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.     

The zinc-finger protein CNBP is required for forebrain formation in the mouse

Mouse mutants have allowed us to gain significant insight into axis development. However, much remains to be learned about the cellular and molecular basis of early forebrain patterning. We describe a lethal mutation mouse strain generated using promoter-trap mutagenesis. The mutants exhibit severe forebrain truncation in homozygous mouse embryos and various craniofacial defects in heterozygotes. We show that the defects are caused by disruption of the gene encoding cellular nucleic acid binding protein (CNBP); Cnbp transgenic mice were able to rescue fully the mutant phenotype. Cnbp is first expressed in the anterior visceral endoderm (AVE) and, subsequently, in the anterior definitive endoderm (ADE), anterior neuroectoderm (ANE), anterior mesendoderm (AME), headfolds and forebrain. In Cnbp(-/-) embryos, the visceral endoderm remains in the distal tip of the conceptus and the ADE fails to form, whereas the node and notochord form normally. A substantial reduction in cell proliferation was observed in the anterior regions of Cnbp(-/-) embryos at gastrulation and neural-fold stages. In these regions, Myc expression was absent, indicating CNBP targets Myc in rostral head formation. Our findings demonstrate that Cnbp is essential for the forebrain induction and specification.     

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.     

Distinct enhancer elements control Hex expression during gastrulation and early organogenesis

In the mouse, embryological and genetic studies have indicated that two spatially distinct signalling centres, the anterior visceral endoderm and the node and its derivatives, are required for the correct patterning of the anterior neural ectoderm. The divergent homeobox gene Hex is expressed in the anterior visceral endoderm, in the node (transiently), and in the anterior definitive endoderm. Other sites of Hex expression include the liver and thyroid primordia and the endothelial cell precursors. We have used transgenic analysis to map the cis-acting regulatory elements controlling Hex expression during early mouse development. A 4.2-kb upstream region is important for Hex expression in the endothelial cell precursors, liver, and thyroid, and a 633-bp intronic fragment is both necessary and sufficient for Hex expression in the anterior visceral endoderm and the anterior definitive endoderm. These same regions drive expression in homologous structures in Xenopus laevis, indicating conservation of these regulatory regions in vertebrates. Analysis of the anterior visceral endoderm/anterior definitive endoderm enhancer identifies a repressor region that is required to downregulate Hex expression in the node once the anterior definitive endoderm has formed. This analysis also reveals that the initiation of Hex expression in the anterior visceral endoderm and axial mesendoderm requires common elements, but maintenance of expression is regulated independently in these tissues.     

The mouse secreted frizzled-related protein 5 gene is expressed in the anterior visceral endoderm and foregut endoderm during early post-implantation development

The anterior visceral endoderm (AVE) plays an important role in anterior-posterior axis formation in the mouse. The AVE functions in part by expressing secreted factors that antagonize growth factor signaling in the proximal epiblast. Here we report that the Secreted frizzled-related protein 5 (Sfrp5) gene, which encodes a secreted factor that can antagonize Wnt signaling, is expressed in the AVE and foregut endoderm during early mouse development. At embryonic day (E) 5.5, Sfrp5 is expressed in the visceral endoderm at the distal tip region of the embryo and at E6.5 in the AVE opposite the primitive streak. In Lim1 embryos, which lack anterior neural tissue and sometimes form a secondary body axis, Sfrp5-expressing cells fail to move towards the anterior and remain at the distal tip of E6.5 embryos. When compared with Dkk1, which encodes another secreted Wnt antagonist molecule present in the visceral endoderm, Sfrp5 and Dkk1 expression overlap but Sfrp5 is expressed more broadly in the AVE. Between E7.5 and 8, Sfrp5 is expressed in the foregut endoderm underlying the cardiac mesoderm. At E8.5, Sfrp5 is expressed in the ventral foregut endoderm that gives rise to the liver. Additional domains of Sfrp5 expression occur in the dorsal neural tube and in the forebrain anterior to the optic placode. These findings identify a gene encoding a secreted Wnt antagonist that is expressed in the extraembryonic visceral endoderm and anterior definitive endoderm during axis formation and organogenesis in the mouse.     

