Implantation and placentation
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CHAPTER
10
Cell populations at gastrulation
Extraembryonic mesoblast
CONCEPTUS WITH A BILAMINAR
Epiblast
EMBRYONIC DISC
Primitive node
At stage 6, the conceptus is composed of the walls of three cavities: the Primitive streak
large chorionic cavity is surrounded by a meshwork of trophoblast and
Amniotic
developing villi, and lined with extraembryonic mesoblast. The chorion,
cavity
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 Yolk sac Hypoblast Allantois
layers. Epiblast and hypoblast contain mixed populations of cells with Mesenchyme Notochord
little restriction, which establish the placental structures and extraem Fig. 10.1 A longitudinal section through an early conceptus. Ingression of
bryonic tissues before the production of embryonic cell lines at gastrula mesoblast is occurring at the primitive streak and the notochord is


tion. The older terminology depicting three germ layers that give rise to ingressing via the primitive (Hensen’s) node.
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
Primitive streak
cause confusion and inhibit the development of more pertinent descrip
tive language to describe these early events. The earliest three cell line Epiblast
ages, not layers, that give rise to the extraembryonic membranes and
the embryo are trophoblast, hypoblast and epiblast; each expresses dif
ferent lineagespecific 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, Hypoblast
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 Endodermal and
ment during which significant rearrangements, migrations and folding notochord cells
of the early embryo occur. The primitive streak is the site of organizer Mesenchyme
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 Extraembryonic mesoblast
will give rise to a complex multilaminar structure with a defined cranio Fig. 10.2 A transverse section through the embryonic plate at the level of
caudal axis. By the end of gastrulation, cell populations from different, the primitive streak to show the early movement of mesoblast between
often widely separated, regions of the embryonic disc will become the epiblast and underlying hypoblast.
spatially related and the embryonic shape will have been produced.
Primitive streak and node
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
Seen from the dorsal (epiblastic) aspect, at stage 6, the embryonic disc ment of the streak, the terms medial and lateral can be used. The relative
appears elongated. The primitive streak is first seen in the caudal region dimensions of the primitive streak and the fates of the cells that pass
of the embryonic disc at this stage as a collection of pluripotent cells, through it change with the developmental stage. Thus, the streak
orientated along its long axis in the median position, conferring extends half way along the disc in the stage 6 embryo, reaching its
the future craniocaudal axis of the embryo (Figs 10.1–10.2). Although greatest relative length in stage 7 and its maximum length in stage 8.
the future cranial and caudal regions of the embryo are well within the Formation of the primitive streak is induced by the underlying vis
boundaries of the embryonic disc, it has become the practice to term ceral hypoblast, which remains beneath the streak even at later stages. 181


Cell populations at gastrulation
182
2
noitCes
The primitive streak may be considered to be generally homologous Neural ectoderm
with the blastopore of lower vertebrates (e.g. amphibia), with the nodal
region corresponding to the dorsal lip. Experiments clearly show the Surface ectoderm
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
Notochord
analogous to ingression through the node of the prechordal plate and
endoderm. The primitive streak similarly may be considered analogous Endoderm Medial halves
of the somites
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 Lateral halves
proliferation, the rate of division being much faster than that of blasto of the somites
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
Mesoblast
vimentin, and loss of cytokeratins by the emerging cells.
As the epiblast cells proliferate, two ridges are formed on each side Primordial germ cells
of the primitive streak, which appears to sink between them. The lower
midline portion of the streak is termed the primitive groove. The process Extraembryonic mesoblast
by which cells become part of the streak and then migrate away from
it beneath the epiblast is termed ingression. Fig. 10.3 The predictive fates of the epiblast cell population at the time
The primitive node, or Hensen’s node, is the most rostral region of the primitive streak is present.
the primitive streak. It appears as a curved ridge of cells similar in shape
to the top of an oldfashioned keyhole. Cells ingressing from the ridge
pass into the primitive pit (the most rostral part of the primitive groove),
along the long axis of the embryonic disc. By stages 9 and 10, the cells
and then migrate rostrally beneath the epiblast. The primitive node has


at the lateral edge of the plate have begun to migrate laterally as free
been recorded in all stage 7 human embryos; it produces axial cell
mesenchymal cells and the plate reduces in height to two cells deep. At
populations, the prechordal plate, notochord, embryonic endoderm
stage 11, the migrating prechordal mesenchyme forms bilateral pre
and the medial halves of the somites. Experimental removal of the
mandibular mesenchymal condensations and is no longer a median
node results in complete absence of the notochord and a failure of
structure. The extent of prechordal cells remaining within the endoderm
neurulation.
is not clear.
Recent studies on the dynamics of ingression at the primitive streak
have shown movement of the underlying extracellular tissues as well as Notochord
of the overlying cells. Reexamination of the processes of cell movement
relative to the surrounding extracellular molecules in embryos has
The notochord, also called chordamesoderm, the head process or
found that vertical and convergent extension motion patterns, previ
chorda, arises from epiblast cells of the medial part of the primitive
ously thought to be limited to the epiblast, also occur in subepiblastic
node. It passes through several stages during development. The cells of
extracellular matrix fibrils of fibronectin (Zamir et al 2008, Szabó et al
the early notochordal process express myogenic markers transitorily as
2011).
they migrate beneath the epiblast, but later they become epithelial,
Position and time of ingression through the forming junctions and a basal lamina. The notochordal cells are inti
mately mixed with endodermal cells, as both cell lines ingress at the
primitive streak
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
Studies of cell fate have shown that epiblast cells that will pass through caudal axis. They form a rostral part, which is composed of a cell mass
the streak are randomly located within the epiblast layer before their continuous with the prechordal mesenchyme; a mid portion, in which


ingression, and that epiblast fate is determined at or before the time of cells are arranged in a tube with a central notochordal canal; and a
ingression through the streak, indicating that passage through the prim caudal epithelial layer of cells, the notochordal plate, which is contigu
itive streak is the most important factor for future differentiation. ous with the embryonic endoderm and forms a roof to the secondary
The position and time of ingression through either streak or node yolk sac. There is a transitory opening between the primitive node (and
directly affect the developmental fate of cells. Passage through the streak amniotic cavity) and the secondary yolk sac called the neurenteric canal
is specified according to position, e.g. via the node, or rostral, middle (so named because its upper opening is in the future caudal floor of
or caudal regions of the streak. Cells that ingress through the primitive the neural groove, and its lower opening is into the archenteron, which
node give rise to the axial cell lines, the prechordal mesenchyme and is the primitive gut); it may still be found at stage 9, and the site of the
notochord, and to the endoderm and the medial halves of the somites. neurenteric canal can be recognized in stage 10 embryos. The ingression
The rostral portion of the primitive streak produces cells for the lateral of notochordal cells at the primitive node is matched by specification
halves of the somites, whereas the middle streak produces the lateral of the overlying neural ectodermal cells, and the notochordal plate is
plate mesoblast. The adjacent caudal portion of the streak gives rise to thus matched in length by the future neural floor plate. Both the noto
the primordial germ cells, which can be distinguished histologically and chord and the region of the floor plate of the neural tube may arise
histochemically, and the most caudal portion of the streak contributes from a common progenitor cell. The early notochord is important for
cells to the extraembryonic mesoblast until the somites are visible. A the maintenance and subsequent development of the neural floor plate
composite of the information on the position of ingression through the and the induction of motor neurones. Removal of the notochord results


streak and node is shown in Figure 10.3. The epiblast cells that do not in elimination of the neural floor plate and motor neurones, and
pass through the streak but instead remain within the epiblast popula expression of sensory cell types.
tion give rise to the neural and surface ectoderm of the embryo.
Caudal eminence
Prechordal plate
From stages 9 and 10, the region between the neurenteric canal and the
The earliest cells migrating through the primitive node and streak give cloacal membrane (see below), including the primitive streak, is termed
rise to both the embryonic endoderm and the notochord. The pre the caudal eminence. It consists of the caudal region of the trunk, com
chordal plate is first seen at stage 7. It has been defined as a localized posed of mesenchyme derived from the primitive streak and epiblast,
thickening of the endoderm rostral to the notochordal process, although and covered with surface ectoderm. Whereas ingression of cells through
it is seen as a highly developed mesenchymal mass in contact with the the primitive streak gives rise to the prechordal and notochordal plates,
floor of the neural groove, rostral to the notochordal process, rather and cells rostral to the neurenteric canal (see below), the cells of the
than as an epithelial layer. The prechordal plate is a temporary collec caudal eminence arise from local division of a mesenchymal popula
tion of cells that underlies the neural plate during stage 9. It is com tion positioned caudal to the neurenteric canal. The caudal portions of
posed of cells that are similar to, or larger and more spherical than, the the notochord, which form later in development when secondary neur
ingressing endodermal cells (Müller and O’Rahilly 2003). In stage 8 ulation processes begin, arise from these cell populations, sometimes
embryos, the prechordal plate is up to eight cells deep and extends termed the caudoneural hinge or junction. This tissue is thicker and


