NEUROPILIN-2 AND THE SYMPATHETIC NERVOUS SYSTEM

信号蛋白家族包括跨膜蛋白和分泌蛋白，它们已被证明在多个系统中作为轴突排斥信号起作用。neuropilin最近被鉴定为分泌信号素的受体：neuropilin-1 （Npn-1）是Sema3A（信号蛋白III）的受体，neuropilin-2 （Npn-2）是Sema3F（信号蛋白IV）的受体。Sema3C （Semaphorin E）在体外能以相同的亲和力与这两种神经肽结合。然而，关于配体-受体相互作用发挥排斥作用的机制，以及它们在发育中的神经系统投射模式中的作用，我们所知甚少。先前的研究发现Sema3F和Npn-2之间存在互补的表达模式。特别有趣的是，在外周，Sema3F在接受交感神经支配的器官中强烈表达，而Npn-2在交感神经元中高度表达，包括那些细胞体位于颈上神经节（SCG）内的神经元、交感神经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与已知分泌信号蛋白之间的具体相互作用，并确定它们在交感神经系统发育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性受体，将通过体外排斥试验确定，使用从Npn-2 -/-小鼠中分离的Npn-1功能性阻断抗体处理的scg，或两者的组合。其次，分泌信号素的时空表达模式将通过原位杂交确定，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进行检查。最后，我们将利用最近生成的Npn-2 -/-小鼠来评估目标交感神经支配对Npn-2的需求。一个特别的重点将放在通过酪氨酸羟化酶（TH）和儿茶酚胺组织荧光技术在一系列靶器官的神经支配缺陷的识别。总的来说，这些体外和体内分析的结果将更好地理解Sema3B、3C、3E、3F和nnp -2在交感靶神经支配中的作用，以及特定的信号素-神经匹林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.