Anterior identity is established in chick epiblast by hypoblast and anterior definitive endoderm

Previous studies of head induction in the chick have failed to demonstrate a clear role for the hypoblast and anterior definitive endoderm (ADE) in patterning the overlying ectoderm, whereas data from both mouse and rabbit suggest patterning roles for anterior visceral endoderm (AVE) and ADE. Based on similarity of gene expression patterns, fate and a dual role in 'protecting' the prospective forebrain from caudalising influences of the organiser, the chick hypoblast has been suggested to be the homologue of the mouse anterior visceral endoderm. In support of this, when transplanted to chick embryos, the rabbit AVE induces anterior markers in the chick epiblast. To reevaluate the role of the hypoblast/ADE (lower layer) in patterning the chick ectoderm, we used rostral blastoderm isolates (RBIs) as an assay, that is, rostral regions of blastoderms transected at levels rostral to the node. RBIs are, therefore, free from the influences of Hensen's node and ingressing axial mesoderm - tissues that are able to induce Ganf, the earliest specific marker of anterior neural plate. We demonstrate, using such RBIs (or RBIs dissected to remove the lower layer with or without tissue replacement), that the hypoblast/ADE (lower layer) is required and sufficient for patterning anterior positional identity in the overlying ectoderm, leading to expression of Ganf in neuroectoderm. Our results suggest that patterning of anterior positional identity and specification of neural identity are separable events operating to pattern the rostral end of the early chick embryo. Based on this new evidence we propose a revised model for establishing anteroposterior polarity, neural specification and head patterning in the early chick that is consonant with that occurring in other vertebrates.     

Origin and role of distal visceral endoderm, a group of cells that determines anterior-posterior polarity of the mouse embryo

Anterior-posterior polarity of the mouse embryo has been thought to be established when distal visceral endoderm (DVE) at embryonic day (E) 5.5 migrates toward the future anterior side to form anterior visceral endoderm (AVE). Lefty1, a marker of DVE and AVE, is asymmetrically expressed in implanting mouse embryos. We now show that Lefty1 is expressed first in a subset of epiblast progenitor cells and then in a subset of primitive endoderm progenitors. Genetic fate mapping indicated that the latter cells are destined to become DVE. In contrast to the accepted notion, however, AVE is not derived from DVE but is newly formed after E5.5 from Lefty1(-) visceral endoderm cells that move to the distal tip. Concomitant with DVE migration, all visceral endoderm cells in the embryonic region undergo global movement. In embryos subjected to genetic ablation of Lefty1-expressing DVE cells, AVE was formed de novo but the visceral endoderm including the newly formed AVE failed to migrate, indicating that DVE guides the migration of AVE by initiating the global movement of visceral endoderm cells. Future anterior-posterior polarity is thus already determined by Lefty1(+) blastomeres in the implanting blastocyst.     

Getting your head around Hex and Hesx1: forebrain formation in mouse

An increasing amount of evidence suggests that in mouse there are two signalling centres required for the formation of a complete neural axis: the anterior visceral endoderm (AVE), and the node and its derivatives. Embryological and genetic studies suggest that the AVE has a head-inducing activity. In contrast, the node appears to act first as a head inducer in synergy with the AVE initiating anterior neural patterning at early stages of mouse development, and later, node derivatives are necessary for maintenance and embellishment of anterior neural character. Hex and Hesx1 are homeobox genes that are expressed in relevant tissues involved in anterior patterning. The analysis of the Hex and Hesx1 mutant mice has revealed that the lack of these genes has little or no effect on the early steps of anterior neural induction. However, both genes are required subsequently for the proper expansion of the forebrain region. We suggest that disturbance in the specification of an Fgf8 signalling centre in the anterior neural ridge may account for the anterior defects observed in these mutants.     

