outcomes but is
associated with increased risk of respiratory distress syndrome due to
slower postnatal movement of fluid out of the lungs (Werner et al 2012,
Bhatta and Keriakos 201 1, Bellini et al 2006). The dramatic rise of this
delivery method is not necessarily driven by improved neonatal out -
comes because caesarean section has been shown to increase the odds
of developing asthma and type 1 diabetes in later life by 20%. Meta-
analyses indicate that the endocrine milieu during caesarean section
may lead to epigenetic effects on hepatic and metabolic function and
so affect immune function in adult life, leading, in some instances, to
food allergy and obesity (Steer and Modi 2009, Hyde and Modi 2012,
Song et al 2013).
Prior to birth, the gut is exposed to hundreds of proteins, metabo -
lites and cytokines that are swallowed in amniotic fluid; there is some evidence for the presence of bacterial species in amniotic fluid prior
to delivery. Establishment of the gut microbiota by exposure to mater -
nal vaginal and colonic bacteria during vaginal delivery leads to normal
maturation of the gut wall. Gut colonization in preterm infants is
delayed (Cilieborg et al 2012). Infants born via caesarean section may
have primary gut flora disturbance for up to 6 months after birth and
associated delay in postnatal immune development (Neu and Rushing
201 1, Neu and Mai 2012, Matamoros et al 2013).
The establishment of enteral feeding has profound effects on early
postnatal maturation. Human breast milk contains many bioactive sub -
stances that enhance gut maturity. It improves gastric emptying, encour -
ages the growth of the microbiotome and contains a range of proteins,
including large amounts of secretory immunoglobulin A and cytokines
(e.g. interleukin (IL)-10) (Jakaitis and Denning 2014). Feeding preterm
infants human milk is associated with less feeding intolerance and is
thought to provide protection against diabetes and obesity in later life
(Valentine and Morrow 2012). The rate of growth of infants fed breast
milk for the first 6 months of postnatal life is different to that of
formula-fed infants. Delivery of low-weight preterm babies, followed
by parenteral feeding with no enteral nutrition, disrupts normal gut
maturation and may lead to necrotizing enterocolitis (Wynn and Neu
2012). However, a balance with the gut microbiome is necessary;
although full-term neonates born in tropical countries have similar
villous height to those born in temperate climates, villous length in the
small intestine shortens within 2–12 months of birth as a consequence
of tropical enteropathy (Ramakrishna et al 2006).
The time of transition to extrauterine life is permanently recorded
in the primary teeth and can be reliably demonstrated in forensic
dental identification. The circadian growth rhythm of dentine and
enamel deposition in tooth germs is temporarily blocked by the meta -
bolic stress of delivery; the resultant change in enamel prism deposi -
tion is seen as a neonatal line or ring that can be detected in all
deciduous teeth and permanent first molar teeth of live births. The
thickness of the line is related to birth difficulties, being thinner in
caesarean section and thicker in vaginal delivery (Sabel et al 2008,
Canturk et al 2014).
NEONATE
The neonatal period extends from birth to 28 days postnatally; it is
divided into an early neonatal period from birth to 7 days, and a late
neonatal period from 7 to 28 days. Technological advances have enabled
successful management of preterm infants, many at ages that were
considered non-viable a decade or two previously. Maturational pro -
cesses involving local interactions and pattern formation still drive
development at local and body-system levels in preterm infants. The sudden release of such fetuses into a gaseous environment of varying temperature, with full gravity and a range of microorganisms, promotes | 304 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Pre- and postnatal development
210.e1
CHaPter 14Animal studies have verified that maternal starvation during preg -
nancy decreases fetal intrauterine growth factor (IGF)-I concentrations
and, together with a general hypoglycaemia, impairs the development
of the fetal β cells of the pancreas. Moreover, fetal undernutrition
induces insulin resistance in the tissues. The coexistence of insulin resistance and impaired β-cell development in the fetus appears to be
important in the pathogenesis of type 2 diabetes. The risk of developing this type of diabetes is greatest in those individuals with low weight at
birth and at 1 year, and who become obese as adults, thus challenging
an already impaired glucose–insulin metabolism. Fetal IGF-I concentra -
tions are also lower in infants who are short at birth as a result of a
long period of maternal undernutrition; these individuals have an exag -
gerated response to growth hormone-releasing factor, which, together with low IGF-I concentrations, suggests a degree of growth hormone
resistance.
It is now thought that the balance of hormonal environment in
intrauterine and early postnatal life is necessary for future adult health.
The presence of altered concentrations of hormones during critical
periods of development may act as endogenous functional teratogens
(Plagemann 2004).
Different birth phenotypes have been correlated with different path -
ological sequelae. Infants who are thin at birth, with a low ponderal
index (weight/length
3), tend to develop a combination of insulin resist -
ance, hypertension, type 2 diabetes and lipid disorders, whereas those who are short in relation to head size tend to develop hypertension and
high plasma fibrinogen concentrations. These associations have been
reported in babies born small for dates, rather than in those born pre -
maturely. Some babies of average weight also develop cardiovascular pathology; they are either thin at birth and small in relation to the size
of their placenta, or of average weight but short in relation to head size
and have below average weight gain during the first year (Barker et al
1993). For more recent views on this concept, the reader is directed to consult Godfrey and Barker (2000), Ross and Beal (2008), and Kel-
ishadi and Poursafa (2014).
Obesity has become increasingly prevalent since the end of the
twentieth century, leading to an increase in type 2 diabetes, hyperten-sion, hyperlipidaemia, atherosclerosis and inflammation in later life. In
pregnancy, an observed decrease in insulin sensitivity as pregnancy
advances leads to increased nutrient transfer to the fetus. In response,
the fetus increases insulin secretion, stimulates IGF-I production, and
fetal growth and fat deposition are promoted (Vrachnis et al 2012).
Obese females start their pregnancy with greater insulin resistance than
females of normal weight, which means that their fetuses produce more
IGF-I and continue to grow. The risk of fetal macrosomia is three times
higher in females with poorly controlled gestational diabetes because
their increased weight reflects fat mass rather than lean body mass
(Catalano and Hauguel-De Mouzon 201 1). Adiposity at birth is related
to obesity and metabolic dysfunction in childhood, which may be
perpetuated through adulthood in an ongoing cycle through the genera -
tions (Catalano and Hauguel-De Mouzon 201 1, Poston 2012). A range
of risk factors for pre-, peri- and postnatal issues in maternal obesity is
given in McGuire et al (2010). | 305 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Growth in infancy and childhood
211
CHaPter 14
Fig. 14.6 A–C, Topographical representations of the anatomy of a full-term neonate. The surface markings of all organs are shown, with some coloured
and others only in outline. The female genital tract is shown on the right of the body, and the male tract is shown on the left.
A B C
From cranial to caudal, the following structures are indicated:
Superior sagittal sinus
Brain Thyroid gland Thymus Liver Gallbladder Spleen Pancreas Blood vessels
Oxygenated blood (red) is returned to the fetal heart via the
umbilical vein, which passes from the umbilicus to the liver .
The right atrium contains oxygenated blood, which mainlypasses to the left atrium. The right ventricle receives some
oxygenated blood from this flow and also the deoxygenated
blood from the head and neck. Blood is returned to theplacenta via two umbilical arteries.Note that the apex of the bladder continues as the urachusto the umbilicus.Note that the lower border of the lung is below the central,upper border of the liver .Note that the suprarenal glands are relatively large andsuperomedial to the lobulated kidneys.From cranial to caudal, the following structures are indicated:
Larynx and trachea
Gastrointestinal tract Pancreas Urinary bladder Prostate glandFrom cranial to caudal, the following structures are indicated:
Maxillae and mandible Costal cartilages Lungs Suprarenal glands Lobulated kidneys Ureter passing to the posterior
surface of the urinary bladder
Right uterine tube, uterus and vagina
Left testis and vas deferens | 306 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Pre- and Postnatal develo Pment
212seCtIon 2
Fig. 14.8 The extent of the ossified skeleton in a full-term neonate. Note
the derivation of the parts of the skeleton: the skull is derived from
paraxial mesenchyme and neural crest mesenchyme; the axial skeleton,
vertebrae and ribs are derived from paraxial mesenchyme; and the
skeletal elements in the limbs are derived from somatopleuric
mesenchyme, which forms the limb buds.
