Convergent and divergent signaling mechanisms of growth cone collapse by ephrinA5 and slit2.

EphrinA5 and slit2 are important repulsive guidance cues in the developing retinotectal system. Both ephrinA5 and slit2 cause growth cone collapse of embryonic chick retinal ganglion growth cones cultured on EHS laminin. However, the signaling mechanism that these guidance cues initiate to cause collapse remains unclear. Here we provide evidence that while both ephrinA5 and slit2 cause collapse in morphologically similar ways, the intracellular signaling leading to the collapse involves shared as well as divergent paths. Pharmacological inhibition of either phosphatidylinositol 3-kinase (PI3K) or src family kinases prevented both ephrinA5-mediated and slit2-mediated growth cone collapse. In contrast, the inhibition of nonclassical protein kinase C (PKC) isoforms blocked ephrinA5-mediated collapse, but did not interfere with slit2-mediated collapse. PI3K was copurified by affinity chromatography with either the ephrinA5 receptors (ephAs) or the slit2 receptor (roundabout). Colocalization studies have also shown that src family kinase members are recruited to the ephA and roundabout receptors upon activation. In contrast, PKC members are recruited to the ephA receptors, but not to the roundabout receptors, upon activation. This demonstrates distinct points of convergence and divergence between the two signaling molecules, ephrinA5 and slit2, and their repulsive guidance in the chick retinotectal system.