Anterior visceral endoderm SMAD4 signaling specifies anterior embryonic patterning and head induction in mice

SMAD4 serves as a common mediator for signaling of TGF-β superfamily. Previous studies illustrated that SMAD4-null mice die at embryonic day 6.5 (E6.5) due to failure of mesoderm induction and extraembryonic defects; however, functions of SMAD4 in each germ layer remain elusive. To investigate this, we disrupted SMAD4 in the visceral endoderm and epiblast, respectively, using a Cre-loxP mediated approach. We showed that mutant embryos lack of SMAD4 in the visceral endoderm (Smad4(Co/Co);TTR-Cre) died at E7.5-E9.5 without head-fold and anterior embryonic structures. We demonstrated that TGF-β regulates expression of several genes, such as Hex1, Cer1, and Lim1, in the anterior visceral endoderm (AVE), and the failure of anterior embryonic development in Smad4(Co/Co);TTR-Cre embryos is accompanied by diminished expression of these genes. Consistent with this finding, SMAD4-deficient embryoid bodies showed impaired responsiveness to TGF-β-induced gene expression and morphological changes. On the other hand, embryos carrying Cre-loxP mediated disruption of SMAD4 in the epiblasts exhibited relatively normal mesoderm and head-fold induction although they all displayed profound patterning defects in the later stages of gastrulation. Cumulatively, our data indicate that SMAD4 signaling in the epiblasts is dispensable for mesoderm induction although it remains critical for head patterning, which is significantly different from SMAD4 signaling in the AVE, where it specifies anterior embryonic patterning and head induction.     

Active cell migration drives the unilateral movements of the anterior visceral endoderm

The anterior visceral endoderm (AVE) of the mouse embryo is a specialised extra-embryonic tissue that is essential for anterior patterning of the embryo. It is characterised by the expression of anterior markers such as Hex, Cerberus-like and Lhx1. At pre-gastrula stages, cells of the AVE are initially located at the distal tip of the embryo, but they then move unilaterally to the future anterior. This movement is essential for converting the existing proximodistal axis into an anteroposterior axis. To investigate this process, we developed a culture system capable of imaging embryos in real time with single cell resolution. Our results show that AVE cells continuously change shape and project filopodial processes in their direction of motion, suggesting that they are actively migrating. Their proximal movement stops abruptly at the junction of the epiblast and extra-embryonic ectoderm, whereupon they move laterally. Confocal microscope images show that AVE cells migrate as a single layer in direct contact with the epiblast, suggesting that this tissue might provide directional cues. Together, these results show that the anteroposterior axis is correctly positioned by the active movement of cells of the AVE in response to cues from their environment, and by a 'barrier' to their movement that provides an endpoint for this migration.     

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.     

Dynamic morphogenetic events characterize the mouse visceral endoderm

Several lines of evidence suggest that the extraembryonic endoderm of vertebrate embryos plays an important role in the development of rostral neural structures. In mice, neural inductive signals are thought to reside in an area of visceral endoderm that expresses the Hex gene. Here, we have conducted a morphological and lineage analysis of visceral endoderm cells spanning pre- and postprimitive streak stages. Our results show that Hex-expressing cells have a tall, columnar epithelial morphology, which distinguishes them from other visceral endoderm cells. This region of visceral endoderm thickening (VET) is found overlying first the distal and then one side of the epiblast at stages between 5.5 and 5.75 days post coitum (d.p.c.). In addition, we show that the epiblast has an anteroposterior-compressed appearance that is aligned with the position of the VET. Intracellular labeling of VET/Hex-expressing cells reveals an anterior and anterolateral shift from their distal epiblast position. VET/Hex-expressing cells are first localized to the anterior side of the epiblast by 5.75 d.p.c. and form a crescent on the anterior half of the embryo at the onset of gastrulation. Subsequently, VET descendants are distributed along the embryonic/extraembryonic boundary by headfold stages at 7.5 d.p.c. The morphological characteristics and position of VET/Hex-expressing cells distinguishes the future anteroposterior axis of the embryo and provide landmarks to stage mouse embryos at preprimitive streak stages. Moreover, the morphological characteristics of pregastrulation mouse embryos together with the stereotyped shift in the position of visceral endoderm cells reveal similarities among amniote embryos that suggest an evolutionary conservation of the mechanisms that pattern the rostral neurectoderm at pregastrula stages.     

