This project examines the nature and properties of the egg cytoplasmic determinants of tissue differentiation that occur in Ascidian embryos. Determinants have been defined operationally by their differential segregation during early cell divisions into restricted cell lineages: cleavage stage blastomeres in isolation develop and self-differentiate only according to already fixed fates. Determinants can be related to early production of apparently histospecific mRNAs and the eventual expression of histospecific proteins and ultrastructural features. The major hypotheses to be examined by this project are (a) the conception that egg cytoplasmic determinants, as defined experimentally, are discrete informational macromolecules that can be isolated from the embryo and identified chemically according to kind and class, (b) that the determinants are either initiators of specific gene functions or are themselves histospecific mRNA molecules for proteins involved in phenotypic expression, and (c) the agents cause expression of new differentiation (transdeterminations) when moved artificially to other lineage cells and this property can be used in devising a biological assay for the factors to aid in their further purification. This proposal is focused on two contrasting determinant systems, muscle expression which seems to depend on maternally-derived gene activator(s), and endodermal alkaline phosphatase development which apparently involves a maternal mRNA for the enzyme. Translational assays (Xenopus oocyte injections, and immunoprecipitation of enzymatically active translation products) are being developed and used to identify mRNAs for acetylcholinesterase and alkaline phosphatase. Other methods of study involve isolating ascidian gene sequences for the muscle acetylcholinesterase and tropomyosin genes and for the alkaline phosphatase gene. cDNA probes will be used to monitor gene expression at certain times and locations in the embryos. The proteins themselves serve as excellent expression markers of differentiation in blastomere isolation experiments that continue to examine determinant segregation patterns, and immunocytochemical methods at the light and electron microscopic levels are being applied to their detection. The project also uses techniques of microinjection and electrofusion transfer of lineage-specific cytoplasm and isolated materials (e.g. poly A RNA) as a basis for devising biological assays of determinants.
