A regulatory network of Sox and Six transcription factors initiate a cell fate transformation during hearing regeneration in adult zebrafish

Using adult zebrafish inner ears as a model for sensorineural regeneration, we performed a targeted ablation of the mechanosensory receptors in the utricle and saccule and characterized the single-cell epigenome and transcriptome at consecutive time-points following hair cell ablation. Using deep learning on the regeneration-induced open chromatin sequences, we were able to identify unique, cell-specific transcription factor (TF) motif patterns enriched in the raw data. We correlated enhancer activity with gene expression to identify gene regulatory networks. A clear pattern of overlapping Sox- and Six-family transcription factor gene expression and binding motifs was detected, suggesting a combinatorial program of TFs driving regeneration and cell identity. Pseudo-time analysis of single-cell transcriptomic data demonstrated that the support cells within the sensory epithelium changed cell identity to a more pluripotent “progenitor” cell population that could either differentiate into hair cells or return to a support cell identity. We showed that sox2 becomes enriched in the progenitor cells and is reduced again when the cells differentiate in either direction. Analysis of the scATAC-seq data identified a 2.6 kb DNA sequence element upstream of the sox2 promoter that dynamically changed in accessibility during hair cell regeneration. When deleted, the upstream regulator of sox2 showed a dominant phenotype that resulted in a hair cell regeneration-specific deficit in both the lateral line and adult inner ear. HIGHLIGHTS ### Competing Interest Statement The authors have declared no competing interest.