RESUMO
The gene encoding the Sox F-group transcription factor Xsox17alpha(1) is specifically expressed throughout the entire region of the Xenopus blastula fated to become endoderm, and is important in controlling endodermal development. Xsox17alpha(1) is a direct target of the maternal endodermal determinant VegT and of Sox17 itself. We have analysed the promoter of the Xenopus laevis Xsox17alpha(1) gene by transgenesis, and have identified two important control elements which reside about 9 kb upstream at the start of transcription. These elements individually drive transgenic endodermal expression in the blastula and gastrula. One contains functional, cooperating VegT and Sox-binding consensus sites. The Sox sites in this region are occupied in vivo. The other responds to TGF-beta signals like Activin or Nodals that act through Smad2/3. We propose that these two regions co-operate in regulating the early endodermal expression of the Xsox17alpha(1) gene.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Grupo de Alta Mobilidade/metabolismo , Regiões Promotoras Genéticas , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Animais , Blástula/embriologia , Blástula/metabolismo , Proteínas de Ligação a DNA/genética , Endoderma/embriologia , Endoderma/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Mutação , Fatores de Transcrição SOXF , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Proteínas de Xenopus/genética , Xenopus laevis/metabolismoRESUMO
Xenopus Nodal-related (Xnr) 5 is one of the earliest expressed components of a network of TGF-beta factors participating in endoderm and mesoderm formation. Zygotic gene expression is not required for induction of Xnr5; rather, expression is dependent on the maternal factors VegT, localised throughout the vegetal pole, and beta-catenin, functional in the future dorsal region of the embryo. Using transient assays with a luciferase reporter in Xenopus embryos, we have defined a minimal promoter, which mimics the response of the endogenous gene to applied factors. Expression of luciferase from the minimal promoter is dorsal-specific and requires two T-box half sites and a functional beta-catenin/XTcf-3 pathway. Mutation of two Tcf/Lef sites in the minimal promoter permits induction by VegT to wild-type promoter levels in the presence of a dominant-negative XTcf-3, indicating that beta-catenin/XTcf-3 are repressive and are not required as transactivators of Xnr5 transcription. The activity of the Tcf/Lef mutant promoter is similar in both ventral and dorsal sides of the embryo. In transgenic experiments, the dorsal specificity of expression of a beta-gal reporter driven by the wild-type minimal promoter is abolished upon mutation of these Tcf/Lef sites. We propose a model in which XTcf-3 functions as a repressor of Xnr5 throughout the blastula embryo, except where repression is lifted by the binding of beta-catenin in the dorsal region. This removal of repression allows activation of the promoter by VegT in the dorsal vegetal region. Subsequently, zygotically expressed LEF1 supersedes the role of beta-catenin/XTcf-3.
Assuntos
Blástula/metabolismo , Proteínas com Domínio T/metabolismo , Proteínas de Xenopus/genética , Animais , Sequência de Bases , Sítios de Ligação , Proteínas do Citoesqueleto/metabolismo , Genes Reporter , Proteínas HMGB/metabolismo , Dados de Sequência Molecular , Mutação , Ligantes da Sinalização Nodal , Regiões Promotoras Genéticas , Fatores de Transcrição TCF , Transativadores/metabolismo , Fator 3 de Transcrição , Proteína 1 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/metabolismo , Xenopus , Proteínas de Xenopus/metabolismo , beta CateninaRESUMO
We have investigated the induction of the six Xenopus nodal-related genes, Xnr1-Xnr6, by maternal determinants. The beta-catenin pathway was modelled by stimulation using Xwnt8, activin-like signalling was modelled by activin, and VegT action was studied by overexpression in animal cap explants. Combinations of factors were examined, and previously unrecognised interactions were revealed in animal caps and whole embryos. For the induction of Xnr5 and Xnr6 in whole embryos, using a beta-catenin antisense morpholino oligonucleotide or a dominant negative XTcf3, we have demonstrated an absolute permissive requirement for the beta-catenin/Tcf pathway, in addition to the requirement for VegT action. In animal caps Xnr5 and Xnr6 are induced in response to VegT overexpression, and this induction is dependent upon the concomitant activation of the beta-catenin pathway that VegT initiates in animal caps. For the induction of Xnr3, VegT interacts negatively so as to inhibit the induction otherwise observed with wnt-signalling alone. The negative effect of VegT is not the result of a general inhibition of wnt-signalling, and does not result from an inhibition of wnt-induced siamois expression. A 294 bp proximal promoter fragment of the Xnr3 gene is sufficient to mediate the negative effect of VegT. Further experiments, employing cycloheximide to examine the dependence of Xnr gene expression upon proteins translated after the mid-blastula stage, demonstrated that Xnrs 4, 5 and 6 are 'primary' Xnr genes whose expression in the late blastula is solely dependent upon factors present before the mid-blastula stage.