Signalling pathways mediating ephrin-A-induced growth cone collapse
The ephrin-A family of axon guidance cues, which activate the EphA family of receptor tyrosine kinases, guide the axons of many types of neuron to the correct target during embryonic development. One particularly well-studied example is the projection of RGC processes to precise positions in the midbrain target that reflect the position of the RGC in the retina. Ephrin-As are membrane-tethered molecules expressed in a gradient in the midbrain, and they govern the formation of the retinotectal map by differential, contact-mediated repulsion of Eph-A-expressing RGC axons. In order to identify signalling molecules that mediate ephrin-A induced repulsion of RGCs, I have developed a novel co-culture assay in which contact with a single non-neuronal cell that expresses endogenous levels of ephrin-A induces rapid loss of RGC growth cone lamella, followed by axon retraction. I have confirmed that these cellular responses are mediated by neuronal EphA receptor signalling and, in combination with the traditional soluble collapse assay, have used this physiologically relevant co-culture assay to identify a more specific role for the Rho effector ROCK in ephrin-A-induced RGC responses than has previously been published. Specifically ROCK activity mediates ephrin-A-induced RGC axon retraction, but not loss of growth cone lamella. I have also identified the non-receptor tyrosine kinase Abl as having a major role in the ephrin-A-induced RGC repulsive response, as the Abl kinase inhibitor STI571 prevents both the ephrin-A-induced loss of RGC lamella and axon retraction. I have demonstrated the existence of a protein complex containing active Eph receptors, Abl and Mena, and shown that disruption of this complex correlates with STI571-dependent inhibition of the ephrin-A-induced RGC repulsive responses. These results comprise the first evidence that Abl plays a role in mediating Eph receptor signals, and is involved in the cytoskeletal rearrangements that underlie ephrin-A-induced growth cone collapse in vitro, and thus both complement and extend the published evidence demonstrating a role for Abl in mediating axon guidance in vivo.