Download Origin, Early Patterning, and Fate of the Mouse Epiblast

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Origin, Early Patterning, and Fate
of the Mouse Epiblast
Anne Camus, Aitana Perea-Gomez, and Jérôme Collignon
Introduction
The epiblast can first be identified as a tissue at the late blastocyst stage, at embryonic day 4.0 (E4.0), when it consists
of no more than 30 apolar cells. The epiblast is known to
generate extraembryonic mesoderm and all fetal cell lineages,
including the germ line. This pluripotency is its most distinctive property. It has to be distinguished from the totipotency
of the blastomeres of earlier cleavage-stage embryos, which
can produce all embryonic and extraembryonic cell lineages
of the conceptus, including the trophectoderm. This chapter
reviews what is known about the formation, the patterning,
and the fate of the epiblast in the mouse embryo. It presents
the latest findings in the field and attempts to complement
earlier reviews.1–6 An important aspect of the establishment of
the epiblast lineage, no doubt critical in the regulation of its
differentiation, is the role of chromatin modifications in the
regulation of gene expression. This is not covered in this
chapter, but relevant information can be found in Chapter 6 of
this book and in several reviews.7–9
Origin and Properties of the Mouse
Epiblast
FORMATION OF THE EPIBLAST
The major differences between the development of mice and
that of other vertebrates at early stages is the slow pace of the
first cleavages and their asynchrony. The first plane of cleavage is meridional, more or less parallel to the animal–vegetal
(AV) axis, which is marked by the position of the second polar
body at the animal pole of the zygote (Fig. 11–1). The zygotic
genome starts to be expressed at the end of the two-cell stage,
36 hours after fertilization. At the beginning of the eight-cell
stage, individual blastomeres are still clearly visible, but as
they become polarized and flattened in a process called
compaction, the whole embryo takes a more spherical shape.
The compaction results in the fourth and fifth division cleavage producing either outer polar cells or inner apolar cells.
Aggregation experiments have shown that unlike inner cells,
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outer cells rapidly lose their totipotence (reviewed by
Pedersen10). They will essentially form the trophectoderm,
which will contribute exclusively to extraembryonic structures. This is the first apparent lineage segregation in mouse
development. Trophectoderm cells secrete a fluid that, trapped
inside by tight junctions established between outer cells,
contributes to the formation and the expansion of a cavity
termed the blastocoel. The inner cells remain together, positioned on one side of the hollow sphere of trophectoderm
cells, where they form the inner cell mass (ICM). The trophectoderm overlying the ICM is called the polar trophectoderm. Interaction with the ICM is critical for polar
trophectoderm cells to remain diploid and to proliferate. In
contrast, cells, from the mural component of the trophectoderm, lining the blastocoel, stop dividing and become polyploid. The embryo at this stage is called a blastocyst. Its AV
axis is inherited from the zygote. Its embryonic–abembryonic
axis is perpendicular to the AV axis, and together they define
the plane of bilateral symmetry of the blastocyst (Fig. 11–1,
reviewed by Gardner5 and Zernicka-Goetz6). Between E3.5
and E4.5, the primitive endoderm (PrE) differentiates at the
blastocoelic surface of the ICM. The remainder of the ICM can
then be called epiblast, or embryonic ectoderm. The polar trophectoderm will produce the extraembryonic ectoderm (ExE).
At this stage, the embryo hatches from the zona pellucida and
implants in the uterine wall. PrE cells see their developmental
fates restricted to extraembryonic tissues (visceral and parietal
endoderm), whereas epiblast cells retain the potential to
generate all embryonic cell lineages. By the late gastrula stage,
the epiblast will have produced both embryonic and extraembryonic mesoderm, germ cells, definitive endoderm, neuroectoderm, and surface ectoderm (reviewed by Tam and Behringer1).
EARLY ALLOCATION OF CELLS TO THE ICM
The potency of cleavage-stage blastomeres was examined in the
mouse either by reducing their number using disaggregation–
reaggregation techniques, or aggregating morulae to form
giant embryos. These classic studies showed that despite drastic alterations, development could proceed and lead to normal
animals (reviewed by Pedersen10). These regulative abilities
suggested that early blastomeres, at least up to the differentiation of the trophectoderm, were equivalent. This data, with
the apparent late onset of embryonic polarity, seemed to make
the possibility of an early specification of the blastomeres
irrelevant to the actual patterning of the embryo (reviewed by
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Animal
Vegetal
2nd polar body
2-cell
E1.5 (20–38h)
Fetrilized egg
E0.0
3-cell
4-cell
E2.0 (38–50 h)
8-cell
E2.5 (50–62 h)
Zygotic transcription
Embryonic
Polar TE
Epiblast
PrE
ICM
Mural TE
Blastocoel
Abembryonic
Compacted 8-cell
E2.5
16-cell/morula
E3.0 (62/74 h)
Compaction
Implantation
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Blastocycst/32 to 64-cell
E3.5
Trophectoderm formation
Primitive endoderm formation
ExE
VE
Hatched blastocyst
256-cell
E4.5
Primitive
streak
Epiblast
PE
AVE
Distal VE
E5.5
(120 epiblast cells)
E6.0/pre-streak
(250 epiblast cells)
Epithelialization/Cavitation
E6.5/early-streak
(660 epiblast cells)
E7.0/mid-streak
(3300 epiblast cells)
Gastrulation
Figure 11–1. Mouse development from fertilization to gastrulation. The first lineage to be determined is the trophectoderm at the morula stage.
At E4.5, a second extraembryonic lineage, the primitive endoderm, is located at the blastocoelic surface of the inner cell mass. After implantation, epiblast cells retain their pluripotency until the mid- to late streak stage and produce the three definitive germ layers during gastrulation (TE,
trophectoderm; PrE, primitive endoderm; PE, parietal endoderm; VE, visceral endoderm; AVE, anterior visceral endoderm; and ExE, extraembryonic ectoderm).
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Origin, Early Patterning, and Fate of the Mouse Epiblast
Gardner5 and Zernicka-Goetz6). However, recent work has
shown that in most embryos, the two blastomeres of the twocell stage do not contribute equally to the different cell lineages that constitute the blastocyst.11,12 The study of this
phenomenon has brought new insights into the early allocation of precursors to the epiblast lineage.
