Development of the vertebrate embryo depends on intercellular signaling to specify the fate of cells comprising the embryonic body. Although many of the intercellular inducing factors involved in these signaling events have now been identified (e.g. FCF, Vg1, Xwnt, noggin), it is not known how intracellular signals triggered by these factors mediate cell fate. Toward this end, we have isolated a SER/THR kinase, Xgsk-3, from the amphibian, Xenopus laevis. Inhibition of Xgsk-3 activity on the ventral side of the embryo with a dominant-negative mutant of Xgsk-3, leads to embryos with completely twinned axes, including secondary heads. This result indicates that Xgsk-3 actively represses axis formation in the early embryo, and that axal development requires the repression of Xgsk-3 activity within the future Spemann organizer, establishing Xgsk-3 as a major intracellular regulator of early axis formation in the Xenopus embryo. In addition, genetic evidence from DrosophiLa, together with localization of the Xgsk-3 gene in Xenopus, suggests that it may also be an important regulator of the neuroectoderm. Three major areas will be pursued: 1. The regulation of Xgsk-3 in early axial patterning will be investigated, by developing biochemical assays for Xgsk-3 function. These experiments will determine which inducing factors have the ability to regulate Xgsk-3, and will ultimately elucidate the mechanism for Xgsk-3 inhibition during early development 2. The role of Xgsk-3 in later axial development and neural development will be determined, using the dominant-negative mutant of Xgsk-3 to block its function during different stages of embryogenesis. 3. A search for the proteins that regulate Xgsk-3 and that are regulated by Xgsk-3 during different developmental stages will be conducted in order to elucidate the intracellular signaling pathway that regulates axis development in the Xenopus embryo. The ultimate goal of this proposal is to provide a dear mechanistic understanding of the intracellular signaling processes that underlie vertebrate embryonic development. Since birth defects are due to a failure in the normal processes of embryogenesis, these studies will delineate the processes that can be perturbed by dysmorphogenic agents and genetic abnormalities.
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