Conceptus with a bilaminar embryonic disc
183
01
retpaHC
A
Amniotic cavity Neural tissue B Intraembryonic coelom
Primitive streak
Ectoderm
Connecting stalk
Bucco-
pharyngeal
membrane Cloacal membrane
Endoderm
Yolk sac
Entrance to
intraembryonic
coelom
C Neural tissue D Primitive streak
Notochordal plate Intraembryonic Intraembryonic coelom
coelom
Primitive streak Allantois
E
Amniotic cavity Intraembryonic coelom
Connecting stalk
Allantois
Yolk sac
Mesenchyme Buccopharyngeal membrane Notochordal plate Cloacal membrane
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.
more advanced in differentiation than the tissues derived from the early Intraembryonic mesoblast (mesenchyme)
primitive streak.
Epiblast cells ingress through the cranial and middle parts of the streak
Embryonic endoderm individually, maintaining their apical epithelial contacts while elong
ating ventrally. The cells become flaskshaped, with thin, attenuated
Before ingression, definitive embryonic endoderm cells are found in the apical necks and broad basal regions. Their basal and lateral surfaces
epiblast, located at the primitive node and rostral primitive streak. In form lamellipodia and filopodia, and the apical contact is released. The
the mouse, the endodermal cells lie beneath the epiblast mainly in the cells are now free mesoblast cells, their fibroblastic, stellate morphology
midline, interspersed with presumptive notochordal cells, forming reflecting the release from the epithelial layer. Once through the streak,
the roof of the secondary yolk sac. The ingressing endoderm displaces the cells migrate away from it, using the basal lamina of the overlying


the visceral hypoblast into the secondary yolk sac wall by a dramatic epiblast and extracellular matrix as a substratum. The cells contact one
territorial expansion that is brought about by a change in the morph another by filopodia and lamellipodia, with which they also contact
ology of the cells (see Figs 10.1–10.2, 10.4). The putative endoderm cells the basal lamina. Gap junctions have been observed between filopodia
are cuboidal epithelial cells within the node but they become squamous and cell bodies. With the appearance of the mesoblast, spaces form
in the endoderm layer; this could result in a fourfold increase in the between the epiblast and visceral hypoblast that are filled with extracel
surface area covered by the cells. A complete replacement of the visceral lular matrix rich in glycosaminoglycans. The migrating mesoblast has a
hypoblast has not yet been confirmed and there may be a mixed popu leading edge of cells that open up the migration routes, and the follow
lation of cells in the endodermal layer in the early stages. Ingression of ing cells seem to be pulled along behind in a coordinated mass move
cells through the streak and node in the human is apparent at stage 6, ment. Mesoblast formed by cells migrating through the primitive node
and, by stage 7, a population of endoderm and notochord cells is and rostral primitive streak will form the paraxial mesenchyme, whereas
present beneath the epiblast (see Figs 10.1–10.2, 10.4). During stages cells migrating through the middle to caudal streak will form the lateral
6–11, the midline roof of the secondary yolk sac becomes populated plate mesenchyme (see Figs 10.1–10.2).
mainly by the notochordal plate, which remains in direct lateral conti
Embryonic ectoderm
nuity with the endodermal cells. It is not until stage 11, 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 When the ingression of cells through the primitive streak is completed,


Figure 12.3. the epithelial cells remaining in the epiblast layer are termed embryonic


Cell populations at gastrulation
184
2
noitCes
ectoderm cells. This layer still contains a mixed population because Mesoblast that passes in a cranial direction flanks the notochordal
both surface ectoderm cells and neural ectoderm cells are present. It is plate and passes around the prechordal plate region, converging medi
believed that these cells were originally in the cranial half of the disc ally to fuse in the midline beyond its cephalic border. This transmedian
when the primitive streak first appeared, at which time the neuralfated mass, in which the heart and pericardium will develop, is initially
cells were closest to the streak, and the surface ectoderm cells were most termed the cardiogenic mesoblast. It fuses with the junctional zone of
cranial (see Fig. 10.3). The process of primary neurulation relocates extraembryonic mesoblast around the extreme cephalic margin of the
most of the neuroepithelial cells (see below). embryonic area. This region will eventually form the septum transver
sum and primitive ventral mesentery of the foregut. Mesoblast passing
Primordial germ cells 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
Although early studies on human embryos have reported primordial
onic mesoblast merge. Mesoblast that streams caudally from the primi
germ cells, and described their development from the early endoderm
tive streak skirts the margins of the cloacal membrane and then
of the yolk sac and allantois, it is now clear from animal experimenta
converges towards the caudal midline extremity of the embryonic disc
tion that the primordial germ cells arise from epiblast ingressing at the
to become continuous with the extraembryonic mesoblast of the con
caudal end of the primitive streak (see Fig. 10.3). It is not known
necting stalk. It is unclear if the lower layer of the cloacal membrane
whether these cells originate from rostral regions that migrate to the
consists of visceral hypoblast, like the more cranial primitive streak
streak or from local caudal regions. Extremely early segregation of the


(the hypoblast is necessary for maintaining the streak), or if it is
germ cells, when the epiblast layer consists of only 10–13 cells, has been
replaced by migrating embryonic endoderm, or if there is a region for
demonstrated. It has been suggested that the primordial germ cells
ingression of endoderm at the caudal end of the streak, similar to the
remain sequestered in the extraembryonic mesenchyme at the caudal
node cranially.
end of the embryo until the embryonic endoderm has been produced
Still further caudally, the embryonic disc develops a midline diver
and gastrulation completed, and that they start to migrate along the
ticulum adjacent to the cloacal membrane. This diverticulum, the
allantoic and hindgut endoderm as the folding of the embryo begins.
allantois, projects into the extraembryonic connecting stalk (see Figs
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 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,
migrate to the genital ridges. Further development of the germ cells is
parietal hypoblast or embryonic endoderm. The allantois later devel
described in Chapter 72.
ops a rich anastomotic blood supply around it, in the manner of the
yolk sac.
TRILAMINAR DISC 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
Although the stage 8 embryo is termed a trilaminar disc, the concept
floor plate cells until both cell lines extend to the buccopharyngeal
of three epithelial layers forming a trilaminar disc is incorrect; the
membrane. The epiblast lateral to the midline contains both future
middle, mesoblast, layer is several cells thick with intervening extracel
surface and neural ectoderm. The latter becomes arranged between the
lular matrix. The embryo at this stage, approximately 23 days after ovu


