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.     

Correct patterning of the primitive streak requires the anterior visceral endoderm

Anterior-posterior axis specification in the mouse requires signalling from a specialised extra-embryonic tissue called the anterior visceral endoderm (AVE). AVE precursors are induced at the distal tip of the embryo and move to the prospective anterior. Embryological and genetic analysis has demonstrated that the AVE is required for anterior patterning and for correctly positioning the site of primitive streak formation by inhibiting Nodal activity. We have carried out a genetic ablation of the Hex-expressing cells of the AVE (Hex-AVE) by knocking the Diphtheria toxin subunit A into the Hex locus in an inducible manner. Using this model we have identified that, in addition to its requirement in the anterior of the embryo, the Hex-AVE sub-population has a novel role between 5.5 and 6.5dpc in patterning the primitive streak. Embryos lacking the Hex-AVE display delayed initiation of primitive streak formation and miss-patterning of the anterior primitive streak. We demonstrate that in the absence of the Hex-AVE the restriction of Bmp2 expression to the proximal visceral endoderm is also defective and expression of Wnt3 and Nodal is not correctly restricted to the posterior epiblast. These results, coupled with the observation that reducing Nodal signalling in Hex-AVE ablated embryos increases the frequency of phenotypes observed, suggests that these primitive streak patterning defects are due to defective Nodal signalling. Together, our experiments demonstrate that the AVE is not only required for anterior patterning, but also that specific sub-populations of this tissue are required to pattern the posterior of the embryo.     

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.     

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.     

Pten regulates collective cell migration during specification of the anterior-posterior axis of the mouse embryo

Pten, the potent tumor suppressor, is a lipid phosphatase that is best known as a regulator of cell proliferation and cell survival. Here we show that mouse embryos that lack Pten have a striking set of morphogenetic defects, including the failure to correctly specify the anterior-posterior body axis, that are not caused by changes in proliferation or cell death. The majority of Pten null embryos express markers of the primitive streak at ectopic locations around the embryonic circumference, rather than at a single site at the posterior of the embryo. Epiblast-specific deletion shows that Pten is not required in the cells of the primitive streak; instead, Pten is required for normal migration of cells of the Anterior Visceral Endoderm (AVE), an extraembryonic organizer that controls the position of the streak. Cells of the wild-type AVE migrate within the visceral endoderm epithelium from the distal tip of the embryo to a position adjacent to the extraembryonic region. In all Pten null mutants, AVE cells move a reduced distance and disperse in random directions, instead of moving as a coordinated group to the anterior of the embryo. Aberrant AVE migration is associated with the formation of ectopic F-actin foci, which indicates that absence of Pten disrupts the actin-based migration of these cells. After the initiation of gastrulation, embryos that lack Pten in the epiblast show defects in the migration of mesoderm and/or endoderm. The findings suggest that Pten has an essential and general role in the control of mammalian collective cell migration.     

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.     

From fertilization to gastrulation: axis formation in the mouse embryo.

Although much remains unknown about how the embryonic axis is laid down in the mouse, it is now clear that reciprocal interactions between the extraembryonic and embryonic lineages establish and reinforce patterning of the embryo. At early post-implantation stages, the extraembryonic ectoderm appears to impart proximal-posterior identity to the adjacent proximal epiblast, whereas the distal visceral endoderm signals to the underlying epiblast to restrict posterior identity as it moves anteriorward. At gastrulation, the visceral endoderm is necessary for specifying anterior primitive streak derivatives, which, in turn, pattern the anterior epiblast. Polarity of these extraembryonic tissues can be traced back to the blastocyst stage, where asymmetry has been linked to the point of sperm entry at fertilization.     

Coordination of cell proliferation and anterior-posterior axis establishment in the mouse embryo

During development, the growth of the embryo must be coupled to its patterning to ensure correct and timely morphogenesis. In the mouse embryo, migration of the anterior visceral endoderm (AVE) to the prospective anterior establishes the anterior-posterior (A-P) axis. By analysing the distribution of cells in S phase, M phase and G2 from the time just prior to the migration of the AVE until 18 hours after its movement, we show that there is no evidence for differential proliferation along the A-P axis of the mouse embryo. Rather, we have identified that as AVE movements are being initiated, the epiblast proliferates at a much higher rate than the visceral endoderm. We show that these high levels of proliferation in the epiblast are dependent on Nodal signalling and are required for A-P establishment, as blocking cell division in the epiblast inhibits AVE migration. Interestingly, inhibition of migration by blocking proliferation can be rescued by Dkk1. This suggests that the high levels of epiblast proliferation function to move the prospective AVE away from signals that are inhibitory to its migration. The finding that initiation of AVE movements requires a certain level of proliferation in the epiblast provides a mechanism whereby A-P axis development is coordinated with embryonic growth.     

