Developing an expression screen for axonal guidance cues

描述(申请人提供)：发育中的神经系统内的神经元以令人难以置信的精确度和特异性相互连接。神经过程被环境中的线索引导到正确的目标，这些线索要么将它们吸引到适当的路径上，要么阻止它们在不适当的路径上延伸。在过去的十年里，已经确定了几个充当指导信号的信号分子家族。通常，这些线索是通过遗传筛选，通过体外生物测定与生化纯化配对，或通过对选定的候选分子进行表征来识别的。虽然每种方法都有强大的优势，但它们都不构成快速、系统、高通量的指导活动屏幕。这项建议的目标是开发一种实用的轴突排斥剂的体外表达筛选。其目的将是在相对较短的时间内利用生物测定法筛选大量重组蛋白质库中的驱避活性。通过阳性选择标准识别活性分子，这种方法将补充更多依赖功能表型丧失来检测新活动的传统正向遗传筛查。在屏幕上发现的候选线索随后可以通过反向遗传和基因敲除方法来测试指导活动。新的轴突引导线索的发现将促进我们对神经元相互连接形成和可塑性的机制的理解，并最终帮助我们改善受损神经系统中的轴突再生。

DESCRIPTION (provided by applicant): Neurons within the developing nervous system interconnect with incredible precision and specificity. Neuronal processes are guided to their correct targets by cues in the environment that either attract them onto appropriate pathways or discourage them from extending on inappropriate pathways. Several families of signaling molecules that act as guidance cues have been identified over the past decade. Typically, these cues have been identified using genetic screens, through the pairing of in vitro bioassays with biochemical purifications, or by the characterization of selected candidate molecules. Although each approach has powerful advantages, none of them constitute a rapid, systematic, high throughput screen for guidance activities. The goal of this proposal is to develop a practical in vitro expression screen for axonal repellents. The aim would be to screen a large library of recombinant proteins in a relatively short period of time using a bioassay for repellent activity. By identifying active molecules through a positive selection criteria, this approach will complement more traditional forward genetic screens that rely on loss of function phenotypes to detect new activities. Candidate cues discovered in the screen could subsequently be tested for guidance activity by reverse genetic and knockdown approaches. The discovery of novel axonal guidance cues will advance our understanding of the mechanisms underlying the formation and plasticity of neuronal interconnections, and may ultimately help us improve axon regeneration in the damaged nervous system.