Cripto-independent Nodal signaling promotes positioning of the A-P axis in the early mouse embryo

During early mouse development, the TGFβ-related protein Nodal specifies the organizing centers that control the formation of the anterior-posterior (A-P) axis. EGF-CFC proteins are important components of the Nodal signaling pathway, most likely by acting as Nodal coreceptors. However, the extent to which Nodal activity depends on EGF-CFC proteins is still debated. Cripto is the earliest EGF-CFC gene expressed during mouse embryogenesis and is involved in both A-P axis orientation and mesoderm formation. To investigate the relation between Cripto and Nodal in the early mouse embryo, we removed the Nodal antagonist Cerberus 1 (Cer1) and simultaneously Cripto, by generating Cer1;Cripto double mouse mutants. We observed that two thirds of the Cer1;Cripto double mutants are rescued in processes that are severely compromised in Cripto^−/− embryos, namely A-P axis orientation, anterior mesendoderm and posterior neuroectoderm formation. The observed rescue is strongly reduced in Cer1;Cripto;Nodal triple mutants, suggesting that Nodal can signal extensively in the absence of Cripto, if Cer1 is also inhibited. This signaling activity drives A-P axis positioning. Our results provide evidence for the existence of Cripto-independent signaling mechanisms, by which Nodal controls axis specification in the early mouse embryo.     

The Vg1-related protein Gdf3 acts in a Nodal signaling pathway in the pre-gastrulation mouse embryo

The formation of the anterior visceral endoderm (AVE) in the pre-gastrulation mouse embryo represents a crucial event in patterning of the anterior-posterior axis. Here, we show that the transforming growth factor beta (Tgfbeta) family member Gdf3 (growth-differentiation factor 3), a close relative of Xenopus Vg1, resembles the Tgfbeta ligand Nodal in both its signaling activity and its role in AVE formation in vivo. Thus, in cell culture, Gdf3 signaling requires the EGF-CFC co-receptor Cripto and can be inhibited by Lefty antagonists. In Xenopus embryos, Gdf3 misexpression results in secondary axis formation, and induces morphogenetic elongation and mesendoderm formation in animal caps. In mouse embryos, Gdf3 is expressed in the inner cell mass and epiblast, and null mutants frequently exhibit abnormal formation or positioning of the AVE. This phenotype correlates with defects in mesoderm and definitive endoderm formation, as well as abnormal Nodal expression levels. Our findings indicate that Gdf3 acts in a Nodal-like signaling pathway in pre-gastrulation development, and provide evidence for the functional conservation of Vg1 activity in mice.     

A Novel Nodal Enhancer Dependent on Pluripotency Factors and Smad2/3 Signaling Conditions a Regulatory Switch During Epiblast Maturation

During early development, modulations in the expression of Nodal, a TGFβ family member, determine the specification of embryonic and extra-embryonic cell identities. Nodal has been extensively studied in the mouse, but aspects of its early expression remain unaccounted for. We identified a conserved hotspot for the binding of pluripotency factors at the Nodal locus and called this sequence “highly bound element” (HBE). Luciferase-based assays, the analysis of fluorescent HBE reporter transgenes, and a conditional mutation of HBE allowed us to establish that HBE behaves as an enhancer, is activated ahead of other Nodal enhancers in the epiblast, and is essential to Nodal expression in embryonic stem cells (ESCs) and in the mouse embryo. We also showed that HBE enhancer activity is critically dependent on its interaction with the pluripotency factor Oct4 and on Activin/Nodal signaling. Use of an in vitro model of epiblast maturation, relying on the differentiation of ESCs into epiblast stem cells (EpiSCs), revealed that this process entails a shift in the regulation of Nodal expression from an HBE-driven phase to an ASE-driven phase, ASE being another autoregulatory Nodal enhancer. Deletion of HBE in ESCs or in EpiSCs allowed us to show that HBE, although not necessary for Nodal expression in EpiSCs, is required in differentiating ESCs to activate the differentiation-promoting ASE and therefore controls this regulatory shift. Our findings clarify how early Nodal expression is regulated and suggest how this regulation can promote the specification of extra-embryonic precusors without inducing premature differentiation of epiblast cells. More generally, they open new perspectives on how pluripotency factors achieve their function.     