Cell populations and morphogenetic movements underlying formation of the avian primitive streak and organizer

The cell populations and morphogenetic movements that contribute to the formation of the avian primitive streak and organizer-Hensen's node-are poorly understood. We labeled selected groups of cells with fluorescent dyes and then followed them over time during formation and progression of the primitive streak and formation of Hensen's node. We show that (1) the primitive streak arises from a localized population of epiblast cells spanning the caudal midline of Koller's sickle, with the mid-dorsal cells of the primitive streak arising from the midline of the epiblast overlying Koller's sickle and the deeper and more lateral primitive streak cells arising more laterally within the epiblast overlying the sickle, from an arch subtending about 30 degrees; (2) convergent extension movements of cells in the epiblast overlying Koller's sickle contribute to formation of the initial primitive streak; and (3) Hensen's node is derived from a mixture of cells originating both from the epiblast just rostral to the incipient (stage 2) primitive streak and later from the epiblast just rostral to the elongating (stage 3a/b) primitive streak, as well as from the rostral tip of the progressing streak itself. Collectively, these results provide new information on the formation of the avian primitive streak and organizer, increasing our understanding of these important events of early development of amniotes.     

Functional redundancy of EGF-CFC genes in epiblast and extraembryonic patterning during early mouse embryogenesis

During early mouse embryogenesis, multiple patterning and differentiation events require the activity of Nodal, a ligand of the transforming growth factor-beta (TGFβ) family. Although Nodal signaling is known to require activity of EGF-CFC co-receptors in many contexts, it has been unclear whether all Nodal signaling in the early mouse embryo is EGF-CFC dependent. We have investigated the double null mutant phenotypes for the EGF-CFC genes Cripto and Cryptic, which encode co-receptors for Nodal, and have found that they have partially redundant functions in early mouse development. Expression of Cripto and Cryptic is non-overlapping prior to gastrulation, since Cripto is expressed solely in the epiblast whereas Cryptic is expressed in the primitive endoderm of the late blastocyst and the visceral endoderm after implantation. Despite these non-overlapping expression patterns, Cripto; Cryptic double mutants display severe defects in epiblast, extraembryonic ectoderm, and anterior visceral endoderm (AVE), resulting in phenotypes that are highly similar to those of Nodal null mutants. Our results indicate that both Cripto and Cryptic function non-cell-autonomously during normal development, and that most if not all Nodal activity in early mouse embryogenesis is EGF-CFC-dependent.     

Uterine-embryonic interaction in pig: activin, follistatin, and activin receptor II in uterus and embryo during early gestation

The mRNA expression patterns of activin beta(A) and follistatin in the uterus and embryo, the mRNA expression of the activin receptor II in the embryo, and the localization in the uterus of the immunoreactive activin beta(A) and the receptor II proteins in the uterus were examined at gestation days 7-12 after ovulation in pig. Activin was located predominantly at the mesometrial side of the uterus during all stages of pregnancy studied. Follistatin mRNA was absent in the uterus during these stages, suggesting that activin of uterine origin is not inhibited by intra-uterine follistatin. The receptor was localized throughout the glandular and luminal epithelium of the uterus. In the embryo, activin was expressed predominantly in the epiblast before unfolding, but after unfolding of the epiblast activin expression shifted to the trophoblast. The expression pattern of follistatin mRNA was contrarily to that of activin, i.e., before unfolding predominantly in the trophoblast (days 8-9), and shifted to the epiblast at day 10. During streak stages, follistatin was detected in the node and primitive streak. Activin receptor II mRNA was first detected at day 8 in the embryoblast. At day 11, it was expressed in trophoblast cells near the epiblast, and in the first ingressing mesoderm cells. During the streak stages, it was expressed predominantly in the trophoblast. The presence of activin and its receptor in uterine epithelium and early embryonic tissues indicate that both embryonic and uterine activin are involved in intra-uterine processes, such as attachment and early embryonic development. Mol. Reprod. Dev. 59: 390-399, 2001.     

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     

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.     

Changes in the expression of the carbohydrate epitope HNK-1 associated with mesoderm induction in the chick embryo

We report that a monoclonal antibody, HNK-1, identifies specific regions and cell types during primitive streak formation in the chick blastoderm. Immunohistochemical studies show that the cells of the forming hypoblast are HNK-1 positive from the earliest time at which they can be identified. Some cells of the margin of the blastoderm are also positive. The mesoderm cells of the primitive streak stain strongly with the antibody from the time of their initial appearance. In the epiblast, some cells are positive and some negative at pre-primitive-streak stages, but as the primitive streak develops a gradient of staining intensity is seen within the upper layer, increasing towards the primitive streak. At later stages of development, the notochord and the mesenchyme of the headfold are positive, while the rest of the mesoderm (lateral plate) no longer expresses HNK-1 immunoreactivity. This antibody therefore reveals changes associated with mesodermal induction: before induction, it recognizes the 'inducing' tissue (the hypoblast) and reveals a mosaic pattern in the responding tissue (the epiblast); after primitive streak formation, the mesoderm of the primitive streak that results from the inductive interactions expresses the epitope strongly. Affinity purification of HNK-1-related proteins in various tissues was carried out, followed by SDS-PAGE to identify them. The hypoblast, mesoderm and epiblast of gastrulating chick embryos have some HNK-1-related proteins in common, while others are unique to specific tissues. Attempts have been made to identify these proteins using Western blots and antibodies known to recognize HNK-1-related molecules, but none of the antibodies used identify the bands unique to any of the tissues studied. We conclude that these proteins may be novel members of the HNK-1/L2 family, and that they may have a role in cell interactions during early development.     

