The overall objective of the research proposed in this grant is to elucidate the molecular mechanisms which enable axons to find and connect with their appropriate target cells. It is expected that the results obtained from these studies will be helpful in understanding disorders of the human nervous system. We propose to use the powerful molecular genetic techniques available in Drosophila to study the molecular basis of neuronal specificity in a simple model system, the visual system of the Drosophila larva. We have previously identified a gene, named disconnected (disco), which is required for the establishment of specific synaptic connections of the larval optic nerve during embryonic development. The experiments proposed here are aimed at determining the biochemical function of the disconnected gene product(s). We also propose to identify and characterize additional genes which control neuronal specificity. In order to accomplish our overall objectives we will: (1) Determine the structure and DNA sequence of the disco gene and thereby obtain the deduced amino acid sequence of the encoded protein(s). (2) Determine the spatial localization of disco mRNA by in situ hybridization. (3) Generate antibodies against disco protein and use them to determine the distribution and subcellular localization of the disco protein(s). (4) Determine which cells require disco gene function by generating very small genetic mosaic patches using nuclear transplantation techniques. (5) Express the disco protein in ectopic sites by fusing its gene to heterologous promoters and reintroducing these fusion genes into the Drosophila germ line by P-element mediated gene transfer. The consequences of such an ectopic expression on the projection pattern of Bolwig's nerve will be analysed in transformed embryos. (6) Investigate the evolutionary conservation of the disco protein. This will involve the isolation of disco genes from different species and the determination of the corresponding DNA sequences. (7) Test the functional significance of conserved domains in the disco polypeptide(s) by mutating selected amino acids using site-directed in vitro mutagenesis and by constructing chimeric disco genes composed of segments derived from different species and introducing them into flies by P-element- mediated gene transfer. (8) Isolate and catalog additional mutants which affect the projection pattern of the larval photoreceptor neurons by insertional mutagenesis with efficient P element vectors. (9) Isolate some of the corresponding genes which, based on genetic and anatomical criteria, most warrant further work. (10) Finally, for all the genes that we study in detail we will ask whether homologous genes can be isolated from vertebrates using methods of nucleic acid sequence cross- homology.
