In vivo analysis of the developing vertebrate retina

描述(申请人提供)：视觉依赖于神经视网膜对图像的准确处理。视网膜的发育涉及一系列事件，这些事件导致视网膜电路的正确连接。建立视网膜回路的第一步是产生每一类主要的视网膜神经元，随后在细胞组件之间建立适当的连接。我们对管理发展中这些主要步骤的机制的了解还不完整。特别是，还不可能确定在体内视网膜神经元整个发育过程中动态塑造视网膜神经元的数量、位置和连接模式所涉及的细胞过程。这一知识很重要，因为当我们实时观察发生在活体动物身上的发育事件时，更容易确定发育过程中细胞功能中断的后果。现在有可能跟踪视网膜神经元的发育，从它们发生的时候到它们在快速发育和基本上透明的斑马鱼胚胎中形成回路的时候。在这项提议中，我们将专注于外视网膜的一个主要细胞成分，水平细胞，并确定：(目标1)非传统的神经发生模式如何产生合适数量的水平细胞，以及子细胞的迁移如何导致其镶嵌状网络的形成，以及(目标2)确定涉及神经元和Muller胶质细胞的细胞-细胞相互作用对于建立水平细胞与视锥感光细胞的连接是重要的。我们的方法是使用荧光标记的视网膜神经元的延时多光子成像来跟踪它们在几个小时到几天的时间内的行为，以及使用钛：蓝宝石红外激光来解开细胞与细胞之间的相互作用的靶向细胞消融方法。总之，拟议的体内研究将增加我们对建立正常视网膜网络所需的细胞行为和相互作用的了解，并为未来视网膜疾病和损伤的研究提供基础。

DESCRIPTION (provided by applicant): Vision depends on the accurate processing of the image by the neural retina. The development of the retina involves a series of events that result in the proper wiring of its circuitry. The first step in building retinal circuits is the generation of each major class of retinal neuron and subsequently, establishing the appropriate connectivity between the cell components. Our understanding of the mechanisms that regulate these major steps in development is not yet complete. In particular, it has not been possible to determine the cellular processes involved in dynamically shaping the number, location and wiring patterns of retinal neurons throughout their entire time-course of development in vivo. This knowledge is important as the consequences of disrupting cellular function during development is more readily identified when we have a real-time view of the developmental events as they occur in the live animal. It is now possible to follow the development of retinal neurons from the time of their genesis to when they form circuits in the rapidly developing and largely transparent zebrafish embryo. In this proposal, we will focus on a major cell component of the outer retina, the horizontal cells, and determine: (Aim 1) how an unconventional mode of neurogenesis produces the right number of horizontal cells, and how migration of the daughter cells lead to the formation of their mosaic-like network, and (Aim 2) determine what cell-cell interactions, involving neurons and Muller glial cells, are important for establishing horizontal cell connectivity with cone photoreceptors. Our approach is to use time-lapse multiphoton imaging of fluorescently labeled retinal neurons to track their behavior over hours to days, and a targeted cell ablation approach using the titanium:sapphire infrared laser to unravel cell-cell interactions. Together, the proposed in vivo studies will increase our understanding of the cellular behaviors and interactions necessary for establishing normal retinal networks and provide the foundation for future investigations on retinal disease and injury.