The overall goal of the proposed study is to exploit the extensive lineage maps and simple embryonic development of the Ascidian, Ciona intestinalis, to characterize the regulatory pathway underlying notochord differentiation. Ciona is a simple chordate that possesses a small genome (comparable in size to the genomes of C. elegans and Drosophila). This simplicity suggests that common features of all chordates, such as the notochord, might be specified by "stripped down" pathways that are directly applicable to vertebrate systems. A detailed notochord lineage has been characterized; after just nine embryonic cleavages the fully differentiated notochord contains only forty cells (about 10-12 hours after fertilization). Dr. Levine believes that Ciona is well poised for molecular analyses, particularly the elucidation of developmental pathways. Much of the project's effort will be devoted to the establishment of a blastomere disaggregation assay, whereby the embryonic activities of potentially interesting regulatory proteins are defined within the context of detailed lineages. This assay will involve the microinjection of early embryos with appropriate mRNAs. Individual, defined blastomeres will be cultured after disaggregating staged embryos, thereby permitting an assessment of autonomous versus nonautonomous regulatory interactions. Special efforts will focus on three regulatory proteins that have been implicated in notochord development in other systems, including axial/pintallavis, floating head/Xnot, and brychyury/T.
