Netrin1 establishes multiple boundaries for axon growth in the developing spinal cord

- Multiple netrin1-mediated hederal boundaries arise in the early spinal cord.
- A haptotactic netrin1+ substrate directs pioneering spinal axons to grow ventrally.
- Netrin1-expressing, not netrin1+, domains, may promote fasciculation and prevent local innervation.
- Spinal axons grow on different netrin1+ substrates as development proceeds.

The canonical model for netrin1 function proposed that it acted as a long-range chemotropic axon guidance cue. In the developing spinal cord, floor-plate (FP)-derived netrin1 was thought to act as a diffusible attractant to draw commissural axons to the ventral midline. However, our recent studies have shown that netrin1 is dispensable in the FP for axon guidance. We have rather found that netrin1 acts locally: netrin1 is produced by neural progenitor cells (NPCs) in the ventricular zone (VZ), and deposited on the pial surface as a haptotactic adhesive substrate that guides Dcc+ axon growth. Here, we further demonstrate that this netrin1 pial-substrate has an early role orienting pioneering spinal axons, directing them to extend ventrally. However, as development proceeds, commissural axons choose to grow around a boundary of netrin1 expressing cells in VZ, instead of continuing to extend alongside the netrin1 pial-substrate in the ventral spinal cord. This observation suggests netrin1 may supply a more complex activity than pure adhesion, with netrin1-expressing cells also supplying a growth boundary for axons. Supporting this possibility, we have observed that additional domains of netrin1 expression arise adjacent to the dorsal root entry zone (DREZ) in E12.5 mice that are also required to sculpt axonal growth. Together, our studies suggest that netrin1 provides “hederal” boundaries: a local growth substrate that promotes axon extension, while also preventing local innervation of netrin1-expressing domains.