Neural crest mesenchyme
Paraxial mesenchymeSomatopleuric mesenchyme
From cranial to caudal, the following structures are indicated:
Superior sagittal sinus Brain Larynx and trachea Maxillae and mandible Thyroid gland Thymus Sternum Heart Liver Vertebrae Pancreas Blood vessels Gastrointestinal tract Visceral peritoneum Urinary bladder and urethra Uterus and vagina
Fig. 14.7 A topographical representation of the anatomy of a full-term
female neonate. The cut surfaces of the organs are the same colours as
in Figure 14.6. | 307 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Growth in infancy and childhood
213
CHaPter 14
Growth has always been regarded as a regular process. The rates of
prenatal and postnatal growth can be indicated by increments in body
length or weight, which, when plotted, form a growth curve (see Fig.
14.9). Growth curves can be plotted for individuals if accurate measure -
ments are taken, preferably by the same person, for the entire period
of growth, i.e. a longitudinal study. An alternative method is to collect
a series of averages for each year of age obtained from different indi -
viduals, i.e. a cross-sectional study. Cross-sectional studies are valuable for the construction of standards for height and weight attained by
healthy children at specific ages, and can establish centile limits of
normal growth, but they cannot reveal individual differences in either
the rate of growth or the timing of particular phases of growth.
The data from longitudinal and cross-sectional studies can also be
used to plot the increments in height or weight from one age to the
next. This produces a velocity curve, which reflects a child’s state at any
particular time much better than the growth curve, in which each point
is dependent on the preceding one. The oldest published longitudinal
study, still of great value today, was made by Count Philibert de Mont -
beillard on his son ( Fig. 14.1 1). It shows that the velocity of growth in
height decreases from birth onwards, and that a marked acceleration of
growth, the adolescent growth spurt, occurs from 13 to 15 years (see
below; see Fig. 14.14).
Cross-sectional data permit comparison of prenatal and postnatal
growth. Childhood growth charts are used to predict normal childhood development. The velocity curve for the prenatal and postnatal period
(Fig. 14.12) shows that the peak velocity for length is reached at about
4 months (note that these prenatal charts use the obstetric measure -
ments of gestational time, in which fetal age is estimated from the last
menstrual period, 2 weeks before fertilization). Growth in weight
usually reaches its peak velocity after birth.
Tanner (in Harrison et al (1964)) noted that the more carefully
measurements are taken, the more regular is the succession of points
on a growth curve. However, a longitudinal study of growth measured
weekly, semi-weekly and daily recorded that growth in length and head
circumference occurred by saltatory increments, with a mean amplitude
of 1.01 cm for length (Lampl 2002); growth stasis, steep changes in
growth and incremental growth have all been recorded in infancy, child -
hood and adolescence (Caino et al 2006).
Charts of height and weight correlated to age are compiled from
extensive cross-sectional growth studies. Such charts show the mean
height or weight attained at each age, termed the fiftieth centile, and also the centile lines for the seventy-fifth, ninetieth and ninety-seventh centiles, in addition to the twenty-fifth, ninth and second centiles. The data shown in Figure 14.13 are derived from United Kingdom cross-
sectional references. Any comparison of an individual growth curve Growth rates of infants born prematurely differ from those born
naturally at term. Normally, fetuses grow rapidly between 20 and
40 weeks. Optimum growth of premature infants is considered to be
equivalent to intrauterine rates. However, whereas, in utero, fetuses show
a slowing growth velocity before term, preterm infants show linear growth. Growth charts for preterm infants have been constructed (Fenton and Kim 2013). The INTERGROWTH-21st project is collecting data for preterm growth (see above).Fig. 14.9 Standardized graphs of ( A) fetal length and ( B) weight from 24 weeks of pregnancy, showing the tenth, fiftieth and ninetieth centiles. 4600
4400
42004000380036003400320030002800260024002200200018001600140012001000
800600400200
24 26 28 30
Preterm Term Post-term32 34 36 38 40 4290%
50%
10%
0
Week of gestationB
Weight (g)53
52
515049484746454443424140393837363534333231
24 26 28 30
Preterm Term Post-term32 34 36 38 40 4290%
50%
10%
Week of gestationA
Length (cm)
Fig. 14.10 Intrauterine growth status and its appropriateness for
gestational age. Gestational age is more closely related to maturity than
birth weight. The mortality for the weight ranges is indicated. 25–50% mortality4600
440042004000380036003400320030002800260024002200200018001600140012001000
800600400200
24 26 28 30
Preterm Term Post-term32 34 36 38 40 4290%
50%
10%
0
Week of gestationWeight (g)
More than 50% mortalityLess than 4% mortality
Appropriate for
gestational age
Small for
gestational age4–25%
mortalityLarge for
gestational age | 308 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Pre- and Postnatal develo Pment
214seCtIon 2
with these data must also take into account ethnicity and the nutritional
and family history of that individual.
Plotting the growth of children on such charts provides guidelines
for the prediction of normal growth and indicates when investigation
of possible growth anomalies should occur. Children who grow in an
environment that does not constrain their growth exhibit a pattern of
growth that is mainly parallel to a particular centile, a phenomenon
that has been termed homeorhesis or canalization (following the same
imaginary ‘canal’ on the growth chart). After deviation from this centile
as a consequence of the adolescent growth spurt, most children return
to the same centile position in adulthood, a finding that suggests that
this pattern is genetically determined within individuals (Cameron
2002). For a comprehensive account of all aspects of postnatal growth,
the reader is directed to consult Human Growth and Development
(Cameron 2002).
The WHO published growth charts for birth to 5 years and 5–19 years
(2006). The charts were derived from children considered to be growing
under optimal environmental conditions: they were breastfed, raised in
environments that minimized poor diets and infection, and had non-smoking mothers. Similar growth charts have been published by WHO
for specified regions (Mansourian et al 2012).
ADOLESCENT GROWTH SPURT AND ADULT SIZE
Growth charts reveal that body length increases from a neonatal range
of 48–53 cm to about 75 cm during the first year after birth, and
increases by 12–13 cm in the second year. Thereafter, 5–6 cm is added
each year. In individual longitudinal growth curves, an increase in the
velocity of growth occurs between 10.5 and 1 1 years in girls, and 12.5
and 13 years in boys. This rapid increase in growth is the adolescent
growth spurt (Fig. 14.14; see Fig. 14.13). In both sexes, it lasts for
2–2.5 years. Girls gain about 16 cm in height during the spurt, with a
peak velocity at 12 years of age. Boys gain about 20 cm in height (mostly
by growth of the trunk), with a peak velocity at 14 years of age, during
which time they may be growing at the rate of 10 cm a year.
Humans seem to be the only species to have a long quiescent interval
between the rapid growth that takes place immediately after birth and
the adolescent growth spurt. This type of growth has been termed the
Infancy/Childhood/Puberty (ICP) growth model (Stevens et al 2013).
It has been suggested that the quiescent interval allows the brain to
mature, and complex social learning to take place, before individuals
pass through puberty and become sexually active.
