Sfrp5 is not essential for axis formation in the mouse

Secreted frizzled related protein (Sfrp) genes encode extracellular factors that can modulate Wnt signaling. During early post-implantation mouse development Sfrp5 is expressed in the anterior visceral endoderm (AVE) and the ventral foregut endoderm. The AVE is important in anterior-posterior axis formation and the ventral foregut endoderm contributes to multiple gut tissues. Here to determine the essential role of Sfrp5 in early mouse development we generated Sfrp5-deficient mice by gene targeting. We report that Sfrp5-deficient mice are viable and fertile. To determine whether the absence of an axis phenotype might be due to genetic redundancy with Dkk1 in the AVE we generated Sfrp5;Dkk1 double mutant mice. AVE development and primitive streak formation appeared normal in Sfrp5(-/-);Dkk1(-/-) embryos. These results indicate that Sfrp5 is not essential for axis formation or foregut morphogenesis in the mouse and also imply that Sfrp5 and Dkk1 together are not essential for AVE development.     

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.     

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.     

Bmpr1a is required for proper migration of the AVE through regulation of Dkk1 expression in the pre-streak mouse embryo

Here, we report a novel mechanism regulating migration of the anterior visceral endoderm (AVE) by BMP signaling through BMPRIA. In Bmpr1a-deficient (Bmpr-null) embryos, the AVE does not migrate at all. In embryos with an epiblast-specific deletion of Bmpr1a (Bmpr1a^null/flox; Sox2Cre embryos), the AVE cells migrate randomly from the distal end of embryos, resulting in an expansion of the AVE. Dkk1, which is normally expressed in the anterior proximal visceral endoderm (PxVE), is downregulated in Bmpr-null embryos, whereas it is circumferentially expressed in Bmpr1a^null/flox; Sox2Cre embryos at E5.75-6.5. These results demonstrate an association of the position of Dkk1 expressing cells with direction of the migration of AVE. In Bmpr1a^null/flox; Sox2Cre embryos, a drastic decrease of WNT signaling is observed at E6.0. Addition of WNT3A to the culture of Bmpr1a^null/flox; Sox2Cre embryos at E5.5 restores expression patterns of Dkk1 and Cer1. These data indicate that BMP signaling in the epiblast induces Wnt3 and Wnt3a expression to maintain WNT signaling in the VE, resulting in downregulation of Dkk1 to establish the anterior expression domain. Thus, our results suggest that BMP signaling regulates the expression patterns of Dkk1 for anterior migration of the AVE.     

Canonical Wnt signaling and its antagonist regulate anterior-posterior axis polarization by guiding cell migration in mouse visceral endoderm

The mouse embryonic axis is initially formed with a proximal-distal orientation followed by subsequent conversion to a prospective anterior-posterior (A-P) polarity with directional migration of visceral endoderm cells. Importantly, Otx2, a homeobox gene, is essential to this developmental process. However, the genetic regulatory mechanism governing axis conversion is poorly understood. Here, defective axis conversion due to Otx2 deficiency can be rescued by expression of Dkk1, a Wnt antagonist, or following removal of one copy of the beta-catenin gene. Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis rotation. Moreover, asymmetrical distribution of beta-catenin localization is impaired in the Otx2-deficient and Wnt-misexpressing visceral endoderm. Concurrently, canonical Wnt and Dkk1 function as repulsive and attractive guidance cues, respectively, in the migration of visceral endoderm cells. We propose that Wnt/beta-catenin signaling mediates A-P axis polarization by guiding cell migration toward the prospective anterior in the pregastrula mouse embryo.     

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

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

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.     

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.     

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.     

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.     

The mouse frizzled 8 receptor is expressed in anterior organizer tissues

Wnt signaling has been shown to be important for axis formation in vertebrates. However, no Wnt ligand or receptor has been shown to be specifically expressed in all the organizer tissues in the mouse embryo. Here we report that the mouse frizzled 8 (mfz8) gene, a Wnt receptor, is expressed in the anterior visceral endoderm (AVE) and the anterior primitive streak, which have been shown to possess organizer activity. mFz8 is also expressed in the descendents of the anterior streak that comprise the anterior mesendoderm (AME) at midgastrulation, with subsequent expression in the anterior neurectoderm, which is specified and patterned by the AVE and AME. Thus, mfz8 is specifically expressed in the organizer tissues that establish the anterior-posterior axis in the mouse embryo.     

Sfrp1, Sfrp2, and Sfrp5 regulate the Wnt/beta-catenin and the planar cell polarity pathways during early trunk formation in mouse

Sfrp is a secreted Wnt antagonist that directly interacts with Wnt ligand. We show here that inactivation of Sfrp1, Sfrp2, and Sfrp5 leads to fused somites formation in early-somite mouse embryos, simultaneously resulting in defective convergent extension (CE), which causes severe shortening of the anteroposterior axis. These observations indicate the redundant roles of Sfrp1, Sfrp2, and Sfrp5 in early trunk formation. The roles of the Sfrps were genetically distinguished in terms of the regulation of Wnt pathways. Genetic analysis combining Sfrps mutants and Loop-tail mice revealed the involvement of Sfrps in CE through the regulation of the planar cell polarity pathway. Furthermore, Dkk1-deficient embryos carrying Sfrp1 homozygous and Sfrp2 heterozygous mutations display irregular somites and indistinct intersomitic boundaries, which indicates that Sfrps-mediated inhibition of the Wnt/beta-catenin pathway is necessary for somitogenesis. Our results suggest that Sfrps regulation of the canonical and noncanonical pathways is essential for proper trunk formation.     

