How extracellular signals are transduced into changes in developmental fate during early vertebrate embryogenesis is largely unknown. The primary focus of this proposal will be on understanding signal transduction during the very earliest patterning event in the development of the frog embryo, the induction of mesoderm. This study will provide important insights into the molecular mechanisms by which intercellular interactions establish the body plan of a vertebrate embryo. Signaling by two major classes of polypeptide factors, the fibroblast growth factor (FGF) and transforming growth factor beta (TGFbeta) superfamilies of factors have been implicated in the induction of mesoderm in frogs. Several putative signal transducers of FGF have been identified both in frog embryos and in other systems, including the proto-oncogenes p21ras and raf-1, and the protein kinases MAP kinase and MAP kinase kinase (MEK). The endogenous function and state of activation of this pathway will be studied by focusing on MAP kinase, a downstream step in this pathway necessary for the induction of mesoderm. The mechanism and pattern of regulation of this kinase will be examined, as well as potentially important substrates for its activity. Less is known about the early intracellular steps transducing TGFbeta superfamily signals than about FGF signal transduction. To characterize components of this pathway, a factor which binds to the promoter of a gene responsive to a putative TGFbeta superfamily member, activin, will be studied. This factor is activated rapidly following activin stimulation of embryonic cells and is the earliest response identified to a TGFbeta superfamily mesodermal inducer. The response of early embryonic cells to this factor will be characterized, its components purified and cloned, and activation of the factor will be used as an assay for the identification of upstream components of the activin signal transduction pathway in early embryos. Although a number of receptors for members of the TGFbeta superfamily have been identified, their functional roles in early embryos have not been characterized. To begin such a characterization, antisense oligonucleotides will be used for the targeted depletion of maternal mRNAs encoding TGFbeta superfamily receptors. Receptor depleted embryos will then be analyzed for responsiveness to exogenous and endogenous inducing signals and for specific types of transcriptional responses.
