Polarity in the rabbit embryo

The main aim of the gastrulation process is commonly regarded to be the generation of the definitive germ layers known as mesoderm, endoderm and ectoderm. Here we discuss how the topography of gene expression, cellular migration and proliferative activity in the preliminary germ layers (hypoblast and epiblast) of the rabbit embryo reveal the sequence of events that establishes the three major body axes. We present a testable model in which a combination of cellular movement in the hypoblast with a morphogen gradient created by the (extraembryonic) trophoblast creates morphological polarity in the embryo and, hence, the co-ordinates for germ layer formation.     

Hypoblast controls mesoderm generation and axial patterning in the gastrulating rabbit embryo

Gastrulation in higher vertebrate species classically commences with the generation of mesoderm cells in the primitive streak by epithelio-mesenchymal transformation of epiblast cells. However, the primitive streak also marks, with its longitudinal orientation in the posterior part of the conceptus, the anterior-posterior (or head-tail) axis of the embryo. Results obtained in chick and mouse suggest that signals secreted by the hypoblast (or visceral endoderm), the extraembryonic tissue covering the epiblast ventrally, antagonise the mesoderm induction cascade in the anterior part of the epiblast and thereby restrict streak development to the posterior pole (and possibly initiate head development anteriorly). In this paper we took advantage of the disc-shape morphology of the rabbit gastrula for defining the expression compartments of the signalling molecules Cerberus and Dickkopf at pre-gastrulation and early gastrulation stages in a mammal other than the mouse. The two molecules are expressed in novel expression compartments in a complementary fashion both in the hypoblast and in the emerging primitive streak. In loss-of-function experiments, carried out in a New-type culturing system, hypoblast was removed prior to culture at defined stages before and at the beginning of gastrulation. The epiblast shows a stage-dependent and topographically restricted susceptibility to express Brachyury, a T-box gene pivotal for mesoderm formation, and to transform into (histologically proven) mesoderm. These results confirm for the mammalian embryo that the anterior-posterior axis of the conceptus is formed first as a molecular prepattern in the hypoblast and then irrevocably fixed, under the control of signals secreted from the hypoblast, by epithelio-mesenchymal transformation (primitive streak formation) in the epiblast.Edited by D. Tautz     

Paracrine effects of embryo-derived FGF4 and BMP4 during pig trophoblast elongation

The crosstalk between the epiblast and the trophoblast is critical in supporting the early stages of conceptus development. FGF4 and BMP4 are inductive signals that participate in the communication between the epiblast and the extraembryonic ectoderm (ExE) of the developing mouse embryo. Importantly, however, it is unknown whether a similar crosstalk operates in species that lack a discernible ExE and develop a mammotypical embryonic disc (ED). Here we investigated the crosstalk between the epiblast and the trophectoderm (TE) during pig embryo elongation. FGF4 ligand and FGFR2 were detected primarily on the plasma membrane of TE cells of peri-elongation embryos. The binding of this growth factor to its receptor triggered a signal transduction response evidenced by an increase in phosphorylated MAPK/ERK. Particular enrichment was detected in the periphery of the ED in early ovoid embryos, indicating that active FGF signalling was operating during this stage. Gene expression analysis shows that CDX2 and ELF5, two genes expressed in the mouse ExE, are only co-expressed in the Rauber's layer, but not in the pig mural TE. Interestingly, these genes were detected in the nascent mesoderm of early gastrulating embryos. Analysis of BMP4 expression by in situ hybridisation shows that this growth factor is produced by nascent mesoderm cells. A functional test in differentiating epiblast shows that CDX2 and ELF5 are activated in response to BMP4. Furthermore, the effects of BMP4 were also demonstrated in the neighbouring TE cells, as demonstrated by an increase in phosphorylated SMAD1/5/8. These results show that BMP4 produced in the extraembryonic mesoderm is directly influencing the SMAD response in the TE of elongating embryos. These results demonstrate that paracrine signals from the embryo, represented by FGF4 and BMP4, induce a response in the TE prior to the extensive elongation. The study also confirms that expression of CDX2 and ELF5 is not conserved in the mural TE, indicating that although the signals that coordinate conceptus growth are similar between rodents and pigs, the gene regulatory network of the trophoblast lineage is not conserved in these species.     

