The notochord provides a """"""""scaffolding"""""""" that shapes much of early chordate embryogenesis. In addition to its structural role in morphogenesis, the inductive activity of the vertebrate notochord is essential for the proper development of the nervous system, skeletal muscle, skeleton, heart, and pancreas. The molecular pathway leading from the initial steps of notochord induction in the blastula embryo to the fully formed and functional notochord is only partially understood. The goal of the research proposed here is to gain a better understanding of the development and function of the notochord using genetic methodology. The proposed experiments will take advantage of a recently developed model system for genetic analysis that uses the ascidian Ciona savignyi. Ascidians have many desirable features that aid in genetic analysis, including a small genome, simple morphology and fixed cell lineage. Chemical mutagenesis screens have yielded several zygotically acting recessive mutations that disrupt notochord development in C. saviguyi. Experiments in this proposal will focus on three mutant lines: chongmague (chm) chobi (chb) and 17.3. Chm disrupts early notochord development, and the cells of the notochord rudiment fail to differentiate properly. Both chb and 17.3 disrupt later stages of notochord development involved in extending the body axis. Experiments specifically directed at characterizing chm will first seek to address whether presumptive notochord cells in embryos homozygous for chm have adopted an alternative differentiated cell fate. Additional experiments will determine whether the mutation responsible for the chm phenotype is in one of several candidate genes. Time lapse video recording experiments on all three mutations will allow for a fuller characterization of when and how the mutations disrupt notochord development. Long term objectives of this project include exploiting the small genome size of C. saviguyi to aid in positional cloning of mutant loci disrupting morphogenesis. Towards this goal, experiments proposed here will examine the degree of genetic polymorphism among wild C. savignyi populations, and will then establish inbred polymorphic lines. These lines will be used in future gene mapping experiments. Finally, after preliminary experiments aimed at optimizing the mutagenesis protocol in C. savignyi, a new and larger scale mutational screen will be performed with the goal of isolating new alleles of the existing mutations and of finding novel alleles that disrupt notochord development.
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