NEUROPILIN-2 AND THE SYMPATHETIC NERVOUS SYSTEM

信号素家族包括跨膜和分泌蛋白，已被证明在多种系统中起轴突排斥信号的作用。最近发现神经毛膜蛋白是分泌信号素的受体：神经毛膜蛋白-1(Npn-1)是Sema3A(信号素III)的受体，神经毛细蛋白-2(Npn-2)是Sema3F(信号素IV)的受体。Sema3C(Semaphorin E)能以相同的亲和力与两种神经毛蛋白结合。然而，关于配体-受体相互作用发挥排斥作用的机制，以及它们在治疗发育中的神经系统投射模式方面的作用，人们知之甚少。以往的研究揭示了Sema3F和Npn-2之间的互补表达模式。特别有趣的是，在外周，Sema3F在接受交感神经支配的器官中强表达，而Npn-2在交感神经元中高表达，包括那些胞体位于颈上神经节(SCG)内的交感神经元、交感神经链中的节后神经元和不同的外周神经丛。这项研究将进一步描述Npn-2和已知的分泌信号素之间的特定相互作用，并确定它们在发育中的交感神经系统中的轴突寻路和靶标识别中的作用。首先，Npn-2是否作为其他分泌信号素的功能受体将通过使用从Npn-2-/-小鼠分离的Npn-1功能性阻断抗体处理的SCG或两者的组合进行病毒排斥试验来确定。其次，分泌信号素的时空表达模式将通过原位杂交来确定，详细的Npn-2蛋白表达模式将通过使用抗Npn-2抗体的免疫细胞化学染色来检测。最后，将使用最近产生的Npn-2-/-小鼠来评估靶交感神经支配中对Npn-2的需求。将特别强调通过酪氨酸羟化酶(TH)和儿茶酚胺组织荧光技术识别一系列靶器官的神经支配缺陷。总的来说，这些在体内和体内的分析结果将使我们更好地理解Sema3B、3C、3E、3F和Npn-2在交感目标神经支配中所扮演的角色，以及特定的信号素-神经粘连蛋白相互作用如何控制交感神经系统的轴突引导和发育。

The semaphorin family includes both transmembrane and secreted proteins that have been shown to function as axon repulsion cues in multiple systems. Neuropilins were recently identified as the receptors for secreted semaphorins: neuropilin-1 (Npn-1) is a receptor for Sema3A (semaphorin III) and neuropilin-2 (Npn-2) is a receptor for Sema3F (semaphorin IV). Sema3C (Semaphorin E) can bind to both of the neuropilins with the same affinity in viro. Little is known, however, about the mechanisms by which ligand-receptor interactions exert their repulsive effect, and their roles curing patterning of projections of the developing nervous system. Previous studies revealed a complementary expression pattern between Sema3F and Npn-2. Especially interesting is that in the periphery, Sema3F is strongly expressed in organs that receive sympathetic innervation, whereas Npn-2 is expressed highly in the sympathetic neurons including those whose cell bodies are within the superior cervical ganglion (SCG), postganglionic neurons in the sympathetic chain, and distinct peripheral plexuses. The studies outlined in this proposal will further characterize the specific interactions between Npn-2 and the known secreted semaphorins, as well as to define their roles in axonal pathfinding and target recognition in the developing sympathetic nervous system. First, whether Npn-2 serves as a functional receptor for other secreted semaphorins will be determined by an in viro repulsion assay using SCGs either treated with Npn-1 functional blocking antibodies, isolated from Npn-2 -/- mice, or a combination of these two. Second, the spatial and temporal patterns of expression of secreted semaphorins will be defined by in situ hybridization, and a detailed Npn-2 protein expression pattern will be examined by immunocytochemical staining using anti-Npn-2 antibodies. Finally, the requirement for Npn-2 in sympathetic innervation of targets will be assessed using recently generated Npn-2 -/- mice. A particular emphasis will be placed on the identification of innervation defects in a series of target organs by Tyrosine Hydroxylase (TH) and catecholamine histofluorescence techniques. As a whole, results from these in viro and in vivo analyses will provide a better understanding of the roles played by Sema3B, 3C, 3E, 3F and Npn-2 in sympathetic target innervation, and how specific semaphorin-neuropilin interactions control axon guidance and development of the sympathetic nervous system.