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

描述（由申请人提供）：动物的行为复杂性和可塑性受到有利于增加中枢神经系统神经系统复杂性的进化趋势的促进，并且需要精确发育控制CMS内的轴突和树突位置。轴突导向受体的Roundabout（Robo）家族在响应于分泌的排斥性Slit而调节中线穿越中起着古老的作用。在果蝇中，Robo 2和RoboS指定了中枢神经系统中轴突的中外侧位置，这是Robo没有的一种新功能。该建议旨在了解果蝇中枢神经系统中Robo受体控制横向位置的分子和功能基础以及进化起源。具体目标是：1.确定Robo和Robo 2在侧向定位中差异活性的分子基础。Robo 2轴突导向受体能够决定果蝇CNS内轴突的中外侧位置，而密切相关的Robo受体则不能。为了定义区分Robo和Robo 2在侧向定位中的活性的最小序列元件，我将测定一系列嵌合Robo受体在体内支配侧向位置的能力。2.确定Robo受体控制侧卧位的生物学基础。为了检验不同的Slit亲和力是Robo和Robo 2在侧向定位中不同活动的原因这一假设，我将（1）通过检查Slit的膜系形式对侧向定位的影响来检验Slit的分级分布是Robo受体控制侧向定位所必需的这一假设;（2）使用定点突变来测试破坏的Slit结合对Robo 2和Robo 3的侧向移位活性的影响;以及（3）通过比较野生型和嵌合Robo受体的Slit亲和力，评估Robo受体横向定位活性是否与Slit亲和力相关，表面等离子体共振3.研究Robo受体控制侧位的进化起源。第三个目标将测试的假设，即控制的横向位置是一个新的功能获得的机器人受体在昆虫的进化。Robo 2和Robo 3调节果蝇中枢神经系统轴突的侧向位置。值得注意的是，Robo 2和Robo 3的直系同源物似乎仅限于昆虫，这增加了控制侧向位置是昆虫进化过程中首次出现的新活动的可能性。为了深入了解Robo受体控制侧向位置的进化起源，我将：（1）分析节肢动物和线虫Robo受体在果蝇中保守的侧向定位活性，（2）研究Robo直系同源物在粉甲虫Tribolium中的功能，Tribolium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.