Apart from the expected change in growth velocity that occurs during
puberty, it is now apparent that taller child height is prospectively
associated with elevated risk of obesity (Johnson et al 2012, Van Dom -
melen 2014). Growth in height continues at a slower rate after the
adolescent growth spurt. Noticeable growth is said to stop at 18 years
in females and 20 years in males; longitudinal studies have indicated
an average figure of 16.25 years (females) and 17.75 years (males) with
a normal variation of ±2 years (Harrison et al 1964). After this time, any
increments that occur as a result of appositional growth at the cranial
and caudal ends of the vertebral bodies and intervening intervertebral Fig. 14.11 A longitudinal study of growth. A, The height of de Montbeillard’s son (1759–1777) from birth to 18 years. B, A growth velocity curve, plotting
increments in height from year to year. (After D’Arcy Thompson, On Growth and Form, 1942, Cambridge University Press.)Height (cm)
Age (years)A B
de Montbeillard’s son
1759–1777
60100
80120160
140180200 24
22
201816141210
86420
B 2 4 6 8 10 12 14 16 18
Height gain (cm per year)
Age (years)de Montbeillard’s son
1759–1777
B 2 4 6 8 10 12 14 16 18
Fig. 14.12 Cross-sectional data showing growth. A, Growth in length in
the prenatal and early postnatal periods. B, The corresponding velocity
curve for this period. (After D’Arcy Thompson, On Growth and Form,
1942, Cambridge University Press.)cm
Birth
Months020
103050
40607080
2 4 6 8 10 12 14 16 18 20 22cm per month
Birth
Months04
2610
812
2 4 6 8 10 12 14 16 18 20 22A
B | 309 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
adolescent growth spurt and adult size
215
CHaPter 14
Fig. 14.13 Standard growth charts of British boys and girls showing the ninetieth, fiftieth and tenth centiles. (© Child Growth Foundation.)63
57514587
817569
39
33272115
93
0
0 2 4 6 8 10 12 14 16 1890%
50%
10%
Age (years) BoysWeight (kg)150
140130120190
180170160
110
100
9080706050
0 2 4 6 8 10 12 14 16 1890%
50%
10%
Age (years) BoysHeight (cm)
63
57514587
817569
39
33272115
93
0
0 2 4 6 8 10 12 14 16 1890%
50%
10%
Age (years) GirlsWeight (kg)150
140130120190
180170160
110
100
9080706050
0 2 4 6 8 10 12 14 16 1890%
50%
10%
Age (years) GirlsHeight (cm)
discs are so small as to be difficult to measure. There is a loss of height
after middle age.
Development-related gene expression correlates with three phases of
human growth (infancy/early childhood; childhood/puberty; final
height); the expression of clusters of growth-related, evolutionarily con -
served genes varies in a development-dependent manner in human
tissues (Stevens et al 2013). The outcomes of the Human Genome
Project have been linked to anthropomorphic traits in meta-analyses of
genome-wide association data that have identified genetic variants at hundreds of loci relevant to growth in pre-puberty, puberty and adult -
hood, particularly those associated with childhood and adult obesity (Genome-wide Investigation of ANThropometric measures – GIANT). A genome-wide association study has shown genetic loci linking pubertal timing, height and growth with childhood obesity (Cousminer
et al 2013).
The phenomenal growth rates of adolescence are most obvious in
the increase in height. Weight gain is more variable. The birth weight
is normally tripled by the end of the first year, and quadrupled by the
end of the second year. Thereafter, weight increases by 2.25–2.75 kg
annually until the adolescent growth spurt, when boys may add 20 kg
to their weight and girls 16 kg. The peak velocity for weight gain lags
behind the peak velocity for height by about 3 months. Body weight
does not reach adult values until some time after adult height is
attained. In recent years, the global estimates of the numbers of adults who are overweight or obese in adult life has exceeded the numbers who do not have sufficient food (Mascie-Taylor and Goto 2007). | 310 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Pre- and Postnatal develo Pment
216seCtIon 2
all organs and systems with time; it does not occur in the developing
embryo, which displays differential rates of growth. Allometric growth
describes the differences in the relative rates of growth between one part
of the body and another, and is most clearly seen in the changes in
body proportion between fetuses, neonates, children and adults.
Between 6 and 7 weeks after fertilization, the head is nearly one-half of
the total embryonic length. It subsequently grows proportionally more
slowly and, at birth, it is one-quarter of the entire length. During child -
hood, this pattern of growth continues with lengthening of the torso and limbs until, in adults, the head is one-eighth of the total length (Fig. 14.16).
Growth of the liver, spleen, kidneys, skeletal and muscular tissues
generally follows pre- and postnatal growth curves given for the INTEGRATION OF TYPES OF GROWTH DURING
DEVELOPMENT AND LIFE
In the later prenatal months and in the postnatal period, the various types of growth occur in differing patterns. The extent of tissue growth in organs depends on the specific duration of multiplicative growth for the cell types ( Fig. 14.15). Different cell populations complete their
initial developmental proliferation and become differentiated at differ -
ent times; the final stage of differentiation is usually the cessation of cell division.
Growth of a body can be described in two ways: isometric and allo-
metric. Isometric growth implies a progressive proportional increase in Fig. 14.14 Typical individual velocity curves for height: British boys and
girls. (After Tanner JM, Whitehouse RH, Takaishi IM 1966 Standards from
birth to maturity for height, weight, height velocity and weight velocity.
Arch Dis Child 41:454–71, with permission from BMJ Publishing.)
Height gain (cm per year)
Girls Boys
Age (years)04
2610
81216
141822
2024
2 4 6 8 10 12 14 16 18
Fig. 14.15 The duration of multiplicative growth for various human
tissues. Prenatal months Postnatal years
Duration of hyperplasia1 90 60 30 10 9 6 3 1 9 3 6Determinate tissues
(number fixed near birth)Indeterminate tissues
(number not fixed near birth)
Birth MaturityNeurones
Skeletal muscle fibres
Seminiferous tubules
Renal nephrons
Heart muscle fibres
Pulmonary alveoli
Ovarian follicles
Thyroid follicles
Hepatocytes
Exocrine acini
Endocrine cells
Blood cellsIntestinal villi
Fig. 14.16 Allometric growth in humans. The head is very large in proportion to the rest of the body during the embryonic period. After this time, the
head grows more slowly than the torso and limbs and, by adulthood, the head is only one-eighth of the body length.
9 16 Neonate 2 5 15 Adult
Age in yearsWeeks of development | 311 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
217
CHaPter 14Key references
entire body. Other tissues have very different growth rates; the brain,
skull, lymphoid tissues and reproductive organs all show differing
growth rates during childhood and adolescence (Kappelou et al 2006)
(Fig. 14.17).
Changes in growth at a tissue level are complex; differential growth
rates during puberty can lead to temporal-specific associated pathology. For example, 30% of childhood fractures affect the radius, mostly the
distal metaphysis, during the pubertal growth spurt, after which the
incidence of these fractures decreases rapidly (Rauch 2012). Local dif -
ferential rates of bone growth are seen in bone growth plates, peripheral and central trabecular bone, and in bone remodelling. During prepu -
bertal growth, the distal radial growth plate adds about 9 mm to the
length of the radius per year; the spaces between the peripheral trabecu -
lae are filled with mineralized bone. It is suggested that incomplete
trabecular coalescence during this rapid growth, rather than bone
remodelling (which takes at least 6 months to complete), leads to lower
bone mineral density, especially in boys, (which decreases rapidly after
the pubertal growth spurt), and higher local metaphysial cortical por -
osity (Wang et al 2010, Rauch 2012).
Fig. 14.17 Growth curves of different tissues, regions of the body and
systems. Note that the growth of lymphoid tissue, thymus, lymph nodes
and intestinal lymph masses decreases after puberty. (Adapted with
permission from Tanner JM 1962 Growth at Adolescence, 2nd ed. Oxford:
Blackwell Publishing.)Size attained as percentage of total postnatal growthLymphoid
Brain and head
General
Reproductive
Age (years)040
2060100%
80120160
140180200%
2 4 6 8 10 12 14 16 18 20
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218CHAPTER 15 Development of the limbs
dermis; and paraxial mesenchyme cells that have migrated from the
somites, and which give rise to the myogenic cells of the muscles and
to some of the endothelial cells that produce an extensive vascular
network in the early limb bud. The ectodermal epithelium covering the
limb bud gives rise to the epidermis of the skin. Motor and sensory
nerves and their associated Schwann cells, together with melanocytes
destined for the skin, migrate into the developing limb somewhat later;
the Schwann cells and melanocytes are derived from the neural crest.
