The long-term objective of this proposal is to understand the cellular and molecular mechanisms that govern the reorganization of neuronal processes. Reorganization of neuronal processes occurs commonly in the wiring of nervous system of both invertebrates and vertebrates, yet little is known about the underlying mechanisms. We have developed the MARCM system (for Mosaic Analysis with a Repressible Cell Marker), in which a small subset of identifiable neurons are both specifically labeled and made homozygous mutant for a candidate gene of interest. Using the MARCM system, we found that the y neurons of the Drosophila mushroom body (MB) undergo major reorganization of their neuronal processes during metamorphosis. This includes the precise pruning of larval-specific axonal brunches and complete pruning of larval dendrites, followed by the extension of adult-specific axonal and dendritic projections. Preliminary results suggest that the steroid hormone ecdysone directly acts on MB y neurons to regulate their process reorganization. The striking and specific reorganization of the processes of MB y neurons, along with the possibility of studying the effects of loss- and galn-of- function mutations in candidate and novel genes using the MARCM system, offer us the unique opportunity to unravel the cellular and molecular mechanisms that regulate neuronal process reorganization. We will determine whether pruning of MB axonal and dendritic processes is via retraction or local degeneration, and whether dendritic reorganization is regulated by their axonal contact and input. We will then focus on identifying proteins that execute the reorganization process by: I) a top- down candidate gene approach from ecdysone regulatory hierarchy; 2) a genetic screen that inactivates genes only in a small population of uniquely labeled MB y neurons; 3) cDNA microarray approaches to identify genes that are differentially expressed in neurons that undergo process reorganization. Our studies promise to identify general principles and molecular mechanisms of neuronal process reorganization, which ensure the proper wiring of the nervous system essential for our mental function. These mechanisms may also be used for experience-dependent reshaping of neuronal connections, and may be misregulated during aging and neurodegenerative diseases to cause degeneration of neuronal processes.
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