REPULSIVE CUES AND THE DEVELOPING VISUAL SYSTEM

视网膜定位图的准确形成对我们的视力至关重要。在这个过程中，视网膜轴突被引导形成大脑中连接的精确地形图，从而使视觉空间被感知。这些引导机制的分子基础尚不清楚。这项建议的长期目标是了解排斥性线索如何引导轴突形成视网膜定位图。最近发现了多种排斥信号引导轴突形成视网膜异位图。最近发现的各种排斥信号改变了我们对轴突如何重新引导的理解。现在是研究这些线索在体内如何发挥作用的时候了。排斥性引导分子（RGM）和ephrin-A5在低等脊椎动物视网膜顶突形成过程中呈高后低前梯度表达。目前的进展已经提出了这两种线索如何在体内作用以形成视网膜顶图的具体假设：1)RGM引导视网膜顶轴突的初始生长。2) Ephrin-A5阻止视网膜轴突离开顶盖，并调节轴突初始生长完成后的外侧分支。拟议中的实验将检验这些假设。在体内观察干扰RGM和ephrin-A5对视网膜直肠项目的影响。小鸡和斑马鱼的胚胎将被研究；它们都有很好的特点，每一个都有不同的优势来实现我们的目标。在视网膜顶图形成过程中，RGM和ephrin-A5的急性和局部失活将通过发色团辅助激光失活（CALI）在最初的轴突生长和后来的侧分支过程中实现。重组逆转录病毒感染可引起鸡耳盖内ephrin-A5的慢性错表达。通过高分辨率轴突追踪和实时成像来评估视网膜顶项目的变化。这两种方法的结合将提供强有力的互补信息，以检验所提出的假设。由于RGM和ephrin-A5可能用于轴突的引导和地形秩序的形成，因此本实验具有重要的临床意义。

Accurate formation of the retinotopic map is critical for our ability to see. During this process, retinal axons are guided to form a precise topographic map of connections in the brain that allow visual space to be perceived. The molecular basis of these guidance mechanisms is not yet known. The long term objective of this proposal is to understand how repulsive cues act to guide axons to form the retinotopic map. The recent discovery of a variety of repulsive cues act to guide axons to form the retinotopic map. The recent discovery of a variety of repulsive cues has modified our understanding of how axons re guided. It is timely to address how these cues function in vivo. Repulsive guidance molecule (RGM) and ephrin-A5 are expressed in high posterior to low anterior gradients in the tectum of lower vertebrates during the formation of the retinotectal projection. Current advances have suggested specific hypotheses of how these two cues act in vivo to shape the retinotectal map: 1) RGM guides initial retinotectal axon outgrowth. 2) Ephrin-A5 prevents retinal axons from exiting the tectum and modulates lateral branching after initial axon outgrowth is completed. The proposed experiments will test these hypotheses. The changes in retinotectal projects caused by perturbing RGM and ephrin-A5 in vivo will be observed. Chick and zebrafish embryos will be studied; they are well characterized and each offers distinct advantages to achieve our goals. Acute and local inactivation of RGM and ephrin-A5 during retinotectal map formation will be achieved by chromophore-assisted laser inactivation (CALI) during initial axon outgrowth and later during lateral branching. Infection with recombinant retrovirus will generate chronic misexpression of ephrin-A5 in the chick tectum. The changes in retinotectal projects will be assessed by high resolution axon tracing and live imaging. The combination of these two approaches will provide strong complementary information that will test the proposed hypotheses. As RGM and ephrin-A5 are likely used for axon guidance and formation of topographic order, the proposed experiments are of significant clinical relevance.