Robo receptor control of lateral position of axons in the Drosophila CNS

描述（由申请人提供）：动物行为的复杂性和可塑性是由中枢神经系统复杂性增加的进化趋势促进的，这需要在中枢神经系统中对轴突和树突位置进行精确的发育控制。轴突引导受体的Roundabout （Robo）家族在调节中线交叉中发挥着古老的作用，以响应分泌的排斥物Slit。在果蝇中，Robo2和RoboS指定中枢神经系统轴突的中外侧位置，这是Robo不具有的新功能。本研究旨在了解机器人受体控制果蝇中枢神经系统侧位的分子和功能基础及其进化起源。具体目标是：1。确定Robo和Robo2在横向定位中的差异活性的分子基础。在果蝇中枢神经系统中，Robo2轴突引导受体能够指示轴突的中外侧位置，而与其密切相关的Robo受体则不能。为了确定区分Robo和Robo2在横向定位中的活性的最小序列元素，我将分析一系列嵌合Robo受体在体内决定横向位置的能力。2. 确定机器人受体控制侧向位置的生物学基础。为了验证不同的Slit亲和力是Robo和Robo2在侧位中的不同活动的原因这一假设，我将(1)通过检查膜系住形式的Slit对侧位的影响来验证机器人受体控制侧位所需的Slit分级分布的假设；(2)利用位点定向突变检测断裂的Slit结合对Robo2和Robo3横向移动活性的影响；(3)利用表面等离子体共振技术比较野生型和嵌合型机器人受体的狭缝亲和力，评估机器人受体横向定位活性是否与狭缝亲和力相关。3. 研究机器人受体控制侧向位置的进化起源。第三个目标将测试一个假设，即控制侧向位置是机器人受体在昆虫进化过程中获得的一种新功能。Robo2和Robo3调节果蝇中枢神经系统轴突的侧向位置。值得注意的是，Robo2和Robo3同源基因似乎仅限于昆虫，这提出了一种可能性，即控制侧向位置是昆虫进化过程中首次出现的一种新活动。为了深入了解Robo受体控制侧位的进化起源，我将：(1)分析节肢动物和线虫的Robo受体在果蝇中保守的侧位活性，(2)研究Robo同源物在面粉甲虫Tribolium中的功能，这是一种保留原始Robo受体的昆虫。

DESCRIPTION (provided by applicant): The behavioral sophistication and plasticity of animals has been facilitated by evolutionary trends favoring increased central nervous system nervous system complexity and requires precise developmental control of axon and dendrite position within the CMS. The Roundabout (Robo) family of axon guidance receptors performs an ancient role in regulating midline crossing in response to the secreted repellant Slit. In Drosophila, Robo2 and RoboS specify mediolateral position of axons in the CNS, a novel function not shared by Robo. This proposal seeks to understand the molecular and functional bases and evolutionary origins of Robo receptor control of lateral position in the Drosophila CNS. The specific aims are to: 1. Identify the molecular basis for differential activity of Robo and Robo2 in lateral positioning. The Robo2 axon guidance receptor is able to dictate mediolateral position of axons within the Drosophila CNS, while the closely related Robo receptor cannot. To define the minimal sequence elements distinguishing Robo and Robo2 activity in lateral positioning, I will assay a comprehensive series of chimeric Robo receptors for their ability to dictate lateral position in vivo. 2. Determine the biological basis for Robo receptor control of lateral position. To test the hypothesis that differential Slit affinity is responsible for the distinct activities of Robo and Robo2 in lateral positioning, I will (1) test the assumption that a graded distribution of Slit is required for Robo receptor control of lateral position by examining the effects of a membrane-tethered form of Slit on lateral position; (2) use sitedirected mutations to test the effects of disrupted Slit binding on lateral shifting activity of Robo2 and Robo3; and (3) assess whether Robo receptor lateral positioning activity correlates with Slit affinity by comparing the Slit affinities of wild type and chimeric Robo receptors using surface plasmon resonance. 3. Examine the evolutionary origins of Robo receptor control of lateral position. The third aim will test the hypothesis that control of lateral position is a novel function acquired by Robo receptors during the evolution of insects. Robo2 and Robo3 regulate lateral position of CNS axons in Drosophila. Notably, Robo2 and Robo3 orthologs appear restricted to insects, raising the possibility that control of lateral position is a novel activity that first appeared during insect evolution. To gain insight into the evolutionary origins of Robo receptor control of lateral position, I will: (1) assay arthropod and nematode Robo receptors for conserved lateral positioning activity in Drosophila, and (2) investigate the function of Robo orthologs in the flour beetle Tribolium, an insect that retains a primitive set of Robo receptors.