Lineage studies using nonintrusive labeling techniques
demonstrated that the embryonic–abembryonic axis is set up
at the two-cell stage, orthogonal to the first plane of cleavage
of the zygote.11,12 This suggested that when development
proceeds unperturbed, the embryonic and abembryonic halves
of the blastocyst are predominantly made of descendants from
either one or the other two-cell stage blastomere. Labeling
studies showed that the clonal boundary between descendants
of the two-cell blastomeres is maintained at least up to the
early blastocyst stage.12,13 What can account for the different
fates of the two-cell blastomeres? Because cell divisions
become asynchronous as early as the two-cell stage, an early
dividing and a late dividing blastomere can be distinguished.
Piotrowska’s labeling study confirmed an earlier hypothesis
that early dividing blastomeres contributed more descendants
to the ICM than late dividing ones.12,14,15 This suggested that
a shorter cell cycle inherited by the descendants of the early
dividing blastomere could result in an apparent prepattern of
the two-cell stage embryo. Labeling studies showed that the
sperm entry position (SEP) often predicted the early dividing
blastomere as well as the position of the first plane of cleavage.12,16,17 The possibility that the sperm may contribute to the
patterning of a mammalian embryo has some appeal because
it echoes its role in some other vertebrates and could be seen
as a trace of an ancient patterning mechanism. In contrast to
what happens in wild-type embryos, clonal analysis of twocell blastomere descendants in parthenogenetic embryos
found no difference in their respective fates.13 This further
supported the notion that sperm contributes to the patterning
of the blastocyst. In addition, ablation of the cortical region at
the SEP disturbed the customary embryonic–abembryonic
patterning of two-cell blastomeres descendants.13 The role of
the SEP in embryo patterning, however, is disputed. Davies
and Gardner found no consistent relationship between the
SEP and the orientation of the first plane of cleavage.18 Other
studies suggest that a shorter cell cycle may not be determinant for the preferential contribution of one blastomere to the
ICM.13,19 Live imaging of developing in vitro–fertilized
embryos may help to resolve these issues.
SEGREGATION OF THE EPIBLAST LINEAGE
The PrE appears as an epithelium on the blastocoelic surface
of the ICM at the late blastocyst stage (E4.5). A basal lamina
separating the PrE from the epiblast is promptly synthesized
(Fig. 11–1). Cell lineage–tracing studies in chimeric embryos
have found that by this stage, the potential of PrE cells and
epiblast cells has become restricted to their respective lineages.20,21 This is the second lineage segregation event in
mouse development. Labeling studies have shown that 1 day
earlier, at the early blastocyst stage, ICM cells lining the
blastocoel frequently comprise descendants from both
blastomeres of the two-cell stage embryo.12 Cell lineage studies showed that these ICM cells produce either PrE descendants or epiblast, mixed clones remaining a rarity.22,23 This
implies that PrE specification is nearing completion but also
that the ICM is still a mixture of both types of precursors. This
would suggest that the formation of the PrE does not result
from a simple induction of the top layer of the ICM to adopt
an endodermal fate. Instead, it could involve an early specification event in a subset of ICM cells and a subsequent cellsorting mechanism, bringing endoderm precursors to the
blastocoelic surface. Genetic analysis may support this
hypothesis. Gata6 is a zinc-finger transcription factor placed
by functional studies at the top of the genetic cascade that
controls the establishment of the PrE and the differentiation of
its derivatives.24,25 Gata6 is expressed at the early blastocyst
stage in a subset of ICM cells, salt-and-pepper fashion, before
becoming uniformly expressed in the PrE layer when it forms
at E4.5.25 Although the dynamic of Gata6 expression in the
ICM is suggestive of a cell-sorting mechanism, this has not
been formally proven. The inactivation of the signal transduction adapter protein encoded by Disabled2 (Dab2), a direct
target of Gata6,26 however, completes the picture. In Dab2
mutant embryos, PrE cells are specified, but they do not form
an epithelial layer separated from the epiblast.27 Instead, they
are found embedded in the epiblast, suggesting they failed to
reach its blastocoelic surface. The authors propose that Dab2mutant PrE cells fail to respond to extracellular cues normally
involved in positioning them. Interestingly, embryos mutant
for γ1-laminin, which cannot assemble a basal lamina, have a
similar phenotype.28
MOLECULAR CONTROL OF PLURIPOTENTIALITY
Embryonic stem (ES) cells are pluripotent cells derived from
cultured blastocysts. They can be maintained undifferentiated
in culture for extended periods of time, expanded, reintroduced in an embryonic context, and found to contribute to all
embryonic lineages (reviewed by Smith29). Their pluripotency
corresponds to that of the epiblast at the late blastocyst stage,
when it looses the ability to form PrE. They have been used
extensively to investigate the molecular basis of pluripotentiality. A specific feature of mouse gestation may have facilitated the derivation of ES cell lines in this species. Female
mice can delay the implantation of blastocysts and keep their
development on hold for up to 3 or 4 weeks, a situation termed
diapause. This occurs when fertilization happens while they
are still nursing a litter, or it can be induced experimentally by
a postfertilization ovariectomy. The molecular pathway
involved in this phenomenon also operates in ES cells. The
self-renewing capacity of ES cells has been found to depend
on the secretion by cocultured feeder cells of a cytokine,
called LIF, that signals via the gp130/LIF receptor complex.
The transduction of this signal operates through a JAK pathway to activate the transcription regulator Stat3, which suppress differentiation in ES cells (Fig. 11–2). Interestingly, the
inactivation of LIF or gp130/LIFR in the embryo does not
result in early developmental defects, but it prevents mutant
blastocysts from recovering from diapause (reviewed by
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Lineage
Induction
Repression
Hypothetical
ICM
Morula
Oct3/4
Mature epiblast
Epiblast
Nanog
Stat3
ES cells
PrE
Trophectoderm
Differentiated ES cells
Figure 11–2. Molecular control of pluripotentiality. The names of the genes are underlined. See the
section “Molecular Control of Pluripotentiality” for comments.
