This proposal outlines a series of experiments whose purpose is to investigate the environmental cues by which growing axons are guided to their target tissues. It uses as its model system the sensory neurons found on the wing of the fruitfly, Drosophila melanogaster. I will attack this problem using two rather different approaches: I) Patterns of gene expression in the wing-Because the pattern of axon guidance cues in the wing has been so well characterized, there are strong constraints upon the way candidate guidance molecules could be distributed. I will exploit this by a) using the newly developed enhancer trap technique, and b), examining the distribution of known gene products, such as hairy, to search for patterns of gene expression and cell differentiation which correspond to our expectations for guidance cues. Mutations will then be used or generated, and antigens detected using immunosuppressive techniques. II) Perturbation of guidance in vitro- A more directed, cellular approach to axon guidance will be used, which depends upon the ability of fixed wing fragments to guide growing dissociated neurons along normal axon pathways. This will be used as a bioassay to test the previously demonstrated ability of polyclonal antiserum to wing tissue to block normal guidance. Inhibitory antiserum will be generated and the inhibitory activity isolated by successive steps of affinity purification using an immunoblotting system, and bioassay. Additional side projects are proposed using the in vitro system.
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