Genetic interaction screen to analyze Robo signaling

描述（由申请人提供）：Slit-Robo信号传导是主要轴突引导信号传导途径之一，其作用是塑造神经系统的回路。最近的工作表明Slit-Robo信号在模型生物的视觉系统和脊髓以及人类皮质脊髓束的发育中起着关键作用。然而，只有少数效应基因参与了这一信号传导途径，这些效应基因中没有一个在脊椎动物中进行了遗传学研究。此外，还不清楚Robo受体的哪些结构域对其体内功能至关重要。该提案将使用正向遗传筛选搜索与斑马鱼误入歧途（robo 2）突变体的相互作用（非互补）。通过筛选标记有GFP转基因的单个F1幼虫，我们将能够在3年内筛选75，000个基因组。对5700个基因组的试验性筛选已经产生了12个假定的突变体。该筛选将（1）鉴定Slit-Robo通路中基因的突变体，然后（2）对这些突变体进行遗传作图，以及（3）进行初步的表型评估，分析多个轴突通路。我们预计会发现3类突变体：（I）robo 2的新突变等位基因，我们将对其进行鉴定和测序;（II）温度敏感的robo 2等位基因，我们将对其进行鉴定和表征;（III）与robo 2表现出强烈遗传相互作用的基因突变，如狭缝配体或下游效应子，我们将对其进行遗传图谱和表型分析。斑马鱼系统为研究脊椎动物轴突引导提供了独特的优势，包括在固定组织中和使用延时电影追踪单个轴突轨迹的能力;在脊椎动物中产生温度敏感等位基因的能力;以及进行正向遗传筛选的能力。在该筛选中分离的robo 2等位基因对于理解Robo 2功能的时间（温度敏感性等位基因）和Robo 2信号传导所需的蛋白质结构域（错义或无义等位基因）将是有价值的。分离的相互作用基因将定义Robo 2信号通路中的一组基因，并使我们能够在体内研究它们的功能。

DESCRIPTION (provided by applicant): Slit-Robo signaling is 1 of the main axon guidance signaling pathways that act to shape the circuitry of the nervous system. Recent work shows the critical role of Slit-Robo signaling in the development of the visual system and spinal cord in model organisms, and the corticospinal tract in humans. However, only a few effector genes have been implicated in this signaling pathway, and none of these effector genes have been studied genetically in vertebrates. In addition, it is poorly understood which structural domains of Robo receptors are critical for their in vivo function. This proposal will use a forward genetic screen searching for interactions (noncomplementation) with the zebrafish astray (robo2) mutant. By screening individual F1 larvae labeled with a GFP transgene, we will be able to screen 75,000 genomes in 3 years. A pilot screen of 5700 genomes has already yielded 12 putative mutants. This screen will (1) identify mutants in genes in the Slit-Robo pathway, then (2) genetically map these mutants and (3) conduct a preliminary phenotypic assessment analyzing multiple axon pathways. We expect to find 3 classes of mutants: (I) new mutant alleles of robo2, which we will identify and sequence; (II) temperature-sensitive robo2 alleles, which we will identify and characterize; and (III) mutations in genes that show strong genetic interactions with robo2, such as slit ligands or downstream effectors, which we will genetically map and analyze phenotypically. The zebrafish system offers unique advantages for studying vertebrate axon guidance, including the ability to trace the trajectories of single axons both in fixed tissue and using timelapse movies; the ability to generate temperature-sensitive alleles in a vertebrate; and the ability to carry out forward-genetic screens. The robo2 alleles isolated in this screen will be valuable for understanding the timing of Robo2 function (temperature-sensitive alleles) and the protein domains necessary for Robo2 signaling (missense or nonsense alleles). The interacting genes isolated will define a set of genes in the Robo2 signaling pathway, and allow us to study their function in vivo.