Dickkopf1 is required for embryonic head induction and limb morphogenesis in the mouse.

Dickkopf1 (Dkk1) is a secreted protein that acts as a Wnt inhibitor and, together with BMP inhibitors, is able to induce the formation of ectopic heads in Xenopus. Here, we show that Dkk1 null mutant embryos lack head structures anterior of the midbrain. Analysis of chimeric embryos implicates the requirement of Dkk1 in anterior axial mesendoderm but not in anterior visceral endoderm for head induction. In addition, mutant embryos show duplications and fusions of limb digits. Characterization of the limb phenotype strongly suggests a role for Dkk1 both in cell proliferation and in programmed cell death. Our data provide direct genetic evidence for the requirement of secreted Wnt antagonists during embryonic patterning and implicate Dkk1 as an essential inducer during anterior specification as well as a regulator during distal limb patterning.     

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.     

Foxh1 and Foxa2 are not required for formation of the midgut and hindgut definitive endoderm

The definitive endoderm forms during gastrulation and is rapidly transformed into the gut tube which is divided along the anterior-posterior axis into the foregut, midgut, and hindgut. Lineage tracing and genetic analysis have examined the origin of the definitive endoderm during gastrulation and demonstrated that the majority of definitive endoderm arises at the anterior end of the primitive streak (APS). Foxh1 and Foxa2 have been shown to play a role in specification of the APS and definitive endoderm. However, prior studies have focused on the role of these factors in specification of foregut definitive endoderm, while their role in the specification of midgut and hindgut definitive endoderm is less understood. Furthermore, previous analyses of these mutants have utilized definitive endoderm markers that are restricted to the anterior endoderm, expressed in extraembryonic endoderm, or present in other germ layers. Here, we characterized the expression of several novel definitive and visceral endoderm markers in Foxh1 and Foxa2 null embryos. In accordance with previous studies, we observed a deficiency of foregut definitive endoderm resulting in incorporation of visceral endoderm into the foregut. Interestingly, this analysis revealed that formation of midgut and hindgut definitive endoderm is unaffected by loss of Foxh1 or Foxa2. This finding represents a significant insight into specification and regionalization of mouse definitive endoderm.     

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.     

Multi-cellular rosettes in the mouse visceral endoderm facilitate the ordered migration of anterior visceral endoderm cells

The visceral endoderm (VE) is a simple epithelium that forms the outer layer of the egg-cylinder stage mouse embryo. The anterior visceral endoderm (AVE), a specialised subset of VE cells, is responsible for specifying anterior pattern. AVE cells show a stereotypic migratory behaviour within the VE, which is responsible for correctly orientating the anterior-posterior axis. The epithelial integrity of the VE is maintained during the course of AVE migration, which takes place by intercalation of AVE and other VE cells. Though a continuous epithelial sheet, the VE is characterised by two regions of dramatically different behaviour, one showing robust cell movement and intercalation (in which the AVE migrates) and one that is static, with relatively little cell movement and mixing. Little is known about the cellular rearrangements that accommodate and influence the sustained directional movement of subsets of cells (such as the AVE) within epithelia like the VE. This study uses an interdisciplinary approach to further our understanding of cell movement in epithelia. Using both wild-type embryos as well as mutants in which AVE migration is abnormal or arrested, we show that AVE migration is specifically linked to changes in cell packing in the VE and an increase in multi-cellular rosette arrangements (five or more cells meeting at a point). To probe the role of rosettes during AVE migration, we develop a mathematical model of cell movement in the VE. To do this, we use a vertex-based model, implemented on an ellipsoidal surface to represent a realistic geometry for the mouse egg-cylinder. The potential for rosette formation is included, along with various junctional rearrangements. Simulations suggest that while rosettes are not essential for AVE migration, they are crucial for the orderliness of this migration observed in embryos. Our simulations are similar to results from transgenic embryos in which Planar Cell Polarity (PCP) signalling is disrupted. Such embryos have significantly reduced rosette numbers, altered epithelial packing, and show abnormalities in AVE migration. Our results show that the formation of multi-cellular rosettes in the mouse VE is dependent on normal PCP signalling. Taken together, our model and experimental observations suggest that rosettes in the VE epithelium do not form passively in response to AVE migration. Instead, they are a PCP-dependent arrangement of cells that acts to buffer the disequilibrium in cell packing generated in the VE by AVE migration, enabling AVE cells to migrate in an orderly manner.     

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.     

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.     

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.