Motor axons are guided to exit points in the spinal cord by Slit and Netrin signals

In the spinal cord, motor axons project out the neural tube at specific exit points, then bundle together to project toward target muscles. The molecular signals that guide motor axons to and out of their exit points remain undefined. Since motor axons and their exit points are located near the floor plate, guidance signals produced by the floor plate and adjacent ventral tissues could influence motor axons as they project toward and out of exit points. The secreted Slit proteins are major floor plate repellents, and motor neurons express two Slit receptors, Robo1 and Robo2. Using mutant mouse embryos at early stages of motor axon exit, we found that motor exit points shifted ventrally in Robo1/2 or Slit1/2 double mutants. Along with the ventral shift, mutant axons had abnormal trajectories both within the neural tube toward the exit point, and after exit into the periphery. In contrast, the absence of the major ventral attractant, Netrin-1, or its receptor, DCC caused motor exit points to shift dorsally. Netrin-1 attraction on spinal motor axons was demonstrated by in vitro explant assays, showing that Netrin-1 increased outgrowth and attracted cultured spinal motor axons. The opposing effects of Slit/Robo and Netrin-1/DCC signals were tested genetically by combining Netrin-1 and Robo1/2 mutations. The location of exit points in the combined mutants was significantly recovered to their normal position compared to Netrin-1 or Robo1/2 mutants. Together, these results suggest that the proper position of motor exit points is determined by a "push-pull" mechanism, pulled ventrally by Netrin-1/DCC attraction and pushed dorsally by Slit/Robo repulsion.