The role of the zebrafish unplugged gene in motor axon pathfinding

During embryonic development, motor axons navigate long distances through a complex environment to form precise connections with their target muscles. This accurate pathfinding process is facilitated by specialized cells scattered along motor axon pathways. These specialized cells, serving as intermediate targets or choice points, provide molecular guidance cues that instruct all motor growth cones to enter and migrate on a common path as well as cues that control individual population of motor growth cones to select divergent pathways. Previous studies in invertebrates and vertebrates have identified numerous genes involved in motor axon guidance. However, the molecular identities of guidance cues remain largely elusive. To identify the guidance cues controlling motor axon pathway selection, I used well-characterized zebrafish primary motor neurons as a model system. I show that in the unplugged mutants of zebrafish, pathway selections of two primary motor neurons, RoP and CaP, are specifically affected. Analyses of chimeric embryos demonstrate that the unplugged gene acts cell non-autonomously for motor axon guidance and the guidance defect can be rescued by a subgroup of wild-type derived adaxial cells. These cells are located dorsal to the choice point and influence pathway selection of motor axons through a contact-independent mechanism. Thus, the unplugged gene may encode a guidance cue controlling motor axon pathway selection. Furthermore, I examined the axonal trajectories of secondary motor neurons in unplugged mutants. Secondary motor axons display similar, but not identical, defects as primary motor axons. Due to the novel phenotype of unplugged mutants, there is no good candidate for the mutated gene. Therefore, I undertook a positional cloning approach to identify the unplugged locus, unplugged maps to the centromere of LG 10. I tested 9,380 meioses to generate a high-resolution map around the unplugged locus. To date, I have covered part of the critical interval of the unplugged locus with BAC/PAC clones. Systematic isolation of transcription units in the critical region and test of their candidacy for the unplugged gene are underway. These efforts provide a foundation for future studies aiming at identification of the unplugged gene and investigation of its mechanism in motor axon guidance