Mechanisms of Roundabout Repulsive Axon Guidance

迂回排斥轴突引导机制

• 批准号: 7262460
• 负责人: Greg J. Bashaw
• 金额: $ 35.14万
• 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/7262460
• 关键词: Accounting; Actins; Adaptor Signaling Protein; Address; Axon; Biochemical Genetics; Biological Assay; Cell Culture Techniques; Class; Co-Immunoprecipitations; Complex; Cues; Cytoplasmic Tail; Cytoskeleton; Data; Defect; Development; Docking; Dose; Drosophila genus; Embryo; Event; Family; Floor; Genes; Genetic; Genetic Screening; Growth Cones; Individual; Left; Ligand Binding; Ligands; Link; Mediating; Metalloproteases; Molecular; Monomeric GTP-Binding Proteins; Mutate; Nerve Regeneration; Nervous system structure; Neurobiology; Numbers; Phenotype; Play; Protein Family; Protein-Serine-Threonine Kinases; Proteins; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Protein; Structure; System; Techniques; Testing; Therapeutic; Transgenic Organisms; Yeasts; analog; axon guidance; base; design; fly; forging; gene function; insight; interest; member; mutant; prevent; receptor; response; 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地导航到其正确的靶点是神经生物学中的一个基本问题。轴突由吸引和排斥的线索引导，这些线索是进化上保守的蛋白质家族的成员。我们感兴趣的是在吸引和排斥轴突引导过程中发挥作用的信号机制。果蝇胚胎中枢神经系统的中线为解决这些问题提供了一个理想的系统。与其结构类似的脊椎动物底板一样，果蝇中线是许多类别的导航轴突的中间目标，这些轴突必须决定是否穿过中线。在果蝇中枢神经系统中，保守的引导信号Slit的作用是防止轴突异常穿过中线。狭缝排斥是由保守的Roundabout（Robo）受体家族介导的。本申请的主要目的是：1）界定和表征响应于Slit配体的Robo介导的轴突排斥所必需和足够的Robo受体胞质结构域的区域，2）评估SH 3-SH 2衔接蛋白Dreadlocks（Dock）和相关蛋白在促进Robo排斥中的潜在作用，3）使用果蝇遗传筛选鉴定参与Slit和Robo信号传导的其他组分。一个完善的果蝇转基因方法将被用来确定哪些区域的机器人的细胞质结构域所需的排斥。经典的遗传和生物化学技术，包括遗传相互作用测试，突变分析，酵母双杂交和免疫共沉淀将被用来研究Dock和相关蛋白在Robo排斥中的潜在作用。此外，将使用已建立的细胞培养技术来确定Robo受体的Slit刺激是否调节Dock/Robo相互作用。为了鉴定参与Robo功能的其他分子，将进行致敏遗传筛选。所提出的遗传筛选策略已经在小规模上成功地用于识别可能在Robo排斥过程中发挥重要作用的基因。破译介导Robo排斥性轴突引导的机制将对神经系统在发育过程中如何正确连接提供重要的见解，并可能对神经再生产生影响。