This project is to study the dauer formation pathway in the nematode C. elegans. The dauer is an alternative L3 larval stage that the developing nematode follows under conditions of crowding and starvation. Previous work in several laboratories has resulted in the identification of a dauer-inducing pheromone and sensory neurons in the head of the nematode that regulate entrance into the dauer pathway. Genetic epistasis analysis of mutants that inappropriately enter the dauer pathway in the absence of pheromone, or alternatively that fail to enter the dauer pathway in the presence of pheromone, has allowed description of a genetic pathway of dauer regulation.
The aim of the present proposal is to further the understanding of the dauer pathway at the cellular and molecular levels. This research is a model for understanding chemosensory transduction and regulation of development by the environment.
The specific aims are as follows: 1) Further characterize the product of the daf-11 gene. This has been shown to be a transmembrane quanylyl cyclase thought to be involved in transducing the dauer pheromone signal in sensory neurons. Experiments include localization with antibodies and transgenes, structure/function analysis, and exploration of genetic interactions with a possible target gene, tax-4, which encodes a cGMP- activated ion channel. 2) Molecularly clone and analyze two genes of the dauer pathway: daf-21, thought to have a function closely related to daf-11, and daf-14, thought to act in a branch of the dauer pathway, and elsewhere, as part of a TGFbeta signalling pathway. 3) Extend the known cellular focus of the dauer pathway downstream of the sensory neurons by laser ablation of candidate interneurons. Interneurons selected will be those with an appropriate pattern of synaptic connectivity to sensory neurons, and those which are found to express molecular components of the dauer genetic pathway. 4) New genes of the dauer pathway will be sought in a genetic screen and placed in the dauer pathway by epistasis analysis. 5) Electrophysiological studies to test models of signal transduction in response to dauer pheromone will be carried out by developing patch clamping methodology for sensory neurons, or by studying individual molecular components in human epithelial kidney cells.
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