primitive node and buccopharyngeal membrane; cells destined to be in
lation, is pearshaped, and broader cranially than caudally (Fig. 10.5).
the neural plate lie medially, and those destined for the neural crest lie
The upper epiblast cells are tall and form a pseudostratified columnar
along the junction between the neural plate and surface ectoderm (see
epithelial layer with a basal lamina, except at the primitive streak, where
Fig. 10.5). A smaller subpopulation of neuronal cells, the ectodermal
the cells are ingressing to form the other layers. The more centrally
placodes, are arranged either close to the neural crest or within the
placed epiblast will give rise to neural ectoderm (neurectoderm) and
rostral limit of the neural plate itself.
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 FOLDING OF THE EMBRYO
prechordal plate. The lower embryonic endoderm, a simple squamous
layer with a developing basal lamina, is not always complete at this
In a diagrammatic representation of the trilaminar disc prior to folding
stage, particularly in the midline caudal to the prechordal plate, which
and viewed from the ectodermal aspect, all of the future external surface
is still occupied by the notochordal process or plate.
of the body is delimited (see Fig. 10.5). The ends of the gut tube are
The middle, mesoblast, layer is composed of free cells migrating
specified on the ectodermal surface at the buccopharyngeal and cloacal
cranially, laterally and caudally from the primitive streak (see Fig. 10.4).
membranes, which are regions where the ectoderm and underlying
They produce extracellular matrix, which separates the epiblast and
endoderm are apposed without intervening mesoblast. In the midline
endoderm of the embryonic area and permits their passage. The streams
between these membranes, proliferation of the neural ectoderm matches


of mesoblast extend between the epiblast and endoderm over all of the
the underlying migration of mesoblast from the primitive streak, so
disc area except cranially at the buccopharyngeal membrane (where the
that the neural plate covers the paraxial mesenchyme on each side of
endoderm and ectoderm become apposed once the prechordal mesen
the notochord (see Fig. 10.6E). As the paraxial mesenchyme segments,
chyme has migrated laterally), and caudally at the cloacal membrane
the formation of the epithelial somites elevates the edges of the neural
(a patch of thickened endoderm, similar to the buccopharyngeal mem
plate and initiates primary neurulation (see Fig. 10.6F; Figs 10.7–10.8).
brane, caudal to the primitive streak). The mesoblast on each side of
The neural plate itself undergoes concurrent morphological changes.
the notochord is termed paraxial mesenchyme.
The most medial cells become wedgeshaped, 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
Buccopharyngeal membrane
(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
Neural plate
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 bucco
Cloacal membrane pharyngeal 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
Fig. 10.5 The extent and shape of the neural plate in an unfolded embryo. the disc will become the lateral and ventral abdominal walls of the


Formation of the intraembryonic coelom
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A Neural tissue Amniotic cavity B C
Intraembryonic Connecting stalk
coelom Allantois
Bucco- Cloacal membrane
pharyngeal
membrane Notochord
Yolk sac
D Foregut Midgut Hindgut E Neural plate Amniotic cavity Neural groove
F
Paraxial mesenchyme Somite
Notochord
Intraembryonic coelom
Yolk sac
Split lateral plate
Notochordal plate
G
Neural crest
Neural tube
Somite
Notochord Fig. 10.6 Head and tail (A–D) and lateral (E–G) folding of the embryo. A–D, Median sagittal
Intraembryonic coelom (longitudinal or axial) sections through the embryonic disc at successive stages; the relative
Midgut 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.
trunk. The portion of the disc beyond the cloacal membrane will form The midline portion of the originally flat, horseshoeshaped coelom
the ventral abdominal wall caudal to the umbilicus. The circumference moves ventrally, leaving the caudal arms of the horseshoe in their origi
of the disc, where the embryonic tissue meets the extraembryonic mem nal position. In this way, the midline part of the coelom, which was
branes, will become restricted to the connection between the ventral originally just rostral to the buccopharyngeal membrane, comes to lie
abdominal wall and the umbilical cord, i.e. the umbilicus. ventral to the foregut (caudal to the buccopharyngeal membrane), and


Head folding begins at stage 9, when the fusing cranial neural plate the two lateral extensions of the coelom pass close to the lateral walls
rises above the surface ectoderm and the portion of the disc rostral to of the foregut on each side. The caudal portions of the coelom (the two
the buccopharyngeal membrane (which contains the cardiogenic mes arms of the horseshoe), which, in the unfolded disc, communicated
enchyme) moves to lie ventral to the developing brain (see Fig. 10.6). laterally with the extraembryonic coelom, turn 90° to lie lateral to the
The prosencephalon and buccopharyngeal membrane are now the most gut, and communicate with the extraembryonic coelom ventrally.
rostral structures of the embryo. The previously flat region of endoderm, Compartments of the coelom that will give rise to the body cavities
which may contain cells from the prechordal plate, is now modified later in development can already be seen. The midline ventral portion,
into a deep tube, the primitive foregut. Tail folding can be seen in stage caudal to the buccopharyngeal membrane, becomes the pericardial
10 embryos, when the entire embryo comes to rise above the level of cavity. The canals lateral to the foregut (pericardioperitoneal canals)
the yolk sac. Similar movement of the part of the disc caudal to the become the pleural cavities and the uppermost part of the peritoneal
cloacal membrane results in its repositioning ventral to the neural plate. cavity. The remaining portion of the coelom becomes the peritoneal
Generally, as the embryo rises above the edges of the disc, the lateral cavity. By stage 11, the intraembryonic coelom within the lateral plate
regions of the disc are drawn ventrally and medially, contributing to mesenchyme extends caudally to the level of the caudal wall of the yolk
the lateral folding of the embryo. 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).
FORMATION OF THE INTRAEMBRYONIC COELOM


In the early embryo, the intraembryonic coelom provides a route for
the circulation of coelomic fluid and, with the beating of the heart tube,
At and just before stage 9 (before formation of the head fold), vesicles functions as a primitive circulation that takes nutritive fluid deep into
appear between the mesenchymal cells cranial to the buccopharyngeal the embryo until it is superseded by the blood vascular system. The
membrane and within the cranial lateral plate mesenchyme. At the coelomic channel, and the primitive circulation that passes through it,
periphery of the vesicles, the mesenchymal cells develop junctional is of paramount importance up to stage 13. Whereas the superficial
complexes and apical polarity, and form an epithelium. The vesicles tissues of the embryo can receive nutrients via the amniotic sac and yolk
become confluent to form a horseshoeshaped tube, the intraembryonic sac fluids, the deeper tissues are, until the formation of the coelom,
coelom, which extends caudally to the level of the first somite and lat under conditions similar to those found in tissue culture. However,
erally into the lateral plate mesenchyme towards the extraembryonic from stage 10, exocoelomic fluid, propelled by the first contractions of
mesenchyme. The lateral plate mesenchyme thus develops somatopleu the developing heart, is brought into contact with the deeply placed
ric coelomic epithelium subjacent to the ectoderm, and a splanchno mesenchyme. This early ‘circulation’ ensures that an adequate supply
pleuric coelomic epithelium next to the embryonic endoderm (see Fig. of nutrients reaches the rapidly increasing amount of embryonic tissue,
12.2C(iv)). At this stage, the intra and extraembryonic coeloms do not and meets most of the requirements of the deeper mesenchymal deriva
communicate. tives. From stage 12, the endothelial system expands and fills rapidly
During development of the head fold, the morphological move with plasma, which passes across the locally thinned coelomic epithe
ments that organize the foregut and buccopharyngeal membrane have lium into the large hepatocardiac channels that project into the peri


a similarly profound effect on the shape of the intraembryonic coelom. cardioperitoneal canals at the level of the seventh somite.