AXES OF LIMBS
For descriptive, experimental and conceptual purposes, various ‘axes’, borders, surfaces and lines in relation to the developing limb bud are
defined and named (Fig. 15.2). An imaginary line from the centre of
the elliptical base of the bud, through the centre of its mesenchymal
core to the centre of the apical ectodermal ridge, defines the proximo
distal axis of the limb bud (previously known in descriptive embryology simply as the axis). Named in relation to the proximodistal axis, the LIMB BUDS
The limbs develop from the lateral body wall; their outgrowth is initi
ated at defined positions along the embryonic axis where cells continue
to proliferate, giving rise to local thickenings that soon develop into
limb buds. These buds are encased in an ectodermal epithelium rimmed
by a longitudinal ridge of columnar epithelial cells: the apical ectoder
mal ridge. Figure 15.1 shows the main stages in the development of a
human upper limb; the stages in development of the lower limb are
basically the same. The early limb bud elongates and, gradually, the
different limb regions become apparent. A broad plate forms at the tip
and, within it, digital rays develop that mark the position of the forming
digits. The digits later separate and become tipped with nails. For
further details of upper and lower limb development, see the appropri
ate regional chapters.
The early limb bud contains a mixed population of mesenchymal
cells: somatopleuric mesenchyme cells that give rise to the connective
tissues, including cartilage, bone, tendon, loose connective tissue and
Fig. 15.1 Scanning electron micrographs to show the development of the upper limb. A, An early limb bud viewed from the postaxial border. B, A limb
bud viewed from the postaxial border; the apical ectodermal ridge (arrows) can be seen. C, A limb bud, dorsolateral view; the shoulder and elbow region
can be discerned, and a hand plate has formed. The apical ectodermal ridge is still obvious at the margin of the hand plate. D, Digital rays are present in
the hand plate and the margin of the plate is becoming notched. E, The fingers are nearly separated and proliferation is commencing at the distal end of
each digit to form the nail bed. F, The fingers each have tactile pads distally, and nail development continues. (Photographs courtesy of P Collins;
printed by S Cox, Electron Microscopy Unit, Southampton General Hospital.)
C
FB
E DA | 315 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Limb buds
219
CHAPTER 15
curve around the body wall; the ventral surface of the limb (closest to
the body wall) remains relatively flat, but the dorsal surface bulges into
the amniotic cavity. The apical ectodermal ridge, originally facing lat
erally, becomes increasingly directed caudally (upper limb) and ven
trally (lower limb) as the limbs extend around the body.
Apical ectodermal ridge
The apical ectodermal ridge is required for limb bud outgrowth. When it is removed from a chick wing bud, outgrowth ceases and a truncated
wing results, whereas when a second apical ectodermal ridge is grafted,
a new outgrowth is induced that develops digits (Saunders et al 1976)
(Fig. 15.3). The molecular basis of the cell–cell interactions between
apical ridge and underlying mesenchyme is conserved between different
vertebrates; the apical ectodermal ridge of a mouse limb bud can
promote outgrowth of a chick wing bud.
There is evidence that the limb mesenchyme beneath the apical
ectodermal ridge provides a factor that is essential for maintaining
activity of the ridge (the so called apical ridge maintenance factor).
Thus, the craniocaudal extent of production of apical ectodermal ridge
maintenance factor determines the length of the apical ectodermal
ridge. When chick leg bud cells are grafted to the tip of a wing bud beneath the apical ectodermal ridge, the ridge is maintained and out
growth continues. It should be noted that the leg cells, even though placed in a wing bud, still form distal leg structures, i.e. toes (Saunders
and Gasseling 1959) (see Fig. 15.3). In addition, leg mesenchyme
will pass information to local wing ectoderm, eliciting appropriate epidermal development: in this case, formation of scales rather than
feathers.
For many years, it was thought that a timing mechanism linked to
limb bud outgrowth specified structures along the proximodistal axis. The timing mechanism was postulated to operate at the tip of a devel
oping limb bud beneath the apical ectodermal ridge in the zone of proliferating undifferentiated mesenchyme cells, which were therefore called the progress zone. It was assumed that, as cells left the progress cranially placed limb border is the preaxial border and the caudally
placed limb border is the postaxial border. (In vertebrate embryos used
as experimental models, e.g. chick embryos, the pre and postaxial
borders are termed anterior and posterior borders, respectively.) Any line that passes through the limb bud from preaxial to postaxial border,
orthogonal to the proximodistal axis, constitutes a craniocaudal axis.
The dorsal and ventral ectodermal surfaces clothe their respective
aspects from preaxial to postaxial borders, and any line that passes from
dorsal to ventral aspect, orthogonal to both proximodistal and cranio
caudal axes, constitutes a dorsoventral axis. It should be noted here that the terms dorsal and ventral axial lines are to be used exclusively in
relation to developing and definitive patterns of cutaneous innervation
of the limbs and their associated levels of the trunk.
The three developmental axes (proximodistal, craniocaudal and
dorsoventral) can be identified in the human developing limb bud by
stage 13. Experiments on chick embryos have shown that a different set
of cell–cell interactions control the development of structures in rela
tion to each of the three principal axes of the limb bud: proximodistal (e.g. humerus to fingers), craniocaudal (e.g. thumb to little finger) and
dorsoventral (e.g. extensors to flexors) (see Fig. 15.2). These experi
ments are described in detail in Hinchcliffe and Johnson (1980).
Outgrowth of the limb bud along its proximodistal axis is controlled
by interactions between the apical ectodermal ridge and underlying somatopleuric mesenchyme, and is accompanied by the sequential for
mation of limb structures along the proximodistal axis. Growth and development along its craniocaudal axis involves an interaction between
a small population of mesenchyme cells on the postaxial border of the
limb bud, termed the zone of polarizing activity, or polarizing region,
and adjacent mesenchyme. The development of the dorsoventral axis
of the limb involves an interaction between the surface ectoderm on the sides of the limb bud and the underlying mesenchyme. These sets of cell–cell interactions are coordinated so that the developing limb develops properly with respect to all three axes.
Early differential growth of parts of the limb bud results in two main
changes to the originally symmetrical axes of the limb. The dorsal aspect of the limb grows faster than the ventral, which causes the limb bud to Fig. 15.2 Axes of the developing limb and specification of structures along the different axes. A, Proximodistal; limb bud viewed from the dorsal side.
B, Craniocaudal; limb bud viewed from the dorsal side. C, Dorsoventral; limb bud viewed from the distal end. The development of limb structures
and the ectodermal specializations are controlled by the limb mesenchyme. Outgrowth of limb bud is controlled by the apical ectodermal ridge and
accompanied by the laying down of structures along the proximodistal axis; craniocaudal pattern is controlled by the zone of polarizing activity
(polarizing region); dorsoventral pattern is controlled by signals from dorsal and ventral ectoderm, shown in cross-section; the section taken in the plane
is shown by the dotted line. The limb bud in the diagram is equivalent to that of a human embryo at approximately stage 14. Apical ectodermal ridge (AER)
Mesenchyme MesenchymeAER
Dorsal ectoderm Ventral ectoderm
Zone of polarizing acivity (ZPA)ZPA AERAERProximalA Proximodistal B Craniocaudal C Dorsoventral
Distal
PostaxialDorsal VentralPreaxial | 316 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
DEvELoPmEnT of THE Limbs
220sECTion 2
ulna) and a mirror image pattern of six digits (the normal chick wing
has three digits (see Fig. 15.3)). The digit closest to the polarizing region
is always the most postaxial digit (in the chick wing, traditionally des
ignated as digit 4), while more preaxial digits develop further away.
