Mechanisms of slit-roundabout mediated axon repulsion

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。轴突受到吸引性和排斥性线索的引导，这些线索是进化保守蛋白质家族的成员。我们建议研究在排斥性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（CNS）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，苍蝇中线是一个中间目标，许多类别的导航轴突，必须决定是否跨越。在果蝇中枢神经系统中，保守的引导因子Slit及其神经元受体Roundarium（Robo）在中线的轴突连接模式中发挥多种作用，主要作为轴突排斥剂。该提议的主要目的是1）测试蛋白水解加工直接有助于Robo排斥和信号终止的假设，2）研究所鉴定的Robo信号传导组分Abelson、Son of Sevenless和CrossGAP如何一起起作用以协调Robo下游的信号传导，以及3）剖析三个果蝇Robo家族成员的不同排斥功能的机制。我们已经建立了一个特定的金属蛋白酶和调节机器人排斥之间的遗传和直接的生化联系，并有必要的遗传，生化和细胞生物学试验，以研究蛋白水解加工影响排斥的机制。我们实验室中建立的类似方法将允许我们继续研究Robo下游的信号分子如何协调调节轴突排斥。一个以前成功的嵌合受体的方法，其中不同部分的Robo 1，Robo 2和Robo 3受体相互交换，然后在转基因果蝇的功能进行测定，将用于了解不同的Robo受体如何导致不同的排斥事件。这些研究有望丰富我们对正常发育过程中Slit-Robo信号传导的理解，并可能为各种人类健康问题提供新的治疗靶点，从神经系统发育障碍到脊髓损伤和中风。

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 evolutionary conserved protein families. We propose to study the signaling mechanisms that function during repulsive axon guidance. The midline of the Drosophila embryonic central nervous system (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. In the Drosophila CNS, the conserved guidance cue Slit and its neuronal receptors the Roundabouts (Robo), play multiple roles in patterning axonal connections at the midline, acting primarily as axonal repellants. The primary aims of this proposal are to 1) test the hypothesis that proteolytic processing directly contributes to Robo repulsion and to signal termination, 2) to investigate how the identified Robo signaling components Abelson, Son of Sevenless and CrossGAP function together to coordinate signaling downstream of Robo and 3) to dissect the mechanisms underlying the distinct repulsive functions of the three Drosophila Robo family members. We have established genetic and direct biochemical links between a specific metalloprotease and regulation of Robo repulsion and have the necessary genetic, biochemical and cell biological assays to investigate the mechanism by which proteolytic processing influences repulsion. Similar kinds of approaches well established in our laboratory will allow for our continued investigation of how signaling molecules that function downstream of Robo coordinately regulate axon repulsion. A previously successful chimeric receptor approach where different portions of the Robo1, Robo2 and Robo3 receptors are exchanged with each other and then assayed for function in transgenic flies will be used to understand how different Robo receptors lead to distinct repulsive events. Together these studies promise to enrich our understanding of Slit-Robo signaling during normal development and may provide new therapeutic targets for diverse human health problems, ranging from developmental disorders of the nervous system to spinal cord injury and stroke.