Cell populations at gastrulation
186
2
noitCes
Position of the
pericardial coelom
NF
H
A
Fig. 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.
B
Fig. 10.7 Scanning electron micrographs of rat embryos at the time of In spite of the importance of the coelom in defining the body cavi
neurulation. A, Ventral view, showing the neural fold (NF), and the heart ties, and of the coelomic epithelium in the production of the major
(H), with the somatopleuric pericardial membrane and surface ectoderm mesenchymal populations of the trunk (Streeter 1942, Langemeijer
removed; the arrow indicates the entrance to the foregut via the cranial 1976, O’Rahilly and Müller 1987) (see Fig. 12.2), until recently the
intestinal portal. B, Dorsolateral view; the arrows indicate the extent of overall contribution of the coelom and its epithelium to the embryo
rostral (to the right) and caudal (to the left) neural tube formation. has received relatively little attention (Carmona et al 2013). The coelom
(Photographs by P Collins; printed by S Cox, Electron Microscopy Unit, is a single, tubular organ that may be compared to the neural tube, in
Southampton General Hospital.) 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
Brain
(early expansion progeny migrate. After the germinal phase, both epithelia ultimately
of neural fold) form the lining of a cavity, ependyma for the neural epithelium, and
mesothelium for the coelomic epithelium. Coelomic epithelium, like
Neural crest First pharyngeal arch, neural epithelium, produces cells destined for different fates from dif
dorsal extremity
ferent sites and at different developmental times. Coelomic cells are like
Rostral neuropore
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
Amnion, cut edges Yolk sac wall covered progeny, similar to the radial glia of the neural tube, has not been
in extraembryonic examined.
mesoderm
Somites (5 and 6) EMBRYONIC CELL POPULATIONS
AT GASTRULATION
After gastrulation, the cells of the embryo contribute to two fundamen
Caudal neuropore tal types of tissue, namely: epithelial and mesenchymal. Differentiation
of specialized circulating blood cells and other cell types occurs in
Unsegmented sequence. Embryonic and fetal cell types are replaced later in develop
paraxial ment or after birth.
mesenchyme
Epithelia
Epithelial populations in the embryo have many of the morphological
Fig. 10.8 A human embryo at stage 10, 2.1 mm long, with nine somites: characteristics of differentiated epithelia, i.e. they are composed of
right lateral and dorsal aspects. Nearly all the yolk sac and the caudal sheets of closely packed, polarized cells, with narrow intercellular clefts
amnion have been excised. containing minimal extracellular material, and a developed basal


embryonic cell populations at gastrulation
187
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A B
Connecting stalk
Trophoblast Embryonic pole Amniotic duct
Amniotic cavity Allantoenteric
diverticulum
Extraembryonic mesoblast
Chorionic
Yolk sac mesoblast
Chorion
Early villous stems
Chorionic cavity/extraembryonic coelom Secondary yolk sac
C D Placental area (chorion frondosum)
Allantoenteric diverticulum
Connecting stalk Cloacal membrane Connecting stalk
attachment becomes
Embryonic area Villous stem Hindgut more ventrally placed
Allantoic canal
Extraembryonic Tail fold
Chorionic mesoblast coelom Midgut
Extraembryonic
Splanchnopleuric Amniotic cavity coelom
Amniotic cavity mesoblast of
a b
yolk sac
Pericardial coelom Secondary yolk sac Pericardial coelom
Septum transversum
Buccopharyngeal
Foregut membrane
Chorion laeve Head fold
F
E
Umbilical cord
Somite Amnion Yolk sac
Neural groove
Chorion Obliterated
yolk duct Buccopharyngeal
Notochord
Intraembryonic membrane
coelom
Amniotic cavity Umbilical
Intercoelomic coelom Pericardial
Midgut communication cavity
Cloacal
Lateral body fold
Yolk duct membrane Septum
Extraembryonic transversum
Yolk sac coelom Allantoic duct
Foregut
Yolk sac wall Extraembryonic Line of fusion of
mesoblast amnion with chorion,
i.e. line of obliterated Amniotic
extraembryonic coelom cavity
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.
lamina containing specific proteins synthesized by the epithelium itself. their early patterning are termed organizers, e.g. the primitive streak.
The cells usually show juxtaluminal lateral surface specializations, such Later germinal epithelia give rise to systemspecific progenitor popula
as desmosomes, tight junctions, gap junctions, and so on, and special tions, e.g. the ventricular zone of the neural tube. All epithelia other
izations of the apical surface, such as microvilli or cilia. than special germinal epithelia divide to produce embryonic growth
Epithelial polarity factors establish the extent of the apical, lateral throughout development and may retain stem cells that will divide
and basal domains of the individual cells forming an epithelium. Three throughout life.
main control pathways have been identified in maintaining apicobasal Characteristically, epithelia clothe internal and external surfaces as
polarity (St Jonston and Sanson 2011). The apical domain is specified simple or compound cellular sheets that separate phases of differing
by the Crumbs transmembrane protein; Baz/PAR3 specifies the posi composition (e.g. the external environment and the subepithelial tissue
tion and extent of the intercellular junction; and Scribble restricts the fluids; intravascular and extravascular fluids, and so on). Traffic of mat
size of the junctional domains. Once established with an underlying erials in the intercellular clefts is limited. Traffic occurs across the cells
basal lamina, the basal surface displays integrins. Changes in the mor because their limiting membranes, which function as energydependent
phology of an epithelial sheet will involve relative changes in the apical, selective barriers, enhance the passage of some materials and impede
lateral and basal domains of all cells. Planar polarity of epithelia is the passage of others.


driven by intercellular cortical actin and myosin. Epithelial cells are
contiguous via actomyosin contractile networks, which contact the
Mesenchyme
lateral cell membrane through Ecadherin adhesion (Gorfinkiel and
Blanshard 2011). Wnt/planar cell polarity signalling pathways are
involved in these epithelial morphogenetic movements and also in The terms mesoblast and mesenchyme are used in this text in a specific
orientated cell division which occurs in epithelial sheets (Tao et al manner and are not interchangeable. Previously, cells forming a popula
2009). tion between the epiblast and hypoblast were termed mesoderm and,
Embryonic epithelia differ from those in the fetus and adult. Two more recently, mesenchyme. The terms primary and secondary mesen
distinct types can be identified. Early germinal epithelia, which give rise chyme have been used to distinguish between those cells that arise
to epithelial or mesenchymal populations of the embryo and confer from ingression through the primitive streak and those that arise from


Cell populations at gastrulation
188
2
noitCes
neural crest ingression, respectively. Primary mesenchymal cells revert routes, removes the support for overlying epithelia, and disrupts the
to epithelia at their destinations. However, whereas some primary normal branching of glandular systems.
mesen chymal cells may become epithelial within a short timeframe, Fibronectin deposited extracellularly along a migration pathway will
e.g. somites and lateral plate, other cells may transform later, e.g. the affect cells that touch it later, causing realignment of their intracellular
epithe lium lining blood vessels. To address these terminological con actin filaments and thus of their orientation; it also induces cell migra
flicts, the mixed population of epiblast cells that ingress through the tion. The receptors for extracellular matrix molecules such as fibronectin
primitive streak and come to lie between the epiblast and embryonic and laminin were originally termed integrins because they integrate
endoderm is termed mesoblast until the cells have migrated to their extracellular proteins and intracellular cytoskeletal elements (via α and
final position, at which time the populations of mesenchyme can be β subunits that span the cell membrane), allowing them to act together;
identified and their fates inferred. the binding preference of integrins depends on their combination of
Mesoblastic and mesenchymal cells have no polarity. They form subunits and environmental conditions.
junctional complexes, which are not exclusively juxtaluminal, and they It is known that extracellular matrix is structured rather than random.
produce extracellular matrix molecules and fibres from the entire cell Epithelial and mesenchymal cell populations can structure the space
surface. Mesenchymal populations are formed from a range of germinal around them by secretion of particular matrix molecules or growth
epithelia and by proliferation of mesenchymal cells directly, and occupy factors, which, in turn, can organize the cells that contact them. Cell–
all the regions between the various epithelial layers described above. matrix interactions and matrix–cell interactions control the position of
The term mesoderm is reserved for the coelomic epithelia that later migration routes and cellular ‘decisions’ to migrate or to begin to dif
form mesothelia. ferentiate. Matrix molecules propagate developmental instructions from


