The nervous system is a complex, highly-ordered cellular network containing many distinct cell types. The broad, long-term objectives of this research are to understand how the diversity and specific connectivity patterns of the nervous system are generated during development. One approach is to identify genes that participate in neuronal development and then analyze their products to discover how they function at molecular and cellular levels. Mutations of the unc-13 gene cause defects including abnormal neuronal connections and modified synaptic transmission in the C. elegans nervous system. The PI has shown that the gene product interacts with second messengers, diacylglycerol (or phorbol ester) and calcium, in the same way as protein kinase C, suggesting that this product is a component of an alternative transduction pathway of the diacylglycerol signal to a different effector function in the nervous system. The gene product has also two regions that are similar to synaptotagmin domains responsible for phospholipid and/or protein binding. This structural feature and phenotypes of the unc-13 mutants suggest that the gene plays a role as a calcium sensor in synaptic transmission.Calcium-sensitive processes in synaptic transmission are important in the regulated exocytosis of neurotransmitter-containing vesicles at nerve terminals, that is a ubiquitous phenomenon throughout the animal kingdom. Analysis of the new signal transduction pathways mediated by the unc-13 product could provide clues for understanding both nervous system development and synaptic transmission.
The aim of the proposed research is to study the novel signal transduction pathway mediated by unc-13 at cellular and molecular levels. The cells expressing unc-13 product at different developmental stages will be identified by immunohistochemistry. The DNA sequences of the promoter region will be analyzed to understand the molecular mechanism that regulates unc-13 gene expression, and the sequence will also be used for construction of transgenic worms expressing reporter genes under the control of the unc-13 promoter. These constructs are useful to identify cells expressing the unc-13 gene product, and to understand the regulatory mechanism of gene expression. In order to find other components of the signal transduction pathway, a genetic approach will be employed to isolate extragenic suppressors of unc-13 mutations. The sup-6 gene, which is a dominant, allele-specific suppressor of unc-13, will be isolated and analyzed by DNA sequencing. In vitro biochemical approaches will also be employed to identify components that directly interact with the unc-13 product. Identification of other members could ultimately link the unc-13 pathway to known signal transduction processes in the nervous system.
