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The SOX family of transcription factors has been implicated in cell fate specification during embryogenesis. One member of this family, Sox9, has been shown to regulate both chondrogenesis and sex determination in the mouse embryo. Heterozygous mutations in Sox9 result in Campomelic Dysplasia (CD), a lethal human disorder characterized by autosomal XY sex reversal, severe skeletal malformations and several craniofacial defects. Sox9 is also expressed in neural crest progenitors but very little is known about the function of Sox9 in the neural crest. We have cloned the Xenopus homolog of the Sox9 gene. It is expressed maternally and accumulates shortly after gastrulation at the lateral edges of the neural plate, in the neural crest-forming region. As development proceeds, Sox9 expression persists in migrating cranial crest cells as they populate the pharyngeal arches. Depletion of Sox9 protein in developing embryos, using morpholino antisense oligos, causes a dramatic loss of neural crest progenitors and an expansion of the neural plate. Later during embryogenesis, morpholino-treated embryos have a specific loss or reduction of neural crest-derived skeletal elements, mimicking one aspect of the craniofacial defects observed in CD patients. We propose that Sox9 is an essential component of the regulatory pathway that leads to cranial neural crest formation.

The SOX family of transcription factors has been implicated in cell fate specification during embryogenesis. One member of this family, Sox9, has been shown to regulate both chondrogenesis and sex determination in the mouse embryo. Heterozygous mutations in Sox9 result in Campomelic Dysplasia (CD), a lethal human disorder characterized by autosomal XY sex reversal, severe skeletal malformations and several craniofacial defects. Sox9 is also expressed in neural crest progenitors but very little is known about the function of Sox9 in the neural crest. We have cloned the Xenopus homolog of the Sox9 gene. It is expressed maternally and accumulates shortly after gastrulation at the lateral edges of the neural plate, in the neural crest-forming region. As development proceeds, Sox9 expression persists in migrating cranial crest cells as they populate the pharyngeal arches. Depletion of Sox9 protein in developing embryos, using morpholino antisense oligos, causes a dramatic loss of neural crest progenitors and an expansion of the neural plate. Later during embryogenesis, morpholino-treated embryos have a specific loss or reduction of neural crest-derived skeletal elements, mimicking one aspect of the craniofacial defects observed in CD patients. We propose that Sox9 is an essential component of the regulatory pathway that leads to cranial neural crest formation.

Wnts are a family of secreted glycoproteins that are important for multiple steps in early development. Accumulating evidence suggests that frizzled genes encode receptors for Wnts. However, the mechanism through which frizzleds transduce a signal and the immediate downstream components that convey that signal are unclear. We have identified a new protein, Kermit, that interacts specifically with the C-terminus of Xenopus frizzled-3 (Xfz3). Kermit is a 331 amino acid protein with a central PDZ domain. Kermit mRNA is expressed throughout Xenopus development and is localized to neural tissue in a pattern that overlaps Xfz3 expression temporally and spatially. Co-expression of Xfz3 and Kermit results in a dramatic translocation of Kermit to the plasma membrane. Inhibition of Kermit function with morpholino antisense oligonucleotides directed against the 5' untranslated region of Kermit mRNA blocks neural crest induction by Xfz3, and this is rescued by co-injection of mRNA encoding the Kermit open reading frame. These observations suggest that Kermit is required for Wnt/frizzled signaling in neural crest development. To the best of our knowledge, Kermit is the first protein identified that interacts directly with the cytoplasmic portion of frizzleds to modulate their signaling activity.

Wnts are a large family of secreted molecules implicated in numerous developmental processes. Frizzled proteins are likely receptors for Wnts and are required for Wnt signaling in invertebrates. A large number of vertebrate frizzled genes have also been identified, but their roles in mediating specific responses to endogenous Wnts have not been well defined. Using a functional assay in Xenopus, we have performed a large screen to identify potential interactions between Wnts and frizzleds. We find that signaling by Xwnt1, but not other Wnts, can be specifically enhanced by frizzled 3 (Xfz3). As both Xfz3 and Xwnt1 are highly localized to dorsal neural tissues that give rise to neural crest, we examined whether Xfz3 mediates Xwnt1 signaling in the formation of neural crest. Xfz3 specifically induces neural crest in ectodermal explants and in embryos, similar to Xwnt1, and at lower levels of expression, synergizes with Xwnt1 in neural crest induction. Furthermore, loss of Xfz3 function, either by depletion with a Xfz3-directed morpholino antisense oligonucleotide or by expression of an inhibitory form of Xfz3 (Nfz3), prevents Xwnt1-dependent neural crest induction in ectodermal explants and blocks neural crest formation in whole embryos. These results show that Xfz3 is required for Xwnt1 signaling in the formation of the neural crest in the developing vertebrate embryo.