Mesenchymal cells support epithelia throughout the developing cell to cell and form a farreaching fourdimensional (spatial and tem
body, both locally where they contribute to the basement membrane poral) mechanism of communication. Mapping the molecular path
and form the lamina propria and smooth muscle of tubes, and ways and the biomechanical processes that underlie morphogenesis, the
generally where they differentiate into connective tissue. Specific dynamic epigenetics of early mesenchyme cells and their secreted three
mesenchymal populations control the patterning of local regions of dimensional extracellular matrices, and the mechanisms by which the
epithelium (e.g. the zone of polarizing activity on the postaxial limb stiffness of the surrounding matrix affects the actions of the cells within
border posterior to the apical ectodermal ridge). 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.
Extracellular matrix Davidson et al 2010, Loganathan et al 2012).
The space beneath epithelia and between mesenchyme cells is filled
Transition between epithelial and
with extracellular matrix; both epithelial and mesenchymal cells syn
mesenchyme states
thesize extracellular matrix molecules and their receptors. Epithelial
cells produce a twodimensional basal lamina, which contains a variety
of matrix molecules, including laminin, fibronectin, type IV collagen Transformations of cell morphology from epithelium to mesenchyme,
and various proteoglycans. The particular molecules can vary during and vice versa, occur in specific places and times during development,
development according to spatial and temporal patterns, resulting in and can be seen as ways of dispersing germinal centres with increasing
changes in the behaviour of the underlying mesenchymal cells (e.g. in restriction. The first epithelialtomesenchyme transition occurs at the
patterning of the basal regions of the skull). Mesenchymal cells produce primitive streak, a germinal epithelium that confers embryonic specifi
extracellular matrix molecules in three dimensions. Those adjacent to cation on the resultant mesoblast population. The mesoblast so formed
an epithelial layer will connect with its basal lamina, forming a base migrates and the cells undergo mesenchymetoepithelial transitions


ment membrane that secures the epithelial layer to the underlying when they reach their final destinations. Series of small epithelial ger
tissue. Cells deep within a mesenchymal population may synthesize minal centres, the somites, are formed, as are larger, more extensive,
matrix molecules (fibrillar or granular) to separate cells locally, open germinal epithelial sheets that line the walls of the intraembryonic
up migration routes or leave information within the matrix to act on coelom. The coelomic walls, especially those derived from somato
cell populations passing at a later time. pleure and splanchnopleure, form germinal epithelia that give rise to
Molecules of the extracellular matrix are complex; they include more the major mesenchymal populations that form the viscera. The early
than 19 individual types of collagen (some of which are capable of epithelial somites undergo further local epithelialtomesenchyme tran
being individually spliced to give more than 100 variants), proteogly sitions to form the sclerotomes, and subsequently form several germinal
cans and glycoproteins (which come in a wide variety of forms, with epithelia in the epithelial plate of each somite. Later mesenchymeto
and without binding proteins), and elastic fibres (see Ch. 2). Hyaluronic epithelium transitions are not associated with the formation of germi
acid, a glycosaminoglycan, has a vast capacity to bind water molecules nal epithelia; the most common involve the transition of mesenchyme
and so create and structure the space between the mesenchymal cells, into the endothelium of the vascular system. The nephrons of the mes
thereby producing much of the overall shape of an embryo. Experimen onephric and metanephric systems also form from mesenchymeto
tal removal of hyaluronic acid prevents the formation of cell migration epithelial transition.
KEY REFERENCES
Müller F, O’Rahilly R 2003 The prechordal plate, the rostral end of the St Jonston D, Sanson B 2011 Epithelial polarity and morphogenesis. Curr
notochord and nearby median features in staged human embryos. Cells Opin Cell Biol 23:540–6.


Tissues Organs 173:1–20. Streeter GL 1942 Developmental horizons in human embryos. Descriptions
O’Rahilly R, Müller F 1987 Developmental Stages in Human Embryos. of age group XI, 13 to 20 somites, and age group XII, 21 to 29 somites.
Washington: Carnegie Institution. Contrib Embryol Carnegie Inst Washington 30:211–45.


Cell populations at gastrulation
188.e1
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REFERENCES
Artus J, Hadjantonakis A 2012 Troika of the mouse blastocyst: lineage seg O’Rahilly R, Müller F 1987 Developmental Stages in Human Embryos.
regation and stem cells. Curr Stem Cell Res Ther 7:78–91. Washington: Carnegie Institution.
Carmona R, Cano E, Mattiotti A et al 2013 Cells derived from the coelomic St Jonston D, Sanson B 2011 Epithelial polarity and morphogenesis. Curr
epithelium contribute to multiple gastrointestinal tissues in mouse Opin Cell Biol 23:540–6.
embryos. PLoS One 8:e55890. Streeter GL 1942 Developmental horizons in human embryos. Descriptions
Davidson LA, Joshi SD, Kim HY et al 2010. Emergent morphogenesis: elastic of age group XI, 13 to 20 somites, and age group XII, 21 to 29 somites.
mechanics of a selfdeforming tissue. J Biomech 43:63. Contrib Embryol Carnegie Inst Washington 30:211–45.
Gorfinkiel N, Blanshard GB 2011 Dynamics of actomyosin contractile activ Szabó A, Rupp PA, Rongish BJ et al 2011 Extracellular matrix fluctuations
ity during epithelial morphogenesis. Curr Opin Cell Biol 23:531–9. during early embryogenesis. Phys Biol 8:045006.
Langemeijer RA 1976 Le Coelome et son revêtement comme organoblast Tao H, Suzuki M, Klyonarl H et al 2009 Mouse prickle 1, the homolog of
ème. Bull Assoc Anat 60:547–58. a PCP gene is essential for epiblast apicalbasal polarity. PNAS 106:
Loganathan R, Potetz BR, Rongish BJ et al 2012 Spatial anisotropies and 14426–31.
temporal fluctuations in extracellular matrix network texture during Zamir EA, Rongish BJ, Little CD 2008 The ECM moves during primitive
early embryogenesis. PLoS One 7:e38266. streak formation – computation of ECM versus cellular motion. PLoS


Müller F, O’Rahilly R 2003 The prechordal plate, the rostral end of the Biol 6:e247.
notochord and nearby median features in staged human embryos. Cells
Tissues Organs 173:1–20.


CHAPTER
11
Embryonic induction and cell division
(further restrictions), they are said to be determined. Determined cells
EMBRYONIC INDUCTION AND CELL DIVISION
are programmed to follow a process of development that will lead to
differentiation. The determined state is a heritable characteristic of cells
The stages of genomic activity that unfold in the fertilized zygote from and is the final step in restriction. Once a cell has become determined,
cleavage onwards are not yet fully elucidated. An admittedly simplistic it will progress to a differentiated phenotype if the environmental
but useful approach likens developmental processes to a series of binary factors are suitable.
choices (see Fig. 8.1), the first being the choice between trophoblast The process of determination and differentiation within embryonic
and inner cell mass, which specifies if the future tissue will be placenta cell populations is reflected by the ability of these populations to
and fetal membranes or embryo. produce specific proteins. Primary proteins (colloquially termed house-
Bioinformatic databases use embryological ontologies wherein the keeping proteins) are considered essential for cellular metabolism,
lineage of organs and tissues to be retraced to the earliest time (see whereas proteins synthesized as cells become determined, those specific
Commentary 2.1). They provide a temporal and spatial framework to the state of determination, are termed secondary proteins; for
against which the sequential activation of genes preceding changes in example, liver and kidney cells, but not muscle cells, produce arginase.
cell and tissue morphology can be matched. They also provide a means Fully differentiated cells produce tertiary proteins, which no other cell
of viewing all of the complex partonomy, lineage data and temporal line can synthesize, e.g. haemoglobin in erythrocytes. The range of
information occurring within embryos within a specific timeframe and housekeeping, regulatory, and tissue-specific proteomes in adult cells is
of linking these to adult anatomical tissue types. As our knowledge of presented in the Human Protein Atlas (www.proteinatlas.org).
the complexity of early developmental events increases, ontological As populations of cells become progressively determined, they can
databases will assist the correlation of embryological information: be described within a hierarchy of cellular development as transiently
current limitations are discussed in Bard (2012). amplifying cells, progenitor cells, stem cells and terminally differenti-
Our knowledge of the early events that occur in a range of mam- ated cells.


