Choosing The Right Path: Analysis Of Axon Guidance In Peripheral Nerve Regeneration

After injury, axons of the peripheral nervous system (PNS) regenerate, and yet functional recovery from peripheral nerve injury is rare. This is because PNS axons regrow slowly and often toward inappropriate targets. Peripheral nerves are composed of bundles of axons that exit the spinal cord via a shared path and then diverge toward different targets forming a complex meshwork of nerve branches. These branched bundles of axons are encased in layers of glia, endothelial cells, and associated extracellular matrix (ECM). After nerve injury, severed axons degenerate and are cleared away, but the encasing cells and ECM beyond the injury site remain as branched tube-like structures that lead to nerve targets. To reconnect with their pre-injury targets, regenerating axons must navigate through these nerve tubes. Importantly, at points where nerve tubes diverge into multiple branches (branch-points), regenerating axons must select the branch that leads to their pre-injury target. Despite important implications for functional recovery, the mechanisms that guide regenerating axons at nerve branch-points are poorly understood. To probe the cellular and molecular mechanisms that guide regenerating axons, we exploit the simple architecture of spinal motor nerves in larval zebrafish, which are composed of two axonal populations that initially share a common path but diverge at a stereotyped branch-point to innervate dorsal or ventral muscles. After laser nerve transection, axons regenerate along their original nerve branch >80% of the time. Using genetic mutants and in vivo time-lapse imaging, we demonstrate that the repulsive axon guidance receptor robo2 is necessary and sufficient to promote axon regeneration along the dorsal branch. During regeneration, a small subset of glia at the nerve branch-point upregulate the Robo-ligand slit1a and the ECM component col4a5. We demonstrate that robo2 functions in a common molecular pathway with col4a5 to guide regenerating axons dorsally, and that the spatiotemporal restriction of col4a5 to the nerve branch-point during regeneration is required to guide regenerating dorsal axons. Our results provide the first cell-autonomous mechanism by which regenerating axons select between nerve branches during regeneration and provide a molecular pathway by which glia at a nerve branch-point guide regenerating axons via local ECM modifications.