Semaphorin-mediated sensory and motor axon guidance

信号蛋白介导的感觉和运动轴突引导

• 批准号: 6886356
• 负责人: Tracy S Tran
• 金额: $ 4.3万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6886356
• 关键词: afferent nerve; alkaline phosphatase; antibody; cell communication molecule; confocal scanning microscopy; developmental neurobiology; gene targeting; genetic models; genetically modified animals; green fluorescent proteins; growth cones; immunocytochemistry; in situ hybridization; laboratory mouse; motor neurons; mutant; neuronal guidance; neuropilins; postdoctoral investigator; proprioception /kinesthesia; protein structure function; spinal cord

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.

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。本提案中概述的研究将描述 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发挥作用。然而，关于这些引导信号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，还有很多东西有待了解。该提案的目标是了解信号蛋白在脊髓感觉和运动神经元群体特定亚群的轴突寻路中功能的细胞和分子机制。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毡蛋白小鼠突变体。我将首先使用碱性磷酸酶 (AP) 标记的配体结合测定以及针对分泌型信号蛋白受体 Neuropilin-1 (Npn-1) 和 Neuropilin-2 (Npn-2) 的抗体，对 3 类信号蛋白及其内源配体进行全面的表达模式分析。为了确定 3 类信号蛋白-Npn 信号传导如何有助于脊髓感觉和运动轴突的正确接线，将在适合此目的的信号蛋白和神经毡蛋白突变小鼠中进行免疫细胞化学和染料标记实验。为了询问适当的目标肌肉神经支配是否需要信号蛋白介导的脊髓运动神经元轴突引导，将使用 HB9:GFP 转基因报告小鼠（其中 HB9 启动子驱动所有运动神经元中的 GFP 表达）在 npn 和信号蛋白突变小鼠中对轴向和肢体运动神经元投影进行详细分析，该小鼠表达 GFP LacZ 专门存在于支配背肌群的外侧运动神经元中，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，该系统与现有的条件 npn-1 突变体相结合，将使我能够解决细胞自主建立脊髓神经回路是否需要信号蛋白-神经毡蛋白信号传导。总之，这些实验将深入了解发育中脊髓的神经连接的建立，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。