The project described in this proposal is directed towards defining the mechanism by which retinoic acid regulates the differentiation of F9 cells. F9 cells provide a useful model system for studying events normally occurring in the preimplantation mouse embryo. When treated with retinoic acid (RA), these cells form a primitive endoderm which becomes competent to differentiation into either parietal or visceral endoderm. Subsequent differentiation depends on a second stimulus: cAMP induces parietal endoder formation, whereas aggregation leads to visceral endoderm formation. I hypothesize that RA induces the acquisition of signal responsiveness during primitive endoderm formation by controlling the expression of genes for constituents of signal transduction pathways. The proposed experiments are designed to identify RA regulated intracellula signal transduction pathways in F9 cells. Specifically the effects of RA on the expression of three classes of protein kinases, which are critical in signal transduction in other systems, will be measured. These are the cyclic AMP dependent protein kinases, the Ca2+, phospholipid dependent protein kinases and the tyrosine specific protein kinase pp60c-src. Studie are proposed that will 1) measure retinoic acid induced changes in the activity, synthesis and mRNA levels for these enzymes; 2) determine the mechanism by which retinoic acid regulates the expression of these kinases; 3) to explore whether a correlation exists between changes in kinase activity and differentiation into either parietal or visceral endoderm; and 4) to manipulate the expression of these enzymes using a molecular genetic approach, to test direct causal relationships between kinase activity and specific differentiated phenotypes.
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