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

Abstract

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.

Single-Cell Transcriptomic Profiling of the Zebrafish Inner Ear Reveals Molecularly Distinct Hair Cell and Supporting Cell Subtypes

“…Recent single-cell and single-nucleus RNA-sequencing efforts have generated a wealth of transcriptomic data from hair cells in several model systems, facilitating more direct comparison of cell types and gene regulatory networks between species. Although single-cell transcriptomic data have recently been published for the zebrafish inner ear (Jimenez et al, 2022; Qian et al, 2022), the diversity of hair cell and supporting cell subtypes has not been thoroughly analyzed. In order to better understand the diversification of cell types in the zebrafish inner ear, and their relationships to those in mammals, here we perform single-cell RNA sequencing (scRNA-seq) of the zebrafish inner ear from embryonic through adult stages.…”