During embryogenesis, motor neurons extend stereotypic axonal projections to axial and limb muscles. En route to their muscle targets, individual motor axons encounter designated choice points at which they split away from the nerve and select a route specific for their cell type. This process of axonal pathway choice depends on extrinsic cues provided by the local environment, but little is known about the molecules or mechanisms by which extrinsic cues direct motor axons. One of the few extrinsic cues known to direct motor axons at individual choice points is the zebrafish unplugged gene. In unplugged null mutants two pioneering motor growth cones reach the choice point but make inappropriate pathway decisions. Through positional cloning we have identified the unplugged gene to encode a muscle specific kinase (MUSK) like receptor tyrosine kinase. In mammals, MuSK has been extensively studied in the context of synapse formation. There, the prevailing model is that Agrin signals through MuSK to recruit the intracellular linker Rapsyn, which """"""""clusters acetylcholine receptors (AChRs) on the postsynaptic membrane of neuromuscular junctions. Unplugged null mutants are motile and viable, and display only a mild reduction in AChRs clustering, suggesting that unplugged function is not absolutely essential for synapse formation. Rather, our results suggest a previously not recognized role of MuSK-like genes in axonal pathway selection. We have compelling evidence that axonal pathway selection through unplugged is activated independently of Agrin, and initiates a signaling cascade independent of the MuSK downstream component Rapsyn. We propose that hitherto unknown ligands signal through the unplugged receptor tyrosine kinase to activate a yet undefined downstream cascade that directs motor axon pathfinding locally at choice points. Here, we propose to 1) identify ectodomain binding proteins as potential ligand(s) that activate unplugged mediated pathway selection, 2) determine the intracellular mechanisms by which unplugged directs motor axons at choice points, and 3) determine the role of the sidetracked gene in pathway selection. These studies are directly relevant to the study of human disease, since genes known to direct axonal growth are implicated in the cause of human disease states and human inherited disorders, and might also play a role in regeneration after nerve injury.
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