Nodal cis-regulatory elements reveal epiblast and primitive endoderm heterogeneity in the peri-implantation mouse embryo

Nodal, a secreted factor known for its conserved functions in cell-fate specification and the establishment of embryonic axes, is also required in mammals to maintain the pluripotency of the epiblast, the tissue that gives rise to all fetal lineages. Although Nodal is expressed as early as E3.5 in the mouse embryo, its regulation and functions at pre- and peri-implantation stages are currently unknown. Sensitive reporter transgenes for two Nodal cis-regulatory regions, the PEE and the ASE, exhibit specific expression profiles before implantation. Mutant and inhibitor studies find them respectively regulated by Wnt/β-catenin signaling and Activin/Nodal signaling, and provide evidence for localized and heterogeneous activities of these pathways in the inner cell mass, the epiblast and the primitive endoderm. These studies also show that Nodal and its prime effector, FoxH1, are not essential to preimplantation Activin/Nodal signaling. Finally, a strong upregulation of the ASE reporter in implanting blastocysts correlates with a downregulation of the pluripotency factor Nanog in the maturing epiblast. This study uncovers conservation in the mouse blastocyst of Wnt/β-catenin and Activin/Nodal-dependent activities known to govern Nodal expression and the establishment of polarity in the blastula of other deuterostomes. Our results indicate that these pathways act early on to initiate distinct cell-specification processes in the ICM derivatives. Our data also suggest that the activity of the Activin/Nodal pathway is dampened by interactions with the molecular machinery of pluripotency until just before implantation, possibly delaying cell-fate decisions in the mouse embryo.     

Analysis of extraembryonic mesodermal structure formation in the absence of morphological primitive streak

During mouse gastrulation, the primitive streak is formed on the posterior side of the embryo. Cells migrate out of the primitive streak to form the future mesoderm and endoderm. Fate mapping studies revealed a group of cell migrate through the proximal end of the primitive streak and give rise to the extraembryonic mesoderm tissues such as the yolk sac blood islands and allantois. However, it is not clear whether the formation of a morphological primitive streak is required for the development of these extraembryonic mesodermal tissues. Loss of the Cripto gene in mice dramatically reduces, but does not completely abolish, Nodal activity leading to the absence of a morphological primitive streak. However, embryonic erythrocytes are still formed and assembled into the blood islands. In addition, Cripto mutant embryos form allantoic buds. However, Drap1 mutant embryos have excessive Nodal activity in the epiblast cells before gastrulation and form an expanded primitive streak, but no yolk sac blood islands or allantoic bud formation. Lefty2 embryos also have elevated levels of Nodal activity in the primitive streak during gastrulation, and undergo normal blood island and allantois formation. We therefore speculate that low level of Nodal activity disrupts the formation of morphological primitive streak on the posterior side, but still allows the formation of primitive streak cells on the proximal side, which give rise to the extraembryonic mesodermal tissues formation. Excessive Nodal activity in the epiblast at pre‐gastrulation stage, but not in the primitive streak cells during gastrulation, disrupts extraembryonic mesoderm development.     

Regulation of extra-embryonic endoderm stem cell differentiation by Nodal and Cripto signaling

The signaling pathway for Nodal, a ligand of the TGFβ superfamily, plays a central role in regulating the differentiation and/or maintenance of stem cell types that can be derived from the peri-implantation mouse embryo. Extra-embryonic endoderm stem (XEN) cells resemble the primitive endoderm of the blastocyst, which normally gives rise to the parietal and the visceral endoderm in vivo, but XEN cells do not contribute efficiently to the visceral endoderm in chimeric embryos. We have found that XEN cells treated with Nodal or Cripto (Tdgf1), an EGF-CFC co-receptor for Nodal, display upregulation of markers for visceral endoderm as well as anterior visceral endoderm (AVE), and can contribute to visceral endoderm and AVE in chimeric embryos. In culture, XEN cells do not express Cripto, but do express the related EGF-CFC co-receptor Cryptic (Cfc1), and require Cryptic for Nodal signaling. Notably, the response to Nodal is inhibited by the Alk4/Alk5/Alk7 inhibitor SB431542, but the response to Cripto is unaffected, suggesting that the activity of Cripto is at least partially independent of type I receptor kinase activity. Gene set enrichment analysis of genome-wide expression signatures generated from XEN cells under these treatment conditions confirmed the differing responses of Nodal- and Cripto-treated XEN cells to SB431542. Our findings define distinct pathways for Nodal and Cripto in the differentiation of visceral endoderm and AVE from XEN cells and provide new insights into the specification of these cell types in vivo.     

