The question addressed in this proposal is a fundamental one in the field of neurobiology, namely how complex neural circuits are assembled during development. Although poorly understood at the molecular level, it is known that developing neurons of both vertebrates and invertebrates are able to recognize and follow specific pathways which lead them to their appropriate synaptic targets. From mutant analyses in Drosophila, we have found that a family of transcription factors, called the LIM-homoedomain (LIM-HD) proteins, control neuronal pathway selection. We have further proposed that a code of LIM-HD expression within the nervous system dictates the specific pathways that deveoping neurons will choose by regulating the expression of genes involved in pathway recognition. Our goal in this project is to elucidate this code and to identify the genes regulated by LIM-HD proteins. Using germline transformation and GAL4-medicated transcriptional activation techniques, we will misexpress and swap specific LIM-HD proteins in identified neurons. Alterations in their axonal projections will be assayed using a set of tau-based axon-targeted reporters that allow the visualization of entire cell morphology. These experiments will test the hypothesis that specific LIM-HD proteins instruct neurons to choose particular pathways. We will use a number of approaches to isolate genes regulated by LIM-HD proteins. Theses include 1) a genetic screen in yeast to select Drosophila genomic DNA fragments that bind specific LIM-HD proteins, 2) the isolation of genetic modifiers of LIM- HD function in Drosophila, 3) screening a collection of tau-lacZ enhancer traps lines for regulation by LIM-HD members, and 4) isolating LIM-HD-depedent genes from cDNA libraries made from single identified LIM-HD-expressing neurons. In our last aim, we will identify new genes involved in neuronal pathfinding. Using a novel misexpression technique, we will screen for genes that when mis- or over-expressed in a particular subset of neurons causes specific alternations in their pathway selection. Since members of the LIM-HD family are highly conserved throughout the animal kingdom, collectively these studies will provide us with a better understanding of neuronal pathway recognition not only in Drosphila, but in higher vertebrates as well.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Genetics Study Section (GEN)
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Small, Judy A
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Salk Institute for Biological Studies
La Jolla
United States
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