malian species and our interpretation of the significance of particular
sequences of gene expression is limited. The upregulation of early genes Transiently amplifying cells Transiently amplifying cells undergo
appears to reflect the time period of implantation. In the mouse, proliferative cell mitosis and produce cells that are equally determined.
implantation occurs one day after blastocyst formation, thus genes At some stage, and as a result of an inductive stimulus, these cells will
relevant to implantation are expressed early, whereas in cattle, implan- enter a quantal cycle that culminates in a quantal mitosis. This will
tation occurs after 14 days, by which time gastrulation has occurred and result in an increase in the restriction of their progeny, which continue
neurulation has been initiated (Oron 2012). to undergo proliferative mitoses at a progressive level of determination.
Within the developing blastocyst, the position of cells relative to The quantal mitosis corresponds to the time of binary choice when the
each other leads to epigenetic changes. The acquisition of cell polarity commitment of the progeny is different from that of the parent.
is fundamental, because this specifies apical and basolateral domains,
cell surface specializations, the position and nature of cell:cell junctions Progenitor cells Progenitor cells are already determined along a
and the concomitant positioning of intracellular organelles and particular pathway. They may individually follow that differentiation
cytoskeletal elements, which in turn leads to the location of future pathway, or may proliferate and produce large numbers of similarly
deposition of extracellular proteins. The two cell phenotypes, epithelial determined progenitor cells that subsequently differentiate; neuroblasts
and non-epithelial cells, are specified during cleavage and early compac- or myoblasts are examples of progenitor cells.
tion within the zona pellucida. All relevant genes required to maintain
these cell shapes and behaviours are upregulated at these very early Stem cells Either individually or as a population, stem cells can both
stages. During the formation of trophoblast, hypoblast (primitive endo- produce determined progeny and reproduce themselves (see Commen-
derm) and epiblast, the cells respond locally to their environment, tary 1.2). It is generally believed that stem cells undergo asymmetric


specifically to other cells that are positioned lateral and basal to them. divisions, in which one daughter cell remains as a stem cell, while the
The genes operating at these early times in the mouse embryo are given other proceeds along a differentiation pathway; in marked contrast,
in Takaoka and Hamada (2012). proliferative cell division may be symmetrical, producing derived cells
Cell populations within the embryo interact to provide the develop- with an identical determination. Human embryonic stem cells (hESCs)
mental integration and fine control necessary to achieve tissue-specific are pluripotent cells that can be derived from the inner cell mass of
morphogenesis. In the early embryo, such interactions may occur only human blastocysts in vitro; or obtained surplus to in vitro fertilization
if particular regions of the embryo are present, e.g. signalling centres or fertility programmes; or created from oocytes donated and fertilized for
organizers. As the embryo matures, some interactions tend to occur that purpose. Although not yet achieved, it is hoped that hESCs can be
between adjacent cell populations, e.g. epithelium and mesenchyme, coaxed down particular pathways under appropriate pharmaceutical
and later between adjacent differentiating tissues, e.g. between nerves conditions to produce differentiated cells that will be effective in revers-
and muscle, or between muscle and skeletal elements. The interactions ing some degenerative diseases (e.g. dopamine-producing neurones for
between adjacent epithelia and underlying connective tissue continue Parkinson’s disease, insulin-producing islet cells for diabetes), or to
throughout embryonic and fetal life and extend into postnatal life. In replace acutely damaged tissues (e.g. motor neurones for acute spinal
the adult, these interactions also permit the metaplastic changes that cord injury, cardiomyocytes in acute myocardial infarction). Proof of
tissues can undergo in response to local environmental conditions. principle has been demonstrated in some animal models, and multipo-
Tissue interactions result in changes or reorganization of one or tent haemopoietic progenitor stem cells from human umbilical cord
both tissues, which would not have occurred in the absence of the blood are now used as an alternative treatment to bone marrow trans-
tissue interactions. The process of tissue interaction is also called induc- plantation for the treatment of some inherited genetic disorders (thalas-


tion, i.e. one tissue is said to induce another. The ability of a tissue to saemia) and blood malignancies (leukaemias).
respond to inductive signals is called competence, and denotes the
ability of a cell population to develop in response to the environments Terminally differentiated cells By virtue of their extreme special-
present in the embryo at that particular stage. After a cell population ization, terminally differentiated cells can no longer divide, e.g. eryth-
has been induced to develop along a certain pathway, it will lose com- rocytes and neurones. Apoptosis is a particular variety of terminal
petence and become restricted. Once restricted, cells are set on a par- differentiation in which the final outcome is the death of the individual
ticular pathway of development; after a number of binary choices cells or cell populations. It occurs in the developing limb, where cells 189


Embryonic induction and cEll division
190
2
noitcEs
die along the pre- and postaxial limits of the apical ectodermal ridge, mesenchyme, which results in the morphogenesis of mammary
limiting its extent, and also between the digits, permitting their gland-like structures; chickens do not normally develop mammary
separation. glands.
Tissue interactions continue into adult life and are probably respon-
Tissue interactions 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.
There are two types of cell and tissue interaction, namely: permissive
The junctions between the mucosa of the vestibule and the lip, and
and instructive.
between the vermilion border and the facial skin, are distinct bound-
In a permissive interaction, a signal from an apposing tissue is neces-
aries of specific epithelial and mesenchymal differentiation, and are
sary for the successful self-differentiation of the responding tissue. This
almost certainly maintained by continuing epithelial–mesenchymal
means that a particular cell population (or the matrix molecules
interactions in adult life. Perturbation of these interactions throughout
secreted by the cells that it contains) will maintain mitotic activity in
the body may underlie a wide variety of adult diseases, including sus-
an adjacent cell population. Since a variety of different cell populations
ceptibility to cancer and proliferative disorders.
may permit a specific cell population to undergo mitosis and cell dif-
ferentiation, no specific instruction or signal that may limit the devel-
Signalling between embryonic cells
opmental options of the responding tissue is involved; this signal
and tissues
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 Cellular interactions may be signalled by four principal mechanisms:
order to express this capacity. Permissive interactions often occur later direct cell–cell contact; cell adhesion molecules and their receptors;
in development, when a tissue whose fate has already been determined extracellular matrix molecules and their receptors; and growth factors
is maintained and stabilized by another. and their receptors. Many of these mechanisms interact, and it is likely


An instructive (directive) interaction (induction) changes the cell that combinations of them are involved in development. Figure 11.1
type of the responding tissue, so that the cell population becomes illustrates some ways by which mesenchymal cells could signal to epi-
restricted. Wessells (1977) proposed four general principles in most thelial cells. An additional set of identical mechanisms could operate
instructive interactions: in the presence of tissue A, responding tissue B for epithelial–mesenchymal cell signalling. Clearly, the complexity of
develops in a certain way; in the absence of tissue A, responding tissue these mechanisms will increase in reciprocal interactions; moreover, a
B does not develop in that way; in the absence of tissue A, but in the single molecule may have different effects on epithelial and mesen-
presence of tissue C, tissue B does not develop in that way; and in the chymal cells.
presence of tissue A, a tissue D, which would normally develop differ- Direct cell–cell contact permits the construction of gap junctions,
ently, is changed to develop like tissue B. which are important for communication and the transfer of informa-
These principles are exemplified during induction of the lens vesicle tion between cells. The transient production of gap junctions is seen as
by the optic cup. An example of last-mentioned principle is the experi- epithelial somites are formed, between neuroepithelial cells within
mental association of chicken flank ectoderm with mouse mammary rhombomeres, and in the tunica media of the outflow tract of the heart.
Epithelium Basal lamina Mesenchyme cell
1
1 Direct cell–cell contact by gap junctions.
2
2 Cell–cell contact by cell adhesion molecules.
3
3 A soluble factor (growth factor) reacting with a receptor for that factor on the epithelial cells.
4
4 Extracellular matrix molecule secreted by the mesenchyme cells interacting with a receptor on the epithelial cell.
5
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


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
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 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
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 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.
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.