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.     

Non-cell-autonomous role for Cripto in axial midline formation during vertebrate embryogenesis

Several membrane-associated proteins are known to modulate the activity and range of potent morphogenetic signals during development. In particular, members of the EGF-CFC family encode glycosyl-phosphatidylinositol (GPI)-linked proteins that are essential for activity of the transforming growth factor beta (TGFbeta) ligand Nodal, a factor that plays a central role in establishing the vertebrate body plan. Genetic and biochemical studies have indicated that EGF-CFC proteins function as cell-autonomous co-receptors for Nodal; by contrast, cell culture data have suggested that the mammalian EGF-CFC protein Cripto can act as a secreted signaling factor. Here we show that Cripto acts non-cell-autonomously during axial mesendoderm formation in the mouse embryo and may possess intercellular signaling activity in vivo. Phenotypic analysis of hypomorphic mutants demonstrates that Cripto is essential for formation of the notochordal plate, prechordal mesoderm and foregut endoderm during gastrulation. Remarkably, Cripto null mutant cells readily contribute to these tissues in chimeras, indicating non-cell-autonomy. Consistent with these loss-of-function analyses, gain-of-function experiments in chick embryos show that exposure of node/head process mesoderm to soluble Cripto protein results in alterations in cell fates toward anterior mesendoderm, in a manner that is dependent on Nodal signaling. Taken together, our findings support a model in which Cripto can function in trans as an intercellular mediator of Nodal signaling activity.     

The Wnt co-receptors Lrp5 and Lrp6 are essential for gastrulation in mice

Recent work has identified LDL receptor-related family members, Lrp5 and Lrp6, as co-receptors for the transduction of Wnt signals. Our analysis of mice carrying mutations in both Lrp5 and Lrp6 demonstrates that the functions of these genes are redundant and are essential for gastrulation. Lrp5;Lrp6 double homozygous mutants fail to establish a primitive streak, although the anterior visceral endoderm and anterior epiblast fates are specified. Thus, Lrp5 and Lrp6 are required for posterior patterning of the epiblast, consistent with a role in transducing Wnt signals in the early embryo. Interestingly, Lrp5(+/-);Lrp6(-/-) embryos die shortly after gastrulation and exhibit an accumulation of cells at the primitive streak and a selective loss of paraxial mesoderm. A similar phenotype is observed in Fgf8 and Fgfr1 mutant embryos and provides genetic evidence in support of a molecular link between the Fgf and Wnt signaling pathways in patterning nascent mesoderm. Lrp5(+/-);Lrp6(-/-) embryos also display an expansion of anterior primitive streak derivatives and anterior neurectoderm that correlates with increased Nodal expression in these embryos. The effect of reducing, but not eliminating, Wnt signaling in Lrp5(+/-);Lrp6(-/-) mutant embryos provides important insight into the interplay between Wnt, Fgf and Nodal signals in patterning the early mouse embryo.     

The nodal precursor acting via activin receptors induces mesoderm by maintaining a source of its convertases and BMP4

During early mouse development, the subtilisin-like proprotein convertases (SPC) Furin and PACE4 pattern the primitive ectoderm and visceral endoderm, presumably by activating the TGFss-related Nodal precursor. Here, mutation of the SPC motif provides direct evidence that Nodal processing is essential to specify anterior visceral endoderm and mesendoderm. Surprisingly, however, the Nodal precursor binds and activates activin receptors to maintain expression of Furin, PACE4, and Bmp4 in extraembryonic ectoderm at a distance from the Nodal source. In return, Bmp4 induces Wnt3, which amplifies Nodal expression in the epiblast and mediates induction of mesoderm. We conclude that uncleaved Nodal sustains the extraembryonic source of proprotein convertases and Bmp4 to amplify Nodal signaling in two nonredundant feedback loops with dual timescales and to localize primitive streak formation at the posterior pole. Based on mathematical modeling, we discuss how these sequential loops control cell fate.     

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.     