Complementary functions of Otx2 and Cripto in initial patterning of mouse epiblast.

The development of the mammalian antero-posterior (A-P) axis is proposed to be established by distinct anterior and posterior signaling centers, anterior visceral endoderm and primitive streak, respectively. Knock-out studies in mice have shown that Otx2 and Cripto have crucial roles in the generation and/or functions of these anterior and posterior centers, respectively. In both Otx2 and Cripto single mutants, the initial formation of the A-P axis takes place in a proximal-distal (P-D) orientation, but subsequent axis rotation fails to occur. To examine the developmental consequences of the lack of these two genes, we have analyzed the Otx2(-/-);Cripto(-/-) double homozygous mutant phenotype. In the double mutants, the expression of the A-P axis markers Cer-l, Lim1, and Wnt3 was not induced, while expression of Fgf8 and T was expanded throughout the epiblast, indicating that the double mutants could not form the A-P axis even in its initial P-D orientation. In addition, the double mutants displayed defects in differentiation of the visceral endoderm overlying the epiblast, as well as in the extraembryonic ectoderm. Furthermore, differentiation of neuroectoderm was accelerated as judged by the reduction of Oct4 expression and emergence of Sox1 and Gbx2 expression in the double mutant epiblast. The resulting ectoderm only displayed characteristics of anterior hindbrain, implicating it as a ground state in the mammalian body plan. Our results indicate that complementary functions of Otx2 and Cripto are essential for initial patterning of the A-P axis in the mouse embryo.     

FGF signalling through RAS/MAPK and PI3K pathways regulates cell movement and gene expression in the chicken primitive streak without affecting E-cadherin expression

Background  

FGF signalling regulates numerous aspects of early embryo development. During gastrulation in amniotes, epiblast cells undergo an epithelial to mesenchymal transition (EMT) in the primitive streak to form the mesoderm and endoderm. In mice lacking FGFR1, epiblast cells in the primitive streak fail to downregulate E-cadherin and undergo EMT, and cell migration is inhibited. This study investigated how FGF signalling regulates cell movement and gene expression in the primitive streak of chicken embryos.     

A role for the hypoblast (AVE) in the initiation of neural induction, independent of its ability to position the primitive streak

The mouse anterior visceral endoderm (AVE) has been implicated in embryonic polarity: it helps to position the primitive streak and some have suggested that it might act as a "head organizer", inducing forebrain directly. Here we explore the role of the hypoblast (the chick equivalent of the AVE) in the early steps of neural induction and patterning. We report that the hypoblast can induce a set of very early markers that are later expressed in the nervous system and in the forebrain, but only transiently. Different combinations of signals are responsible for different aspects of this early transient induction: FGF initiates expression of Sox3 and ERNI, retinoic acid can induce Cyp26A1 and only a combination of low levels of FGF8 together with Wnt- and BMP-antagonists can induce Otx2. BMP- and Wnt-antagonists and retinoic acid, in different combinations, can maintain the otherwise transient induction of these markers. However, neither the hypoblast nor any of these factors or combinations thereof can induce the definitive neural marker Sox2 or the formation of a mature neural plate or a forebrain, suggesting that the hypoblast is not a head organizer and that other signals remain to be identified. Interestingly, FGF and retinoids, generally considered as caudalizing factors, are shown here to play a role in the induction of a transient "pre-neural/pre-forebrain" state.     

Visceral endoderm mediates forebrain development by suppressing posteriorizing signals

The anterior visceral endoderm (AVE) has attracted recent attention as a critical player in mouse forebrain development and has been proposed to act as "head organizer" in mammals. However, the precise role of the AVE in induction and patterning of the anterior neuroectoderm is not yet known. Here we identified a 5'-flanking region of the mouse Otx2 gene (VEcis) that governs the transgene expression in the visceral endoderm. In transgenic embryos, VEcis-active cells were found in the distal visceral endoderm at 5.5 days postcoitus (dpc), had begun to move anteriorly at 5.75 dpc, and then became restricted to the AVE prior to gastrulation. The VEcis-active visceral endoderm cells exhibited ectodermal morphology distinct from that of the other endoderm cells and consisted of two cell layers at 5.75 dpc. In the Otx2(-/-) background, the VEcis-active endoderm cells remained distal even at 6.5 dpc when a primitive streak was formed; anterior definitive endoderm was not formed nor were any markers of anterior neuroectoderm ever induced. The Otx2 cDNA transgene under the control of the VEcis restored these Otx2(-/-) defects, demonstrating that Otx2 is essential to the anterior movement of distal visceral endoderm cells. In germ-layer explant assays between ectoderm and visceral endoderm, the AVE did not induce anterior neuroectoderm markers, but instead suppressed posterior markers in the ectoderm; Otx2(-/-) visceral endoderm lacked this activity. Thus Otx2 is also essential for the AVE to repress the posterior character. These results suggest that distal visceral endoderm cells move to the future anterior side to generate a prospective forebrain territory indirectly, by preventing posteriorizing signals.