morphogenesis and pattern formation
191
11
rEtPaHc
Endogenous electrical fields are also believed to have a role in cell–cell at the tip of the acinus is therefore split into two, and two branches
communication. Such fields have been demonstrated in a range of develop from this point.
amphibian embryos, and in vertebrate embryos during primitive streak Pattern formation concerns the processes whereby the individual
ingression. Neuroepithelial cells are electrically coupled, regardless of members of a mass of cells, initially apparently homogeneous, follow
their position relative to inter-rhombomeric boundaries. a number of different avenues of differentiation that are precisely
The spatial and temporal distribution of a variety of cell adhesion related to each other in an orderly manner in space and time. The pat-
molecules has been localized in the early embryo. The appearance of terns embraced by the term apply not only to regions of regular geo-
these molecules correlates with a variety of morphogenetic events that metric order, e.g. the crystalline lens, but also to asymmetric structures
involve cell aggregation or disaggregation; e.g. an early response of such as the limb. For such a process to occur, individual cells must be
groups of cells to embryonic inductive influences is the expression of informed of their position within the embryo, and utilize that informa-
cadherins, calcium-dependent adhesion molecules typically found in tion for appropriate differentiation. Patterning of regions is seen in: the
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-
Extracellular matrix Blood vessel Mesenchyme cell
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 1
for extracellular matrix molecules give rise to a number of congenital
disorders, e.g. mutations in type I collagen produce osteogenesis imper-
2
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, 3
namely: endocrine, autocrine, paracrine, intracrine, juxtacrine and 4
matricrine (Fig. 11.2). 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
5
protein, in the case of insulin-like growth factors. 6
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 1 Endocrine Delivery of growth factor by the blood stream from a distant biosynthetic site to the
embryo, particularly during early development. target mesenchyme cell.
The most obvious examples of morphogenesis are the large migra-
2 Autocrine Synthesis of growth factor by the cell, its secretion, binding and activation of a surface
tions that occur during gastrulation; local examples include branching receptor elsewhere on its own surface.
morphogenesis, which occurs in the developing lungs and kidneys, for
example, and in most glandular organs. The development of branches 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.
from a tubular duct occurs over a period of time. An interaction between
the proliferating epithelium of the duct and its surrounding mesen-
4 Juxtacrine Synthesis of growth factor by the cell. The growth factor remains on the cell surface
chyme and extracellular matrix results in a series of clefts that produce and binds to and activates a receptor on an immediately adjacent cell.
a characteristic branching pattern (Fig. 11.3). During tubular and acinar
development, hyaluronidase secreted by the underlying mesenchymal 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.


cells breaks down the basal lamina produced by the epithelial cells; this
increases epithelial mitoses locally and results in an expanding acinus. 6 Matricrine Synthesis and export of growth factor from the cell. The growth factor binds to the
Cleft formation is initiated by the mesenchyme, which produces col- extracellular matrix where it remains active and subsequently binds to and activates a receptor for
lagen III fibrils within putative clefts. (If the collagen is removed, no that growth factor on the same or a different cell.
clefts develop, whereas if excess collagen is not removed, supernumer- Fig. 11.2 In addition to the mechanisms described in Figure 11.1, cells
ary clefts appear.) The collagen acts to protect the basal lamina from may also communicate by the reception, production and secretion of
the effects of the hyaluronidase, which means that the overlying epithe- growth factors. A typical embryonic mesenchyme cell may receive and
lia have a locally reduced rate of mitosis. The region of rapid mitoses produce growth factors in this way.
A B
Collagen
Narrow cleft Hyaluronidase produced
by mesenchyme cells
Collagen fibrils
Mesenchyme cells
Hyaluronidase
Epithelial cells
Collagen fibrils
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.)


Embryonic induction and cEll division
192
2
noitcEs
progress zone and zone of polarizing activity within the limbs; the fates Wnt family and hedgehog signalling molecules. A range of cell receptor
of the medial and lateral, and later the cranial and caudal, halves of the molecules are also required. For details on individual members within
somites; and the neural crest mesenchyme within the pharyngeal this range of factors, see Carlson (2014).
arches. For details of patterning in vertebrate development, see Tickle
(2003). Experimental approaches to embryology
Genes in development
One of the most exciting techniques to provide information on cell
movements and fates during development is the use of chimeric
Two related themes have emerged from experimental studies of devel- embryos. Small portions of an embryo are excised and replaced with
opment: first, that the control of embryonic morphology has been similar portions of an embryo from a different species at the same stage,
highly conserved in evolution between vertebrates and invertebrates; and the resulting development is then studied. This technique has been
and second, that this control involves families of genes coding for particularly effective using chick and quail embryos because the nucle-
proteins that act as transcriptional regulators. olus is especially prominent in all quail cells, whereas it is not promi-
Homeobox genes are believed to be responsible, at least in part, for nent in chick cells, which means that quail cells may be easily identified
the evolutionary origin of the embryonic body plan (Robert 2001). within a chick embryo after chimeric transplantation (Le Douarin
Experimental study of transgenic animals in which the homeobox genes 1969). The technique has also confirmed the reciprocity of tissue inter-
have been knocked out provide some evidence of their function; action between the embryonic species, a phenomenon that had previ-
however, because developmental processes permit significant recovery ously been illustrated, for a limited period, in co-cultures of embryonic
from insult, some of the outcomes cannot be directly interpreted as avian and mammalian tissues. Somite development and vertebral for-
demonstrating the effect of such gene loss. mation have been studied in mouse–chick chimeras (Fontaine-Pérus


Other gene families required for normal development include the 2000). The production, in vitro, of human–animal chimeric cell lines is
T-box family, Helix-Loop-Helix transcription factors and Sox genes; the providing new ways of studying cellular pathways, as is the introduction
signalling factors, transforming growth factor beta (TGF-β), bone mor- of human artificial chromosome vectors into animal cells to study their
phogenetic proteins (BMPs), fibroblast growth factor family (FGF), the interaction.
KEY REFERENCES
Bard J 2012 A new ontology (structured hierarchy) of human developmental Oron E, Ivanova N 2012 Cell fate regulation in early mammalian develop-
anatomy for the first 7 weeks (Carnegie stages 1–20). J Anat 221: ment. Phys Biol 9:045002.
406–16. Robert JS 2001 Interpreting the homeobox: metaphors of gene action and
Carlson BM 2014 Human Embryology and Developmental Biology, 5th ed. activation in development and evolution. Evol Dev 3:287–95.Takaoka
Philadelphia: Elsevier, Saunders; Ch. 4. K, Hamada H 2012 Cell fate decisions and axis determination in the
Fontaine-Pérus J 2000 Mouse–chick chimera: an experimental system for early mouse embryo. Development 139:3–14.
study of somite development. Curr Top Dev Biol 48:269–300. Tickle C 2003 Patterning in Vertebrate Development. Oxford: Oxford Uni-
Le Douarin NM 1969 Particularités du noyau interphasique chez la caille versity Press.
japonaise (Coturnix coturnix japonica). Utilisation de ces particularités Wessells NK 1977 Tissue Interaction and Development. Menlo Park, CA:
comme ‘marquage biologique’ dans les recherches sur les interactions Benjamin.
tissulaires et les migrations cellulaires au cours de l’ontogenèse. Bull Biol
Fr Belg 103:435–52.


Embryonic induction and cell division
192.e1
11
rEtPaHc
REFERENCES
Bard J 2012 A new ontology (structured hierarchy) of human developmen- Oron E, Ivanova N 2012 Cell fate regulation in early mammalian develop-
tal anatomy for the first 7 weeks (Carnegie stages 1–20). J Anat 221: ment. Phys Biol 9:045002.
406–16. Robert JS 2001 Interpreting the homeobox: metaphors of gene action and
Carlson BM 2014 Human Embryology and Developmental Biology, 5th ed. activation in development and evolution. Evol Dev 3:287–95.
Philadelphia: Elsevier, Saunders; Ch. 4. Takaoka K, Hamada H 2012 Cell fate decisions and axis determination in
Fontaine-Pérus J 2000 Mouse–chick chimera: an experimental system for the early mouse embryo. Development 139:3–14.
study of somite development. Curr Top Dev Biol 48:269–300. Tickle C 2003 Patterning in Vertebrate Development. Oxford: Oxford Uni-
Le Douarin NM 1969 Particularités du noyau interphasique chez la caille versity Press.
japonaise (Coturnix coturnix japonica). Utilisation de ces particularités Wessells NK 1977 Tissue Interaction and Development. Menlo Park, CA:
comme ‘marquage biologique’ dans les recherches sur les interactions Benjamin.
tissulaires et les migrations cellulaires au cours de l’ontogenèse. Bull Biol
Fr Belg 103:435–52.


