The overall goal of this proposal is to elucidate the in vivo function of a zebrafish semaphorin, semaZ2. Semaphorins are putative inhibitory axon guidance molecules, and semaZ2 has an in vivo expression pattern that suggests it may act to guide the formation of specific axon pathways in the zebrafish brain. Transgenic zebrafish that ectopically express semaZ2 under control of the zebrafish hsp70 heat shock promoter will be used to analyze the effects of semaZ2 in growing axons. The heat inducibility of the hsp70 promoter allows both temporal and spatial control from ectopic transgene expression. First, the effects of ubiquitous semaZ2 expression at select stages of development will be analyzed by heating entire embryos to induce expression in all cells, and then examining axon trajectories. Second, semaZ2 will be misexpressed in specific cells at select time points to analyze the effects of axons growing the region of the expression cells. Third, growth cones extending near semaZ2 expressing cells will be observed with time lapse video microscopy to investigate whether SemaZ has inhibitory or repulsive activity on particular growth cones. An understanding of the in vivo function of molecules like semaphorins that regulate axon outgrowth will be crucial for understanding diseases of human neural development and problems with regeneration after injury to the CNS.
