Mechanisms of Roundabout Repulsive Axon Guidance

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正确的目标是神经生物学中的一个基本问题。轴突受到吸引和排斥信号的引导，这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起作用的信号机制。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。就像它的结构类似物——脊椎动物的底板一样，苍蝇的中线是许多种类的导航轴突的中间目标，它们必须决定是否越过中线。在果蝇中枢神经系统中，保守的引导信号Slit起着防止轴突异常越过中线的作用。狭缝排斥是由Roundabout （Robo）受体保守家族介导的。该应用程序的主要目的是：1)划分和表征Robo受体细胞质结构域的区域，这些区域对于响应Slit配体的Robo介导的轴突排斥是必要的和充分的，2)评估SH3-SH2接头蛋白Dreadlocks （Dock）和相关蛋白在促进Robo排斥中的潜在作用，3)使用果蝇遗传筛选识别参与Slit和Robo信号传导的其他成分。一种成熟的果蝇转基因方法将用于确定Robo细胞质域的哪些区域需要排斥力。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，酵母双杂交和共免疫沉淀将被用来研究Dock和相关蛋白在机器人排斥中的潜在作用。此外，已建立的细胞培养技术将用于解决Robo受体的Slit刺激是否调节Dock/Robo相互作用。为了确定参与Robo功能的其他分子，将进行增敏基因筛选。提出的基因筛选策略已经成功地在小范围内用于鉴定可能在机器人排斥过程中发挥重要作用的基因。破译机器人排斥性轴突引导的调节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。

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.