AXON GUIDANCE IN FLIES--SCREENING AND IMAGING WITH GFP

California Institute Of Technology, Pasadena CA

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Abstract

Our goal is to find genes in Drosophila involved in axon pathfinding and synaptogenesis in the neuromuscular system; to determine the mutant phenotypes of a selection of these genes and assess their roles in a known network of genes; to analyze novel mutants in live pre-parations, using new methods of marking and imaging tissue; to analyze the phosphotyrosine phosphatase mutants (PTPs) with these methods in order to gain information about how their axonogenesis defects come about. An overexpression screen will be carried out with the P-element insertion lines of the Rorth collection using an induced, tissue-specific promoter (GAL 4 system), here expressing in the nervous system. Genes will be identified and isol-ated based on P-element flanking sequences obtained from the BDGP (Berkeley Dros. Genome Project). Overexpression phenotypes will first be analyzed by traditional methods (nervous system-specific antibody stains) in embryos and larvae, and based on these results, P-ex-cision null mutants can be generated in those lines which appear to be of most interest, based on sequence and phenotype. Mutants can then be combined with other known pathfinding mutants to find potential interactions. Novel and known mutants (PTPs) will be analyzed using GFP and GFP fusions as markers expressed in axons, growth cones, and (at larval stages) in synapses, using specific drivers for all or subsets of motorneurons, allowing live visualization by 2-photon confocal microscopy of the genesis of axon pathfinding and synaptogenesis phenotypes.