Understanding the molecular mechanisms that govern nervous system patterning is a central focus of developmental neurobiology and, importantly, will result in the identification of molecular mechanisms relevant to many disease processes. The studies outlined in this proposal will characterize the role of class 3 secreted semaphorin axon guidance molecules in axonal pathfinding and target recognition during development. Secreted class 3 semaphorin proteins have been shown to function in axon repulsion both in vivo and in vitro. Much remains to be learned, however, about the precise mechanisms used by these guidance cues and their receptors to establish neuronal connectivity in select neural systems. The goal of this proposal is to understand the cellular and molecular mechanisms underlying semaphorin function in axonal pathfinding of specific subsets of spinal sensory and motor neuronal populations. Our strategy utilizes immunocytochemical and molecular approaches, and also existing and new semaphorin and neuropilin mouse mutants. I will first conduct a comprehensive expression pattern analysis of class 3 semaphorins and their endogenous ligands, employing alkaline phosphatase (AP)-tagged ligand binding assays and also antibodies to the secreted semaphorin receptors neuropilin-1 (Npn-1) and neuropilin-2 (Npn-2). In order to determine how class 3 semaphorin-Npn signaling contributes to the proper wiring of spinal sensory and motor axons, immunocytochemical and dye labeling experiments will be carried out in the both semaphorin and neuropilin mutant mice appropriate for this purpose. To ask whether semaphorin-mediated guidance of spinal motor neuron axons is required for appropriate target muscle innervation, a detailed analysis of both axial and limb motor neuron projections will be conducted in npn and semaphorin mutant mice using an HB9:GFP transgenic reporter mouse where the HB9 promoter drives GFP expression in all motor neurons, a LIM1-tauLacZ reporter mouse that expresses LacZ exclusively in lateral motor neurons which innervate dorsal muscle groups, and retrograde tracing techniques. Finally, I will generate an npn-2 conditional knockout using the LoxP recombinase system which, in combination with a conditional npn-1 mutant already in hand, will allow me to address whether semaphorin-neuropilin signaling is required cell autonomously for the establishment of spinal neural circuits. Together, these experiments will provide insight into the establishment of neural connectivity of the developing spinal cord, and they may have important implications for influencing regeneration of spinal neurons following injury.
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