Cell populations at the start CHAPTER
12
of organogenesis
contains the transmedian pericardial cavity, which communicates
SPECIFICATION OF THE BODY AXES AND THE
dorsocaudally with right and left pericardioperitoneal canals. These
BODY PLAN pass dorsally to the transverse septum mesenchyme and open caudally
into the extraembryonic coelom on each side of the midgut. The intra
Embryos may be thought of as being constructed with three orthogonal embryonic mesenchyme has begun to differentiate and the paraxial
spatial axes (cephalocaudal, dorsoventral and laterolateral), plus a tem mesenchyme is being segmented into somites. Neural groove closure is
poral axis. In mammalian embryos, axes cannot be specified at very progressing caudally, so that a neural tube is forming between the newly
early stages; embryonic axes can be defined only after the early extra segmenting somites. Rostrally, the early brain regions, which have not
embryonic structures have been formed and the inner cell mass can be yet fused, can be discerned. The neuroepithelium is separated from the
seen. The position of the future epiblast can be predicted in human dorsal aspect of the gut by the notochord. The earliest blood vessels
embryos when the hollow blastocyst has formed. The inner cell mass have appeared and a primitive tubular heart occupies the pericardium.
becomes (seemingly) randomly located on the inside of the trophecto The chorionic circulation is soon to be established, after which the
derm and forms a population of epiblast cells subjacent to the tropho embryo rapidly becomes completely dependent on the maternal blood
blast. This region implants first. It is not known whether the stream for its requirements. The embryo is connected to the developing
trophectoderm in contact with the inner cell mass initiates implanta placenta by a mesenchymal connecting stalk in which the umbilical
tion, so that the future dorsal surface of the embryo is closest to the vessels develop, and which also contains the allantois, a hindgut diver
disrupted maternal vessels at the implantation site, or whether the inner ticulum. The lateral body walls are still widely separated. The embryo


cell mass can travel around the inside of the trophoblast to gain a posi has contact with three different vesicles: the amnion, which is in contact
tion subjacent to the implantation site once implantation has started. with the surface ectoderm; the yolk sac, which is in contact with the
Axes may be conferred on the whole embryonic disc, which is ini endoderm; and the chorionic cavity, containing the extraembryonic
tially flat and mainly twodimensional. However, their subsequent ori coelom, which is in contact with the intraembryonic coelomic lining
entation in the folded threedimensional embryo will be completely (see Fig. 10.10).
different. The dorsal structures of the folded embryo form from a cir The early body plan of the embryo is segmented. The boundaries
cumscribed central ellipse of the early flat embryonic disc (see Fig. between the segments are maintained by the differential expression of
10.5). Lateral and ventral structures form from the remainder of the genes and proteins that restrict cell migration in these regions. Organo
disc, and the peripheral edge of the disc eventually becomes constricted genetic processes either retain the segmental plan, e.g. spinal nerves, or
at the umbilicus (see Figs 10.9–10.10). Although the appearance of part replace it locally, e.g. the modifications of somatic intersegmental
of the epiblast is taken to specify the dorsal surface of the embryo, the vessels by the development of longitudinal anastomoses. Abnormalities
inner layer, i.e. the hypoblast, is not, by default, a ventral embryonic may result from improper specification of segments along the cephalo
structure. caudal axis and may fail to produce the appropriately modified segmen
The primary, cephalocaudal, axis is conferred by the appearance of tal plan.
the primitive streak in the bilaminar disc. The primitive streak patterns The degree to which vertebrate embryos are developmentally con
cells during ingression, and so also specifies the dorsoventral axis, strained at this period of development is controversial. Comparative
which becomes apparent after embryonic folding. The position of studies on the timing at which specific embryonic structures appear,


ingression through the streak confers axial, medial or lateral character heterochrony, have shown that other embryonic species do not follow
istics on the forming mesenchyme cells. The axial and medial popula the same developmental sequence as humans (Richardson and Keuck
tions remain as dorsal structures in the folded embryo, and the surface 2002). Although some developmental mechanisms are highly con
ectoderm above them will exhibit dorsal characteristics. The lateral plate served, e.g. the homeobox gene codes, others may have been dissociated
mesenchyme will assume lateral and ventral positions after embryonic and modified in different vertebrate species during evolution.
folding, and the surface ectoderm above this population will gain Organogenesis, the further development of body regions and organs
ventral characteristics. that is described elsewhere in this book, starts from about stage 10
The third and last spatial axis is the bilateral, or laterolateral, axis, (approximately 28 days). Although it is both conventional and conven
which appears as a consequence of the development of the former two ient to consider the further development of each body system on an
axes. Initially, the right and left halves of the embryonic body are bilat individual basis, not only do all systems develop simultaneously, but
erally symmetric. Lateral projections, the upper and lower limbs, also they interact and modify each other as they develop. This necessary
develop in two places on each side of the body wall (somatopleure). interdependence is supported by the evidence of experimental embry
With the last axis established, the temporal modification of the ology and reinforced by the phenomena of growth anomalies, which
original embryonic axes can be seen. The segmental arrangement of the cut across the artificial boundaries of systems in most instances. For
cephalocaudal axis is very obvious in the early embryo and is retained these reasons, it is recommended that the development of an individual
in many structures in adult life. Similarly, dorsal embryonic structures system or body region should be studied in relation to others, especially
remain dorsal and undergo relatively little change. However, structures those most closely associated with it, whether spatiotemporally or
that were originally midline and ventral, especially those derived from causally.
splanchnopleuric mesenchyme, e.g. the cardiovascular system and the


gut, are subject to extensive shifts, and change from a bilaterally sym
EMBRYONIC CELL POPULATIONS AT THE START
metric arrangement to an entire body that is now chiral, i.e. has distinct
OF ORGANOGENESIS
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 The developmental processes operating in the embryo between stages
attained a recognizable body plan. In human embryos, this corresponds 5 and 9 enabled the construction of the bi and trilaminar embryonic
to the end of stage 10 (Fig. 12.1). The head and tail folds are well disc, the intraembryonic coelom and new proliferative epithelia. From
formed, with enclosure of the foregut and hindgut (proenteron and the end of stage 10, a range of local epithelial and mesenchymal popula
metenteron), although the midgut (mesenteron) is only partly con tions now interact to produce viscera and appendages. The inductive
stricted from the yolk sac. The forebrain projection dominates the influences on these tissues and their repertoire of responses are very
cranial end of the embryo, and the buccopharyngeal membrane and different from those seen at the onset of gastrulation. The range of
cardiac prominence are caudal and ventral to it. The cardiac prominence tissues present at the start of organogenesis, when the body plan is clear, 193


Cell populations at the start of organogenesis
194
2
noitCes
A B
Neural tube
Pharynx
Pericardial cavity
Amnion Pericardial cavity
Pericardioperitoneal canal
Foregut
Yolk sac wall Entrance to pericardioperitoneal canal
Neural tube
Somite
Midgut
Yolk sac wall
Left umbilical vein
Peritoneal cavity
Hindgut
Umbilical cord
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.
is given below and shown in Figures 12.1 and 12.2. For a summary of then passes over the occipital and cervicothoracic parts of the embryo,
the fates of the embryonic cell populations, see Figure 12.3. superficial to the four occipital somites and later to the occipitocervical
junction. Further caudally, it is associated with the upper limb field,
Epithelial populations in the embryo 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
Surface ectoderm
intraembryonic coelom, and later (between stages 12 and 13), the
During embryogenesis, the surface ectoderm shows regional differences
mesonephric duct and ridge. In stages 14 and 15, this portion of the
in thickness. Ectoderm over the dorsal region of the head and trunk is
ectodermal ring gives rise to the mammary line. Caudal to the lower
thin, as is the pericardial covering; this has been interpreted as a con
limb field, in the unfolded embryo, the ring passes distal to the cloacal