Axial differentiation and early gastrulation stages of the pig embryo

Differentiation of the principal body axes in the early vertebrate embryo is based on a specific blueprint of gene expression and a series of transient axial structures such as Hensen's node and the notochord of the late gastrulation phase. Prior to gastrulation, the anterior visceral endoderm (AVE) of the mouse egg-cylinder or the anterior marginal crescent (AMC) of the rabbit embryonic disc marks the anterior pole of the embryo. For phylogenetic and functional reasons both these entities are addressed here as the mammalian anterior pregastrulation differentiation (APD). However, mouse and rabbit show distinct structural differences in APD and the molecular blueprint, making the search of general rules for axial differentiation in mammals difficult. Therefore, the pig was analysed here as a further species with a mammotypical flat embryonic disc. Using light and electron microscopy and in situ hybridisation for three key genes involved in early development (sox17, nodal and brachyury), two axial structures of early gastrulation in the pig were identified: (1) the anterior hypoblast (AHB) characterised by increased cellular height and density and by sox17 expression, and (2) the early primitive streak characterised by a high pseudostratified epithelium with an almost continuous but unusually thick basement membrane, by localised epithelial–mesenchymal transition, and by brachyury expression in the epiblast. The stepwise appearance of these two axial structures was used to define three stages typical for mammals at the start of gastrulation. Intriguingly, the round shape and gradual posterior displacement of the APD in the pig appear to be species-specific (differing from all other mammals studied in detail to date) but correlate with ensuing specific primitive streak and extraembryonic mesoderm development. APD and, hence, the earliest axial structure presently known in the mammalian embryo may thus be functionally involved in shaping extraembryonic membranes and, possibly, the specific adult body form.     

The hypoblast (visceral endoderm): an evo-devo perspective

When amniotes appeared during evolution, embryos freed themselves from intracellular nutrition; development slowed, the mid-blastula transition was lost and maternal components became less important for polarity. Extra-embryonic tissues emerged to provide nutrition and other innovations. One such tissue, the hypoblast (visceral endoderm in mouse), acquired a role in fixing the body plan: it controls epiblast cell movements leading to primitive streak formation, generating bilateral symmetry. It also transiently induces expression of pre-neural markers in the epiblast, which also contributes to delay streak formation. After gastrulation, the hypoblast might protect prospective forebrain cells from caudalizing signals. These functions separate mesendodermal and neuroectodermal domains by protecting cells against being caught up in the movements of gastrulation.     

Wnt3 signaling in the epiblast is required for proper orientation of the anteroposterior axis

The establishment of anteroposterior (AP) polarity in the early mouse epiblast is crucial for the initiation of gastrulation and the subsequent formation of the embryonic (head to tail) axis. The localization of anterior and posterior determining genes to the appropriate region of the embryo is a dynamic process that underlies this early polarity. Several studies indicate that morphological and molecular markers which define the early AP axis are first aligned along the short axis of the elliptical egg cylinder. Subsequently, just prior to the time of primitive streak formation, a conformational change in the embryo realigns these markers with the long axis. We demonstrate that embryos lacking the signaling factor Wnt3 exhibit defects in this axial realignment. In addition, chimeric analyses and conditional removal of Wnt3 activity reveal that Wnt3 expression in the epiblast is required for induction of the primitive streak and mesoderm whereas activity in the posterior visceral endoderm is dispensable.     

Specific Epiblast Loss and Hypoblast Impairment in Cattle Embryos Sensitized to Survival Signalling by Ubiquitous Overexpression of the Proapoptotic Gene BAD

Early embryonic lethality is common, particularly in dairy cattle. We made cattle embryos more sensitive to environmental stressors by raising the threshold of embryo survival signaling required to overcome the deleterious effects of overexpressing the proapoptotic protein BAD. Two primary fibroblast cell lines expressing BAD and exhibiting increased sensitivity to stress-induced apoptosis were used to generate transgenic Day13/14 BAD embryos. Transgenic embryos were normal in terms of retrieval rates, average embryo length or expression levels of the trophectoderm marker ASCL2. However both lines of BAD-tg embryos lost the embryonic disc and thus the entire epiblast lineage at significantly greater frequencies than either co-transferrred IVP controls or LacZ-tg embryos. Embryos without epiblast still contained the second ICM-derived lineage, the hypopblast, albeit frequently in an impaired state, as shown by reduced expression of the hypoblast markers GATA4 and FIBRONECTIN. This indicates a gradient of sensitivity (epiblast > hypoblast > TE) to BAD overexpression. We postulate that the greater sensitivity of specifically the epiblast lineage that we have seen in our transgenic model, reflects an inherent greater susceptibility of this lineage to environmental stress and may underlie the epiblast-specific death seen in phantom pregnancies.     

