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.     

Bone morphogenetic protein 4 signaling regulates development of the anterior visceral endoderm in the mouse embryo

The extraembryonic ectoderm (ExE) of the mouse conceptus is known to play a role in embryo patterning by signaling to the underlying epiblast and surrounding visceral endoderm. Bmp4 is one of the key ExE signaling molecules and has been recently implicated to participate in regulating development and migration of the anterior visceral endoderm (AVE). However, it remains unclear when exactly BMP4 signaling starts to regulate AVE positioning. To examine this, we have chosen to affect BMP4 function at two different time points, at embryonic day 5.25 (E5.25), thus before AVE migration, and E5.75, just after AVE migration. To this end, an RNAi technique was used, which consisted of the injection of Bmp4 dsRNA into the proamniotic cavity of the egg cylinder followed by its targeted electroporation into the ExE. This resulted in specific knockdown of Bmp4 . It was found that Bmp4 RNAi at E5.25, but not at E5.75, led to an abnormal pattern of expression of the AVE marker Cerberus-like . Thus, BMP4 signaling appears to affect the expression of Cer1 at a specific time window. This RNAi approach provides a convenient means to study spatial and temporal function of genes shortly after embryo implantation.     

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.     

Heading forwards: anterior visceral endoderm migration in patterning the mouse embryo

The elaboration of anterior–posterior (A–P) pattern is one of the earliest events during development and requires the precisely coordinated action of several players at the level of molecules, cells and tissues. In mammals, it is controlled by a specialized population of migratory extraembryonic epithelial cells, the anterior visceral endoderm (AVE). The AVE is a signalling centre that is responsible for several important patterning events during early development, including specifying the orientation of the A–P axis and the position of the heart with respect to the brain. AVE cells undergo a characteristic stereotypical migration which is crucial to their functions.     

The different roles of cyclinD1-CDK4 in STP and mGluR-LTD during the postnatal development in mice hippocampus area CA1

Background

The extraembryonic tissues, visceral endoderm (VE) and extraembryonic ectoderm (ExE) are known to be important for the induction of primordial germ cells (PGCs) in mice via activation of the bone morphogenetic protein (BMP) signalling pathway. We investigated whether the VE and ExE have a direct role in the specification of PGCs, or in an earlier event, namely the induction of the PGC precursors in the proximal posterior epiblast cells.

Results

We cultured embryonic day (E) 5.75 to E7.0 mouse embryos in an explant-assay with or without extraembryonic tissues. The reconstituted pieces of embryonic and extraembryonic tissues were assessed for the formation of both PGC precursors and specified PGCs. For this, Blimp1:gfp and Stella:gfp transgenic mouse lines were used to distinguish between PGC precursors and specified PGC, respectively. We observed that the VE regulates formation of an appropriate number of PGC precursors between E6.25–E7.25, but it is not essential for the subsequent specification of PGCs from the precursor cells. Furthermore, we show that the ExE has a different role from that of the VE, which is to restrict localization of PGC precursors to the posterior part of the embryo.

Conclusion

We show that the VE and ExE have distinct roles in the induction of PGC precursors, namely the formation of a normal number of PGC precursors, and their appropriate localization during early development. However, these tissues do not have a direct role during the final stages of specification of the founder population of PGCs.     

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.     

Novel gene expression patterns along the proximo-distal axis of the mouse embryo before gastrulation

Background  

To date, the earliest stage at which the orientation of the anterior-posterior axis in the mouse embryo is distinguishable by asymmetric gene expression is shortly after E5.5. At E5.5, prospective anterior markers are expressed at the distal tip of the embryo, whereas prospective posterior markers are expressed more proximally, close to the boundary with the extraembryonic region.     

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.     

Crosstalk between Nodal/activin and MAPK p38 signaling is essential for anterior-posterior axis specification

Nodal/activin signaling plays a key role in anterior-posterior (A-P) axis formation by inducing the anterior visceral endoderm (AVE), the extraembryonic signaling center that initiates anterior patterning in the embryo. Here we provide direct evidence that the mitogen-activated protein kinase (MAPK) p38 regulates AVE specification through a crosstalk with the Nodal/activin signaling pathway. We show that p38 activation is directly stimulated by Nodal/activin and fails to be maintained upon inhibition of this pathway both in vivo and in vitro. In turn, p38 strengthens the Nodal signaling response by phosphorylating the Smad2 linker region and enhancing the level of Smad2 activation. Furthermore, we demonstrate that this p38 amplification loop is essential for correct specification of the AVE in two ways: first, by showing that inhibiting p38 activity in 5.5 days postcoitum embryo cultures leads to a switch from AVE to an extraembryonic visceral endoderm cell identity, and second, by demonstrating that genetically reducing p38 activity in a Nodal-sensitive background leads to a failure of AVE specification in vivo. Collectively, our results reveal a novel role for p38 in regulating the threshold of Nodal signaling and propose a new mechanism by which A-P axis development can be reinforced during early embryogenesis.     

