NEUROPILIN-2 AND THE SYMPATHETIC NERVOUS SYSTEM

脑信号蛋白家族包括跨膜和分泌蛋白，已被证明在多个系统中起轴突排斥线索的作用。 神经纤毛蛋白最近被鉴定为分泌的脑信号蛋白的受体：神经纤毛蛋白-1（Npn-1）是Sema 3A（脑信号蛋白III）的受体，神经纤毛蛋白-2（Npn-2）是Sema 3F（脑信号蛋白IV）的受体。 Sema 3C（Semaphorin E）可以在体内以相同的亲和力结合两种神经纤毛蛋白。 然而，人们对配体-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中的作用知之甚少。 先前的研究揭示了Sema 3F和Npn-2之间的互补表达模式。 特别令人感兴趣的是，在外周，Sema 3F在接受交感神经支配的器官中强烈表达，而Npn-2在交感神经元中高度表达，所述交感神经元包括其细胞体位于上级颈神经节（SCG）内的交感神经元、交感链中的节后神经元和不同的外周神经丛。 本提案中概述的研究将进一步表征Npn-2和已知分泌的信号蛋白之间的特定相互作用，以及定义它们在发育中的交感神经系统中轴突寻路和靶点识别中的作用。 首先，Npn-2是否充当其它分泌的脑信号蛋白的功能性受体将通过使用用Npn-1功能性阻断抗体处理的、从Npn-2 -/-小鼠分离的SCG或这两者的组合的体外排斥测定来确定。第二，分泌的脑信号蛋白的表达的空间和时间模式将被定义通过原位杂交，和一个详细的Npn-2蛋白的表达模式将被检查通过免疫细胞化学染色使用抗Npn-2抗体。 最后，将使用最近产生的Npn-2 -/-小鼠评估靶的交感神经支配中对Npn-2的需求。 一个特别的重点将放在识别神经支配缺陷的一系列靶器官酪氨酸羟化酶（TH）和儿茶酚胺组织荧光技术。 作为一个整体，从这些在体内和体外分析的结果将提供一个更好的理解Sema 3B，3C，3E，3F和Npn-2在交感神经靶神经支配中发挥的作用，以及如何特定的脑信号蛋白-神经纤毛蛋白相互作用控制轴突的指导和交感神经系统的发展。

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.