Smith29). The epiblast may therefore rely on a gp130 independent pathway for its expansion during unperturbed development but switches to the gp130 pathway when implantation
must be delayed. This suggests that ES cells may represent a
specific state of the epiblast. ES cells have nevertheless
helped to demonstrate that growth and differentiation can be
separated and the critical role of extrinsic factors in controlling progression of the latter.
Cellular pluripotentiality is, however, clearly dependent
on the presence of intrinsic factors. The expression of
Oct-3–4, a POU family transcription factor, in early blastomeres, ICM cells, epiblast cells, germ cells, and ES cells was
suggestive of a possible role in determining pluripotentiality.
In its absence, ICM cells become nonproliferating trophoblast
cells.30 Oct-3–4, therefore, acts in ICM cells to prevent
their differentiation into trophectoderm (Fig. 11–2). In vitro
studies suggest the Oct-3–4 relationship with pluripotentiality
is complex, as its presence is required to maintain the selfrenewing capacity of ES cells, but that it promotes their
differentiation into extraembryonic endoderm when transiently expressed at higher levels.31 Another function of
Oct-3–4 in ICM cells is to activate Fgf4 production to promote in a paracrine fashion the maintenance and proliferation
of a trophoblast stem-cell population in the adjacent polar
trophectoderm.
Nanog, a divergent homeodomain-containing transcription
factor, was recently identified as another determinant of
pluripotentiality. Its expression is first detected in inner
cells of morula-stage embryos, remains on in ICM cells and
off in trophoblast cells at the blastocyst stage, becomes
restricted to the epiblast after PrE differentiation, and is downregulated at implantation.32,33 It was also found in primordial
germ cells and in some cultured pluripotent cell lines. The
ICM of embryos deficient for Nanog differentiate completely
into parietal endoderm (PE) but not into trophoblast.33
Thus, Nanog may be required later than Oct-3–4 for maintenance of pluripotency in epiblast progenitors (Fig. 11–2). ES
cells deficient for Nanog also lost pluripotentiality and produced extraembryonic endoderm.32,33 However, forced
expression of Nanog in ES cells could bypass their requirement for the LIF/Stat3 pathway. These cells maintained Oct3–4 expression and a self-renewing capacity that resisted
attempts to promote differentiation.32,33 The induction of
Nanog expression is independent of that of Oct-3–4 as it was
readily detected in Oct-3–4−/− mutant embryos. The main
function of Nanog seems to be to fend off PrE differentiation.
The onset of its expression fittingly corresponds to the timing
of specification of this tissue, as suggested by the expression
of Gata6. Given the opposite effects of Oct-3–4 and Nanog
regarding PrE differentiation, it will be interesting to investigate how they might regulate Gata6 expression.
Two other transcription factors have been found to play
roles in the maintenance of the epiblast, but genetic studies
suggest their activity is required somewhat later. Sox2 is an
HMG box-containing transcription factor. Its inactivation
leads to a failure to maintain the epiblast beyond the time of
PrE differentiation.34 As a result, the mutant conceptus contains only PE cells and trophoblast cells. The activation of
Fgf4 transcription in the epiblast, possibly required for the
maintenance of the polar trophectoderm lineage, is dependent
on the association of Oct-3–4 with Sox2.35 An earlier role of
Sox2 within the ICM could be masked by the persistence of
maternal protein up to the blastocyst stage. Foxd3 is a winged
helix–forkhead family transcription factor also known to
interact with Oct-3–4. Foxd3–/– mutant blastocysts look normal
and still express Oct-3–4, Sox2, and Fgf4, but their ICM cells
fail to expand when developed in culture.36 However, mutant
embryos do not present overt abnormalities before E6.5. The
role of Foxd3 seems to be in the maintenance of epiblast
progenitors.
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Origin, Early Patterning, and Fate of the Mouse Epiblast
EPITHELIALIZATION OF THE EPIBLAST
The PrE differentiates to form the visceral endoderm (VE)
and PE. PE cells migrate out of the PrE to line the entire blastocoelic cavity (Fig. 11–1). They secrete extracellular matrix
components that assemble to form a specialized membrane
called Reichert’s membrane, which surrounds the embryo. VE
cells cover the epiblast and ExE. At early postimplantation
stages, the trophectoderm, Reichert’s membrane, and the VE
constitute an ensemble that filters and transports nutrients and
waste, essential for the survival and growth of the embryo
(reviewed by Bielinska et al.37). Complex interactions take
place between the VE and the underlying ExE and epiblast
that drive reciprocal maintenance and differentiation.
Functional studies of genes involved in the differentiation of
the VE have helped to characterize this interdependency.
Thus, deficiencies for the nuclear factors Gata6, vHNF1, and
HNF4 all result in early embryonic lethality caused by a
primary defect in VE differentiation and an associated
degenerescence of the epiblast.24,25,38–40 Failure to assemble or
differentiate a proper VE also results in cavitation defects. In
both the Dab2 and the γ1-laminin mutants, for example, the
proamniotic cavity doesn’t form in the epiblast.27,28 The
proamniotic cavity is normally formed shortly after implantation by a process that involves epithelialization of the epiblast
cells attached to the basal lamina and possibly apoptosis of
medial epiblast cells (Fig.11–1).41 Embryoid bodies have been
used to model the formation of the proamniotic cavity in vitro
to identify the interactions and the molecules involved.
Embryoid bodies form when aggregates of ES cells are
cultured in suspension for a few days.42 They have an outer
layer of endoderm surrounding a core of epiblast-like cells,
separated by a basal lamina. After a few days in culture, they
cavitate in a fashion similar to that of the embryo, except that
once epithelialization has occurred, a greater number of cells
are left in the middle to undergo apoptosis.41 Mutant studies
and in vitro studies have shown that the differentiation of the
VE, which depends both directly and indirectly on bone
morphogenetic protein (BMP) and Indian hedgehog signaling, is required for the cavitation of the epiblast.25,27,43,44
Defective interactions with the basal lamina, caused by lossof-function mutations in γ1-laminin, β1-integrin, or Integrin
Linked Kinase (ILK), prevent epiblast cells from becoming
polarized and forming an epithelium.28,45–47 The assembly of
the basal lamina also has a positive feedback effect on the differentiation of the VE.48 It had been postulated that a signal
promoting cell death in epiblast cells was delivered by the
VE,41 but mutant studies have brought little evidence to support this hypothesis or even warrant the necessity for such a
signal. It seems possible that the epithelialization of the epiblast cells could create a barrier, lowering the flow of nutrients
for the few epiblast cells remaining unattached to the basal
lamina, thereby triggering their apoptosis.
