Deconstructing Retinal Ganglion Cell Axonal Regeneration in Xenopus laevis

The retinal ganglion cells (RGCs) of the optic nerve are essential for transmission of visual information to the brain. Diverse insults to the optic nerve result in partial to total vision loss as the axons of RGCs are destroyed. In glaucoma, axons are injured at the optic nerve head; in other optic neuropathies, axons can be damaged along the entire visual pathway. In all cases, as mammals cannot regenerate injured central nervous system cells, once the axons are lost, vision loss is irreversible.However, RGC axons of the African clawed frog Xenopus laevis are capable of regeneration and functional reinnervation of central brain targets following injury. In this dissertation, I describe a novel tadpole optic nerve crush (ONC) procedure and assessments of axon growth and brain innervation based on live imaging of RGC-specific transgenes which can be used to assay putative regeneration-associated genes in vivo. Using these assays with a CRISPR/Cas9-based F0 knockout screen, I report that the MAPKKK dual leucine zipper kinase (dlk) is necessary for regeneration of RGC axons following injury. Loss of Dlk does not affect vision as assessed by a behavioral assay but does block functional vision restoration after ONC. Dlk absence does not affect axonal outgrowth of RGCs either during development or from RGCs generated in the retina after the injury, but only affects the axonal regeneration of those RGCs whose axons were injured. While Dlk loss does not alter the acute change in mitochondria movement that occurs within RGC axons soon after injury, it does completely block the activation of the transcription factor c-Jun within RGCs days after the injury. Taken together, these results show that Dlk is essential for the axonal injury signal to reach the nucleus, suggesting that the difference between species that can and cannot regenerate their RGC axons after injury is likely the transcriptional response downstream of a MAPK cascade. While mechanisms intrinsic to retinal ganglion cells, such as Dlk-dependent signal activation, are critical to successful RGC regeneration, RGC interactions with myeloid and glial cell populations in the retina and optic nerve are also highly likely to affect RGC survival and regeneration. Thus, using the same tadpole optic nerve crush assay, I further report that ablation of myeloid cells using a novel cell-type specific inducible transgene also delays optic nerve regeneration and reinnervation of the optic tectum following ONC. The absence of myeloid cells also results in a significant delay in clearance of cellular debris derived from the injury site. Additionally, removal of cellular debris immediately follows myeloid cell influx into both brain and optic nerve, indicating that debris removal by myeloid cells may be required for axonal regeneration. My work elucidates two key aspects of an evolutionarily conserved successful regeneration response, one cell intrinsic and one cell extrinsic. Understanding the molecular mechanisms utilized by a regeneration-capable species may be essential to the rational design of future clinical interventions to regrow the optic nerve. My work also suggests that a combination of different molecular and cellular interventions will likely be the only way to achieve axonal regeneration sufficient for functional recovery of vision