| The focus of this thesis research has been to examine the intracellular pathways through which FGF acts to specify developmental fate during Xenopus development, in order to better understand the role FGF-generated signals play endogenously during mesoderm induction and axial patterning. It is demonstrated that a functional FGF signaling pathway is required for the initial response of cells to activin-like signals, as well as for subsequent maintenance of mesodermal gene expression. FGF signals are also found to be required for responsiveness to endogenous mesoderm inducing signals. Downstream components of the FGF signaling pathway which are necessary and/or sufficient for mesoderm induction are identified. Overexpression of activated mutants of RAF, MEK and MAP kinase is found to be sufficient for induction of mesoderm in explanted animal caps. In addition, overexpression of a dominant inhibitory mutant of RAF, or of the MAP kinase phosphatase MKP-1, is found to block mesoderm induction by both FGF and activin.;A sensitive assay for MAP kinase activity is used as a tool for investigating the spatial and temporal distribution of endogenous FGF signaling. MAP kinase activity is found to be low maternally, and to increase significantly at gastrula stages. The temporal profile of this activity correlates well with the expression pattern of Xenopus eFGF, a secreted member of the FGF family. Spatially, MAP kinase activity is found to be lowest in animal pole tissue, and higher in vegetal pole cells and the marginal zone. Endogenous MAP kinase activity is shown to be FGF receptor dependent, demonstrating that FGF signaling is active in all three germ layers. A maternally expressed transcription factor, XETS-1, is identified as a MAP kinase substrate which can induce expression of mesodermal markers in animal cap assays. Overexpression of XETS-1 is found to inhibit anterior development in a manner reminiscent of XeFGF overexpression. Expression of XETS-1 during gastrula stages, when it normally is not expressed, permits FGF-mediated mesoderm induction past the normal period of competence. |