Attached to the basal lamina, with their apical side bordering the proamniotic cavity, epiblast cells form a tall, columnar
pseudo-stratified epithelium. When they divide, epiblast cells
have to relinquish contact with the basal lamina, and mitosis
occurs at the apical surface of the layer. Lineage tracing of
sister cells has shown that they easily become separated when
they reestablish contact with the basal lamina, and clonal
analysis of their descendants found that this results in
the absence of coherent clonal growth in the epiblast up to the
gastrulation stage.49 This cell-mingling effect is linked to
the high proliferation rate of the epiblast, which numbers 30
cells at the late blastocyst stage (E 4.0) and a few thousand at
the midstreak stage (E 6.75).50 In contrast, VE cells form a
shorter epithelium, which maintains coherent clonal growth
throughout these stages.23,49,51 These data suggest that any
possible positional cue to embryonic polarity, inherited from
preimplantation stages, is more likely to be maintained in an
extraembryonic tissue than in the epiblast at the early eggcylinder stage. Clonal analysis of descendants from cells of
the top layer of the ICM at the early blastocyst stage has been
informative in that respect. Labeled cells that became PrE
cells were more likely to contribute to distal VE if they were
originally close to the animal pole of the blastocyst.22
Conversely, endoderm precursors close to the vegetal pole
were more likely to contribute to VE covering the extraembryonic region. This study suggests that the spatial organization of the blastocyst prefigures the proximal–distal polarity
of the postimplantation embryo.22
Postimplantation Patterning
of Epiblast Cells
LATE COMMITMENT OF EPIBLAST CELLS
Fate mapping by clonal analysis in the early gastrula has
demonstrated that the developmental fates and morphogenetic
movements of cell populations in different regions of the
epiblast are predictable during gastrulation. However, the
progeny of individual cells can contribute to a variety of
embryonic and extraembryonic tissues in all three germ
layers.1,52–54 Therefore, at this stage, epiblast cells are not
irreversibly committed or restricted to any tissue lineage.
Acquisition of a more restricted cell fate and lineage determination is likely to take place when gastrulation is completed
(from E7.5 onward). Cells exhibit progressive restriction in
their potency as they become committed to a precise developmental fate and differentiate.
Major decisions about lineage allocation are made during
gastrulation when the single-layered epiblast is transformed
into the three primary germ layers of the embryo: the endoderm, the mesoderm, and the ectoderm (Fig. 11–1, reviewed
by Tam and Behringer1). Posterior and proximal–lateral epiblast cells delaminate and ingress through the primitive streak
(PS) as it forms proximally at the posterior side of the
embryo. They are subsequently allocated to either the mesoderm or the endoderm germ layers. Fate mapping studies of
the PS have revealed a regionalization of cell fate. The type of
mesoderm produced depends on the time and the position at
which cells ingress though the PS (reviewed by Tam and
Behringer1). The first cells to go through the streak mainly
generate extraembryonic mesoderm. Subsequently, as the PS
elongates toward the distal tip of the embryo, cells emerging
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from the anterior, the middle, and the posterior region of the
streak, respectively, contribute to anterior mesoderm and
definitive endoderm, paraxial mesoderm, and lateral mesoderm (Fig. 11–3A). This sequential recruitment of epiblast
cells to distinct mesodermal fates between the mid- and late
gastrula stage also reflects the regionalized expression of
mesoderm-inducing and -regionalizing factors (FGF, Wnt,
and TGF-β) along the proximal–distal axis of the PS.
Progenitors of different mesodermal lineages may therefore
be differently specified, depending on the combination of
mesoderm-inducing factors they are exposed to during their
passage through the PS. Whether this regionalization reflects
any restriction in cell potency can be directly tested by heterotopic transplantations. Such experiments assess the developmental plasticity of the transplanted cells when confronted
with a different environment. When reintroduced into the epiblast, newly formed mesodermal cells can reingress through
the PS and produce all mesoderm types formed by pluripotent
epiblast cells apart from lateral mesoderm.55 These experiments indicate that cellular ingression through the PS does not
result in a dramatic restriction of lineage potency.
Distal and anterior epiblast cells do not migrate through
the PS. They expand anteriorly and laterally and form the
ectoderm germ layer.52,56,57 At early gastrulation stages, these
epiblast cells are not committed to a particular fate as descendants of a single cell can colonize the neuroectoderm, the
surface ectoderm, and the amnion ectoderm (Fig. 11–3A).
Heterotopic transplantations during early gastrulation stages
further revealed their developmental plasticity. Distal epiblast
cells of early streak embryos, fated to become ectoderm, can
contribute to extraembryonic mesoderm, lateral mesoderm,
and primordial germ cells when grafted in the proximal
region.58 In contrast, anterior epiblast cells of late streak
embryos grafted in ectopic positions mainly produce neural
tissue, indicating that neuroectoderm specification might have
occurred at the late streak stage.56 In vitro culture of germ
layer explants provides a useful assay to test ectoderm specification, defined as the behavior of this tissue when grown in
isolation.59,60 The homeobox gene Otx2 is widely expressed in
the epiblast prior to gastrulation but becomes progressively
restricted to the anterior end of the embryo by late gastrulation and ultimately marks the anterior neuroectoderm.
