Date of Award
Doctor of Philosophy (PhD)
Axons of the peripheral nervous system have retained the remarkable ability to regenerate. However, after peripheral nerve injury, patients often suffer from the inability to properly localize sensation and/or the loss of fine motor control, suggesting that regenerating peripheral axons reinnervate ectopic targets. Despite decades of research in myriad model systems, whether PNS axons regenerate randomly or navigate selectively to their original targets remains controversial. Moreover, while some studies suggest that regenerating axons can choose specific paths, the cell and molecular mechanisms underlying target-selective regeneration have remained elusive. Using live-cell imaging in larval zebrafish we find that after complete nerve transection regenerating motor axons exhibit a strong preference for their original muscle territory, and that axons probe both correct and incorrect trajectories extensively before selecting their original path. We demonstrate that the glycosyltransferase lysyl hydroxylase 3 is required to modify extracellular matrix collagens to provide growth and guidance to regenerating axons. Using transgenic rescue experiments, we determine that post-injury expression of lh3 and lh3 expression in Schwann cells is sufficient to restore target-selective regeneration. Moreover, we show that Schwann cells neighboring the transection site upregulate the lh3 substrate collagen4a5 and that during regeneration collagen4a5 destabilizes axons probing inappropriate trajectories to ensure target-selective regeneration. These results demonstrate that selective ECM components match subpopulations of regenerating axons with their original targets.
It has long been hypothesized that regenerating axons might reuse developmental guidance cues to reestablish synaptic connections after peripheral nerve injury. Intriguingly, Collagen4 can bind the canonical axon repellants Slit and Netrin, and we find that slit1a is upregulated with collagen4a5 in the same population of Schwann cells after injury. Additionally, we demonstrate that the slit receptors robo2 and robo3 are both required for target-selective regeneration. Together these results demonstrate that regenerating peripheral nerves reemploy developmental guidance molecules and reveal a possible mechanistic framework by which the ECM constituent collagen4a5 binds and presents the repellant slit1a to convey synaptic target selection through robo2 and robo3 repulsion of regenerating axons in vivo.
Isaacman-Beck, Jesse, "Molecular Analysis of Target Specific Peripheral Nerve Regeneration" (2015). Publicly Accessible Penn Dissertations. 1778.