The broad objective of this proposal is to elucidate the processes that underlie cell lineage specification during early embryogenesis, a central problem of immense significance in developmental biology. The system bhosen for study is the actin family of the sea urchin embryo, a well characterized gene family and a very popular embryonic system suitable for the proposed experimentation. The sea urchin Strongylocentrotus purpuratus actin gene family consists of eight members; six genes that are active at different developmental stages of the animal life and two pseudogenes. The five cytoskeletal type actin genes are organized into two genomic linkage groups. Both coordinate as well as independent regulation of individual genes within a linkage group has been observed. The spatial patterns of expression of individual actin genes are distinct during embryogenesis. Actin mRNAs transcribed from two linked genes, CyIIIa and CyIIIb, accumulate exclusively in the cells of the aboral ectoderm, although at quite distinct embryonic stages. The temporal as well as the cell lineage specific regulation of the actin genes and the function of their respective proteins in the developing animal is not understood. The project deals with the sequence characterization of the CyIIIb gene, the identification of its regulatory elements using gene transfer into sea urchin embryos, the comparison of such ontogenic signals between CyIIIa and CyIIIb and the study of the physiological function of these ectodermal actins during embryonic development. Specifically, to gain more insight into the structural/functional similarities and/or differences of the ectodermal actins, the complete nucleotide sequence of the CyIIIb gene will be determined, and the coding region will be compared to the respective sequence of the CyIIIa gene. To identify possibly similar regulatory elements of the two genes, the sequencing of the CyIIIb gene will be extended to the flanking regions. To study the ontogenic regulation of this gene, fusion constructs of the chloramphenicol acetyl transferase and the Beta-galactosidase genes with CyIIIb 5' flanking sequences will be injected into sea urchin eggs and their expression monitored. Injections of such constructs bearing different lengths and deletions of the CyIIIb 5' flanking sequences will allow the identification of temporal and tissue specific cis acting regulatory elements. To study the physiological function of these ectodermal specific actin genes, reverse actin gene constructs coding for antisense RNAs will be injected into the sea urchin eggs. Targeted expression and therefore cell lineage specific blocking of actin expression will be accomplished using the tissue specific regulatory elements of the two genes or the CyIIIa and CyIIIb gene specific noncoding segments.