Thus, the signal from the polarizing region appears to control both digit
number and digit pattern. Digit number is controlled by regulating the
width of the limb bud: both directly, via promoting cell proliferation,
and indirectly, by controlling production of the apical ectodermal ridge
maintenance factor. Digit pattern is specified by concentration of a dif
fusible morphogen produced by the polarizing region. The width of the
limb bud is also limited by zones of programmed cell death on the preaxial and postaxial borders. (It should be noted that, in the literature
relating to work on animal embryos, pre and postaxial borders are
termed anterior and posterior borders, respectively, and therefore the regions in which programmed cell death occurs are termed the anterior and posterior necrotic zones.) When the length of the apical ectodermal ridge is reduced, fewer digits form (oligodactyly), while, when the apical zone during limb bud outgrowth, their proximodistal fate became fixed.
Thus, cells spending a short time at the tip of the limb bud would be
specified to form proximal structures, while cells spending a longer time
would be specified to form distal structures (Summerbell et al 1973).
However, it now seems that the proximodistal pattern is specified in the
very early limb bud (Dudley et al 2002) and that the prespecified struc
tures are progressively elaborated as the bud grows out.
ZONE OF POLARIZING ACTIVITY
The zone of polarizing activity (or polarizing region) at the postaxial
border was discovered by a grafting experiment on chick wing buds (Saunders and Gasseling 1968). When a polarizing region is grafted to the preaxial border of an early chick wing bud, so that there are polar
izing region cells at both borders, duplication of distal limb structures occurs, with two ulnae (instead of an anterior radius and posterior Fig. 15.3 A–C, Experimental manipulations that identified the fundamental cell–cell interactions involved in patterning the three main axes of the limb in
the chick. Abbreviation: ZPA, zone of polarizing activity. Apical ectodermal ridge
Leg mesenchyme
ZPAPolarizing region graft (ZPA)
RightLeft
Ventral
Ventral
DorsalDorsalSecond wing axis with digits
Dorsoventral inversion
of distal wing structuresDistal leg structures
Mirror-image,
duplicated patternof wing digitsA Proximodistal
B Craniocaudal
C Dorsoventral | 317 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
Development of limb tissues
221
CHAPTER 15DEVELOPMENT OF LIMB TISSUES
DEVELOPMENT OF BONE AND CONNECTIVE TISSUE
IN THE LIMB
The first signs of the formation of skeletal elements are regions of
increased cell density in the core of the limb bud. The cells in these
regions go on to differentiate into chondrocytes, which produce large
quantities of extracellular matrix, in which they become embedded,
giving rise to a cartilage model of the bone to be developed. One centre
of chondrogenesis forms in the proximal region of an early human limb
bud; two centres of chondrogenesis then form more distally as the limb
bud lengthens, followed by five centres in the broadened distal tip. The
Sox9 transcription factor is essential for chondrocyte differentiation in
mouse embryos; the Sox9 gene is expressed before and during deposi
tion of cartilage (Wright et al 1995). Sox9 gene transcripts are expressed
in the humerus and forearm skeletal elements of human embryos at
approximately 44 days, and in the carpals, metacarpals and phalanges,
in addition to the more proximal elements, at approximately 52 days
(Fig. 15.4). Mutations in human Sox9 are associated with campomelic
dysplasia, a skeletal dysmorphology syndrome (Foster et al 1994).
Some bony eminences, which are sites of entheses, have been shown to derive from chondrogenic foci which form separately and later than
the cartilage model of the long bones (Blitz et al 2013). The cells co
express Sox9 and the transcription factor scleraxis (Scx). Blockage of Scx
or Bmp4 expression in the limb arrests eminence development. It is
suggested that as the fully formed eminences are subjected to tension whereas joint surfaces are subjected to compression, then different
developmental pathways are in operation to cope with different func
tional parameters (Blitz 2013). Scx encoding the transcription factor ectodermal ridge becomes longer, more digits form (polydactyly). Other regions of apoptotic cell death occur between the digits and result
in digital separation, but these appear later than the anterior and pos
terior necrotic zones; the resultant debris in all of the necrotic zones is removed by macrophages.
Grafts of the zone of polarizing activity only generate mirror image
digit patterns when placed beneath or adjacent to the apical ectoder
mal ridge, suggesting that the ridge produces a factor that maintains polarizing region activity. A positive feedback loop, where the polariz
ing region and the apical ectodermal ridge mutually maintain each other’s activity, would ensure that development of craniocaudal and
proximodistal axes is coordinated. Like that of the apical ectodermal
ridge, the function of the polarizing region is conserved across species.
Thus, grafts of the postaxial border of limb buds from mammals,
including human limb buds (Fallon and Crosby 1977), generate addi
tional chick digits, showing that the polarizing region produces the same molecular signal, although its interpretation depends on the
responding cells.
ECTODERMAL INTERACTION
Signals from the ectoderm control the dorsoventral axis. It is possible to remove the surface ectodermal epithelium (like peeling off a glove)
from the mesenchymal core of an early chick limb bud. When the
ectodermal epithelium from a left limb bud is recombined with the
mesenchymal core from a right limb bud, keeping the craniocaudal axis
in register, the distal part of the limb that develops conforms to the
polarity of the ectoderm rather than the polarity of the mesenchyme
(MacCabe et al 1974) (see Fig. 15.3). This can be seen in the pattern
of muscles, orientation of the joints and eventual characteristic differ
entiation of the epidermis.
MOLECULAR BASIS OF CELL–CELL INTERACTIONS
The apical ectodermal ridge produces fibroblast growth factors, extracel
lular cell–cell signalling molecules that act on the underlying mesen
chyme cells and are essential for limb bud outgrowth (Niswander et al
1993). Fibroblast growth factors also maintain the activity of the polar
izing region. The latter expresses Sonic hedgehog (Shh), a gene encoding
another class of secreted protein that acts as an extracellular signalling
molecule (Riddle et al 1993). Sonic hedgehog protein (Shh) acts on
adjacent mesenchyme cells, controlling their proliferation and also
maintaining the expression of the Gremlin gene, encoding an extracel
lular antagonist of secreted bone morphogenetic proteins. Gremlin functions as the apical ectodermal ridge maintenance factor (Zuniga
et al 1999). Sonic hedgehog plays a pivotal role in the specification of
the craniocaudal digit pattern: it spreads across the limb bud so that cells at different positions across the craniocaudal axis are exposed to
different Shh concentrations (Tickle and Barker 2013). Responding cells
transduce the signal through the Gli family of transcription factors. (The
Gli proteins are bifunctional effectors of Shh signalling and can lead to
either activation or repression of expression of target genes.) Dorsal
limb ectoderm expresses Wnt7a, encoding another extracellular signal
ling molecule that acts as a dorsalizing signal (Parr and McMahon 1995).
Among the genes expressed in response to cell–cell signalling mol
ecules in the limb are 5′ members of the Hox-a and Hox-d complexes.
Hox
genes code for transcription factors that act in a cell autonomous
fashion to control expression of many target genes. Hox-d gene expres
sion in the distal limb bud shows a nested arrangement similar to that of Russian babushka dolls: the postaxial border of the limb bud tip
expresses five Hox-d genes ( d
13, d12, d1 1, d10 and d9), while in the
adjacent, more preaxial, portion, only four genes are expressed ( d12,
d1 1, d10 and d9), and so on, moving across the limb until only Hoxd 9
is expressed at the preaxial border (Dollé et al 1989). The fact that five
different Hox-d expression domains can be distinguished prompted the
suggestion that this may be why we have five fingers (and toes) (Tabin
1992): an attractive but unlikely explanation because, in the chick wing
bud, the digits all come from the region that expresses all five Hox-d
genes. The gene encoding the transcription factor Lmxb1 is expressed
in the dorsal part of the limb in response to Wnt 7a signalling by dorsal ectoderm.
The molecular basis of limb development is conserved in humans.
