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

Abstract

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.

Slit/Robo signals prevent spinal motor neuron emigration by organizing the spinal cord basement membrane

“…It is interesting to note that the floor plate function was not necessary to retain motor neurons in the spinal cord. Contrast to the previous reports showing a ventral repulsive role in controlling the position of the motor neurons within the neural tube, prevention attraction into the floor plate (Kim et al, 2017a;Kim et al, 2015), and in setting the position of the motor exit point (Kim et al, 2017b), emigration does not neatly fit into a push-pull balance between midline attraction and repulsion. The results instead point to a local role in controlling the ability of motor neurons to either stay in their normal place in the nucleus, or emigrate out.…”

“…Robo1 and Robo2 were expressed by spinal motor neurons and Slit2 were also expressed by the floor plate and spinal motor neurons (Brose et al, 1999;Kim et al, 2017b;Lee et al, 2015). These expression patterns of Slits and Robos suggest that motor neurons have Robo receptors to respond to Slits, which in turn could be derived from motor neurons themselves, or from the floor plate.…”

JNK Signaling Pathway Involvement in Spinal Cord Neuron Development and Death

“…Thus, giving that at least some molecular pattern expression in developing MN axons are shared with the commissural axons, we could speculate that also in this cell population, JNK phosphorylation is mediated by Netrin-1 and/or Wnt signaling to control MN axon pathfinding within and outside the spinal cord, acting at different multiple levels and by an intertwined manner. For instance, DCC and Netrin-1 knockout mice show defects in MN pathfinding: the exit point of the axons of ventral MNs is shifted dorsally, indicating that Netrin-1 normally guides axonal elongation ventrally [44,55].…”

Comparative transcriptomic profiling of peripheral efferent and afferent nerve fibres at different developmental stages in mice

“…Commissural axons grow along stereotyped dorsoventral trajectories in the early developing spinal cord. The projection patterns are encoded by the spatiotemporal distributions of axon guidance cues, specifically netrin-1/ DCC signalling for ventral attraction and SLIT/ROBO signalling for dorsal repulsion 33 . The SLIT proteins are best known for their classic roles in mediating chemorepulsion through the ROBO receptors during axon guidance 34 .…”