Gene Regulatory Mechanisms Driving Cellular Reprogramming for Successful Optic Nerve Regeneration in Zebrafish

Abstract

Mammalian retinal ganglion cells (RGCs) in the central nervous system (CNS) often die after trauma to the optic nerve by either injury or disease. The surviving RGCs fail to regenerate their axons, eventually resulting in irreversible vision loss. Therefore, the functional restoration of eye-to-brain pathways is a significant challenge in the field. Unlike mammals, adult fish can fully regenerate CNS axons, enabling functional recovery from optic nerve damage. Both fish and mammals normally undergo a developmental down-regulation of axon growth activity as neurons mature. Fish are able to undergo damage-induced “reprogramming” through re-expression of genes necessary for axon growth and guidance, however, the gene regulatory mechanisms remain unknown. Our comprehensive analyses describe a hierarchical gene regulatory circuitry that controls successful axon regeneration in the zebrafish optic nerve. We characterized the gene regulatory reprogramming in zebrafish RGCs at specific time points along the axon regeneration continuum from early growth to target re-innervation in the brain. The regeneration program reveals a sequential activation of stage-specific pathways associated with axon regeneration and down-regulation of the mature neuronal state. We demonstrated that stage-specific pathways are regulated by a temporally changing cast of transcription factors that bind to stably accessible DNA regulatory regions. Interestingly, we also found a discrete set of regulatory regions that changed in accessibility, consistent with higher-order changes in chromatin organization that mark (1) the beginning of regenerative axon growth in the optic nerve, and (2) the reestablishment of synaptic connections in the brain. We further revealed the transcriptional mechanism and the putative downstream targets of the regeneration-associated transcription factor encoding gene, Jun. Together, these data provide insight into the gene regulatory logic driving successful CNS axon regeneration. The key gene regulatory candidates will aid in developing nerve repair strategies that stimulate the cell-intrinsic ability of mammalian RGCs following optic neuropathies or injuries.