Semaphorin-mediated sensory and motor axon guidance

描述(由申请人提供)：了解支配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。这项提案中概述的研究将表征在发育过程中分泌的3类信号素轴突引导分子在轴突寻路和目标识别中的作用。分泌的3类信号素蛋白在体内和体外都被证明在轴突排斥中发挥作用。然而，关于这些引导线索和它们的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，仍有许多有待了解。这项建议的目的是了解信号素在脊髓感觉和运动神经元群体特定亚群的轴突路径发现中潜在的细胞和分子机制。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素和神经粘连蛋白小鼠突变体。我将首先通过碱性磷酸酶(AP)标记的配体结合分析以及分泌的信号素受体神经粘蛋白-1(Npn-1)和神经粘连蛋白-2(Npn-2)的抗体，对3类信号素及其内源性配体的表达模式进行全面的分析。为了确定3类信号素-Npn信号如何在脊髓感觉和运动轴突的正确连接中起作用，将在信号素和神经毛膜蛋白突变小鼠中进行免疫细胞化学和染料标记实验。为了探讨是否需要信号素介导的脊髓运动神经元轴突的指导才能进行适当的靶肌肉神经支配，我们将使用HB9：GFP转基因报告鼠(HB9启动子驱动所有运动神经元中GFP的表达)、Lim1-tauLacZ报告鼠(仅在支配背侧肌群的外侧运动神经元中表达LacZ)和逆行追踪技术，对NPN和信号素突变小鼠的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重组酶系统生成NPN-2条件敲除，该系统与已有的条件NPN-1突变体相结合，将使我能够解决是否细胞自主地需要信号素-神经匹林信号来建立脊髓神经回路。综上所述，这些实验将为建立发育中的脊髓的神经连接提供洞察力，并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.