Mouse epiblasts change responsiveness to BMP4 signal required for PGC formation through functions of extraembryonic ectoderm

Mouse primordial germ cells (PGCs) are initially identified as a cluster of alkaline phosphatase (AP)-positive cells within the extraembryonic mesoderm near the posterior part of the primitive streak at embryonic day (E) 7.25. Clonal analysis of epiblast cells has revealed that the putative precursors of PGCs are localized in the proximal epiblast, and we demonstrated that the conditions required for PGC formation are induced in the proximal region of epiblasts by extraembryonic ectoderm. Bone morphogenetic protein (BMP) 4 and BMP8b, which belong to the transforming growth factor-beta (TGF-beta) superfamily, might generate induction signals from extraembryonic ectoderm. Smad1 and Smad5, which are intracellular signaling molecules for BMP4, might also play a critical role in stimulating epiblasts to form PGC. However, how pluripotential epiblasts temporally and spatially respond to BMP signals to form PGCs remains unclear. The present study examines changes of responsiveness to BMP4 for PGC formation in epiblasts and their molecular mechanisms. We initially examined the effect of recombinant human (rh) BMP4 upon cultured epiblasts at different developmental stages, and found that they acquire the ability to respond to BMP4 signals for PGC formation between E5.25 and E5.5. In addition, such competence was conferred upon epiblasts by the extraembryonic ectoderm. We also showed that the increased expression of Smad1 and the onset of Smad5 expression induced by extraembryonic ectoderm might be responsible for quick acquisition of this competence. Furthermore, we show that only proximal epiblast cells maintain responsiveness to BMP4 for PGC formation at E6.0, and that this is associated with the proximal epiblast-specific expression of Smad5. These results explain why only the proximal region of epiblasts can sustain the ability to form PGCs.     

Cell movements during epiboly and gastrulation in zebrafish

Beginning during the late blastula stage in zebrafish, cells located beneath a surface epithelial layer of the blastoderm undergo rearrangements that accompany major changes in shape of the embryo. We describe three distinctive kinds of cell rearrangements. (1) Radial cell intercalations during epiboly mix cells located deeply in the blastoderm among more superficial ones. These rearrangements thoroughly stir the positions of deep cells, as the blastoderm thins and spreads across the yolk cell. (2) Involution at or near the blastoderm margin occurs during gastrulation. This movement folds the blastoderm into two cellular layers, the epiblast and hypoblast, within a ring (the germ ring) around its entire circumference. Involuting cells move anteriorwards in the hypoblast relative to cells that remain in the epiblast; the movement shears the positions of cells that were neighbors before gastrulation. Involuting cells eventually form endoderm and mesoderm, in an anterior-posterior sequence according to the time of involution. The epiblast is equivalent to embryonic ectoderm. (3) Mediolateral cell intercalations in both the epiblast and hypoblast mediate convergence and extension movements towards the dorsal side of the gastrula. By this rearrangement, cells that were initially neighboring one another become dispersed along the anterior-posterior axis of the embryo. Epiboly, involution and convergent extension in zebrafish involve the same kinds of cellular rearrangements as in amphibians, and they occur during comparable stages of embryogenesis.     

BMP receptor IA is required in the mammalian embryo for endodermal morphogenesis and ectodermal patterning

BMPRIA is a receptor for bone morphogenetic proteins with high affinity for BMP2 and BMP4. Mouse embryos lacking Bmpr1a fail to gastrulate, complicating studies on the requirements for BMP signaling in germ layer development. Recent work shows that BMP4 produced in extraembryonic tissues initiates gastrulation. Here we use a conditional allele of Bmpr1a to remove BMPRIA only in the epiblast, which gives rise to all embryonic tissues. Resulting embryos are mosaics composed primarily of cells homozygous null for Bmpr1a, interspersed with heterozygous cells. Although mesoderm and endoderm do not form in Bmpr1a null embryos, these tissues are present in the mosaics and are populated with mutant cells. Thus, BMPRIA signaling in the epiblast does not restrict cells to or from any of the germ layers. Cells lacking Bmpr1a also contribute to surface ectoderm; however, from the hindbrain forward, little surface ectoderm forms and the forebrain is enlarged and convoluted. Prechordal plate, early definitive endoderm, and anterior visceral endoderm appear to be expanded, likely due to defective morphogenesis. These data suggest that the enlarged forebrain is caused in part by increased exposure of the ectoderm to signaling sources that promote anterior neural fate. Our results reveal critical roles for BMP signaling in endodermal morphogenesis and ectodermal patterning.     

Angiomotin regulates visceral endoderm movements during mouse embryogenesis

In pregastrula stage mouse embryos, visceral endoderm (VE) migrates from a distal to anterior position to initiate anterior identity in the adjacent epiblast. This anterior visceral endoderm (AVE) is then displaced away from the epiblast by the definitive endoderm to become associated with the extra-embryonic ectoderm and subsequently contributes to the yolk sac. Little is known about the molecules that regulate this proximal displacement. Here we describe a role for mouse angiomotin (amot) in VE movements. amot expression is initially detected in the AVE and subsequently in the VE associated with the extra-embryonic ectoderm. Most amot mutant mice die soon after gastrulation with distinct furrows of VE located at the junction of the embryonic and extra-embryonic regions. Mutant analysis suggests that VE accumulation in these furrows is caused by defects in cell migration into proximal extra-embryonic regions, although distal-to-anterior movements associated with the epiblast, definitive endoderm formation, and anterior specification of the epiblast appear to be normal. These results suggest that amot acts within subregions of the VE to regulate morphogenetic movements that are required for embryo viability.     

