RETINAL AXON PATHFINDING MUTANTS IN ZEBRAFISH

当轴突在发育中的神经系统中找到它们的目标时，它们受到一个由积极和消极信号组成的复杂环境的指导，这些信号由参与寻找路径的生长分子的机械感知和转导，但其中许多仍然未知。斑马鱼的大规模基因筛查已经分离出大约25个基因，这些基因是视网膜轴突投射到顶盖所必需的。其中，烟灰缸突变体表现出最严重和特殊的寻路表型。烟灰缸中的视网膜轴突似乎忽略了许多引导信号：它们反复越过中线，向前脑投射，向后投射到后脑，以及向正常目标投射。没有普遍的大脑模式错乱，早期的轴突支架没有受到干扰。胚胎眼移植表明，烟灰缸的作用是眼睛自主的。因此，烟灰缸基因产物很可能是视网膜轴突所需的轴突寻路分子。我们已经在高分辨率下绘制了烟灰缸的遗传图谱，并发现它与新的斑马鱼环形交叉路口同源物zRobo1A非常接近。这里提出的实验旨在了解烟灰缸在视网膜轴突寻路过程中的作用，并阐明该基因的分子性质。首先，将在固定胚胎中标记轴突，并在活体胚胎中观察到正在生长的轴突，以确定烟灰缸轴突何时何地走错了路。细胞类型特定的标记也将被用来检查烟灰缸是否改变视网膜神经节细胞的命运。其次，在寻路过程中，需要在不同的选择点放置单细胞烟灰缸。第三，通常表现出各种各样行为的六组特定的非视网膜轴突将被标记，以测试它们是否在烟灰缸中受到影响。第四，将克隆烟灰缸基因。我们将首先测试它是否是zRobo1A中的缺陷。如果没有，我们已经开始了一次基因组行走，并将对该基因进行定位克隆。综上所述，该项目将促进我们对野生型视网膜轴突如何发现路径的理解；阐明单个斑马鱼视网膜轴突的行为在哪里、何时以及如何需要烟灰缸的功能，为未来对所有斑马鱼视网膜顶盖突变体的分析奠定基础，这将为视觉系统发育的遗传控制提供重要的见解。

As axons pathfind to their targets in the developing nervous system, they are guided by a complex environment of positive and negative cues, which are sensed and transduced by the machinery of the growth molecules involved in pathfinding, but many remain unknown. A large- scale genetic screen in zebrafish has isolated approximately -25 genes required for the projection of retinal axons to the tectum. Of these, the ashtray mutant shows the most severe and specific pathfinding phenotype. Retinal axons in ashtray seem to ignore many guidance cues: they cross the midline repeatedly, and project anteriorly to forebrain and posteriorly to hindbrain as well as to their normal target. There is no general derangement of brain patterning, and the early axon scaffold is undisturbed. Embryonic eye transplants show that ashtray acts eye- autonomously. Thus, the ashtray gene product is likely to be an axon pathfinding molecule required in retinal axons. We have genetically mapped ashtray at high resolution, and found it maps very close to a novel zebrafish roundabout homolog, zRobo1A. The experiments proposed here are designed to understand how ashtray acts during retinal axon pathfinding and to elucidate the molecular nature of the gene. First, axons will be labeled in fixed embryos and growing axons observed in live embryos to determine when and where ashtray axons misroute. Cell type-specific markers will also be used to check whether ashtray acts to change retinal ganglion cell fate. Second, single- cell ashtray is required at distinct choice points during pathfinding. Third, six specific sets of non-retinal axons that normally exhibit a wide variety of behaviors will be labeled to test whether they are affected in ashtray. Fourth, the ashtray gene will be cloned. We will first test whether it is a defect in zRobo1A. If not, we have begun a genomic walk and will clone the gene positionally. In summary, this project will advance our understanding of how wildtype retinal axons pathfind; illuminate where, when, and how the behavior of single zebrafish retinal axons requires the function of ashtray, be established for the future analysis of all the zebrafish retinotectal mutants, which promise to yield important insights into the genetic control of visual system development.