Profiling Dynamic Changes in DNA Accessibility During Axon Regeneration After Optic Nerve Crush in Adult Zebrafish.

A time-course series utilizing assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) can be used to detect changes in accessibility of DNA regulatory elements such as promoters and enhancers over the course of regeneration. This chapter describes methods for preparing ATAC-seq libraries from isolated zebrafish retinal ganglion cells (RGCs) following optic nerve crush at selected post-injury time points. These methods have been used for identifying dynamic changes in DNA accessibility that govern successful optic nerve regeneration in zebrafish. This method may be adapted to identify changes in DNA accessibility that accompany other types of insults to RGCs or to identify changes that occur over the course of development.

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.

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.