The goal of the proposed research is to understand the cellular, genetic, and ultimately molecular mechanisms underlying nervous system control of defecation behavior in the nematode Caenorhabditis elegans. The primary focus will be on motor control of the specialized anal muscles that mediate defecation. The study will extend the current knowledge of two GABA-containing putative motor neurons, called AVL and DVB, that are required for anal muscle contraction. This will be accomplished by using a laser microbeam to functionally eliminate identified neurons, individually or in groups, in order to determine their role in control of defecation. These results will lead to a tentative determination of the complete network of neurons important in this behavior. The study will also extend the identification of genes important for defecation, adding to the 23 such genes already identified. To this end a large set of mutants defective in control of anal muscle contraction will be isolated. This part of the study will also use classical genetic methods of reversion of existing dominant mutants, epistasis tests, and detailed determination of mutant phenotype to characterize the function of these genes.
The final aim of the study will be to extend this genetic analysis to the molecular level. Existing monoclonal antibodies, which are directed against the putative anal muscle motor neurons AVL and DVB, will be used as an immunofluorescent stain in C. elegans whole mounts. The detailed structure of AVL and DVB will be compared in the large set of defecation mutants and the wild type. These tests will determine whether the mutants have altered the cell fate or neuronal process guidance of AVL and DVB. One gene, exp-1, is implicated as a possible component of an excitatory GABA neuromuscular junction made by AVL and DVB on the anal muscles. exp-l, and possibly a small set other similar genes identified in this study, will be cloned using the nearly complete physical map of C. elegans and DNA mediated transformation rescue of exp-l mutants. The gene(s) will be sequenced, its gene product localized in vivo, and its expression assessed in the many other mutants that affect control of anal muscle function. These studies will be directed at understanding the molecular function of this gene. Two features of the proposed study are of general health related interest. First, C. elegans provides the only model for the systematic genetic study of defecation, a behavior nearly universal in metazoans, and in which there are a wide range of disorders in humans. Second, motor control of anal muscle contraction in C. elegans provides an unusually good opportunity to identify and analyze mutants affecting an identified GABA synapse.
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