MOLECULAR DEVELOPMENT OF CENTRAL RETINAL PATHWAYS

我研究的长期目标是在分子水平上理解 精确神经元形成的机制 哺乳动物视觉系统中的连接。这里提出的实验 通过研究功能上重要的视网膜是如何 视交叉处的神经节细胞轴突通路是在 胚胎发育。对细胞表面分子的理解 参与视网膜轴突指导将使我们能够制定策略 控制和指导视网膜轴突的生长，因此是 试图在受伤后重新建立视觉联系的努力 疾病。此外，由于细胞表面的相互作用不仅是 对视网膜轴突的引导很重要，但很可能也支配着 其他视网膜神经元之间的连接模式，了解 细胞表面分子如何引导这些发育事件是 利用组织移植修复视神经元的策略 关系。 这里提出的研究形成了一套综合的实验，在这些实验中 我们使用体外和体内两种方法来分析 CD44和L1，两个在胚胎上显著表达的细胞表面分子 视交叉未来区域的视网膜轴突和神经元。我们 测试L1和CD44共同作用的假设，调节相互作用 视网膜轴突和胚胎交叉神经元之间的关系及其调控 形成“X”形视网膜通路的交叉点称为 视交叉。其次，我们还检查了CD44，这是显著的 在中线处表达，直接或与之相关 与其他细胞表面分子一起，在视网膜的特定路线上 轴突进入同侧和对侧视束。这一假设 是基于CD44调节细胞表面配体受体的事实 互动，并被独特地放置在中线，以影响 轴突是否穿过中线进入对侧的决定 视束或避免与同侧交叉和投射。 实验上，CD44和L1作用的分子基础及其作用 在视网膜通路中，用三种方法研究体内发育。 一种是注射对体内CD44和L1功能的直接干扰 将试剂阻断到活的小鼠胚胎中并检查 对视网膜通路形成的影响。二是CD44和CD44的干扰 L1在胚胎视觉系统准备中的作用及分析 延时视频显微镜对生长锥行为的影响。第三, 使用表达截短形式的CD44和L1的细胞系 确定调节它们对视网膜轴突作用的功能域 成长。这些研究解决了我们对视觉的理解上的主要差距 通过测试已定义的表面分子的功能来开发途径 在活体中的重要性并通过解剖机制来了解分子 为中枢视觉通路的发展奠定基础。

The long term objective of my research is to understand at a molecular level the mechanisms governing the formation of precise neuronal connections in the mammalian visual system. The experiments proposed here pursue this goal by investigating how the functionally important retinal ganglion cell axon pathways at the optic chiasm are formed during embryonic development. An understanding of the cell surface molecules involved in retinal axon guidance will enable us to devise strategies to control and direct the growth of retinal axons, and is thus fundamental to efforts attempting to re-establish visual connections following injury and disease. Furthermore, since cell surface interactions are not only important for retinal axon guidance, but very likely also govern the patterns of connectivity among other retinal neurons, an understanding of how cell surface molecules direct these developmental events is central to strategies using tissue transplantation to restore visual neuronal connections. The studies proposed here form an integrated set of experiments in which we use both in vitro and in vivo approaches to analyze the function of CD44 and L1, two cell surface molecules prominently expressed on embryonic retinal axons and neurons at the future region of the optic chiasm. We test the hypotheses that L1 and CD44 acting together, mediate interactions between ingrowing retinal axons and embryonic chiasm neurons and govern the formation of the "X" shaped retinal pathway intersection known as the optic chiasm. Secondly, we also examine whether CD44, which is prominently expressed at the midline, is involved, either directly or in conjunction with other cell surface molecules, in the specific routing of retinal axons into the ipsilateral and contralateral optic tracts. This hypothesis is based on the fact that CD44 regulates cell surface ligand-receptor interactions and is uniquely placed at the midline to influence the decision of axons whether to cross the midline into the contralateral optic tract or avoid crossing and project ipsilaterally. Experimentally, the molecular basis of CD44 and L1 action and their role in retinal pathway development in vivo is studied using three approaches. One, is direct perturbation of CD44 and L1 function in vivo by injections of blocking reagents into living mouse embryos and examination of the effects on retinal pathway formation. Second, is interference of CD44 and L1 function in embryonic visual system preparations and analysis of effects on growth cone behavior using time-lapse videomicroscopy. Third, is the use of cell lines expressing truncated forms of CD44 and L1 to define the functional domains mediating their actions on retinal axon growth. These studies address major gaps in our understanding of visual pathway development by testing defined surface molecules for functional importance in vivo and by dissecting the mechanisms to learn the molecular basis for central visual pathways development.