Sall4 isoforms act during proximal-distal and anterior-posterior axis formation in the mouse embryo

Reciprocal signals from embryonic and extra-embryonic tissues pattern the embryo in proximal-distal (PD) and anterior-posterior (AP) fashion. Here we have analyzed three gene trap mutations of Sall4, of which one (Sall4-1a) led to a hypomorphic and recessive phenotype, demonstrating that Sall4-1a has yet undescribed extra-embryonic and embryonic functions in regulating PD and AP axis formation. In Sall4-1a mutants the self-maintaining autoregulatory interaction between Bmp4, Nodal and Wnt, which determines the PD axis was disrupted because of defects in the extra-embryonic visceral endoderm. More severely, two distinct Sall4 gene-trap mutants (Sall4-1a,b), resembling null mutants, failed to initiate Bmp4 expression in the extra-embryonic ectoderm and Nodal in the epiblast and were therefore unable to initiate PD axis formation. Tetraploid rescue underlined the extra-embryonic nature of the Sall4-1a phenotype and revealed a further embryonic function in Wnt/beta-catenin signaling to elongate the AP axis during gastrulation. This observation was supported through genetic interaction with beta-catenin mutants, since compound heterozygous mutants recapitulated the defects of Wnt3a mutants in posterior development.     

Roles of organizer factors and BMP antagonism in mammalian forebrain establishment

A critical question in mammalian development is how the forebrain is established. In amphibians, bone morphogenetic protein (BMP) antagonism emanating from the gastrula organizer is key. Roles of BMP antagonism and the organizer in mammals remain unclear. Anterior visceral endoderm (AVE) promotes early mouse head development, but its function is controversial. Here, we explore the timing and regulation of forebrain establishment in the mouse. Forebrain specification requires tissue interaction through the late streak stage of gastrulation. Foxa2(-/-) embryos lack both the organizer and its BMP antagonists, yet about 25% show weak forebrain gene expression. A similar percentage shows ectopic AVE gene expression distally. The distal VE may thus be a source of forebrain promoting signals in these embryos. In wild-type ectoderm explants, AVE promoted forebrain specification, while anterior mesendoderm provided maintenance signals. Embryological and molecular data suggest that the AVE is a source of active BMP antagonism in vivo. In prespecification ectoderm explants, exogenous BMP antagonists triggered forebrain gene expression and inhibited posterior gene expression. Conversely, BMP inhibited forebrain gene expression, an effect that could be antagonized by anterior mesendoderm, and promoted expression of some posterior genes. These results lead to a model in which BMP antagonism supplied by exogenous tissues promotes forebrain establishment and maintenance in the murine ectoderm.     

The hypoblast of the chick embryo positions the primitive streak by antagonizing nodal signaling

The hypoblast (equivalent to the mouse anterior visceral endoderm) of the chick embryo plays a role in regulating embryonic polarity. Surprisingly, hypoblast removal causes multiple embryonic axes to form, suggesting that it emits an inhibitor of axis formation. We show that Cerberus (a multifunctional antagonist of Nodal, Wnt, and BMP signaling) is produced by the hypoblast and inhibits primitive streak formation. This activity is mimicked by Cerberus-Short (CerS), which only inhibits Nodal. Nodal misexpression can initiate an ectopic primitive streak, but only when the hypoblast is removed. We propose that, during normal development, the primitive streak forms only when the hypoblast is displaced away from the posterior margin by the endoblast, which lacks Cerberus.     

Activation of epiblast gene expression by the hypoblast layer in the prestreak chick embryo

Axis formation is a highly regulated process in vertebrate embryos. In mammals, inductive interactions between an extra-embryonic layer, the visceral endoderm, and the embryonic layer before gastrulation are critical both for anterior neural patterning and normal primitive streak formation. The role(s) of the equivalent extra-embryonic endodermal layer in the chick, the hypoblast, is still less clear, and dramatic effects of hypoblast on embryonic gene expression have yet to be demonstrated. We present evidence that two genes later associated with the gastrula organizer (Gnot-1 and Gnot-2) are induced by hypoblast signals in prestreak embryos. The significance of this induction by hypoblast is discussed in terms of possible hypoblast functions and the regulation of axis formation in the early embryo. Several factors known to be expressed in hypoblast, and retinoic acid, synergistically induce Gnot-1 and Gnot-2 expression in blastoderm cell culture. The presence of retinoic acid in prestreak embryos has not yet been directly demonstrated, but exogenous retinoic acid appears to mimic the effects of hypoblast rotation on primitive streak extension, raising the possibility that retinoid signaling plays some role in the pregastrula embryo.