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P436643-V1-11 12/24/04 5:47 PM Page 129 11 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, Handbook of Stem Cells Volume 1 Copyright © 2004 by Elsevier Inc. All rights of reproduction in any form reserved. 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 129 30/07/04 12:46 AM Page 130 Anne Camus, Aitana Perea-Gomez, and Jérôme Collignon 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 P436643-V1-11 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). 130 P436643-V1-11 30/07/04 12:46 AM Page 131 11. 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 131 P436643-V1-11 30/07/04 12:46 AM Page 132 Anne Camus, Aitana Perea-Gomez, and Jérôme Collignon 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. 132 P436643-V1-11 30/07/04 12:46 AM Page 133 11. 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 133 P436643-V1-11 30/07/04 12:46 AM Page 134 Anne Camus, Aitana Perea-Gomez, and Jérôme Collignon 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 134 P436643-V1-11 30/07/04 12:46 AM Page 135 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. 135 P436643-V1-11 30/07/04 12:46 AM Page 136 Anne Camus, Aitana Perea-Gomez, and Jérôme Collignon 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. 136 P436643-V1-11 30/07/04 12:46 AM Page 137 11. 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 137 P436643-V1-11 30/07/04 12:46 AM Page 138 Anne Camus, Aitana Perea-Gomez, and Jérôme Collignon 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 138 P436643-V1-11 30/07/04 12:46 AM Page 139 11. 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. 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