This proposal investigates a putative repulsive factor, RAGS, an endogenous chick tectal protein that is believed to be involved in the guidance of retinal ganglion cells during development. During axon growth in vitro, Drescher has shown the axons avoid growing into RAGS labeled cells. The candidate proposes to tested whether the endogenous receptor for RAGS is CEK7, an EPH receptor tyrosine kinase shown to be in chick retina. The experiments are well- conceived with a clear conceptual basis. The techniques are readily available to the sponsor and all necessary reagents have been created. Based on homologies with other species, it seems likely that this proposal will created very useful developmental system in which to study axon guidance. Demonstrating a RAGS/CEK7 functional interaction will add to our expanding knowledge of inhibitory factors that prevent functional regeneration of transplantation following optic nerve damage. The project is divided into two specific aims. First, the candidate proposed to demonstrate the interaction between the ligand and receptor, and to determine which of the receptor isoforms can bind to RAGS. Recombinant sources of RAGS and CEK7 will be used. Activation of the receptor will be measured by autophosphorylation.
The second aim will investigate whether the RAGS/CEK7 interaction is actually repulsive using actual chick retinal ganglion cells. To show the specificity of a repulsive interaction, CEK7 will be inactivated by chromophore-assisted laser inactivation. The CEK7 turnover rate following laser inactivation will be determined so that tests of retinal axon guidance can study axon growth in the presence or absence of active CEK7. A stripe assay will show whether inactive CEK7 axons grow towards RAGS cells and active CEK7 axons avoid RAGS axons.
