Collaborative and specialized functions of Robo1 and Robo2 in spinal commissural axon guidance

Commissural neurons project axons across the floor plate at the spinal cord ventral midline. After crossing, commissural axons turn rostrally, sort into distinct positions within the ventrolateral funiculus, and never reenter the floor plate. Robo1 and Robo2 are receptors for the midline repellents Slit1-Slit3, and upregulation of Robos in post-crossing axons allows expulsion from the floor plate and prevents recrossing. Before crossing, Robo-mediated repulsion is attenuated by the divergent family member Robo3/Rig-1. To define the relative contributions of Robo family members to commissural axon guidance in mice, we studied commissural axon trajectories in combination mutants between Robo1, Robo2, and Robo3. Our results suggest the existence of another receptor contributing to Slit repulsion because the failure of midline crossing in Robo3 mutants is rescued largely but not entirely by loss of both Robo1 and Robo2 and because axon guidance defects in mice lacking both Robo1 and Robo2 are less severe than in mice lacking all Slits. Analysis of post-crossing axon trajectories indicates that Robo1 and Robo2 collaborate to prevent axons from reentering the gray matter and projecting dorsally alongside contralateral pre-crossing axons. We also discovered a previously unappreciated division of labor between Robo1 and Robo2 in post-crossing axons. Robo2 is required for axons to project away from the floor plate into the lateral funiculus. In contrast, Robo1 prevents axonal stalling after crossing. Our results reveal specialized and complementary actions of Robo1 and Robo2 in commissural axon guidance and suggest the existence of an as yet unidentified Slit receptor.