Anterior epiblast explants maintain Otx2 expression in culture
only when dissected from midstreak stage embryos onward,
indicating that anterior neural identity might be specified after
this stage.60 In vitro culture of anterior epiblast explants has
also demonstrated that the expression of engrailed genes is
specified by midstreak stage, at least 12 hours before their
onset of expression in the midbrain region of the neural tube
at the early somite stage.59 In addition explant–recombination
assays provided evidence that anterior mesendoderm-derived
signals are critical for establishing neural regional identity in
the anterior ectoderm at mid- to late streak stages.59,60 These
studies suggest that the specification of anterior epiblast cells
into a neuroectodermal fate occurs around the midstreak
stage. Nevertheless, anterior ectoderm cells retain developmental plasticity after their specification. Indeed, when
recombined with VE, cells of the anterior epiblast at the late
streak stage can be respecified and can adopt posterior mesodermal fates.61 Fgf signaling can also change the fate of anterior
epiblast from ectoderm to mesoderm. Anterior epiblast
explants treated with Fgf2 subsequently express molecular
markers consistent with a differentiation into paraxial and
axial mesoderm.62
In contrast, cells from the organizer are committed to their
fate much earlier. The 1924 pioneering embryological experiments of Spemann and Mangold in the amphibian embryo
first demonstrated that cells of the dorsal blastopore lip have
the ability to induce a complete secondary axis when ectopically grafted onto the ventral side of a host embryo (reviewed
by Harland and Gerhart63). Distinguishable from other cell
populations, the organizer is defined by a unique combination
of inductive, morphogenetic, and patterning properties that
influence the surrounding host tissues to differentiate into a
duplicated axis. Cell populations with developmental and
functional properties similar to the Spemann–Mangold organizer have been identified in other vertebrates, such as zebra
fish, bird, and mouse. In the mouse, heterotopic transplantation of the organizer cell population to the lateral region of the
late streak stage embryo leads to axis duplication65–68
(reviewed by Camus and Tam64). The organizer mainly produces the axial mesoderm (notochord) and the floor plate of
the neural tube. Induced host tissues differentiate into neural
tissue, paraxial mesoderm (somites), and gut endoderm with
different anterior–posterior characteristics. These organizer
transplantation studies provide the ultimate demonstration of
the competence and plasticity of the ectoderm layer of the
embryo at the end of gastrulation.
Based on cell fate and axis-inducing activity, three populations of cells with organizing activities have been identified
in the embryo during gastrulation. They represent the successive identities of the organizer: The early gastrula organizer is
made up of a few cells in the posterior epiblast at the early
streak stage; the midgastrula organizer comprises cells of the
anterior tip of the PS at midstreak stage; and finally, the node,
an ectoderm and endoderm two-layered structure, is found
at the anterior extremity of the fully extended PS65–67,69
(Fig. 11–3A). Regardless of the gastrulation stage the donor
organizer is derived from, transplantations studies reveal that
differentiation of the organizer cells is regulated autonomously. When transplanted to an ectopic site, the organizer
population always self-differentiates and influences neighboring host tissues to change their fate.
Cell fate analysis of these distinct organizer populations
indicates that they undergo significant changes in their cellular
composition during gastrulation.69 This study suggests that
the gastrula organizer is composed of transiently recruited
precursors. Most of them are allocated to various anterior–
posterior levels of the axial mesendoderm as the embryo develops. Gene expression analysis has revealed that the gastrula
organizer expresses different combinations of organizerspecific genes during gastrulation (reviewed by Camus
et al.64).Whether the changes in lineage potency and molecular
properties of the organizer population during gastrulation
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Early-streak
E6.5
PS
Extraembryonic mesoderm
Mesoderm
Ectoderm
Surface ectoderm
Forebrain
Late-streak
E7.5
Midbrain
Hindbrain
Spinal cord
Paraxial mesoderm
PS
Lateral mesoderm
Axial mesoderm and
definitive endoderm
A
Epiblast
Extraembryonic ectoderm (ExE)
Visceral endoderm (VE)
Distal VE and AVE
BMP4 expression in the distal ExE
Nodal and cripto expression
in the epiblast
Proximal
E5.5
E6.5
Posterior
Anterior
B
Distal
Figure 11–3. Fate map and early patterning of epiblast cells. (A) In the fate map of epiblast cells at the early streak and late streak stages, a black
vertical bar represents the extent of the primitive streak (PS). Only the epiblast layer of the embryo is represented. Epiblast cells recruited into the proximal
PS at the early streak stage generate predominantly extraembryonic mesoderm. Lateral mesoderm and paraxial mesoderm cells arise at more distal positions as the PS extends. The anterior–distal region of the PS contains the precursors of axial mesendoderm cells, the organizer derivatives. In the anterior
region, at the late streak stage, epiblast cells have been specified to form neuroectoderm with different anterior–posterior identities. (B) Proximal–distal
and anterior–posterior patterning of the epiblast is shown. At E5.5, Nodal signaling from the epiblast is required to specify distal visceral endoderm (VE)
cells. Distal VE cells express Nodal antagonists that inhibit Nodal action in the distal epiblast. Reciprocal interactions between the epiblast and the extraembryonic ectoderm (ExE) reinforce Nodal signaling in the proximal epiblast. At E6.5, anterior visceral endoderm cells repress Nodal and possibly Wnt
expression in the anterior epiblast. Signals derived from the posterior VE, the posterior ExE, or both may contribute to induce, maintain, or both the PS in
the posterior epiblast region. During gastrulation, signals derived from the organizer are required for the patterning of adjacent tissues.
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reflect significant differences in axis-inducing activity has not
yet been clearly demonstrated. Together, these results indicate
that the mouse gastrula organizer is a dynamic cell population. The chick gastrula organizer, Hensen’s node, is also
composed of transiently recruited precursors that acquire and
lose organizer gene expression as they coast through the node
region. Embryological experiments have demonstrated that
reciprocal interactions between Hensen’s node and its neighboring tissues ensure the maintenance of an organizer cell
state in a fixed spatial domain with dynamic cellular composition.70 Whether similar interactions between the middle
region and the anterior region of the PS regulate the organizer
cell state in the mouse embryo is unknown.
INTERACTIONS WITH EXTRAEMBRYONIC LINEAGES
ESTABLISH EPIBLAST POLARITY BEFORE GASTRULATION
Because of the cell-mingling phenomenon characterized
within the epiblast at pregastrula stages, it is generally
assumed that any spatial information derived from early
polarity cues would be transmitted to the epiblast by extraembryonic tissues.22,49,51 Genetic data, experimental embryology,
and results from chimera experiments have provided compelling evidence that reciprocal interactions between the
extraembryonic and embryonic lineages establish and reinforce early patterning in the mouse embryo (reviewed by
Beddington and Robertson2 and Lu et al.3).
