Mechanisms of slit-roundabout mediated axon repulsion

描述(申请人提供)：如何在发育中的神经系统轴突成功地导航到它们的正确目标是神经生物学的一个基本问题。轴突受吸引力和排斥性线索的引导，后者是进化保守蛋白家族的成员。我们建议研究在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(CNS)的中线为解决这些问题提供了理想的系统。就像它的结构类似于脊椎动物的底板一样，苍蝇中线是许多类别导航轴突的中间目标，这些轴突必须决定是否穿越。在果蝇中枢神经系统中，保守的引导线索缝隙及其神经元受体环形交叉路口(ROBO)在形成中线轴突连接的过程中起着多重作用，主要作为轴突排斥剂。这一建议的主要目的是：1)验证蛋白质降解处理直接促进Robo排斥和信号终止的假说；2)研究已识别的Robo信号成分Abelson、Seven less的儿子和CrossGAP如何共同作用于协调Robo下游的信号；3)剖析三个果蝇Robo家族成员不同排斥功能的机制。我们已经在一种特定的金属蛋白酶和Robo排斥力的调节之间建立了遗传和直接的生化联系，并拥有必要的遗传、生化和细胞生物学分析来研究蛋白质分解处理影响排斥力的机制。我们实验室建立的类似方法将使我们能够继续研究ROBO下游的信号分子是如何协调调节轴突排斥的。一种以前成功的嵌合受体方法，即Robo1、Robo2和Robo3受体的不同部分相互交换，然后在转基因果蝇中进行功能分析，将用于了解不同的Robo受体如何导致不同的排斥事件。总之，这些研究有望丰富我们对Sit-Robo信号在正常发育过程中的理解，并可能为从神经系统发育障碍到脊髓损伤和中风等各种人类健康问题提供新的治疗靶点。

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.