Signaling Mechanisms of Retinal Axon Guidance

描述(申请人提供)：在哺乳动物视觉系统的形成过程中，新生的神经元延伸轴突，与视网膜和大脑中特定的靶区细胞进行固定的连接。在视觉系统发育过程中，Eph受体和它们的肾上腺素配体在视网膜轴突向大脑的正确引导方面发挥着关键作用。然而，介导依赖于Eph的轴突靶向事件的细胞内信号事件仍不清楚。这项研究计划的主要目标是识别和理解将细胞外轴突引导线索转化为适当的轴突转向事件的关键细胞内信号事件。我们的初步研究表明，Vav和Ephexin家族RhoGEF(Rho家族GTP酶鸟嘌呤核苷酸交换因子)在体外和体内都是Eph依赖的轴突排斥的重要调节因子。我们试图探索Vav GEF在适当地将视网膜轴突定向到大脑中的确切作用，并检验Vav GEF调节从吸引信号到排斥信号的转换的假设。为了解决这些观点并开始了解Vav GEF在视网膜发育和轴突靶向中的作用和调控，我们提出了以下具体目标：1)通过分析Vav缺陷的EphB1阳性轴突在视交叉发育过程中的行为，确定Vav GEF在体内视网膜化轴突靶向中的作用，2)确定Vav GEF在体外和体内调节吸引和排斥Eph信号的作用，以及3)通过研究Vav2的EphK依赖的降解来确定Vav GEF在视网膜轴突引导过程中的调节。在这个方案中，将使用体内轴突示踪技术、生化和分子生物学方法以及细胞培养技术来探索Vav GEF在视网膜发育中的作用。此外，我们还开发了一种化学调控的、基于细胞培养的方法，以更容易地测试Eph受体信号事件的作用。这些研究的结果将为控制视觉系统正常发育的细胞信号过程提供有价值的新见解。了解这些视网膜轴突引导机制可能为视网膜发育障碍、视网膜神经损伤后轴突再生以及导致部分或完全失明的神经退行性疾病的治疗方法提供重要的新见解。

DESCRIPTION (provided by applicant): During formation of the mammalian visual system, newly born neurons extend axons to make stereotyped connections with specific target field cells within the retina and brain. Eph receptors and their ephrin ligands play critical roles during visual system development in the proper guidance of retinal axons to the brain. However, the intracellular signaling events that mediate Eph-dependent axon targeting events has remained unclear. The main goal of this research proposal is to identify and understand the critical intracellular signaling events that convert the extracellular axon guidance cue into an appropriate axon turning event. Our preliminary studies have revealed that Vav and Ephexin family RhoGEFs (Rho family GTPase guanine nucleotide exchange factors) are essential regulators of Eph-dependent axon repulsion in vitro and in vivo. We seek to explore the precise role of Vav GEFs in the proper targeting of retinal axons to the brain, and to test the hypothesis that Vav GEFs regulate a switch from attractive to repulsive signaling. To address these ideas and to begin to understand the role and regulation of Vav GEFs in retinal development and axoon targeting, we propose the following specific aims: 1) to determine the role of Vav GEFs in retinogeniculate axon targeting in vivo by analyzing the behavior of Vav-deficient, EphB1 -positive axons at the optic chiasm during development, 2) to determine the role of Vav GEFs in regulating attractive vs. repulsive Eph signaling in vitro and in vivo, and 3) to determine the regulation of Vav GEFs during retinal axon guidance by studying the Eph kinase-dependent degradation of Vav2. In this proposal, in vivo axon tracing techniques, biochemical and molecular biology approaches combined with cell culture techniques will be employed to explore the role of Vav GEFs in retinal development. Moreover we have developed a chemically-regulated, cell culture-based approach to more easily test the role of Eph receptor signaling events. The results of these studies will provide valuable new insights into the cell signaling processes that control proper visual system development. Understanding these retinal axon guidance mechanisms might provide important new insights into therapeutic approaches to retinal development disorders, axon regeneration following retinal nerve injury and neurodegenerative diseases that lead to partial or total blindness.