EPH RECEPTOR-LIGAND INTERACTIONS ARE NECESSARY FOR GUIDANCE OF RETINAL GANGLION-CELL AXONS IN-VITRO

Previous results of an in vitro guidance test, the stripe assay, have demonstrated the presence of a repulsive axon guidance activity for te mporal retinal axons in the posterior part of the vertebrate optic tec tum. Ephrin-A5 and Ephrin-A2 are ligands for the EphA subfamily of Eph receptor tyrosine kinases, which are expressed in overlapping gradien ts in the posterior part of the tectum. When recombinantly expressed, both proteins have been shown to guide retinal ganglion cell axons in the stripe assay. While these results suggest that Ephrin-A5 and Ephri n-A2 form part of the posterior repulsive guidance activity, they do n ot elucidate whether they are necessary components. Here we report tha t soluble forms of the ligands at nanomolar concentrations completely abolish this repulsive activity. Similar results were obtained with th e soluble extracellular domain of EphA3, which is a receptor for Ephri n-A2 and Ephrin-A5, but not with the corresponding domain of EphB3, a receptor for the transmembrane class of Eph ligands. These experiments show that the repulsive axon guidance activity seen in the stripe ass ay is mediated by Ephrin-A ligands.