A subset of VE cells at the distal tip of the embryo starts
to express a specific repertoire of genes at E5.5. It comprises
the homeobox gene Hex as well as Cerl and Lefty1, which
encode secreted proteins.71–74 Between E5.5 and E6.0, their
distal domain of expression is displaced proximally to a
position opposite of where the PS will eventually form,
marking for the first time the anterior of the embryo. Lineagetracing studies demonstrated that this shift in the position of
the expression domain is the consequence of a unidirectional
movement of distal VE cells toward the anterior71,75,76
(Fig. 11–3B). These cells express additional markers once
they have reached their anterior position (reviewed by
Beddington et al.,2 Perea-Gomez et al.,4 and Camus et al.64).
This group of cells was therefore called anterior visceral
endoderm, or AVE (Fig. 11–3B). These findings demonstrated
that the VE is patterned along the proximal–distal axis of the
embryo at E5.5 and along the anterior–posterior axis from
E6.0. Could this proximal–distal patterning of the VE prefigure the establishment of the anterior–posterior axis in the
epiblast? Gene expression analyses indicate that the ExE is
also patterned along the proximal–distal axis at early stages
before gastrulation. The TGF-β-secreted molecule Bmp4 and
the T-box genes Eomesodermin (Eomes) and Brachyury
(T) are expressed specifically in the distal ExE cells abutting
the proximal epiblast at E6.077–79 (Fig. 11–3B). In the epiblast,
two factors required for PS and mesoderm formation, the
TGF-β molecule Nodal and its coreceptor Cripto, first found
throughout the epiblast at E5.0, see their expression progressively restricted to a proximal region by E5.75.80,81 The
secreted molecule Wnt3, involved in PS formation, is also
detected in the proximal epiblast adjacent to the ExE at this
stage.82 By E6.25, the expression of Nodal, Cripto, and Wnt3
becomes circumscribed to the posterior epiblast, where genes
involved in mesoderm migration, such as T and Fgf8, are then
induced and where the PS arises at E6.5 (Fig. 11–3B).
Together, these studies have led to the proposal that the
proximal–distal polarity of the early postimplantation embryo
is transformed into the anterior–posterior polarity of the
gastrulating embryo through asymmetric cell movements in
the VE and posterior restriction of proximal epiblast markers
(reviewed by Beddington et al.2).
The establishment of a proximal–distal pattern and its
transformation into the anterior–posterior axis of the gastrula
stage embryo has been shown to depend on signals from the
ExE and the VE that modulate Nodal signaling in the epiblast.
Nodal is a major player in early embryonic patterning. Mouse
embryos bearing a mutation in the Nodal gene fail to express
other proximal epiblast markers and do not form a PS. In
addition, AVE specification is abolished in Nodal−/−
mutants.80,83–85 Nodal signals via a complex formed by type II
and type I serine-threonine kinase receptors (Act RIIA, Act
RIIB, ALK 4, ALK 7) and EGF-CFC cofactors (cripto or
cryptic). The activated receptor complex phosphorylates
Smad2 and Smad3, which associate with Smad4 and translocate to the nucleus. There, they cooperatively regulate the
transcription of target genes with other DNA-binding
proteins, such as the forkhead transcription factor Foxh1
(reviewed by Whitman86). The expression of Nodal in the
epiblast, extremely dynamic, is under the control of two
cis-regulatory elements characterized by transgenesis. The
5′ proximal epiblast enhancer (PEE) controls Nodal expression in the proximal epiblast and in the PS at later stages. The
intronic asymmetric enhancer (ASE), containing two Foxh1binding sites, is involved in a positive feedback loop that
activates Nodal expression throughout the epiblast and in
the VE.87,88
Recent studies have suggested that reciprocal interactions
between the ExE and the proximal epiblast region are likely to
establish a proximal–distal gradient of Nodal signaling in the
prestreak stage mouse embryo.85,89 Compound mutants,
chimera analysis, and tissue-explants studies have demonstrated that the proprotein convertases Spc1 and Spc4 produced
by distal ExE cells are required to cleave the immature form
of Nodal secreted by adjacent proximal epiblast cells. Spc1
and Spc4 therefore directly potentiate Nodal signaling in the
proximal epiblast of the pregastrula embryo.89 Moreover,
Beck et al. have proposed that Spc1 and Spc4 regulate Nodal
activity in the epiblast indirectly by stimulating the induction
of BMP4 in the ExE. In turn, BMP4 would signal to the epiblast, leading to the amplification of the expression of the
Nodal coreceptor Cripto in cells adjacent to the ExE.89
Previous mutant analysis suggested that Nodal signaling is
involved at early stages in regulating and maintaining BMP4
expression in the ExE.85 These findings therefore indicate that
reciprocal tissue interactions are essential for the establishment of proximal–distal patterning (Fig. 11–3B).
Similar interactions involving the regulation of Nodal
signaling have been described between the epiblast and the VE.
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Origin, Early Patterning, and Fate of the Mouse Epiblast
Analysis of mutant embryos indicates that the initial specification of distal VE cells requires high levels of Nodal signal
produced in the epiblast and transduced by Smad2 in the
VE85,90–92 (Fig. 11–3B). Nodal expression in the VE is also
likely activated, via the autoregulatory element ASE, by
Nodal produced in the epiblast.85,90 In addition to Nodal signals, the transcription factors Otx2, Foxa2, and Lhx1 and the
Wnt signal transducer β-catenin are required for correct specification of distal VE cells.93–96 Little is known about the
mechanisms that drive the distal-to-anterior movement of VE
cells between E5.5 and E6.0.76 However, in mutant embryos in
which Nodal signaling is attenuated, AVE markers such as
Hex are still expressed in distal positions at E6.5. This indicates that distal VE cells fail to move anteriorly in the absence
of high Nodal levels.81,91,92,97 A similar phenotype has been
observed in mouse embryos lacking β-catenin.96 Lineage-tracing
analysis and transgenic rescue experiments have demonstrated that Otx2 is required in the VE for its distal to anterior
movement.94,95
Ablation of the AVE provided evidence of a role for this
tissue in patterning the anterior epiblast fated to generate the
anterior neuroectoderm and surface ectoderm.98 Analyses of
chimeric embryos with a mutant VE and a wild-type epiblast
have demonstrated that Foxa2, Lhx1, Otx2, and Nodal are
required in the AVE to ensure proper development of anterior
neural structures (reviewed by Beddington et al.2). Based on
these findings and with the knowledge that an ectopically
grafted organizer (early gastrula organizer or node) can only
induce a secondary axis lacking anterior neural structures, the
AVE has been proposed as the mouse equivalent of
the amphibian head organizer, likely to play a central role in
the induction of anterior neural tissues. However, transplantation studies, germ-layer explant assays, and genetic studies
have clearly demonstrated that the AVE alone cannot induce
anterior neuroectoderm but rather can impart anterior fate
on the adjacent epiblast by repressing the action of posterior
signals68,82,94 (reviewed by Perea-Gomez et al.4).
