The overall aim is to examine the roles that signal transduction molecules play in determining cell fate in sea urchin embryos. The specific aims are based on experiments showing that agents that alter signalling through protein kinase C (PKC) cause dramatic changes in cell fate, and that these same treatments cause an increase in expression of a transcription factor homologous to the forkhead (FH)class of transcription factors. The PI has cloned a number of cDNAs which encode sea urchin PKCs. The PI intends to isolate more cDNAs encoding PKC isotypes and study the temporal and spatial expression of the different PKC isotypes during sea urchin development. In addition, the PI has designed methods to alter the activity of PKC in vivo, and he will use these methods to study PKC function. These studies will make use of inhibitory pseudosubstrate peptides which will be injected into sea urchin eggs or individual blastomeres of early sea urchin embryos. The effects on cell fate will be assayed using a variety of tissue specific markers of differentiation. Second, the PI will use a sea urchin genomic DNA fragment encoding a FH domain to isolate cDNAs encoding FH transcription factors, and he will study their spatial expression during development and with and without lithium and TPA. The PI will also isolate genomic clones containing FH genes as a first step in linking their expression to PKC activity.
