Project Summary/Abstract During nervous system development and post-injury regeneration, nerve pathways are formed by migrating neuronal axons which are guided by extracellular cues. Although many of the major signaling pathways that regulate axon guidance have been identified, our understanding of the mechanisms by which individual genes influence specific axon guidance outcomes remains limited. In animals with complex nervous systems, such as insects and mammals, individual ligands and receptors from conserved signaling pathways can promote diverse or even opposing outcomes in different populations of neuronal axons. This proposal uses the fruit fly Drosophila melanogaster as a model to address the question of how individual genes can specify diverse axon guidance outcomes. The evolutionarily conserved Roundabout (Robo) family of axon guidance receptors control multiple distinct axon guidance outcomes in the developing Drosophila embryonic central nervous system (CNS). The proposed research takes advantage of the molecular and genetic tools available in Drosophila to dissect the mechanisms underlying the diverse axon guidance activities of individual Robo receptors. The project will use structure/function gene modification approaches to characterize the functional domains that contribute to the axon guidance activities of individual Robo receptors, will investigate how the transcriptional regulation of robo genes in specific neuronal populations contributes to specific axon guidance decisions, and will test candidate mechanisms that may account for the receptors’ role(s) in different axon guidance contexts. The proposed research will provide insight into how individual axon guidance genes can specify diverse developmental outcomes, identify potentially evolutionarily conserved mechanisms that may operate in other animals, including humans, where members of the Robo receptor family are also key regulators of axon guidance, and may suggest novel strategies for restoring proper regulation of axon guidance in the contexts of nervous system injury, repair, and regeneration.