Abstract
Purpose :
Unlike mammalian species, Xenopus laevis can regenerate their retinal ganglion cell (RGC) axons after optic nerve injury. The molecular mechanisms enabling this remain unknown. We performed a pilot CRISPR-screen using a novel moderate-throughput tadpole optic nerve crush (ONC) assay to identify genes necessary for axonal regrowth. Among the genes targeted was Dual leucine zipper kinase (Dlk; i.e. Map3k12).
Methods :
For Dlk knockout (KO) line creation, sgRNAs targeting Dlk were injected into eggs within 30 minutes of fertilization. Dlk KO tadpoles were subjected to ONC using bevelled 50-75 um glass needles mounted on micromanipulators at 8 days post-fertilization. Optic nerves and tecta were live imaged over the next 7 days using a Leica fluorescent stereomicroscope. For retrograde tracing, Mitotracker-soaked gelfoam was inserted into the optic tecta at 6 days post-ONC and eyes were dissected out 24 hours later. Degree of reinnervation was assessed from the live imaging data. Phosphorylation of Jun (pJun) and source of tectal innervating axons were assessed in immunolabeled cryosections imaged using a Leica Dragonfly confocal microscope. Custom IPlab scripts were used for all quantifications. All animals were genotyped via sequencing and TIDE analysis.
Results :
Dlk full KO animals had 27% of the tectal innnervation seen in wildtype animals 6 days post-ONC, and animals with only 2 copies of an in-frame deletion, Dlk(d56-58), had 60% (X. laevis is allotetraploid with 4 copies of Dlk). In wildtype animals, innervation post-ONC was mainly from central (crushed) RGCs; however, in Dlk KOs, tectal innervation was limited to the most peripheral (newly born) RGCs. ONC significantly increased nuclear pJun selectively in RGCs 2d post-ONC, a response blunted but not absent (40% less than control retinas) in Dlk KOs.
Conclusions :
Dlk specifically affects RGC axonal regeneration in a regeneration-capable species. Our assay shows that while new axons are born after injury, most tectal innervation post-ONC comes from preexisting axons, and this innervation is selectively inhibited in the Dlk KO animals. Findings are largely consistent with other literature placing Dlk in a pathway conveying an axonal injury signal to the soma. However, the pJun results show that RGC soma sense axonal injury independent of Dlk. We believe that genes downstream of Dlk in X. laevis offer much promise to promote RGC axonal regeneration in mammals.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.