Mutations associated with many of the genes described above are responsible for human congenital limb malformations. Details can be
found in Ferretti and Tickle (2006), Zeller et al (2009) and Zuniga et al
(2012).Fig. 15.4 Sox9 expression in the skeleton of developing limbs of human
embryos. A, Stage 18; approximately 44 days post ovulation. Key:
1, humerus; 2, ribs; 3, heart; 4, forearm; 5, vertebral body; 6, scapula.
B, Stage 21; approximately 52 days post ovulation. Key: 1, carpal; 2,
metacarpal; 3, phalanges; 4, humerus; 5, scapula; 6, rib. Sox9 transcripts
are detected in sections with radioactively labelled probes followed by
autoradiography. Positive signal is represented by silver grains that
show up as white areas. Scale bars represent 500 µm in both A and B.
(The human tissue was provided by the joint MRC-Wellcome Human Developmental Biology Resource (http://www.hdbr.org) at the Institute
of Human Genetics, Newcastle upon Tyne, UK. By courtesy of Dr Susan Lindsay, Institute of Human Genetics, University of Newcastle upon
Tyne, UK.)
1
2
3
1
24
5
65
6
34
A
B | 318 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
DEvELoPmEnT of THE Limbs
222sECTion 2migration into the limb, probably because of inhibitory signals pro
duced by the somatopleuric mesenchyme. Muscle differentiation
depends on the key activity of the MyoD family of transcription factors
(Weintraub et al 1991).
The myogenic cells colonize the limb bud in a proximodistal direc
tion but never reach the most distal portion of the limb. Experiments in chick embryos revealed that myogenic cells are still indifferent
regarding their region specific determination when they first enter the
limb. Thus, when brachial level somites are grafted opposite the leg
forming region, or when pelvic level somites are grafted opposite the
wing forming region, a normal limb musculature develops, irrespective
of the origin of the somites, i.e. the muscle cells, unlike the somato
pleuric mesenchyme, are not specified to be wing or leg (Chevallier et al
1977). Other experiments show that the muscle pattern developed in
the limb reflects the pattern of the skeletal elements: duplication or lack
of digits is accompanied by the duplication or lack of the corresponding
muscles (Robson et al 1994).
The first myogenic cells to arrive in the limb form the principal
dorsal and ventral premuscle masses. It is believed that, in all classes of tetrapods, limb muscle development begins with these masses, which
produce all the skeletal muscle in the limb. The premuscle masses
undergo a stereotyped spatiotemporal sequence of divisions and sub
divisions as the limb lengthens. In human embryos, this leads to the individualization of about 19 muscles in the upper limb and 14 muscles
in the lower limb. The mechanisms that divide the muscle masses are
not known; one suggestion is that these divisions are created by invad
ing somatopleuric cells. In the upper limb, the premuscle masses first
divide into three masses, the next division gives rise to the muscles
attached to the carpus, and the final division produces the long muscles
of the digits. A similar pattern is seen in the lower limb.
Each anatomical muscle appears as a composite structure. The
muscle cells and myosatellite cells are of somitic origin, and the con
nective tissue envelopes and the tendons are of somatopleuric origin. The precise way in which the muscles become anchored to the develop
ing bones by the tendons is not clear. For further development of skeletal muscle, see page 109.
The literature suggests that all musculoskeletal elements are in their
appropriate positions in human limbs by 10 weeks. For detailed
accounts of the process, consult O’Rahilly and Gardner (1975) and
Uhthoff (1990). For recent reviews on limb development, including tissue formation, see Lane and Tickle (2003).
DEVELOPMENT OF NERVES IN THE LIMB
During development, the spinal cord opposite the limbs becomes spe
cialized, with the formation of longitudinal minicolumns (columels) along its rostrocaudal axis. The columels consist of groups of individual
pools of differentiated motor neurones, precisely organized according
to function (Jessell et al 201 1). Motor neurones within each individual
pool innervate a particular muscle. Neurones in medial pools express the transcription factor Islet1 and their axons innervate ventral limb
muscles, while neurones in lateral pools express the related transcrip
tion factor, Lim1, and innervate dorsal limb muscles. Neurones also express different Hox proteins according to their position along the
rostrocaudal axis of the spinal cord, and this determines their identity
and their position within the spinal cord (Dasen et al 2005).
Motor axons enter the developing limbs earlier than sensory axons
at the time when dorsal and ventral muscle masses have started to form.
Guidance of the growing axons is not dependent on the presence of
migrating myoblasts; when these are absent, the main nerve trunks in
the limb still form, although the smaller motor branches that would
innervate individual muscles do not form (Lewis et al 1981, Honig et al
2005). Motor axons grow into the dorsal and ventral muscle masses
and innervate muscle groups in a proximo–distal progression. Correct
axonal routing depends on both repulsive and attractive cues. The
pathfinding of motor axons is an important model system for under
standing binary axon guidance decisions: glial cell (line) derived neu
rotrophic factor (GDNF) attracts motor neurone growth cones, and
interacts synergistically with ephrin As to determine growth cone direc
tionality (Drescher 201 1). Experimental studies in mice and chickens
have shown that axons of neurones in lateral pools grow into the dorsal
part of the limb as a result of repulsive interactions between the receptor EphA4 (expressed by these motor neurones) and its ligand, ephrinA
(expressed by ventral limb cells). The axons of EphB expressing motor
neurones in medial pools grow into the ventral part of the limb as a
result of repulsive interactions with ephrinB expressing cells in the
dorsal limb (Drescher 2010). The dorso ventral differences in Ephrin
expression in the limb are controlled by the system that establishes the Scleraxis is expressed in the early limb subjacent to the ectoderm.
Studies of Scx knockout mice show that the differentiation of force
transmitting tendons can be distinguished from muscle anchoring
tendons (Muchison et al 2007). A population of Scx+/Sox9+ progenitor
cells have been shown to give rise to Scx−/Sox9+ chondrocytes and Scx+/
Sox9− tenocytes/ligamentocytes. The Scx+/Sox9+ cells contribute particu
larly to establishing the enthesis organ between the cartilage and tendon
(Blitz et al 2013). The closer the tendon is to the cartilage primodium
(anchoring tendon), the more tenocytes arise from the Sox9+ lineage.
In general, the number of such derived tenocytes decreases with increas
ing distance from the cartilage model, although there is variation
between individual force transmitting tendons (Sugimoto et al 2013).
The first evidence of bone formation is seen at the mid section of
the diaphyses of long bones at 7–8 weeks in human embryos. Vascular
invasion of the cartilage matrix precedes the formation of a periostial collar, and subsequently extends proximally and distally until it reaches
the future epiphysial level, where a growth plate will be established. By
10 weeks, columns of chondrocytes can be seen at the epiphysial level
of most bones. However, only the lower end of the femur and upper
end of the tibia develop secondary ossification centres before birth.
For further details describing the development of cartilage, see page
84; for the development of bone, see page 91. For further details describ
ing the development of specific limb bones, see the appropriate regional chapter. It should be noted that maternal nutrition and vitamin status
affects fetal bone growth and that despite vitamin supplementation in
Western countries, pregnant women in northern latitudes may remain
vitamin D deficient in winter pregnancies. Infant length was shorter in
pregnancies with early serum 25 hydroxyvitamin D below the median
value (Walsh et al 2013).
DEVELOPMENT OF JOINTS IN THE LIMB
Regions of developing cartilage are easily recognized histologically in
the developing limb because they consist of widely spaced cells sur
rounded by matrix. These regions are separated by transverse bands of relatively flattened cells – interzones – which mark the sites of future
joints. Their subsequent development varies according to the type of
joint that is formed.
