To establish functional neuronal connections during embryogenesis, axons navigate considerable distances to reach their targets. During this process of axonal pathfinding, the axons are repeatedly confronted with choice points at which they must select their appropriate axonal pathways. Correct pathway selection requires the presence of spatially and temporally orchestrated cues at each choice point and the responsiveness of individual neural growth to only a specific set of cues. The objective of the studies described here is to learn how selection of axonal pathway is achieved during vertebrate embryogenesis. To study axonal pathway selection in vertebrates, the studies use the recently identified unplugged gene which functions in selecting motor axonal pathways in the zebrafish embryo. In unplugged mutants, motor axons exit the spinal cord in a common nerve path, but at a subsequent choice point, an identified subpopulation of motor axons specifically fails to select its appropriate axonal pathway. Analysis of chimeric embryos suggests that unplugged gene activity is required cell non-autonomously, indicating that cells surrounding the neuron provide unplugged gene activity. Genetic mapping places unplugged within a 0.25 centi Morgan chromosomal region (corresponding to about 165 kb) in which no genes implicated in axon guidance have been mapped. Thus, the unplugged gene is a strong candidate to encode a (novel) cue, specialized in vertebrate axonal pathway selection. The experiments in this proposal will: (1) test the hypothesis that unplugged functions in local axonal guidance at the choice point rather than in motor neuron specification (through a detailed analysis of motor neuron identity in unplugged embryos); (2) define the cell type(s) critical for unplugged mediated axonal pathway selection (through transplantation of labeled cells between mutant and wild-type embryos); and (3) identify the molecular nature of the unplugged gene (through positional cloning). The unplugged gene is among the first vertebrate genes known to control motor axonal pathway decisions. Thus, it provides a unique opportunity to understand the biological and molecular mechanisms underlying human hereditary neuropathies, such as MASA and Kallman syndrome.
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