Longitudinal axon guidance by long range cues in the embryonic brain

描述(申请人提供)：本提案的总体目标是确定在胚胎脊椎动物大脑中建立第一条纵向轴突通路的分子机制。纵向轴突构成了中枢神经系统的主要通信通道，在大脑区域和脊髓之间传输所有信号。在大脑发育的早期，先锋轴突通过选择特定的路径和准确地长出很长的距离，建立了一个最初的简单的纵向束阵列。我们的初步结果证实Sit/Robo信号是纵向引导的主要决定因素，特别是Slit1和Slit2分泌的信号蛋白及其Robo1和Robo2受体。我们发现，在携带Sit或Robo突变的小鼠胚胎中，由于广泛的背腹漂移和其他错误，所有先锋纵向束都被严重破坏。该提案的总体目标是定义Sit/Robo信令如何控制纵向制导。该实验系统是小鼠和小鸡早期胚胎中简单有序的先锋轴突阵列，使用了一系列完整胚胎和培养的轴突的功能分析。目的1.确定分泌性缝隙线索在引导纵轴突中的作用。我们对狭缝突变胚胎的初步分析发现，Slit1和Slit2提供了关键线索。我们假设，狭缝的功能要么是直接的远程指导信号，以定向和定位轴突，要么是轴突对其他指导线索做出反应的强制性许可信号。为了区分这些机制，我们将研究Sit突变小鼠的轴突投射，在外植体培养中用Sits挑战轴突，并在体内错误表达Slits。目的2.检测缝隙受体Robo家族在纵向引导中的作用。我们的证据表明，Robo1和Robo2的突变体轴突不能沿着精确的路径导航。这些结果表明，Robo1和Robo2参与了缝隙信号的传递。我们认为，机器人通过调节特定的异构体组合或通过不同的表达水平来设定不同纵向束的位置。为了测试这些Robo机制，我们将研究Robo突变小鼠的轴突，并在特定的轴突群体中错误表达机器人。这项提议的主要目标是定义在胚胎大脑发育期间神经连接是如何形成的。对发育过程中神经元生长机制的新见解将为了解大脑中的出生缺陷以及如何在创伤或疾病后刺激和引导神经系统的功能再生提供重要的见解。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to define the molecular mechanisms that establish the first longitudinal axon pathways in the embryonic vertebrate brain. Longitudinal axons form the main communication highways in the CNS, transmitting all signals between brain regions and spinal cord. During early brain development, pioneer axons establish an initial simple array of longitudinal tracts by choosing specific pathways and accurately growing long distances. Our preliminary results have identified Slit/Robo signaling as a major determinant of longitudinal guidance, specifically the Slit1 and Slit2 secreted signaling proteins and their Robo1 and Robo2 receptors. We show that all pioneer longitudinal tracts are severely disrupted in mouse embryos carrying Slit or Robo mutations, due to extensive dorsal-ventral wandering and other errors. The overall goal of the proposal is to define how Slit/Robo signaling controls longitudinal guidance. The experimental system is the simple organized array of pioneer axons in early mouse and chick embryos, using a range of functional assays in intact embryos and with cultured axons. Aim 1. Determine the function of secreted Slit cues in guiding longitudinal axons. Our initial analysis of Slit mutant embryos identifies Slit1 and 2 as providing critical cues. We hypothesize that Slits function either as direct long-range instructive signals to orient and position axons, or as obligatory permissive signals for axons to respond to other guidance cues. To distinguish between these mechanisms, we will study axon projections in Slit mutant mice, challenge axons with Slits in explant cultures, and mis-express Slits in vivo. Aim 2. Test the function of the Robo family of Slit receptors in longitudinal guidance. Our evidence shows that axons mutant for Robo1 and 2 are unable to navigate along precise pathways. These results indicate that Robo1 and 2 mediate Slit signaling. We propose that Robos set the position of different longitudinal tracts via modulation of either specific combinations of isoforms or by different expression levels. To test these Robo mechanisms, we will study axons in Robo mutant mice, and mis-express Robos in specific axon populations. The main goal of this proposal is to define how neural wiring can form during embryonic brain development. New insights into neuron growth mechanisms during development will provide significant insights into birth defects in the brain, as well as how functional regeneration of the nervous system could be stimulated and guided following trauma or disease.