Mechanisms of Roundabout Repulsive Axon Guidance

迂回排斥轴突引导机制

• 批准号: 6919818
• 负责人: Greg J. Bashaw
• 金额: $ 37.06万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6919818
• 关键词: Drosophilidae; arthropod genetics; binding proteins; biological signal transduction; developmental genetics; developmental neurobiology; genetic screening; genetically modified animals; immunocytochemistry; neuronal guidance; protein structure function; sensory receptors; tissue /cell culture; yeast two hybrid system

DESCRIPTION (provided by applicant): How axons in the developing nervous system successfully navigate to their correct targets is a fundamental problem in neurobiology. Axons are guided by both attractive and repulsive cues, which are members of evolutionarily conserved protein families. We are interested in the signaling mechanisms that function during attractive and repulsive axon guidance. The midline of the Drosophila embryonic CNS provides an ideal system to address these questions. Like its structural analog, the vertebrate floor plate, the fly midline is an intermediate target for many classes of navigating axons, which must decide whether or not to cross the midline. In the Drosophila CNS, the conserved guidance cue, Slit, functions to prevent axons from abnormally crossing the midline. Slit repulsion is mediated by the conserved family of Roundabout (Robo) receptors. The major aims of this application are: 1) to delimit and characterize the regions of the Robo receptor's cytoplasmic domain that are necessary and sufficient for Robo-mediated axon repulsion in response to the Slit ligand, 2) to assess the potential role of the SH3-SH2 adaptor protein Dreadlocks (Dock) and associated proteins in contributing to Robo repulsion, 3) to identify additional components involved in Slit and Robo signaling using a Drosophila genetic screen. A well established Drosophila transgenic approach will be used to determine which regions of Robo's cytoplasmic domain are required for repulsion. Classical genetic and biochemical techniques, including genetic interaction tests, mutant analysis, yeast two hybrid and co-immunoprecipitation will be used to investigate the potential role of Dock and associated proteins in Robo repulsion. In addition, established cell culture techniques will be used to address whether Slit stimulation of the Robo receptor regulates Dock/Robo interactions. To identify additional molecules involved in Robo function, a sensitized genetic screen will be performed. The proposed genetic screening strategy has already been successfully used on a small scale to identify genes that may play important roles during Robo repulsion. Deciphering the mechanisms that mediate Robo repulsive axon guidance will give important insight into how the nervous system is correctly wired during development and may have implications for nerve regeneration.

描述（由申请人提供）：发育中的神经系统中的轴突如何成功导航到其正确的目标是神经生物学中的一个基本问题。轴突以吸引力和排斥性线索为指导，这些提示是进化保守的蛋白质家族的成员。我们对在有吸引力和排斥的轴突指导过程中起作用的信号传导机制感兴趣。果蝇胚胎中枢神经系统的中线提供了解决这些问题的理想系统。就像其结构类似物（脊椎动物地板板）一样，蝇中线是许多导航轴突的中间目标，必须决定是否越过中线。在果蝇中枢神经系统中，保守的引导提示，狭缝，功能，以防止轴突异常越过中线。缝隙排斥是由保守的回旋处受体家族介导的。该应用的主要目的是：1）为了响应缝隙配体而定界和特征机器人细胞质结构域的区域，对于机器人介导的轴突排斥而言是必要和足够的，响应于缝隙配体的轴突排斥，2）评估SH32-SH2适配器蛋白蛋白质dreadlock（dock）和相关的蛋白质的潜在作用，以构成Robo的其他蛋白质和相关的蛋白质，并与之相关的蛋白质，并与之相关的蛋白质，并撰写了RobiN的贡献，并具有贡献的蛋白质，并涉及ROBO的贡献。使用果蝇遗传筛查的机器人信号传导。将使用良好的果蝇转基因方法来确定Robo细胞质结构域的哪些区域需要排斥。经典的遗传和生化技术，包括遗传相互作用测试，突变体分析，酵母两种杂种和共免疫沉淀，用于研究码头和相关蛋白在机器人排斥中的潜在作用。另外，已建立的细胞培养技术将用于解决机器人受体的裂隙刺激是否调节码头/机器人相互作用。为了鉴定Robo功能中涉及的其他分子，将执行敏化的遗传筛选。提出的遗传筛查策略已经在小规模上成功使用，以识别可能在机器人排斥期间起重要作用的基因。解释介导机器人排斥轴突指导的机制将使人们对神经系统在发育过程中的正确连接方式有重要见解，并可能对神经再生产生影响。