Molecular control of neuronal position during retinal development

描述（由申请人提供）：视网膜神经元均匀分布在整个视网膜上，这种模式称为马赛克。在发育过程中，通过接触介导的排斥作用，同一类型的神经元之间会产生均匀的间隔。允许同型神经元识别彼此并因此避免彼此的分子机制尚不清楚。这里的目标是了解同型识别信号是如何启动，接收和翻译成信号，调整细胞位置。中心假设是跨膜蛋白MEGF 10和MEGF 11构成受体-配体系统，其：1）通过在细胞-细胞接触时结合赋予同型识别;和2）触发产生相互细胞-细胞排斥的细胞内信号传导途径，从而产生镶嵌间隔。这项工作的基本原理是，它将通过揭示第一组识别分子（即MEGF 10/11）如何定位神经元，提供马赛克形成的第一个机制解释。由此揭示的机制有望为视网膜神经元如何识别和避免彼此提供一般性的见解，为理解马赛克以及影响视觉功能的其他神经元图案化事件开辟道路。为此，提出了以下具体目标：1）确定启动识别信号的细胞间分子相互作用。初步数据表明，MEGF 10和11介导这些相互作用结合到自己和作为受体和配体。为了检验这一假设，将通过生物化学方法测定每种分子的结合特异性，并使用Megf 10和Megf 11突变小鼠在体内确认其受体/配体功能。2)确定识别信号在细胞中是如何报告的。初步数据显示，MEGF 10是抑制识别信号所必需的。使用生物化学和体内遗传学实验，这个目的将测试的假设，ITAM磷酸酪氨酸基序在MEGF 10细胞内域介导这些识别信号。3)确定识别信号如何改变细胞行为以产生马赛克间距。这一目标将检验认知改变树突行为的假设。具体来说，有人提出，识别会引起同型树突排斥，通过这种排斥，神经元会划出独特的区域，使它们能够避开邻居。在Megf 10; Megf 11双突变小鼠中，识别将被遗传性废除，树突排斥将通过视网膜外植体的活体成像进行评估。总之，这三个目标中提出的实验有望首次揭示：1）当同一类型的细胞接触时，细胞表面分子相互结合; 2）这些分子如何触发排斥，以确定神经元的位置。这种方法是创新的，因为它部署了新的工具和方法，使第一个分子研究的同型识别镶嵌图案。这一贡献将是重要的，因为决定神经元精确位置的分子事件对视网膜和整个神经系统的回路功能都很重要。

DESCRIPTION (provided by applicant): Retinal neurons are evenly spaced across the retina, a pattern known as a mosaic. Even spacing arises during development through contact-mediated repulsion that occurs specifically between neurons of the same type. The molecular mechanisms that allow homotypic neurons to recognize each other, and consequently to avoid each other, are not known. The objective here is to learn how homotypic recognition signals are initiated, received, and translated into signals that adjust cell position. The central hypothesis s that the transmembrane proteins MEGF10 and MEGF11 constitute a receptor-ligand system that: 1) confers homotypic recognition through binding upon cell-cell contact; and 2) triggers intracellular signaling pathways that produce mutual cell-cell repulsion, thereby creating mosaic spacing. The rationale for this work is that it will provide the first mechanistic explanation of mosaic formation, by revealing how the first identified set of recognition molecules (i.e. MEGF10/11) positions neurons. The mechanisms thus revealed are expected to provide general insight into how retinal neurons recognize and avoid each other, opening the way to understanding both mosaics as well as other neuronal patterning events that influence visual function. To this end, the following Specific Aims are proposed: 1) Determine the intercellular molecular interactions that initiate recognition signals. Preliminary data suggest that MEGF10 and 11 mediate these interactions by binding to themselves and acting as both receptors and ligands. To test this hypothesis the binding specificity of each molecule will be determined biochemically, and their receptor/ligand function will be confirmed in vivo using Megf10 and Megf11 mutant mice. 2) Determine how recognition signals are reported in the cell. Preliminary data show that MEGF10 is required to transduce recognition signals. Using biochemical and in vivo genetic experiments, this Aim will test the hypothesis that ITAM phosphotyrosine motifs in the MEGF10 intracellular domain mediate these recognition signals. 3) Determine how recognition signals alter cellular behavior to produce mosaic spacing. This aim will test the hypothesis that recognition alters the behavior of dendrites. Specifically, it is proposed that recognition causes homotypic dendritic repulsion, through which neurons stake out unique territories that allow them to avoid their neighbors. Recognition will be abrogated genetically in Megf10; Megf11 double mutant mice and dendritic repulsion will be assessed by live imaging of retinal explants. Together, the experiments proposed in these three Aims are expected to reveal for the first time 1) the cell-surface molecules that bind to each other when cells of the same type touch; and 2) how these molecules trigger repulsion in order to specify neuronal position. The approach is innovative because it deploys novel tools and methods to enable the first molecular studies of homotypic recognition in mosaic patterning. The contribution will be significant because molecular events that determine the precise locations of neurons are important for circuit function, both in the retina and throughout the nervous system.