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.

Axon pathfinding is orchestrated by numerous guidance cues, including Slits and their Robo receptors, but it remains unclear how information from multiple cues is integrated or filtered. Robo3, a Robo family member, allows commissural axons to reach and cross the spinal cord midline by antagonizing Robo1/2-mediated repulsion from midline-expressed Slits and potentiating deleted in colorectal cancer (DCC)-mediated midline attraction to Netrin-1, but without binding either Slits or Netrins. We identified a secreted Robo3 ligand, neural epidermal growth factor-like-like 2 (NELL2), which repels mouse commissural axons through Robo3 and helps steer them to the midline. These findings identify NELL2 as an axon guidance cue and establish Robo3 as a multifunctional regulator of pathfinding that simultaneously mediates NELL2 repulsion, inhibits Slit repulsion, and facilitates Netrin attraction to achieve a common guidance purpose.

Axons travel to their targets in bundles or fascicles, but the molecules regulating fasciculation remain incompletely characterized. We found that Slit2 and its Robo receptors are expressed by motor axons, and that inactivation of Slit2 or Robo1 and Robo2 in mice caused axons to defasciculate prematurely at muscle targets. In vitro, Slit2 secreted by motoneurons regulated fasciculation through Robo1 and Robo2. These results support the idea that Slit2 promotes axon fasciculation via an autocrine and/or juxtaparacrine mechanism.