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。本提案概述的研究将描述3类分泌信号蛋白轴突引导分子在发育过程中轴突寻路和目标识别中的作用。分泌的3类信号蛋白已被证明在体内和体外的轴突排斥中起作用。然而，关于这些引导线索及其受体在选定的神经系统中建立神经元连接所使用的精确机制，仍有许多有待研究。本研究的目的是了解信号素在脊髓感觉和运动神经元群特定亚群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，以及现有的和新的信号蛋白和神经匹林小鼠突变体。我将首先对第3类信号素及其内源性配体进行全面的表达模式分析，采用碱性磷酸酶（AP）标记的配体结合试验以及分泌信号素受体neuropilin-1 （Npn-1）和neuropilin-2 （Npn-2）的抗体。为了确定3类信号素- npn信号如何促进脊髓感觉轴突和运动轴突的正常连接，将在信号素和神经匹林突变小鼠中进行免疫细胞化学和染料标记实验。为了了解信号蛋白介导的脊髓运动神经元轴突的引导是否需要适当的目标肌肉神经支配，我们将使用HB9:GFP转基因报告小鼠（HB9启动子驱动所有运动神经元中GFP的表达）和LIM1-tauLacZ报告小鼠（仅在支配背肌群的外侧运动神经元中表达LacZ），对npn和信号蛋白突变小鼠的轴向和肢体运动神经元投射进行详细分析。以及逆行追踪技术。最后，我将使用LoxP重组酶系统生成npn-2条件敲除，该系统与已经掌握的条件npn-1突变体相结合，将使我能够解决信号素-神经肽信号是否需要细胞自主地建立脊髓神经回路。总之，这些实验将为发育中的脊髓神经连通性的建立提供见解，并且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.