Extracellular signalling molecules responsible for the establishment of early embryonic pattern in vertebrates have recently begun to be identified and characterized. Little is known, however, about how these signals act on target cells to specify developmental fate. The proposed research will investigate the early, pre-transcriptional events at the plasma membrane and in the cytosol involved in mediating the action of these extracellular signals. The primary focus of this work will be on the signals responsible for the induction of mesoderm during early frog embryogenesis. This study will provide important insights into the molecular mechanisms by which intercellular interactions establish the body plan of a vertebrate embryo. Two agents (fibroblast growth factor, polyoma middle T) known to activate cellular tyrosine kinases during mitogenic stimulation of fibroblasts have previously been found to respecify prospective embryonic ectoderm to form mesoderm. To determine the role of tyrosine phosphorylation in mesoderm induction, anti-phosphotyrosine antibodies will be used to identify substrates for tyrosine phosphorylation in response to mesoderm inducing factors. Preliminary experiments have identified MAP kinase as such a substrate. The phosphorylation and activation of MAP kinase in response to inducing factors will be further characterized. Several approaches to inhibition and activation of MAP kinase will be used to test the importance of its role in inductive signal transduction. The c-ras proto-oncogene product, p2lras, has also been implicated in the action of mitogenic signals in fibroblasts. Overexpression of dominant inhibitory and constitutively active mutants of P21ras by RNA microinjection will be used to investigate the role of this putative signal transducer during mesoderm induction. Immunoprecipitation of endogenous p2l ras from embryos will be used to assess P21ras activation during mesoderm induction. To study endogenous signalling events during early development, both anti-phosphotyrosine antibodies and SH2 domains from several receptor tyrosine kinase associated signalling enzymes will be used as probes for whole mount affinity cytochemistry of embryos. This will provide a novel tool for visualization of spatial and temporal patterns of receptor tyrosine kinase activated signal transduction. This technique will be used to study patterns of signalling during normal and experimentally manipulated early development.
