Mechanisms underlying optic nerve regeneration in zebrafish.

Abstract

Following injury to the central nervous system (CNS), adult mammals are largely unable to regenerate damaged axons resulting in permanent loss of function. Zebrafish, on the other hand, can robustly regenerate damaged CNS axons and regain lost function. This model organism provides an opportunity to study the cellular and molecular mechanisms necessary for successful CNS regeneration. The identification of gene regulatory mechanisms active during successful nerve regeneration may identify targets for therapeutic enhancement of mammalian regeneration. Previously, our lab generated transgennc zebrafish harboring the alpha1 tubulin promoter driving green fluorescent protein expression. This transgene, like the endogenous alpha1 tubulin gene, is specifically expressed in the developing CNS, repressed in the adult CNS, and re-induced during nerve regeneration. Using this alpha1 tubulin promoter as a probe we have identified an enhancer that is necessary for alpha1 tubulin promoter activation following optic nerve injury. Transgenic zebrafish harboring deletions or mutations in the alpha1 tubulin promoter identified a G/C-rich element within this enhancer that is necessary, but not sufficient, for alpha1 tubulin promoter induction during optic nerve regeneration. Electrophoretic mobility supershift assays suggested Sp/KLF transcription factors could to bind the G/C-rich element. Zebrafish DNA microarrays were used to investigate the identity of specific Sp/KLF family members that were induced during optic nerve regeneration. Hundreds of genes, including the Sp/KLF family members KLF6a and KLF7a, were identified. KLF6a and KLF7a are both able to bind to the alpha1 tubulin promoter and transactivate its expression. Morpholino antisense oligos were used to knockdown KLF6a and KLF7a expression in adult zebrafish RGCs. Knockdown of these proteins decreases alpha1 tubulin promoter transgene expression in RGCs during optic nerve regeneration. In addition, retinal explants from KLF6a and KLF7a morpholino treated eyes demonstrated reduced axon regeneration as comoared to controls. These studies establish KLF6a and KLF7a as important transcriptional regulators of optic axon regeneration and provide an approach for investigating the requirement for additional regeneration associated genes in successful re-growth of damaged optic axons.