Differential requirements for Smad4 in TGFbeta-dependent patterning of the early mouse embryo

Genetic and biochemical data have identified Smad4 as a key intracellular effector of the transforming growth factor beta (TGFbeta superfamily of secreted ligands. In mouse, Smad4-null embryos do not gastrulate, a phenotype consistent with loss of other TGFbeta-related signaling components. Chimeric analysis reveals a primary requirement for Smad4 in the extra-embryonic lineages; however, within the embryo proper, characterization of the specific roles of Smad4 during gastrulation and lineage specification remains limited. We have employed a Smad4 conditional allele to specifically inactivate the Smad4 gene in the early mouse epiblast. Loss of Smad4 in this tissue results in a profound failure to pattern derivatives of the anterior primitive streak, such as prechordal plate, node, notochord and definitive endoderm. In contrast to these focal defects, many well-characterized TGFbeta- and Bmp-regulated processes involved in mesoderm formation and patterning are surprisingly unaffected. Mutant embryos form abundant extra-embryonic mesoderm, including allantois, a rudimentary heart and middle primitive streak derivatives such as somites and lateral plate mesoderm. Thus, loss of Smad4 in the epiblast results not in global developmental abnormalities but instead in restricted patterning defects. These results suggest that Smad4 potentiates a subset of TGFbeta-related signals during early embryonic development, but is dispensable for others.     

Mouse embryos lacking Smad1 signals display defects in extra-embryonic tissues and germ cell formation

The Smad proteins are important intracellular mediators of the transforming growth factor beta (TGFbeta) family of secreted growth factors. Smad1 is an effector of signals provided by the bone morphogenetic protein (BMP) sub-group of TGFbeta molecules. To understand the role of Smad1 in mouse development, we have generated a Smad1 loss-of-function allele using homologous recombination in ES cells. Smad1-/- embryos die by 10.5 dpc because they fail to connect to the placenta. Mutant embryos are first recognizable by 7.0 dpc, owing to a characteristic localized outpocketing of the visceral endoderm at the posterior embryonic/extra-embryonic junction, accompanied by a dramatic twisting of the epiblast and nascent mesoderm. Chimera analysis reveals that these two defects are attributable to a requirement for Smad1 in the extra-embryonic tissues. By 7.5 dpc, Smad1-deficient embryos show a marked impairment in allantois formation. By contrast, the chorion overproliferates, is erratically folded within the extra-embryonic space and is impeded in proximal migration. BMP signals are known to be essential for the specification and proliferation of primordial germ cells. We find a drastic reduction of primordial germ cells in Smad1-deficient embryos, suggesting an essential role for Smad1-dependent signals in primordial germ cell specification. Surprisingly, despite the key involvement of BMP signaling in tissues of the embryo proper, Smad1-deficient embryos develop remarkably normally. An examination of the expression domains of Smad1, Smad5 and Smad8 in early mouse embryos show that, while Smad1 is uniquely expressed in the visceral endoderm at 6.5 dpc, in other tissues Smad1 is co-expressed with Smad5 and/or Smad8. Collectively, these data have uncovered a unique function for Smad1 signaling in coordinating the growth of extra-embryonic structures necessary to support development within the uterine environment.     

Combinatorial activities of Smad2 and Smad3 regulate mesoderm formation and patterning in the mouse embryo

TGFbeta/activin/Nodal receptors activate both Smad2 and Smad3 intracellular effector proteins. The functional activities of these closely related molecules have been extensively studied in cell lines. We show both are expressed in the early mouse embryo from the blastocyst stage onwards and mediate Foxh1-dependent activation of the Nodal autoregulatory enhancer in vitro. Genetic manipulation of their expression ratios reveals that Smad3 contributes essential signals at early post-implantation stages. Thus, loss of Smad3 in the context of one wild-type copy of Smad2 results in impaired production of anterior axial mesendoderm, while selective removal of both Smad2 and Smad3 from the epiblast additionally disrupts specification of axial and paraxial mesodermal derivatives. Finally, we demonstrate that Smad2;Smad3 double homozygous mutants entirely lack mesoderm and fail to gastrulate. Collectively, these results demonstrate that dose-dependent Smad2 and Smad3 signals cooperatively mediate cell fate decisions in the early mouse embryo.     

Role of the anterior visceral endoderm in restricting posterior signals in the mouse embryo

Recent genetic and embryological experiments have demonstrated that head formation in the mouse embryo is dependent on signals provided by two organising centers during gastrulation, the anterior visceral endoderm (AVE) and the anterior primitive streak (also called the Early Gastrula Organiser, EGO). However the molecular nature of the signals triggering anterior neural formation from the epiblast is not clearly understood. The analysis of mouse mutants has allowed the identification of some of the molecular players involved in the process of head formation. In this review, we describe different mutant embryos in which impairment of visceral endoderm function leads to similar defects in antero-posterior axis specification. These phenotypes are consistent with a role of the AVE in protecting anterior embryonic regions from signals that promote posterior development. We propose that a genetic cascade in the AVE, involving HNF3beta, Lim1, Otx2, Smad2 and ActRIB, leads to the production of secreted TGFbeta antagonists that protect the anterior epiblast region from Nodal signalling.     

