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

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

Regeneration Rosetta: An Interactive Web Application To Explore Regeneration-Associated Gene Expression and Chromatin Accessibility

Time-course high-throughput assays of gene expression and enhancer usage in zebrafish provide a valuable characterization of the dynamic mechanisms governing gene regulatory programs during CNS axon regeneration. To facilitate the exploration and functional interpretation of a set of fully-processed data on regeneration-associated temporal transcription networks, we have created an interactive web application called Regeneration Rosetta. Using either built-in or user-provided lists of genes in one of dozens of supported organisms, our web application facilitates the (1) visualization of clustered temporal expression trends; (2) identification of proximal and distal regions of accessible chromatin to expedite downstream motif analysis; and (3) description of enriched functional gene ontology categories. By enabling a straightforward interrogation of these rich data without extensive bioinformatic expertise, Regeneration Rosetta is broadly useful for both a deep investigation of time-dependent regulation during regeneration in zebrafish and hypothesis generation in other organisms

Cellular reprogramming for successful CNS axon regeneration is driven by a temporally changing cast of transcription factors

In contrast to mammals, adult fish display a remarkable ability to fully regenerate central nervous system (CNS) axons, enabling functional recovery from CNS injury. Both fish and mammals normally undergo a developmental downregulation 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. Here we present the first comprehensive analysis of gene regulatory reprogramming in zebrafish retinal ganglion cells at specific time points along the axon regeneration continuum from early growth to target re-innervation. Our analyses reveal a regeneration program characterized by sequential activation of stage-specific pathways, regulated by a temporally changing cast of transcription factors that bind to stably accessible DNA regulatory regions. Strikingly, we also find a discrete set of regulatory regions that change 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 re-establishment of synaptic connections in the brain. Together, these data provide valuable insight into the regulatory logic driving successful vertebrate CNS axon regeneration, revealing key gene regulatory candidates for therapeutic development