Recent work has established the role of several extracellular factors in the patterning of early vertebrate embryos. Although much has been learned concerning the mechanism of transduction of extracellular signals from plasma membrane to nucleus in cultured somatic cells, little is known about the intracellular pathways mediating signals in early embryogenesis. The proposed research will examine the role of cytoplasmic serine-threonine specific kinases in the transduction of signals during early frog development. Attention will be focussed on two kinases, Raf-1 and GSK3 beta, which have been implicated as cytosolic signal transducers in both somatic and early embryonic cells., Both the embryological role and biochemical mechanisms of regulation and action of these kinases will be examined using the early fog embryo as an assay system. This study will provide important insights into the mechanisms by which the extracellular signals responsible for establishing the body plan of a vertebrate embryo are transduced through the cytoplasm of their target cells. Xenopus GSK3beta will be cloned by homology with other GSK3betas and its pattern of expression in the early embryo. Microinjection of RNA encoding epitope tagged GSK3beta will be used to study GSK3beta phosphorylation and activity during both normal development and in response to specific signalling molecules. The functional role of GSK3beta in embryonic patterning will be examined by mis-expression of GSK3beta activity in both whole embryos and in defined early embryonic tissues. The regulation of Raf-1 during early embryogenesis will also be examined. Raf-1 phosphorylation, activity towards a physiological substrate, and association with cellular proteins will tested during normal embryogenesis, in response to specific inductive signals, and in response to manipulation of specific signaling pathways upstream to Raf-1, Raf-1 kinase substrates in the embryo which may mediate Raf-1 effects on patterning will be cloned using an expression library method and tested as Raf-1 substrates in vivo by micro-injection into embryos with activated Raf-1.
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