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，它们受到积极和消极线索的复杂环境的引导，这些线索被参与寻路的生长分子的机制感知和转导，但许多仍然未知。在斑马鱼中进行的大规模遗传筛选已经分离出视网膜轴突投射到顶盖所需的约25个基因。在这些突变体中，突变体表现出最严重和最特异的寻路表型。视网膜神经轴突似乎忽略了许多引导线索：它们反复穿过中线，向前投射到前脑，向后投射到后脑以及它们的正常目标。大脑模式没有普遍的紊乱，早期的轴突支架也没有受到干扰。胚胎眼移植表明，胚胎眼自主活动。因此，该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基因图谱，发现它与一种新的斑马鱼迂回同源物zRobo 1A非常接近。这里提出的实验旨在了解在视网膜轴突寻路过程中，基因是如何起作用的，并阐明该基因的分子性质。首先，将在固定的胚胎中标记轴突，并在活胚胎中观察生长的轴突，以确定轴突何时以及在何处错误路由。细胞类型特异性标记物也将用于检查视网膜神经节细胞是否改变视网膜神经节细胞的命运。第二，在寻路过程中，需要在不同的选择点上进行单小区搜索.第三，通常表现出各种行为的六组特定的非视网膜轴突将被标记，以测试它们是否受到视网膜病变的影响。第四，将克隆p53基因。我们将首先测试它是否是zRobo 1A中的缺陷。如果没有，我们已经开始了基因组步行，并将克隆基因的位置。总之，该项目将推进我们对野生型视网膜轴突如何寻路的理解;阐明单个斑马鱼视网膜轴突的行为在何处、何时以及如何需要神经元的功能，为未来分析所有斑马鱼视网膜顶盖突变体奠定基础，这有望对视觉系统发育的遗传控制产生重要的见解。

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.