Epiblast and primitive-streak origins of the endoderm in the gastrulating chick embryo

Gastrulation is characterized by the extensive movements of cells. Fate mapping is used to follow such cell movements as they occur over time, and prospective fate maps have been constructed for several stages of the model organisms used in modern studies in developmental biology. In chick embryos, detailed fate maps have been constructed for both prospective mesodermal and ectodermal cells. However, the origin and displacement of the prospective endodermal cells during crucial periods in gastrulation remain unclear. This study had three aims. First, we determined the primitive-streak origin of the endoderm using supravital fluorescent markers, and followed the movement of the prospective endodermal cells as they dispersed to generate the definitive endodermal layer. We show that between stages 3a/b and 4, the intraembryonic definitive endoderm receives contributions mainly from the rostral half of the primitive streak, and that endodermal movements parallel those of ingressing adjacent mesodermal subdivisions. Second, the question of the epiblast origin of the endodermal layer was addressed by precisely labeling epiblast cells in a region known to give rise to prospective somitic cells, and following their movement as they underwent ingression through the primitive streak. We show that the epiblast clearly contributes prospective endodermal cells to the primitive streak, and subsequently to definitive endoderm of the area pellucida. Finally, the relationship between the hypoblast and the definitive endoderm was defined by following labeled rostral primitive-streak cells over a short period of time as they contributed to the definitive endoderm, and combining this with in situ hybridization with a riboprobe for Crescent, a marker of the hypoblast. We show that as the definitive endodermal layer is laid down, there is cell-cell intercalation at its interface with the displaced hypoblast cells. These data were used to construct detailed prospective fate maps of the endoderm in the chick embryo, delineating the origins and migrations of endodermal cells in various rostrocaudal levels of the primitive streak during key periods in early development.     

Absence of Nodal signaling promotes precocious neural differentiation in the mouse embryo

After implantation, mouse embryos deficient for the activity of the transforming growth factor-beta member Nodal fail to form both the mesoderm and the definitive endoderm. They also fail to specify the anterior visceral endoderm, a specialized signaling center which has been shown to be required for the establishment of anterior identity in the epiblast. Our study reveals that Nodal-/- epiblast cells nevertheless express prematurely and ectopically molecular markers specific of anterior fate. Our analysis shows that neural specification occurs and regional identities characteristic of the forebrain are established precociously in the Nodal-/- mutant with a sequential progression equivalent to that of wild-type embryo. When explanted and cultured in vitro, Nodal-/- epiblast cells readily differentiate into neurons. Genes normally transcribed in organizer-derived tissues, such as Gsc and Foxa2, are also expressed in Nodal-/- epiblast. The analysis of Nodal-/-;Gsc-/- compound mutant embryos shows that Gsc activity plays no critical role in the acquisition of forebrain characters by Nodal-deficient cells. This study suggests that the initial steps of neural specification and forebrain development may take place well before gastrulation in the mouse and highlights a possible role for Nodal, at pregastrula stages, in the inhibition of anterior and neural fate determination.     

Otx2 is required for visceral endoderm movement and for the restriction of posterior signals in the epiblast of the mouse embryo

Genetic and embryological experiments have demonstrated an essential role for the visceral endoderm in the formation of the forebrain; however, the precise molecular and cellular mechanisms of this requirement are poorly understood. We have performed lineage tracing in combination with molecular marker studies to follow morphogenetic movements and cell fates before and during gastrulation in embryos mutant for the homeobox gene Otx2. Our results show, first, that Otx2 is not required for proliferation of the visceral endoderm, but is essential for anteriorly directed morphogenetic movement. Second, molecules that are normally expressed in the anterior visceral endoderm, such as Lefty1 and Mdkk1, are not expressed in Otx2 mutants. These secreted proteins have been reported to antagonise, respectively, the activities of Nodal and Wnt signals, which have a role in regulating primitive streak formation. The visceral endoderm defects of the Otx2 mutants are associated with abnormal expression of primitive streak markers in the epiblast, suggesting that anterior epiblast cells acquire primitive streak characteristics. Taken together, our data support a model whereby Otx2 functions in the anterior visceral endoderm to influence the ability of the adjacent epiblast cells to differentiate into anterior neurectoderm, indirectly, by preventing them from coming under the influence of posterior signals that regulate primitive streak formation.     

Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation

The developmental fate of cells in the epiblast of early-primitive-streak-stage mouse embryos was assessed by studying the pattern of tissue colonisation displayed by lac Z-expressing cells grafted orthotopically to nontransgenic embryos. Results of these fate-mapping experiments revealed that the lateral and posterior epiblast contain cells that will give rise predominantly to mesodermal derivatives. The various mesodermal populations are distributed in overlapping domains in the lateral and posterior epiblast, with the embryonic mesoderm such as heart, lateral, and paraxial mesoderm occupying a more distal position than the extraembryonic mesoderm. Heterotopic grafting of presumptive mesodermal cells results in the grafted cells adopting the fate appropriate to the new site, reflecting a plasticity of cell fate determination before ingression. The first wave of epiblast cells that ingress through the primitive streak are those giving rise to extraembryonic mesoderm. Cells that will form the mesoderm of the yolk sac and the amnion make up a major part of the mesodermal layer of the midprimitive-streak-stage embryo. Cells that are destined for embryonic mesoderm are still found within the epiblast, but some have been recruited to the distal portion of the mesoderm. By the late-primitive-streak-stage, the mesodermal layer contains only the precursors of embryonic mesoderm. This suggests that there has been a progressive displacement of the midstreak mesoderm to extraembryonic sites, which is reminiscent of that occurring in the overlying endodermal tissue. The regionalisation of cell fate in the late-primitive-streak mesoderm bears the same spatial relationship as their ancestors in the epiblast prior to cell ingression. This implies that both the position of the cells in the proximal-distal axis and their proximity to the primitive streak are major determinants for the patterning of the embryonic mesoderm. © 1995 Wiley-Liss, Inc.     

Intercellular Interactions,Position, and Polarity in Establishing Blastocyst Cell Lineages and Embryonic Axes

The formation of the three lineages of the mouse blastocyst provides a powerful model system to study interactions among cell behavior, cell signaling, and lineage development. Hippo signaling differences between the inner and outer cells of the early cleavage stages, combined with establishment of a stably polarized outer epithelium, lead to the establishment of the inner cell mass and the trophectoderm, whereas FGF signaling differences among the individual cells of the ICM lead to gradual separation and segregation of the epiblast and primitive endoderm lineages. Events in the late blastocyst lead to the formation of a special subset of cells from the primitive endoderm that are key sources for the signals that establish the subsequent body axis. The slow pace of mouse early development, the ability to culture embryos over this time period, the increasing availability of live cell imaging tools, and the ability to modify gene expression at will are providing increasing insights into the cell biology of early cell fate decisions.     

The endoderm of the mouse embryo arises by dynamic widespread intercalation of embryonic and extraembryonic lineages

The cell movements underlying the morphogenesis of the embryonic endoderm, the tissue that will give rise to the respiratory and digestive tracts, are complex and not well understood. Using live imaging combined with genetic labeling, we investigated the cell behaviors and fate of the visceral endoderm during gut endoderm formation in the mouse gastrula. Contrary to the prevailing view, our data reveal no mass displacement of visceral endoderm to extraembryonic regions concomitant with the emergence of epiblast-derived definitive endoderm. Instead, we observed dispersal of the visceral endoderm epithelium and extensive mixing between cells of visceral endoderm and epiblast origin. Visceral endoderm cells remained associated with the epiblast and were incorporated into the early gut tube. Our findings suggest that the segregation of extraembryonic and embryonic tissues within the mammalian embryo is not as strict as believed and that a lineage previously defined as exclusively extraembryonic contributes cells to the embryo.     

Cell-surface proteomics identifies lineage-specific markers of embryo-derived stem cells

The advent of reprogramming and its impact on stem cell biology has renewed interest in lineage restriction in mammalian embryos, the source of embryonic (ES), epiblast (EpiSC), trophoblast (TS), and extraembryonic endoderm (XEN) stem cell lineages. Isolation of specific cell types during stem cell differentiation and reprogramming, and also directly from embryos, is a major technical challenge because few cell-surface proteins are known that can distinguish each cell type. We provide a large-scale proteomic resource of cell-surface proteins for the four embryo-derived stem cell lines. We validated 27 antibodies against lineage-specific cell-surface markers, which enabled investigation of specific cell populations during ES-EpiSC reprogramming and ES-to-XEN differentiation. Identified markers also allowed prospective isolation and characterization of viable lineage progenitors from blastocysts by flow cytometry. These results provide a comprehensive stem cell proteomic resource and enable new approaches to interrogate the mechanisms that regulate cell fate specification.     

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.     

Individual blastomeres of 16- and 32-cell mouse embryos are able to develop into foetuses and mice

Cell and developmental studies have clarified how, by the time of implantation, the mouse embryo forms three primary cell lineages: epiblast (EPI), primitive endoderm (PE), and trophectoderm (TE). However, it still remains unknown when cells allocated to these three lineages become determined in their developmental fate. To address this question, we studied the developmental potential of single blastomeres derived from 16- and 32-cell stage embryos and supported by carrier, tetraploid blastomeres. We were able to generate singletons, identical twins, triplets, and quadruplets from individual inner and outer cells of 16-cell embryos and, sporadically, foetuses from single cells of 32-cell embryos. The use of embryos constitutively expressing GFP as the donors of single diploid blastomeres enabled us to identify their cell progeny in the constructed 2n↔4n blastocysts. We showed that the descendants of donor blastomeres were able to locate themselves in all three first cell lineages, i.e., epiblast, primitive endoderm, and trophectoderm. In addition, the application of Cdx2 and Gata4 markers for trophectoderm and primitive endoderm, respectively, showed that the expression of these two genes in the descendants of donor blastomeres was either down- or up-regulated, depending on the cell lineage they happened to occupy. Thus, our results demonstrate that up to the early blastocysts stage, the destiny of at least some blastomeres, although they have begun to express markers of different lineage, is still labile.