This proposal addresses the role of TGFbeta signaling in the formation of the vertebrate mesodermal germ layer. Using Xenopus laevis embryos and embryonic explants in the context of expression cloning, candidate gene approaches and microarray technology, we have identified five genes which, by modulating the TGFbeta pathway provide a strong input in mesoderm formation. The candidate gene approach has lead to the discovery that LTBP-1 is an organizer specific gene which binds and synergizes with all the embryonic functions of activin, including mesoderm and secondary axis induction. I propose to address the specificity of the interaction of LTBP-1 with members of the TGFbeta family, its relationship with organizer specific inhibitors which also bind to activin such as follistatin, and explore the functions of related family members LTBP2 and 3. A gain of function screen has led to the discovery of OS4, an evolutionarily conserved gene of previously unknown function, which is also able to induce mesoderm and secondary axis. I propose to link OS4 to a signaling pathway(s), first by performing an epistatic analysis of OS4 in the TGFB pathway, and second by isolation of binding partners. Our prototype Xenopus microarrays have identified a large number of activin-regulated cDNAs, including three novel genes. I propose, first, to follow on the embryological and biochemical activities of these novel responsive genes. Second, I propose the use of a 5000 gene array available in my laboratory to globally identify genes (including immediate early response) that are regulated by different thresholds of activin protein concentration, with the aim of characterizing and organizing temporally the repertoire of genes regulated by activin. Taken together, the aims of this grant will enrich our knowledge not only about early vertebrate mesoderm formation, but also about the TGFBeta pathway, which is relevant to many other areas of biology and medicine beyond embryology.
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