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.

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. However, fish spontaneously undergo damage-induced "reprogramming" through re-expression of genes necessary for axon growth, guidance, and restoration of functional synaptic connections with target neurons. What remains unknown are the gene regulatory mechanisms that underlie successful reprogramming of adult neurons for functional CNS axon regeneration. Here we present the first comprehensive analysis of gene expression changes (RNA-seq) and DNA regulatory element accessibility (ATAC-seq) in zebrafish retinal ganglion cells (RGCs) at specific time points along the axon regeneration continuum from early growth to target reinnervation. 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 (a) the beginning of regenerative axon growth in the optic nerve, and (b) the reestablishment 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 and gene regulatory candidates for therapeutic development. Significance statementCNS axon damage, due to acute nerve injury or degenerative diseases, often leads to permanent loss of function in human patients. Despite recent discoveries of pathways that promote or inhibit regenerative nerve growth in the mammalian CNS, the capacity for restoring neuronal connections between the retina and the brain after optic nerve damage in mammals remains elusive. Unlike mammals, optic nerve injury in fish induces the re-expression of developmentally downregulated genes that encode proteins important for re-growing and re-establishing axonal connections between the retina and the brain. These studies reveal stage-specific gene regulatory mechanisms associated with regenerating RGCs as they reinitiate axon growth, cross the midline, select appropriate brain targets, and re-establish synapses.

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.

Time-course high-throughput assays of gene expression and enhancer usage in 8 zebrafish provide a valuable characterization of the dynamic mechanisms governing gene 9 regulatory programs during CNS axon regeneration. To facilitate the exploration and functional 10 interpretation of a set of fully-processed data on regeneration-associated temporal transcription 11 networks, we have created an interactive web application called Regeneration Rosetta. Using 12 either built-in or user-provided lists of genes in one of dozens of supported organisms, our web 13 application facilitates the (1) visualization of clustered temporal expression trends; (2) 14 identification of proximal and distal regions of accessible chromatin to expedite downstream motif 15 analysis; and (3) description of enriched functional gene ontology categories. By enabling a 16 straightforward interrogation of these rich data without extensive bioinformatic expertise, 17 Regeneration Rosetta is broadly useful for both a deep investigation of time-dependent regulation 18 during regeneration in zebrafish and hypothesis generation in other organisms. 19 Keywords CNS axon regeneration; gene expression; chromatin accessibility; functional 20 enrichment; zebrafish; R/Shiny 21 22The Regeneration Rosetta interactive web app was built in R using the Shiny and flexdashboard 131 packages. In addition to the other software packages already cited above, it makes use of the 132 data.table and RSQLite R packages for fast data manipulation, DT for rendering HTML tables 133 using JavaScript, readxl for parsing data from Excel spreadsheets, dplyr for data manipulation, 134 and tokenizers to convert user-provided gene IDs into tokens. The Regeneration Rosetta R/Shiny 135 7 app is available at http://ls-shiny-prod.uwm.edu/rosetta/. A FAQ page is available directly on the 136 app website. Source code for the Regeneration Rosetta is available from GitHub: 137 https://github.com/andreamrau/rosetta. The processed data used within the app are directly 138 located in https://github.com/andreamrau/rosetta/tree/master/data; scripts used to process the 139

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