The aim of the proposed research is to determine the specificity of expression of individual genes in various cell lineages of the sea urchin (Stronglylocentrotus purpuratus) embryo. In situ hybridization techniques will be used to detemine the spatial distribution of individual mRNAs, using radioactively labeled RNA probes derived from recombinant DNAs containing individual sea urchin mRNA sequences. Using probes representing mRNAs expressed at blastula stage, the extent of restriction of individual mRNAs to one or more cell lineages during development will be examined, and the time of appearance of specific mRNA sets in different lineages will be analyzed. Cell-lineage-specific mRNAs will be used as markers to construct a fate map of the embryo at the level of mRNA expression. The tissue specificity of expression of different members of 3 multi-gene families will be investigated. Detaled patterns will be established for 6 actin genes which preliminary studies have shown are expressed in a highly lineage- and stage-specific manner. The timing of the switch in expression of histone genes from early to late variants will be compared in different cell lineages. Possible correlations between this switch and other developmental events (expression of cell-type-specific mRNAs, changes in rate of cell division) will be examined. The distributions of different late-variant histone mRNAs of the H2B subclass will be compared. Spatial patterns of expression of Alpha and Beta tubulin mRNAs will be investigated. The timing of appearance of cell-lineage-specific mRNAs will be related to events of differentiation. The correlation of different pathways of differentiation with expression of specific mRNA sets will be examined using procedures which alter the determination/differentiation of cell lineages. These studies are fundamental to understanding the requirements for differential gene expression during early development.
