Maternal Control of Ascidian Development

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0080060SmithZygotic development begins with the fusion of sperm and egg. While sperm provides only the male pronucleus, the egg contributes, in addition to the female pronucleus, the egg cytoplasm which contains molecules essential for proper development of the embryo. These "maternal factors" in the egg control numerous process include sperm recognition, cell-cycle activation, and determination of the primary axes and germ layers. Ascidians, which are primitive members of the chordate phylum, have classically been used to study the role of maternal factors in early development. In fertilized ascidian eggs extensive rearrangement of egg cytoplasm can be observed. The partitioning of distinct cytoplasmic factors specifies many tissue types including muscle and endoderm.This project will use newly developed genetic methods in ascidians to analyze maternal control of early development. Ascidians are particularly well suited for this type of analysis. The species to be used in this project, Ciona savignyi, is a hermaphrodite with the capacity for self fertilization. These properties streamline procedures for identification and recovery of mutants. Briefly, adult C. savignyi are first treated with the chemical mutagen ENU. Sperm from the ENU-treated animals is crossed to wild-type eggs to generate heterozygous F1s. The F1s are grown to maturity (10-12 weeks), and then sperm and eggs are collected from each and mixed to make a self-fertilized F2 generation. One-quarter of the F2s will be homozygous for each mutant locus carried by an F1. When the F2s reach maturity, the eggs are dissected, crossed with wild-type sperm, and then screened for mutant phenotypes. If a potential mutant is observed, sperm from the same animal is crossed to wild-type eggs in order to recover the mutation. A pilot screen has shown that this screen is feasible, and a number of candidate mutants are being characterized. Funding from this proposal will be used to complete the pilot screen, perform an expanded screen, and characterize recovered mutants in order to better understand the nature of the mutations.