Comprehensive analysis for growth cone guidance and axonal branch regulation

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In order for axons to reach their proper targets both spatio-temporal regulation of guidance molecules and stepwise control of growth cone sensitivity to guidance molecules is required. By getting support of this grant, we elucidated that in zebrafish Sema3A1, a secreted class 3 semaphorin, plays an essential role in guiding the Caudal Primary (CaP) motor axon that pioneers the initial portion of the motor pathway. The expression pattern of Sema3A1 suggested that it delimited the pioneer CaP axons to the initial, common pathway via a repulsive action, but then CaP axons become insensitive to Sema3A1 beyond the common pathway. Indeed, nrp-1a that likely encodes a component of the Sema3A1 receptor is specifically expressed by CaP during the early portion of its outgrowth but not during later stages when extending into sema3A1 expressing muscle cells. To directly examine this hypothesis expression of sema3A1 and/or nrp-1a was manipulated in several ways. First, antisense knockdown of Sema3A1 induced CaP axons to branch excessively, stall and/or follow aberrant pathways. Furthermore, dynamic analysis showed they extended more lateral filopodia and often failed to pause at the horizontal myoseptal choice point. Second, antisense knockdown of Nrp〜1a and double knockdown of Nrp-1a/Sema3A1 induced similar outgrowth defects in CaP. Third, CaP axons were inhibited by focally misexpressed sema3A1 along the initial common pathway but not along their pathway beyond the common pathway. Thus, as predicted, Sema3A1 is repulsive to CaP axons in the common portion of the pathway, but not beyond the common pathway. Fourth, induced ubiquitous overexpression of sema3A1 caused the CaP axons but not the other primary motor axons to follow aberrant pathways. These results suggest that the repulsive response to Sema3A1 of the primary motor axons is both cell-type specific and dynamically regulated perhaps via regulation of nrp-1a.