The broad objectives of this proposal are the identification, characterization and functional analysis of embryonic regulatory proteins and their corresponding genes that control early gene activity and thus, elucidate the mechanism responsible for the establishment of the early embryonic cell lineages during the cleavage stages in the sea urchin embryo. The short term goals of the proposed research studies are the purification of two embryonic regulatory factors (CF1 and EFl) that are differentially distributed in time and space within the developing embryo, the cloning and characterization of their corresponding genes, and the functional significance of these regulators in ontogeny of the sea urchin and other embryos. CFI is a maternal, in origin, nuclear protein that is found exclusively in the ectodermal cells of the embryo. EF1 is an early embryonic but not maternal nuclear protein that exhibits two distinct site specific binding forms before and after gastrulation; the early one is present in the ectoderm, whereas the late form is contained in the meso-endodermal germ layer. Both factors have their target sites in the regulatory region of the early cleavage activated cytoskeletal actin gene, CyIIIb. Purification of the two proteins will be carried out by DNA affinity chromatography using early and late embryonic nuclear extracts. The amino acid sequence of the two proteins will be determined and used to synthesize nucleotide probes which will allow the cloning of their corresponding cDNAs. The structure of these proteins will be determined by sequencing full length CDNA clones. The developmental pattern of expression of the corresponding genes will be analyzed in time and space by RNA blot and in situ hybridizations to embryos from different stages. The protein localization within the embryo will be analyzed with the use of antibodies raised against the embryonic or the recombinant proteins. We will study the ontogenic significance of these regulatory factors and their functional domains in vivo by introducing intact and mutant proteins and test fusion genes, by microinjection, into developing sea urchin embryos and Xenopus oocytes.
