Common and Specific Gene Regulatory Programs in Zebrafish Caudal Fin Regeneration at Single-Cell Resolution

Following amputation, zebrafish regenerate their injured caudal fin through lineage-restricted reprogramming. Although previous studies have charted various genetic and epigenetic dimensions of this process, the intricate gene regulatory programs shared by or unique to different regenerating cell types remain under-investigated. Here, we mapped the regulatory landscape of fin regeneration by applying paired snRNA-seq and snATAC-seq on uninjured and regenerating fin. This map delineates the regulatory dynamics of predominant cell populations at multiple stages of regeneration. We observed a marked increase in the accessibility of chromatin regions associated with regenerative and developmental processes at 1 dpa, followed by a gradual closure across major cell types at later stages. This pattern is distinct from that of transcriptomic dynamics which is characterized by several waves of gene upregulation and downregulation. We identified and in vivo validated cell type-specific and position-specific regeneration-responsive enhancers, and constructed regulatory networks by cell type and stage. Our single-cell resolution transcriptomic and chromatin accessibility map across regenerative stages provides new insights into regeneration regulatory mechanisms and serves as a valuable resource for the community. Fin tissue regenerating from adult zebrafish was collected at 1, 2, 4, and 6 days post-amputation (dpa). For the pre-injury (0 dpa) sample, we collected two segments of fin rays from each fish after removing the distal half of their caudal fin. Each time point had two biological replicates. Nuclei were isolated from each fin tissue sample. Libraries were then constructed from these nuclei following the instructions of the Chromium Single Cell Multiome ATAC + Gene Expression (10x Genomics) kit and were subsequently sequenced on NovaSeq 6000 (Illumina). Raw reads were processed by Cell Ranger ARC v2.0.0 (10x Genomics) with default parameters for read tagging, alignment to the zebrafish reference genome (GRCz11), and feature counting based on Ensembl release 104 (cellranger-arc count).