The broad objective of this proposal is to learn how cells interact in the developing embryo to bring about determination and differentiation. the commitment of the progeny of a totipotent ovum to the various differentiation pathways of the developing embryo is a basic biological problem of prime importance. Choices made by cells to follow different paths are central to normal and abnormal embryonic development, to cancer, would repair, and aging. The experiments will focus on the sea urchin embryo, a system very similar to vertebrates in its reliance on cell interactions as a way of bringing about determination of various cell types and lineages in early development. This embryo has various blastomeres that can be separated form one another and recombined in various ways. New tissue specific markers that serve as reliable indicators of specific differentiation pathways are now available. first, these markers will be used to learn the rules that govern specific gene expression. This will involve experiments in which individual blastomeres are cultured alone or in combination with other blastomere types, or with various agents (e.g., Li ion, FGF). The fate of the blastomeres and expression of various genes will be measured by in situ hybridization and immunocytochemistry. Are there localized substances in the vegetal hemisphere of the egg that initiate the lineages that form gut and spicules: Are there ions or agonists that might be effective in causing vegetal differentiation of animal cells? Do these agents use secondary messenger pathways to effect their actions? Second, the effort to develop and characterize tissue specific cDNA clones will be continued. The efforts will concentrate on cloning spicule matrix genes, and on genes expressed solely in descendants of mesomeres. Third, the details of how expression of spicule matrix genes is regulated will be studied. Transcription of these genes will be studied, in vitro, in nuclear extracts that transcribe defined templates. This functional assay will be used to conduct studies on the DNA sequences necessary for promoter activity and to study proteins in the extracts that interact with the DNA.
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