In developing fibrous joints, the interzone is converted into collagen,
which is the definitive medium connecting the bones involved. In
developing synchondroses, the interzone becomes (growth) cartilage of
the modified hyaline type; in developing symphyses, it is predomi
nantly fibrocartilage. In developing synovial joints, the interzonal mes
enchyme becomes trilaminar when a more tenuous intermediate zone
appears, splitting the mesenchyme into two dense strata. As the skeletal
elements chondrify, and in part ossify, the dense strata of the interzonal
mesenchyme also become cartilaginous; subsequent cavitation of the
intermediate zone establishes the cavity of the joint. The loose mesen
chyme around the cavity forms the synovial membrane and probably
also gives rise to all other intra articular structures, such as ligaments,
discs and menisci. In joints containing discs or menisci, and in com
pound articulations, more than one cavity may appear initially, some
times merging later into a complex single cavity. As development proceeds, thickenings in the fibrous capsule can be recognized as the
specializations peculiar to a particular joint. (In some joints, such acces
sions to the fibrous capsule are derived from neighbouring tendons, muscles or cartilaginous elements.) Although the initial stages in the
process of cavitation of joints are independent of movements, a true
joint cavity can generally form only in the presence of movements.
DEVELOPMENT OF MUSCLE IN THE LIMB
It is now well established that all limb myogenic precursor cells origi
nate from the somites (Chevallier et al 1977, Kardon et al 2003). These
cells are committed at an early stage and can be identified in the lateral
halves of the somites. After the mesenchymal sclerotome cells have
migrated from the epithelial somite, the remaining dorsolateral portion
is termed the epithelial plate or dermomyotome of the somite (see Fig.
44.3). Cells from the dorsomedial edge of the dermomyotomes form
the axial musculature, whereas, at limb levels, cells de epithelialize and
migrate from the ventrolateral edges of the dermomyotome into the
limb bud. These precursor limb myoblasts migrate through a non
random, structured network of extracellular fibrils. At their leading
ends, the migrating cells exhibit filopodia, which are in contact with the extracellular fibrils or with other cells; it is believed that the orienta
tion of the extracellular fibrils may direct the migration of the cells. The precursor muscle cells do not differentiate into muscle before their | 319 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
vasculature of the limb
223
CHAPTER 15movements decreases after the thirty fifth week of gestation and, from
this time, there is an increase in the duration of fixed postures. This
restriction of normal fetal movements in late gestation reflects the
degree of compliance of the maternal uterus; there is a slowing of
growth at this time.
In addition to promoting normal musculoskeletal development,
movements of the fetus encourage skin growth and flexibility indirectly.
Fetuses with in utero muscular dystrophies, or other conditions that
result in small or atrophied muscles, have webs of skin, pterygia, which pass across the flexor aspect of the joints and severely limit movement.
Multiple pterygium syndrome is characterized by webbing across the
neck, the axillae and antecubital fossae. Usually, the legs are maintained
straight and webbing is not seen at the hip and knee. A group of con
genital disorders, collectively termed ‘multiple congenital contractures’, may result from genetic causes, or limitations of embryonic and fetal
joint mobility, or may be secondary to muscular, connective tissue,
skeletal or neurological abnormalities. These conditions may be recog
nized on prenatal ultrasound examination by the appearance of fixed, immobile limbs in bizarre positions, or by webbing in limb flexures.
Specific syndromes, lethal multiple pterygium syndrome and congenital
muscular dystrophy, have been described.
The workload undertaken by the musculoskeletal system before
birth is relatively light because the fetus is supported by the amniotic
fluid and, therefore, under essentially weightless conditions. The load
on the muscles and bones is generated by the fetus itself, with little
gravitational effect. The reduction of gravitational force afforded by the
supporting fluid means that all parts of the fetus are subject to relatively
equal forces and that the position assumed by the fetus relative to
gravity is of little consequence. This is important to ensure the normal
modelling of fetal bones, especially the skull. Skulls of premature
babies may become distorted as a result of the weight of the head on
the mattress, despite regular changes in position, and the application
of oxygen therapy via a mask attached by a band around the head can
cause dysostosis of the occipital bone.
VASCULATURE OF THE LIMB
ARTERIES
The early limb bud receives blood via intersegmental arteries that con
tribute to a primitive capillary plexus. At the tip of the limb bud, there
is a terminal plexus, which is constantly renewed in a distal direction
as the limb grows (see Figs 47.1, 79.1). Later, one main vessel, the axial
artery, supplies the limb and the terminal plexus.
The development of the vasculature in the limb precedes the mor
phological and molecular changes that occur within the limb mesen
chyme as tissues begin to form. Cartilage differentiation within the
chick limb bud occurs only after local vascular regression begins, and
only in areas with few or no capillaries (Hallmann et al 1987). It is not
known whether the presence, or lack, of blood vessels affords different
local environmental stimuli for mesenchymal cells (by varying the
supply of nutrients to the tissue), or whether the local environment is
controlled by the endothelial cells. Similarly, it is not clear whether
inductive factors from the limb mesenchyme cause the changes that
occur in the pattern of blood vessels. Work on chick wing buds suggests
that the position of the central artery in the primitive limb bud vascu
lature depends on Shh signalling from the polarizing region (Davey
et al 2007).
VEINS
At the tip of the early limb bud, blood in the terminal capillary plexus
returns to the body via a marginal vein that develops along the pre and
postaxial borders of the limb. As the limb enlarges, the marginal vein
can be subdivided into pre and postaxial veins, which run along their
respective borders and which are the precursors of the superficial veins
of the limb. Generally, the preaxial (superficial) veins join the deep
veins at the proximal joint, and the postaxial (superficial) veins join the
deep veins at the distal joint of the limb. Deep veins develop in situ
alongside the arteries.dorso ventral axis of the limb and that results in dorsal expression of
Lmxb1.
Sensory neurones in the dorsal root ganglia opposite the limbs send
axons peripherally to terminate in the skin and to innervate propriocep
tors in the limb muscles. The trajectories of sensory axons into the limb
can be influenced by interaction with motor axons. When sensory axons
grow into the developing limb, it has already been invaded by motor
axons and the environment will have been altered by any extracellular
factors they may have secreted. If motor axons are removed before
sensory axons extend into the limb, those sensory axons, which would
normally terminate in muscle (Ia afferent axons), appear unable to do
so and instead become cutaneous nerves (Honig et al 1986, Honig and
Rutishauser 1996, Honig et al 1998).
The developing skin (ectoderm and immediately underlying somato
pleuric mesenchyme) is essential for the normal development of cuta
neous sensory axons. If the ectoderm is removed, the cutaneous nerves that would project to it are absent. This can be prevented by applying
the signalling molecule BMP4 (Honig et al 2004, Honig et al 2005).
Cutaneous sensory axons are not matched to particular regions of skin in the limb and can innervate regions outside their usual anatomical
dermatomes, or expand into regions of skin where the cutaneous inner
vation is eliminated (Wang and Scott 2002).
Proprioceptive sensory neurones are characterized by expression of
the transcription factor Runx3 and also express a neurotrophic factor
receptor (TrkC) that binds specifically to the neurotrophic factor NT 3.
NT3 is expressed in the limb bud and may serve as a short distance
guide to the ingrowing axons (Genc et al 2004). There is a reciprocal
interaction with the neuronal soma: in the absence of NT 3, propriocep
tive neurones in the dorsal root ganglia die. The central processes of the proprioceptive sensory neurones innervating limb muscle form mono
synaptic connections to motor neurones in the spinal cord after their peripheral axons have extended into the limbs. However, the pattern
for these sensory/motor connections appears to be specified early. The
proximo–distal position of the sensory endings in the limb is linked to
the dorso–ventral location of the target motor neurones in the spinal
cord, such that proximal sensory axons will connect to ventral motor
columels (Sürmeli et al 201 1). NT 3 is also expressed in ventral neural
tube and, as in the periphery, may help guide the axons to the correct
location in the spinal cord.
The different types of sensory endings show activation at different
developmental times; mechanoreceptors and proprioceptors are active
ahead of nociceptive neurones prenatally. A third wave of mechanosen
sitivity acquisition by the remaining nociceptors occurs just after birth
(Lechner et al 2009).
