Semaphorin-mediated sensory and motor axon guidance

描述（由申请人提供）：了解控制神经系统模式的分子机制是发育神经生物学的中心焦点，重要的是，将导致识别与许多疾病过程相关的分子机制。在这个建议中概述的研究将描述3类分泌的脑信号蛋白轴突导向分子在轴突寻路和目标识别过程中的作用。分泌的3类脑信号蛋白已被证明在轴突排斥在体内和体外的功能。然而，关于这些引导线索及其受体在选择的神经系统中建立神经元连接的精确机制，还有很多东西有待了解。本研究的目的是了解脑信号蛋白在脊髓感觉神经元和运动神经元群体的轴突寻路中的细胞和分子机制。我们的策略利用免疫细胞化学和分子方法，也现有的和新的脑信号蛋白和神经纤毛蛋白小鼠突变体。我将首先进行全面的3类脑信号蛋白及其内源性配体的表达模式分析，采用碱性磷酸酶（AP）标记的配体结合试验，也分泌的脑信号蛋白受体神经纤毛蛋白-1（Npn-1）和神经纤毛蛋白-2（Npn-2）的抗体。为了确定3类脑信号蛋白-Npn信号传导如何有助于脊髓感觉和运动轴突的正确布线，将在适于此目的的脑信号蛋白和神经纤毛蛋白突变小鼠中进行免疫细胞化学和染料标记实验。为了询问是否需要脑信号蛋白介导的脊髓运动神经元轴突的引导用于适当的靶肌肉神经支配，将在npn和脑信号蛋白突变小鼠中使用HB 9：GFP转基因报告小鼠（其中HB 9启动子驱动GFP在所有运动神经元中表达）、仅在支配背肌群的侧向运动神经元中表达LacZ的LIM 1-tauLacZ报告小鼠和逆行追踪技术对轴向和肢体运动神经元投射进行详细分析。最后，我将使用LoxP重组酶系统产生一个npn-2条件性敲除，该系统与已有的条件性npn-1突变体相结合，将使我能够解决是否需要脑信号蛋白-神经纤毛蛋白信号传导细胞自主建立脊髓神经回路。总之，这些实验将提供深入了解发育中脊髓的神经连接的建立，它们可能对影响损伤后脊髓神经元的再生具有重要意义。

DESCRIPTION (provided by applicant): Understanding the molecular mechanisms that govern nervous system patterning is a central focus of developmental neurobiology and, importantly, will result in the identification of molecular mechanisms relevant to many disease processes. The studies outlined in this proposal will characterize the role of class 3 secreted semaphorin axon guidance molecules in axonal pathfinding and target recognition during development. Secreted class 3 semaphorin proteins have been shown to function in axon repulsion both in vivo and in vitro. Much remains to be learned, however, about the precise mechanisms used by these guidance cues and their receptors to establish neuronal connectivity in select neural systems. The goal of this proposal is to understand the cellular and molecular mechanisms underlying semaphorin function in axonal pathfinding of specific subsets of spinal sensory and motor neuronal populations. Our strategy utilizes immunocytochemical and molecular approaches, and also existing and new semaphorin and neuropilin mouse mutants. I will first conduct a comprehensive expression pattern analysis of class 3 semaphorins and their endogenous ligands, employing alkaline phosphatase (AP)-tagged ligand binding assays and also antibodies to the secreted semaphorin receptors neuropilin-1 (Npn-1) and neuropilin-2 (Npn-2). In order to determine how class 3 semaphorin-Npn signaling contributes to the proper wiring of spinal sensory and motor axons, immunocytochemical and dye labeling experiments will be carried out in the both semaphorin and neuropilin mutant mice appropriate for this purpose. To ask whether semaphorin-mediated guidance of spinal motor neuron axons is required for appropriate target muscle innervation, a detailed analysis of both axial and limb motor neuron projections will be conducted in npn and semaphorin mutant mice using an HB9:GFP transgenic reporter mouse where the HB9 promoter drives GFP expression in all motor neurons, a LIM1-tauLacZ reporter mouse that expresses LacZ exclusively in lateral motor neurons which innervate dorsal muscle groups, and retrograde tracing techniques. Finally, I will generate an npn-2 conditional knockout using the LoxP recombinase system which, in combination with a conditional npn-1 mutant already in hand, will allow me to address whether semaphorin-neuropilin signaling is required cell autonomously for the establishment of spinal neural circuits. Together, these experiments will provide insight into the establishment of neural connectivity of the developing spinal cord, and they may have important implications for influencing regeneration of spinal neurons following injury.