Cripto is required for mesoderm and endoderm cell allocation during mouse gastrulation

During mouse gastrulation, cells in the primitive streak undergo epithelial–mesenchymal transformation and the resulting mesenchymal cells migrate out laterally to form mesoderm and definitive endoderm across the entire embryonic cylinder. The mechanisms underlying mesoderm and endoderm specification, migration, and allocation are poorly understood. In this study, we focused on the function of mouse Cripto, a member of the EGF-CFC gene family that is highly expressed in the primitive streak and migrating mesoderm cells on embryonic day 6.5. Conditional inactivation of Cripto during gastrulation leads to varied defects in mesoderm and endoderm development. Mutant embryos display accumulation of mesenchymal cells around the shortened primitive streak indicating a functional requirement of Cripto during the formation of mesoderm layer in gastrulation. In addition, some mutant embryos showed poor formation and abnormal allocation of definitive endoderm cells on embryonic day 7.5. Consistently, many mutant embryos that survived to embryonic day 8.5 displayed defects in ventral closure of the gut endoderm causing cardia bifida. Detailed analyses revealed that both the Fgf8–Fgfr1 pathway and p38 MAP kinase activation are partially affected by the loss of Cripto function. These results demonstrate a critical role for Cripto during mouse gastrulation, especially in mesoderm and endoderm formation and allocation.     

Disruption of early proximodistal patterning and AVE formation in Apc mutants

In the postimplantation mouse embryo, axial patterning begins with the restriction of expression of a set of genes to the distal visceral endoderm (DVE). This proximodistal (PD) axis is subsequently transformed into an anteroposterior axis as the VE migrates anteriorly to form the anterior visceral endoderm (AVE). Both Nodal and Wnt signaling pathways are involved in these events. We show here that loss of function in the adenomatous polyposis coli gene (Apc) leads to constitutive beta-catenin activity that induces a proximalization of the epiblast with the activation of a subset of posterior mesendodermal genes, and loss of ability to induce the DVE. The loss of some DVE genes such as Hex and goosecoid is rescued in chimeras where only the epiblast was wild type; however, these DVE markers were no longer restricted distally but covered the entire epiblast. Thus, the Apc gene is needed in both embryonic and extraembryonic lineages for normal PD patterning around implantation, suggesting that early restricted activation of the Wnt pathway may be important for initiating axial asymmetries. In addition, we found that nuclear beta-catenin and other molecular markers are asymmetrically expressed by 4.5 days.     

Evolutionary origin of the Otx2 enhancer for its expression in visceral endoderm

In the mouse, the Otx2 gene has been shown to play essential roles in the visceral endoderm during anterior-posterior axis formation and head induction. While these are primary processes in vertebrate embryogenesis, the visceral endoderm is a tissue unique to mammals. Two enhancers (VE and CM) have been previously found to direct Otx2 expression during early embryogenesis. This study demonstrates that in anterior visceral endoderm the CM enhancer does not have an activity by itself, but enhances the activity of the VE enhancer. These two enhancers also cooperate for the activities in anterior mesendoderm and cephalic mesenchyme. Comparative studies suggest that VE enhancer function was most likely established before the divergence of sarcopterygians into Actinistia, Dipnoi and tetrapods, while the nucleotide sequence corresponding to the VE enhancer was already present in the last common ancestor of bony fishes. The CM enhancer sequence and function would have been also established in ancestral sarcopterygians. The VE/CM enhancers and their gene cascades in the ancestral sarcopterygian head organizer would then have been co-opted by amphibian deep endoderm cells and mammalian visceral endoderm cells for the head development.     

Differential response of epiblast stem cells to Nodal and Activin signalling: a paradigm of early endoderm development in the embryo