EMBRYONIC MOVEMENTS
Embryonic movements are vital for development of the musculoskel
etal system (Pitsillides 2006, Nowlan et al 2010). Simple movements of
an extremity have been observed sporadically as early as the seventh
week of gestation in human embryos. Combined movements of limb,
trunk and head commence between 12 and 16 weeks of gestation.
Movements of the embryo and fetus encourage normal skin growth and flexibility, in addition to the progressive maturation of the muscu
loskeletal system.
FETAL MOVEMENTS
Fetal movements have been detected by ultrasonography in the second
month of gestation. Those related to trunk and lower limb movements
are perceived consistently by the mother from about 16 weeks’ gestation
(quickening). Movements of the fetus often involve slow and asym
metric twisting and stretching movements of the trunk and limbs,
which resemble athetoid movements. There may also be rapid, repeti
tive, wide amplitude limb movements, similar to myoclonus.
By 32 weeks’ gestation, symmetric flexor movements are most fre
quent. By term, the quality of the movements has generally matured to smooth, alternating movement of the limbs, with medium speed and
intensity. The reduced effect of gravity in utero may cause certain fetal
movements to appear, on ultrasonography, more fluent than the equiva
lent movements observed postnatally. The number of spontaneous | 320 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |
DEvELoPmEnT of THE Limbs
224sECTion 2KEY REFERENCES
Ferretti P, Tickle C 2006 The limbs. In: Ferretti P, Copp A, Tickle C et al (eds)
Embryos, Genes and Birth Defects, 2nd ed. Chichester: Wiley, pp.
123–66.An account of the genetic basis of limb development.
Hinchcliffe JR, Johnson DR 1980 The Development of the Vertebrate Limb.
An Approach through Experiment, Genetics, and Evolution. Oxford:
Clarendon Press.
A presentation of the classical experiments on limb development.
Lane EB, Tickle C 2003 How to make a hand: a 3 day symposium held at
the University of Dundee. J Anat 202:1.
A collection of reviews on limb development and human limb malformations.
Nowlan NC, Sharpe J, Roddy KA et al 2010 Mechanobiology of embryonic
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Development of the limbs
224.e1
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e32COMMENTARY
Human anatomy informatics 2.1
Introduction
Formal description of anatomy, both adult and developmental, has
become a necessary component of informatics in the biomedical sci -
ences because it provides the natural infrastructure for integrating very large amounts of tissue-associated data. This effort started with the
formalizations of human, fruit fly and mouse anatomy in the 1990s
and now includes all the major model organisms (Bard 2005, Druzin -
sky et al 2013). The types of data that are associated with tissues cur -
rently include gene expression, diseases and abnormal phenotypes.
Although most users do not need to know in detail how anatomy is
handled within a computational context, there are two key aspects that
should be appreciated. First, the sort of formal knowledge held online
in databases is structured as a collection of triadic statements (‘triples’)
of the general style:
< > < > < >A B relationship
For an anatomical fact, A and B might represent tissues and typical
relationships would include part_of, derives_from (embryonic lineage),
starts_at and ends_at (developmental stages) and is_a (tissue-type clas -
sification). Part_of, in particular, has several meanings and these may
or may not need to be distinguished (e.g. connected parts such as the
bones of a limb, and distributed parts such as the components of
the glandular system). The great advantage of this approach is that the triples can be easily linked (the interventricular septum is part_of the
heart is part_of the cardiovascular system) to make a hierarchy or
network (more formally, this is a mathematical graph made of nodes
and edges). Such graphs are the normal way of handling complex sets of knowledge in an informatics context, and readers will already have come across them through, for example, Linnaean classifications (rela -
tionship: is_a) and evolutionary clades (relationship: descends_with_
modification_from). These relationships enable specific queries about
the knowledge included in the ontology to be answered (e.g. what are
the parts of the heart?). Graphs that deal with a specific domain of
knowledge (e.g. anatomy, cell or tissue type) are known as ontologies,
and a large number of bio-ontologies are available at the OBO
Foundry site. Ontologies are not meant to be read as text but, if
written in the OBO format, a list of triples based on IDs can be visual -
ized in browsers such as OBO-Edit (see Fig. 2.1.3). Details of all
online resources are included in Table 2.1.1.
Second, every term in such bio-ontologies – be it a tissue, a gene, a
disease or any other entity – has associated with it a unique identity of
the general form abcd:wxyz, where abcd is a unique marker for the ontol-
ogy (its namespace) and wxyz is a number associated with the item
(thus, EHDAA2:0002091 represents the trophectoderm of the human
embryo). Users never need to know such IDs but it is these that are sent from one computer to another when a user wants to access database-
associated information (e.g. gene expression) about, say, a tissue; this
is because databases use these IDs internally instead of names, as they
are unambiguous.
Formalizations of human anatomy
Adult human anatomy has been catalogued in two very different ways. The Federative International Programme on Anatomical Terminologies
(FIPAT) has produced three formal and comprehensive terminologies
Table 2.1.1 Online resources associated with normal and abnormal human anatomy
Resource Namespace Website Description
DECIPHER http://decipher.sanger.ac.uk/ A Sanger-based database that links chromosomal information with disease data
Elements of Morphology http://elementsofmorphology.nih.gov/ A National Institutes of Health (NIH)-funded project aimed at standardization of
terms used to describe human morphology. So far, the head and neck, and hands and feet have been terminologically formalized, with further regions to be developed
Federative International Programme on Anatomical Terminologies (FIPAT)http://www.ifaa.net/index.php/fipat Digital copies of the contents of Terminologia Anatomica, Terminologia Histologica and Terminologia Embryologica as published in books produced by IFAA
Foundational Model of Anatomy (FMA) ExplorerFMAID http://sig.biostr.washington.edu/ A viewer that shows the rich information stored in the FMA
Human Developmental Anatomy EHDAA2 Anatomical and tissue-associated data for Carnegie stage 1–20 embryos
Human Developmental Studies Network (HUDSEN)http://www.hudsen.org/ A source containing human embryological gene expression and image data
Human Phenotype Ontology (HPO) HP http://www.human-phenotype-ontology.org/ A standardized vocabulary of phenotypic abnormalities encountered in human disease
OBO Foundry http://www.obofoundry.org/ A repository for bio-ontologies
OBO-Edit http://oboedit.org/ A downloadable Java-based viewer for analysing ontologies in the OBO format
Online Mendelian Inheritance in Man (OMIM) http://www.omim.org/ An online catalogue of human Mendelian genetic disease with detailed phenotype annotations
Orphanet http://www.orpha.net/ A portal for rare disease information with a classification and encyclopaedia of rare diseases, listing genes involved and detailed phenotypic descriptions
Pictures of Standard Syndromes and Undiagnosed Malformations (POSSUM)http://www.possum.net.au/ A source of information on over 4000 syndromes, including multiple malformations, chromosomal abnormalities, skeletal dysplasias and metabolic disorders, linked to over 30,000 images including photos, X-rays, scans, diagrams and histology
Uber-anatomy Ontology UBERON http://uberon.github.io/ A cross-species anatomy ontology classified according to traditional anatomical
criteria such as structure, function and developmental lineage; includes
comprehensive relationships to taxon-specific anatomical ontologies bridging Drosophila to Homo sapiens
Virtual Physiological Human http://physiomeproject.org/ A project aiming to integrate computer models of the mechanical, physical and biochemical functions of a living human body
Winter–Baraitser Dysmorphology Database (WBBD)http://www.lmdatabases.com/index.html A resource developed initially in 1987 as the London Dysmorphology Database, containing information on over 4700 dysmorphic, multiple congenital anomaly and mental retardation syndromesJonathan B L Bard, Paul N Schofield | 323 | Gray's Anatomy: 41st Edition | grays_anatomy.pdf | https://archive.org/download/GraysAnatomy41E2015PDF/Gray%27s%20Anatomy%2041E%202015.pdf | PyPDF2TextLoader | https://archive.org/details/GraysAnatomy41E2015PDF |