Comprehensive analysis for growth cone guidance and axonal branch regulation

引导分子的时空调控和生长锥对引导分子的敏感性的逐步控制是轴突到达目标的必要条件。通过获得这项资助，我们阐明了斑马鱼分泌的3类信号蛋白Sema3A1在引导尾侧初级（CaP）运动轴突中起重要作用，该轴突是运动通路的初始部分。Sema3A1的表达模式表明，它通过排斥作用将先锋CaP轴突划入初始的共同通路，但随后CaP轴突对共同通路以外的Sema3A1变得不敏感。事实上，可能编码Sema3A1受体成分的nrp-1a在其生长的早期阶段被CaP特异性表达，但在延伸到表达Sema3A1的肌肉细胞的后期阶段则不表达。为了直接检验这一假设，sema3A1和/或nrp-1a的表达被通过几种方式操纵。首先，Sema3A1的反义敲除诱导CaP轴突过度分支、失速和/或遵循异常通路。此外，动态分析显示它们延伸更多的外侧丝足，并且经常不能在水平肌间隔选择点停留。其次，Nrp ~ 1a的反义敲低和Nrp-1a/Sema3A1的双重敲低在CaP中诱导了类似的生长缺陷。第三，沿初始共同途径局部错误表达的Sema3A1抑制了CaP轴突，但不沿共同途径以外的途径。因此，正如预测的那样，Sema3A1在该通路的共同部分对CaP轴突具有排斥性，但不超出共同通路。第四，诱导的sema3A1普遍过表达导致CaP轴突而非其他初级运动轴突遵循异常通路。这些结果表明，初级运动轴突对Sema3A1的排斥反应是细胞类型特异性的，并且可能通过nlp -1a的调节而动态调节。

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.