Topographic-specific axon branching controlled by ephrin-As is the critical event in retinotectal map development

The retinotectal projection is the predominant model for studying molecular mechanisms controlling development of topographic axonal connections. Our analyses of topographic mapping of retinal ganglion cell (RGC) axons in chi ck optic tectum indicate that a primary role for guidance molecules is to r egulate topographic branching along RGC axons, a process that imposes uniqu e requirements on the molecular control of map development. We show that to pographically appropriate connections are established exclusively by branch es that form along the axon shaft. Initially, RGC axons overshoot their app ropriate termination zone (TZ) along the anterior-posterior (A-P) tectal ax is; temporal axons overshoot the greatest distance and nasal axons the leas t, which correlates with the nonlinear increasing A-P gradient of ephrin-A repellents. In contrast, branches form along the shaft of RGC axons with su bstantial A-P topographic specificity. Topography is enhanced through the p referential arborization of appropriately positioned branches and eliminati on of ectopic branches. Using a membrane stripe assay and time-lapse micros copy, we show that branches form de novo along retinal axons. Temporal axon s preferentially branch on their topographically appropriate anterior tecta l membranes. After the addition of soluble EphA3-Fc, which blocks ephrin-A function, temporal axons branch equally on anterior and posterior tectal me mbranes, indicating that the level of ephrin-As in posterior tectum is suff icient to inhibit temporal axon branching and generate branching specificit y in vitro. Our findings indicate that topographic branch formation and arb orization along RGC axons are critical events in retinotectal mapping. Ephr in-As inhibit branching along RGC axons posterior to their correct TZ, but alone cannot account for topographic branching and must cooperate with othe r molecular activities to generate appropriate mapping along the A-P tectal axis.