The first evidence of an early role of the distal VE and the
AVE cells in regulating the expression of posterior markers in
the epiblast came from explant recombination experiments.
The AVE of early to midstreak stage embryos does not induce
anterior neuroectoderm markers but can significantly reduce
the expression of T and Cripto in epiblast explants. This
repressing activity is abolished in Otx2-mutant AVE.94 Results
from genetic analyses are consistent with these observations
(reviewed by Perea-Gomez et al.4). Inactivation of genes
required for AVE specification, such as the signal transducer
Smad2 or the transcription factors Foxa2 and Lhx1, results in
the enlargement of proximal–posterior fates throughout the
epiblast and the severe reduction or absence of distal–anterior
epiblast derivatives.90,93 The action of the AVE in epiblast
patterning is partly mediated by two secreted proteins, Cerl
and Lefty1, that function as extracellular Nodal antagonists
(reviewed by Whitman86). Cerl and Lefty1 expression is first
detected in the distal VE and then in the AVE prior to gastrulation.73,74,99 Interestingly, the expression of these two
Nodal antagonists in the VE is most likely dependent on
Nodal itself as part of a negative feedback loop.85,90,91 Cerl−/−,
Lefty1−/− compound mutants, and chimeric embryos lacking
Cerl and Lefty1 expression in the VE show supernumerary
primitive streaks.99 These results indicate that Cerl and Lefty1
are required in the AVE to restrict the action of Nodal signals
involved in PS induction and mesoderm formation (Fig. 11–3B).
These findings strengthen the hypothesis that the main function of the AVE is to provide repressing signals that prevent
the anterior epiblast from adopting a posterior fate under the
influence of signals such as Nodal. Subsequent interactions
between anterior epiblast and anterior mesendoderm tissues
derived from the organizer allow anterior neural development
to proceed (reviewed by Martinez-Barbera and Beddington100).
Nodal may not be the only signal antagonized by AVE
cells. Wnt3 is required downstream of Nodal for PS formation
in the posterior epiblast. Moreover, ectopic or deregulated
Wnt signaling leads to axis duplication at early gastrulation
stages.101–103 Interestingly, the Wnt antagonist Dkk1 is
expressed in the AVE, and biochemical data suggest that the
secreted protein Cerl might also function as a Wnt antagonist.104,105 This is consistent with the suggestion that another
role of the AVE would be to antagonize Wnt signals in the
epiblast. However the capacity of Dkk1 and Cerl to restrict
the extent of Wnt signaling in the epiblast has not been
demonstrated so far.
Because interactions between AVE and epiblast clearly
contribute to the anterior–posterior patterning, a role for
posterior VE cells in patterning the posterior epiblast is
conceivable. Although asymmetric gene expressions have
been reported in the posterior VE, no evidence so far indicates
that posterior VE cells have such a role.3
COMPLEX INTERACTIONS BETWEEN DISTINCT
SIGNALING PATHWAYS DURING EPIBLAST
PATTERNING
A network of signaling pathways regulates the migration and
the differentiation of epiblast cells during gastrulation.
Genetic studies and experimental embryology have provided
effective tools for investigating connecting points in this network and characterizing some of the interactions that govern
the determination of different cell fates.
In the posterior region, epiblast cells that delaminate
through the PS region produce a variety of mesoderm and
endoderm types under the influence of different signaling
pathways. Recent studies have suggested that, as in other
vertebrate embryos, graded levels of Nodal signaling in the
posterior epiblast could govern the generation of distinct PS
derivatives (extraembryonic mesoderm, embryonic mesoderm, and definitive endoderm).86,106 Characterization of
Nodal hypomorph mutants displaying different degrees of
Nodal activity have indicated that epiblast cells are sensitive
to the level of Nodal signaling.97 Loss of function of its downstream mediator Foxh1, its modulator Arkadia, or of the Spc1
and Spc4 Nodal convertases does not abolish mesoderm
formation, but it impairs the generation of axial mesoderm
and definitive endoderm.89,91,107,108 These results indicate that
Nodal signals for the formation of axial mesendoderm tissues
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derived from the organizer must be higher than those for the
formation of more posterior–lateral mesoderm tissues.
Negative regulators of the pathway are also likely to contribute to the establishment of a gradient of Nodal activity.
Thus, the combined loss-of-function of the Nodal antagonists
Cerl and Lefty1 or of the transcriptional corepressor Drap1
lead to the expansion of axial mesendoderm derivatives at
the expense of paraxial mesoderm.99,109 A recent study provided compelling evidence that graded Nodal signaling is also
required for the specification within the organizer region
of the anterior definitive endoderm and prechordal plate progenitors. When Nodal activity is specifically lowered in the
proximal–posterior epiblast because of the exclusive deletion
of the cis-regulatory element PEE of the Nodal gene, the anterior definitive endoderm and prechordal plate are missing.110
A similar phenotype has been observed when Smad2 activity
is removed from the epiblast by a conditional inactivation
strategy. Interestingly, in both of these mutants, the node, the
notochord, and the floor plate develop normally. In contrast,
inactivation of one copy of Smad3 in the context of the
Smad2-deficient epiblast leads to a failure to specify all
organizer derivatives, including the notochord.110 Together,
these results indicate that graded Nodal signaling in the
posterior epiblast contributes to mesoderm and endoderm patterning, requiring increasing levels of Nodal for posterior–
lateral mesoderm, notochord, prechordal plate, and anterior
endoderm formation. Recent evidence suggests that the Wnt
signaling pathway is also involved in the choice between
endoderm and mesoderm fates. Indeed, conditional deletion
of β-catenin in the epiblast results in endoderm cells adopting
precardiac mesoderm cell fate111 (reviewed by Tam et al.112).