Mouse epiblast stem cells (EpiSCs) display temporal differences in the upregulation of Mixl1 expression during the initial steps of in vitro differentiation, which can be correlated with their propensity for endoderm differentiation. EpiSCs that upregulated Mixl1 rapidly during differentiation responded robustly to both Activin A and Nodal in generating foregut endoderm and precursors of pancreatic and hepatic tissues. By contrast, EpiSCs that delayed Mixl1 upregulation responded less effectively to Nodal and showed an overall suboptimal outcome of directed differentiation. The enhancement in endoderm potency in Mixl1-early cells may be accounted for by a rapid exit from the progenitor state and the efficient response to the induction of differentiation by Nodal. EpiSCs that readily differentiate into the endoderm cells are marked by a distinctive expression fingerprint of transforming growth factor (TGF)-β signalling pathway genes and genes related to the endoderm lineage. Nodal appears to elicit responses that are associated with transition to a mesenchymal phenotype, whereas Activin A promotes gene expression associated with maintenance of an epithelial phenotype. We postulate that the formation of definitive endoderm (DE) in embryoid bodies follows a similar process to germ layer formation from the epiblast, requiring an initial de-epithelialization event and subsequent re-epithelialization. Our results show that priming EpiSCs with the appropriate form of TGF-β signalling at the formative phase of endoderm differentiation impacts on the further progression into mature DE-derived lineages, and that this is influenced by the initial characteristics of the cell population. Our study also highlights that Activin A, which is commonly used as an in vitro surrogate for Nodal in differentiation protocols, does not elicit the same downstream effects as Nodal, and therefore may not effectively mimic events that take place in the mouse embryo.     

Ventral closure, headfold fusion and definitive endoderm migration defects in mouse embryos lacking the fibronectin leucine-rich transmembrane protein FLRT3

The three fibronectin leucine-rich repeat transmembrane (FLRT) proteins contain 10 leucine-rich repeats (LRR), a type III fibronectin (FN) domain, followed by the transmembrane region, and a short cytoplasmic tail. XFLRT3, a Nodal/TGFβ target, regulates cell adhesion and modulates FGF signalling during Xenopus gastrulation. The present study describes the onset and pattern of FLRT1-3 expression in the early mouse embryo. FLRT3 expression is activated in the anterior visceral endoderm (AVE), and during gastrulation appears in anterior streak derivatives namely the node, notochord and the emerging definitive endoderm. To explore FLRT3 function we generated a null allele via gene targeting. Early Nodal activities required for anterior-posterior (A-P) patterning, primitive streak formation and left-right (L-R) axis determination were unperturbed. However, FLRT3 mutant embryos display defects in headfold fusion, definitive endoderm migration and a failure of the lateral edges of the ventral body wall to fuse, leading to cardia bifida. Surprisingly, the mutation has no effect on FGF signalling. Collectively these experiments demonstrate that FLRT3 plays a key role in controlling cell adhesion and tissue morphogenesis in the developing mouse embryo.     

Rac1-Dependent Collective Cell Migration Is Required for Specification of the Anterior-Posterior Body Axis of the Mouse

Cell migration and cell rearrangements are critical for establishment of the body plan of vertebrate embryos. The first step in organization of the body plan of the mouse embryo, specification of the anterior-posterior body axis, depends on migration of the anterior visceral endoderm from the distal tip of the embryo to a more proximal region overlying the future head. The anterior visceral endoderm (AVE) is a cluster of extra-embryonic cells that secretes inhibitors of the Wnt and Nodal pathways to inhibit posterior development. Because Rac proteins are crucial regulators of cell migration and mouse Rac1 mutants die early in development, we tested whether Rac1 plays a role in AVE migration. Here we show that Rac1 mutant embryos fail to specify an anterior-posterior axis and, instead, express posterior markers in a ring around the embryonic circumference. Cells that express the molecular markers of the AVE are properly specified in Rac1 mutants but remain at the distal tip of the embryo at the time when migration should take place. Using tissue specific deletions, we show that Rac1 acts autonomously within the visceral endoderm to promote cell migration. High-resolution imaging shows that the leading wild-type AVE cells extend long lamellar protrusions that span several cell diameters and are polarized in the direction of cell movement. These projections are tipped by filopodia-like structures that appear to sample the environment. Wild-type AVE cells display hallmarks of collective cell migration: they retain tight and adherens junctions as they migrate and exchange neighbors within the plane of the visceral endoderm epithelium. Analysis of mutant embryos shows that Rac1 is not required for intercellular signaling, survival, proliferation, or adhesion in the visceral endoderm but is necessary for the ability of visceral endoderm cells to extend projections, change shape, and exchange neighbors. The data show that Rac1-mediated epithelial migration of the AVE is a crucial step in the establishment of the mammalian body plan and suggest that Rac1 is essential for collective migration in mammalian tissues.     

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.