EphA3 couples attractive guidance to persistent retinal axon growth through modulation of EphA4 forward signaling in a plasma membrane cholesterol-dependent manner

Abstract

Topographic mapping requires retinal axons not only to avoid inappropriate targets but also to advance directionally through permissive and attractive territories. Using chick retinal ganglion cells in stripe and Dunn chamber gradient assays, we investigated how extracellular EphA3 coordinates the directional guidance and growth dynamics of nasal axons. Nasal axons preferentially grew on EphA3-containing stripes and turned toward increasing concentrations of EphA3 in soluble gradients. EphA3 also increased axonal growth velocity, primarily by prolonging extension phases and reducing growth interruptions rather than by increasing maximal extension-phase velocity. Pharmacological inhibition of EphA4 enhanced axonal extension but abolished EphA3-dependent attraction, indicating that basal EphA4 activity is required for spatial guidance even though EphA4 signaling can constrain axonal advance. Plasma membrane cholesterol depletion altered basal growth dynamics and attenuated EphA3-dependent turning and extension persistence. Together, these findings identify EphA3 as a noncanonical attractive cue that coordinates directional guidance with persistent nasal axon growth through mechanisms dependent on EphA4 activity and membrane cholesterol. They also suggest that sustained EphA4 inhibition may promote axonal growth while compromising topographic targeting.