Induction and migration of the anterior visceral endoderm is regulated by the extra-embryonic ectoderm

The anterior visceral endoderm (AVE) is an extra-embryonic tissue required for specifying anterior pattern in the mouse embryo. The AVE is induced at the distal tip of the 5.5 dpc embryo and then migrates to the prospective anterior, where it imparts anterior identity upon the underlying epiblast (the tissue that gives rise to the embryo proper). Little is known about how the AVE is induced and what directs its migration. In this paper, we describe an essential role for another extra-embryonic tissue, the extra-embryonic ectoderm (ExE), in patterning the AVE and epiblast. Removal of the ExE in pre-gastrulation embryos leads to ectopic AVE formation, to a failure of AVE cell migration and to the assumption by the entire epiblast of an anterior identity. Ectopic transplantation of ExE cells inhibits AVE formation and leads to an expansion of the posterior epiblast marker T. These results demonstrate that the ExE restricts the induction of the AVE to the distal tip of the mouse embryo and is required to initiate the migration of these cells to the prospective anterior. Together, these data reveal a novel role for the ExE in the specification of the anteroposterior axis of the mouse embryo.     

Otx2 and HNF3beta genetically interact in anterior patterning

Patterning the developing nervous system in the mouse has been proposed to depend on two separate sources of signals, the anterior visceral endoderm (AVE) and the node or organizer. Mutation of the winged-helix gene HNF3beta leads to loss of the node and its derivatives, while mutation of the homeobox gene Otx2 results in loss of head structures, apparently at least partially because of defects in the AVE. To investigate the potential genetic interactions between the two signaling centers, we crossed Otx2+/- and HNF3beta+/- mice and found that very few Otx2+/-;HNF3beta+/- double heterozygous mutants survived to weaning. Normal Mendelian ratios of genotypes were observed during gestation, but more than half the double heterozygotes displayed a severe anterior patterning phenotype that would be incompatible with postnatal survival. The phenotype was characterized by varying degrees of holoprosencephaly, cyclopia with proboscis-like structures, and anterior forebrain truncations. Regional marker analysis revealed that ventral forebrain structures of Otx2+/-;HNF3beta+/- mutant embryos were most severely affected. Shh expression was completely absent in the anterior region of Otx2+/-;HNF3beta+/- embryos, suggesting that Otx2 and HNF3beta genetically interact, directly or indirectly, to regulate Shh expression in the anterior midline. In addition, the forebrain truncations suggest an involvement of both genes in anterior patterning, through their overlapping expression domains in either the AVE and/or the prechordal mesoderm.     

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.     

Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation in the mouse.

Lim1 is a homeobox gene expressed in the extraembryonic anterior visceral endoderm and in primitive streak-derived tissues of early mouse embryos. Mice homozygous for a targeted mutation of Lim1 lack head structures anterior to rhombomere 3 in the hindbrain. To determine in which tissues Lim1 is required for head formation and its mode of action, we have generated chimeric mouse embryos and performed tissue layer recombination explant assays. In chimeric embryos in which the visceral endoderm was composed of predominantly wild-type cells, we found that Lim1(-)(/)(-) cells were able to contribute to the anterior mesendoderm of embryonic day 7.5 chimeric embryos but that embryonic day 9.5 chimeric embryos displayed a range of head defects. In addition, early somite stage chimeras generated by injecting Lim1(-)(/)(-) embryonic stem cells into wild-type tetraploid blastocysts lacked forebrain and midbrain neural tissue. Furthermore, in explant recombination assays, anterior mesendoderm from Lim1(-)(/)(-) embryos was unable to maintain the expression of the anterior neural marker gene Otx2 in wild-type ectoderm. In complementary experiments, embryonic day 9.5 chimeric embryos in which the visceral endoderm was composed of predominantly Lim1(-)(/)(-) cells and the embryo proper of largely wild-type cells, also phenocopied the Lim1(-)(/)(-) headless phenotype. These results indicate that Lim1 is required in both primitive streak-derived tissues and visceral endoderm for head formation and that its inactivation in these tissues produces cell non-autonomous defects. We discuss a double assurance model in which Lim1 regulates sequential signaling events required for head formation in the mouse.     

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.