Wnts are a family of secreted glycoproteins that are important for multiple steps in early development. Accumulating evidence suggests that frizzled genes encode receptors for Wnts. However, the mechanism through which frizzleds transduce a signal and the immediate downstream components that convey that signal are unclear. We have identified a new protein, Kermit, that interacts specifically with the C-terminus of Xenopus frizzled-3 (Xfz3). Kermit is a 331 amino acid protein with a central PDZ domain. Kermit mRNA is expressed throughout Xenopus development and is localized to neural tissue in a pattern that overlaps Xfz3 expression temporally and spatially. Co-expression of Xfz3 and Kermit results in a dramatic translocation of Kermit to the plasma membrane. Inhibition of Kermit function with morpholino antisense oligonucleotides directed against the 5′ untranslated region of Kermit mRNA blocks neural crest induction by Xfz3, and this is rescued by co-injection of mRNA encoding the Kermit open reading frame. These observations suggest that Kermit is required for Wnt/frizzled signaling in neural crest development. To the best of our knowledge, Kermit is the first protein identified that interacts directly with the cytoplasmic portion of frizzleds to modulate their signaling activity.

Wnts are a large family of secreted molecules implicated in numerous developmental processes. Frizzled proteins are likely receptors for Wnts and are required for Wnt signaling in invertebrates. A large number of vertebrate frizzled genes have also been identified, but their roles in mediating specific responses to endogenous Wnts have not been well defined. Using a functional assay in Xenopus, we have performed a large screen to identify potential interactions between Wnts and frizzleds. We find that signaling by Xwnt1, but not other Wnts, can be specifically enhanced by frizzled 3 (Xfz3). As both Xfz3 and Xwnt1 are highly localized to dorsal neural tissues that give rise to neural crest, we examined whether Xfz3 mediates Xwnt1 signaling in the formation of neural crest. Xfz3 specifically induces neural crest in ectodermal explants and in embryos, similar to Xwnt1, and at lower levels of expression, synergizes with Xwnt1 in neural crest induction. Furthermore, loss of Xfz3 function, either by depletion with a Xfz3-directed morpholino antisense oligonucleotide or by expression of an inhibitory form of Xfz3 (Nfz3), prevents Xwnt1-dependent neural crest induction in ectodermal explants and blocks neural crest formation in whole embryos. These results show that Xfz3 is required for Xwnt1 signaling in the formation of the neural crest in the developing vertebrate embryo.

Mesodermal patterning in the amphibian embryo has been extensively studied in its dorsal aspects, whereas little is known regarding its ventrolateral regionalization due to a lack of specific molecular markers for derivatives of this type of mesoderm. Since smooth muscles (SM) are thought to arise from lateral plate mesoderm, we have analyzed the expression of an alpha-actin isoform specific for SM with regard to mesoderm patterning. Using an antibody directed against alpha-SM actin that recognized specifically this actin isoform in Xenopus, we have found that the expression of alpha-SM actin is restricted to visceral and vascular SM with a transient expression in the heart. The overall expression of the alpha-SM actin appears restricted to the ventral aspects of the differentiating embryo. alpha-SM actin expression appears to be activated following mesoderm induction in animal cap derivatives. Moreover, at the gastrula stage, SM precursor cells are regionalized since they will only differentiate from ventrolateral marginal zone explants. Using the animal cap assay, we have found that alpha-SM actin expression is specifically induced in treated animal cap with bFGF or a low concentration of XTC-MIF, which induce ventral structures, but not with a high concentration of XTC-MIF, which induces dorsal structures. Altogether, these results establish that alpha-SM actin is a reliable marker for ventrolateral mesoderm. We discuss the importance of this novel marker in studying mesoderm regionalization.

Mesodermal patterning in the amphibian embryo has been extensively studied in its dorsal aspects, whereas little is known regarding its ventrolateral regionalization due to a lack of specific molecular markers for derivatives of this type of mesoderm. Since smooth muscles (SM) are thought to arise from lateral plate mesoderm, we have analyzed the expression of an - actin isoform specific for SM with regard to mesoderm patterning. Using an antibody directed against -SM actin that recognized specifically this actin isoform in Xenopus, we have found that the expression of -SM actin is restricted to visceral and vascular SM with a transient expression in the heart. The overall expression of the -SM actin appears restricted to the ventral aspects of the differentiating embryo. -SM actin expression appears to be activated following mesoderm induction in animal cap derivatives. Moreover, at the gastrula stage, SM precursor cells are regionalized since they will only differentiate from ventrolateral marginal zone explants. Using the animal cap assay, we have found that -SM actin expression is specifically induced in treated animal cap with bFGF or a low concentration of XTC-MIF, which induce ventral structures, but not with a high concentration of XTC-MIF, which induces dorsal structures. Altogether, these results establish that -SM actin is a reliable marker for ven-trolateral mesoderm. We discuss the importance of this novel marker in studying mesoderm regionalization.

The appearance and localization of N-CAM during neural induction were studied in Pleurodeles waltl embryos and compared with recent contradictory results reported in Xenopus laevis. A monoclonal antibody raised against mouse N-CAM was used. In the nervous system of Pleurodeles, it recognized two glycoproteins of 180 and 140 xlO3 which are the Pleurodeles equivalent of N-CAM-180 and -140. Using this probe for immunohistochemistry and immunocytochemistry, we showed that N-CAM was already expressed in presumptive ectoderm at the early gastrula stage. In late gastrula embryos, a slight increase in staining was observed in the neurectoderm, whereas the labelling persisted in the noninduced ectoderm. When induced ectodermal cells were isolated at the late gastrula stage and cultured in vitro up to 14 days, a faint polarized labelling of cells was observed initially. During differentiation, the staining increased and became progressively restricted to differentiating neurons.