Cdx1::Cre allele for gene analysis in the extraembryonic ectoderm and the three germ layers of mice at mid‐gastrulation

Transgenic mice with a defined cell‐ or tissues‐specific expression of Cre‐recombinase are essential tools to study gene function. Here we report the generation and analysis of a transgenic mouse line (Cdx1::Cre) with restricted Cre‐expression from Cdx1 regulatory elements. The expression of Cre‐recombinase mimicked the endogenous expression pattern of Cdx1 at midgastrulation (from E7.5 to early headfold stage) inducing recombination in the three germlayers of the primitive streak region throughout the posterior embryo and caudal to the heart. This enables gene modifications to investigate patterning of the caudal embryo during and after gastrulation. Interestingly, we identified Cdx1 expression in the trophectoderm (TE) of blastocyst stage embryos. Concordantly, we detected extensive Cre‐mediated recombination in the polar TE and, although to lesser extent, in the mural TE. In E7.5 postimplantation embryos, almost all cells of the extraembryonic ectoderm (ExE), which are derived from the polar TE, are recombined although the ExE itself is negative for Cdx1 and Cre at this stage. These results indicate that Cdx1::Cre mice are also a valuable tool to study gene function in tissues essential for placental development. genesis 47:204–209, 2009. © 2009 Wiley‐Liss, Inc.     

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.     

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.     

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.     

Gastrula organiser and embryonic patterning in the mouse

Embryonic patterning of the mouse during gastrulation and early organogenesis engenders the specification of anterior versus posterior structures and body laterality by the interaction of signalling and modulating activities. A group of cells in the mouse gastrula, characterised by the expression of a repertoire of "organiser" genes, acts as a source and the conduit for allocation of the axial mesoderm, floor plate and definitive endoderm. The organiser and its derivatives provide the antagonistic activity that modulates WNT and TGFbeta signalling. Recent findings show that the organiser activity is augmented by morphogenetic activity of the extraembryonic and embryonic endoderm, suggesting embryonic patterning is not solely the function of the organiser.     

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.     

A novel role for retinoids in patterning the avian forebrain during presomite stages

Retinoids, and in particular retinoic acid (RA), are known to induce posterior fates in neural tissue. However, alterations in retinoid signalling dramatically affect anterior development. Previous reports have demonstrated a late role for retinoids in patterning craniofacial and forebrain structures, but an earlier role in anterior patterning is not well understood. We show that enzymes involved in synthesizing retinoids are expressed in the avian hypoblast and in tissues directly involved in head patterning, such as anterior definitive endoderm and prechordal mesendoderm. We found that in the vitamin A-deficient (VAD) quail model, which lacks biologically active RA from the first stages of development, anterior endodermal markers such as Bmp2, Bmp7, Hex and the Wnt antagonist crescent are affected during early gastrulation. Furthermore, prechordal mesendodermal and prospective ventral telencephalic markers are expanded posteriorly, Shh expression in the axial mesoderm is reduced, and Bmp2 and Bmp7 are abnormally expressed in the ventral midline of the neural tube. At early somite stages, VAD embryos have increased cell death in ventral neuroectoderm and foregut endoderm, but normal cranial neural crest production, whereas at later stages extensive apoptosis occurs in head mesenchyme and ventral neuroectoderm. As a result, VAD embryos end up with a single and reduced telencephalic vesicle and an abnormally patterned diencephalon. Therefore, we propose that retinoids have a dual role in patterning the anterior forebrain during development. During early gastrulation, RA acts in anterior endodermal cells to modulate the anteroposterior (AP) positional identity of prechordal mesendodermal inductive signals to the overlying neuroectoderm. Later on, at neural pore closure, RA is required for patterning of the mesenchyme of the frontonasal process and the forebrain by modulating signalling molecules involved in craniofacial morphogenesis.     

Specification of an anterior neuroectoderm patterning by Frizzled8a-mediated Wnt8b signalling during late gastrulation in zebrafish

Wnts have been shown to provide a posteriorizing signal that has to be repressed in the anterior neuroectoderm for normal anteroposterior (AP) patterning. We have previously identified a zebrafish frizzled8a (fz8a) gene expressed in the presumptive anterior neuroectoderm as well as prechordal plate at the late gastrula stage. We have investigated the role of Fz8a-mediated Wnt8b signalling in anterior brain patterning in zebrafish. We show that in zebrafish embryos: (1) Wnt signalling has at least two different stage-specific posteriorizing activities in the anterior neuroectoderm, one before mid-gastrulation and the other at late gastrulation; (2) Fz8a plays an important role in mediating anterior brain patterning; (3) Wnt8b and Fz8a can functionally interact to transmit posteriorizing signals that determine the fate of the posterior diencephalon and midbrain in late gastrula embryos; and (4) Wnt8b can suppress fz8a expression in the anterior neuroectoderm and potentially affect the level and/or range of Wnt signalling. In conclusion, we suggest that a gradient of Fz8a-mediated Wnt8b signalling may play crucial role in patterning the posterior diencephalon and midbrain regions in the late gastrula.