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.