The analysis of the targeted disruption of Fgf receptor 1
(Fgfr1) provides a vivid illustration of the interactions
between the major signaling pathways. Fgfr1 expression is
detected throughout the epiblast at pregastrulation stages and
subsequently into the posterior part of the PS. Fgfr1-mutant
embryos display a severe reduction of lateral and paraxial
mesoderm and an apparent expansion of the axial mesendoderm.113,114 Chimeras analyses indicated that Fgfr1−/− cells fail
to undergo the epithelial to mesenchymal transition, accumulate in the PS, and form ectopic neural tubes.115,116 Therefore,
incorrectly specified mesoderm precursors are able to respond
to neuralizing signals and to change their fate accordingly.
Further molecular analysis of the chimeric embryos and
explant assays provided remarkable insights into the downstream targets of the Fgfr1 pathway and the mechanisms by
which Fgfr1 signaling controls both the patterning of mesoderm cells and morphogenesis.117 Expression of the T-box
gene Tbx6 is dramatically reduced in Fgfr1-mutant embryos.
The phenotype of Tbx6 mutants is similar to that of Fgfr1 in
that cells fated to form paraxial mesoderm differentiate into
ectopic neural tubes.118 Fgfr1 might therefore act through
Tbx6 for the patterning of paraxial mesoderm cells. The
expression of the zinc-finger transcription factor Snail is also
missing in Fgfr1 mutant cells. Snail normally represses the
expression of the cell adhesion molecule E-cadherin. As a
result, Fgfr1−/− cells have abnormally high levels of E-cadherin,
which accounts for their failure to achieve the epithelial to
mesenchymal transition. Ciruna and Rossant117 have reported
that this defect also indirectly affects Wnt signaling and the
patterning of mesoderm cells. Indeed, in mutant cells with
high levels of E-cadherin, β-catenin is sequestered at the cell
membrane, and its nuclear function in mediating Wnt signaling
is impaired. In accordance with this finding, Fgfr1-deficient
embryos do not express T, a direct target of Wnt signaling, in
the regions of the PS fated to generate lateral and paraxial
mesoderm. These observations can be related to the phenotype
of Wnt3a mutants in which T expression is also missing and
cells fated to produce the paraxial mesoderm form ectopic
neural tubes.119 These studies provide evidence that interactions between the FGF and the Wnt pathways play an essential
role in morphogenesis and in the patterning of mesoderm cells
by regulating both cell adhesion and cell fate determination.
How cells integrate different signals and modulate their
response accordingly is a key issue. The study of a gene called
Churchill (ChCh), recently characterized in the chick, provides
an example of a possible mechanism.120 It encodes a zincfinger protein that may play a pivotal role in altering the
response of epiblast cells to Fgf signaling. During gastrulation, Nodal signaling and Fgf signaling cooperate to induce
T and Tbx6L in the epiblast and to regulate cell ingression
through the PS. However, continued exposure to Fgf signaling
induces ChCh in the epiblast, subsequently leading to the
activation of Smad-interacting-protein 1 (Sip1) that blocks further mesoderm induction. Concomitantly, ChCh-expressing
epiblast cells become sensitive to neural-inducing signals
emanating from the node. This study therefore suggests that
ChCh functions as a switch determining the response of
epiblast cells to Fgf signaling.
Summary
Although it is difficult to reconstruct past events from today’s
observations, it appears likely that the evolution of viviparity
in the mammalian lineage was made possible by that of
pluripotentiality. To survive and develop in utero, the mammalian embryo has to develop an extraembryonic interface
that feeds and protects embryonic precursors. However, these
embryonic precursors have to possess a protection of their
own, dependent on intrinsic factors, because they must remain
unresponsive to the signals that first promote the differentiation of extraembryonic lineages. A subsequent release of this
intrinsic blockage to differentiation may allow signals from
the extraembryonic tissues to initiate the patterning of the
embryo. On the assumption that positional cues inherited from
the egg play a role in patterning the embryo, extraembryonic
tissues constitute the most likely vessel to carry them until
the epiblast becomes receptive. When it does, a network of
signaling pathways coordinates the growth, the specification,
and the migration of the different cell populations that
compose the embryo. The first overt signs of embryonic
differentiation, marking the establishment of the anterior–
posterior axis, become apparent quite late in development in
comparison to other vertebrates. As differentiation is always
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Origin, Early Patterning, and Fate of the Mouse Epiblast
associated with a loss in developmental potency, this correlates with the prolonged undifferentiated state of the epiblast.
What lies ahead is the relentless characterization of the
molecular and cellular interactions that govern the establishment, the maturation, and the differentiation of the epiblast.
Beyond this, we need a deeper understanding of what goes
on outside of the cell, within its cytoplasm, and in its nucleus
while it is exposed to the signals that impinge on its fate
in the embryo. What is, for example, the range of the secreted
signaling molecules and of their antagonists? How are graded
levels of a particular signal generated, and how are they read
and integrated by individual cells? One key challenge will
be to correlate these events with specific changes in the status
of the chromatin. These studies will no doubt contribute
to the stem cell field, bringing valuable insights into the mechanisms that control their differentiation toward a given
lineage.
ACKNOWLEDGMENTS
We thank Dr. R. Pedersen for the opportunity to write this chapter. We
are grateful to Drs. J. Aghion and D. Saberan-Djoneidi for critical
reading of the manuscript, and to Drs. C. Chazaud and M. ZernickaGoetz for helpful discussions. We would like to acknowledge M.
Barre for help with the figures. Our laboratory receives support from
the Centre National de la Recherche Scientifique, the Ministère de la
Recherche (ACI grant), the Fondation pour la Recherche Médicale,
and the Association pour la Recherche contre le Cancer (ARC 5456).
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