Highlights
•
Integrated scRNA-seq and scATAC-seq of zebrafish inner ears during hearing regeneration•
Sox transcription factors trigger the regeneration response in the support cells•
Sox and Six factors cooperate spatially during hair cell differentiation•
An essential enhancer controls the precise timing of sox2 expression during regeneration
Summary
Using adult zebrafish inner ears as a model for sensorineural regeneration, we ablated the mechanosensory receptors and characterized the single-cell epigenome and transcriptome at consecutive time points during hair cell regeneration. We utilized deep learning on the regeneration-induced open chromatin sequences and identified cell-specific transcription factor (TF) motif patterns. Enhancer activity correlated with gene expression and identified potential gene regulatory networks. A pattern of overlapping Sox- and Six-family TF gene expression and binding motifs was detected, suggesting a combinatorial program of TFs driving regeneration and cell identity. Pseudotime analysis of single-cell transcriptomic data suggested that support cells within the sensory epithelium changed cell identity to a “progenitor” cell population that could differentiate into hair cells. We identified a 2.6 kb DNA enhancer upstream of the sox2 promoter that, when deleted, showed a dominant phenotype that resulted in a hair-cell-regeneration-specific deficit in both the lateral line and adult inner ear.
Introduction
The capacity to regenerate tissues after injury unevenly manifests across the vertebrate lineage.1 In most mammals, consistent cellular renewal is limited to certain cell types, such as skin, gut, and blood, while major tissue regeneration is even further restricted to a small number of organs, such as the liver. Damage to the mammalian inner ear sensory epithelium is irreversible and results in permanent hearing loss or vestibular defects. Interestingly, this is a feature that sets mammals apart from most other vertebrates who can continually produce new hair cells (HCs) throughout their lifetimes and/or can regenerate them in response to trauma.
The HCs are mechanosensory receptors used in the inner ear auditory and vestibular organs of all vertebrates and in the lateral line systems of aquatic vertebrates.2 In fish these organs are the saccule and utricle, respectively (Figure S1A).3 The saccule in fish primarily detects acoustic vibrations (amplified through the body instead of via an eardrum), while the utricle primarily functions as a gravitation sensor but has also shown some auditory potential.2,4 HCs similar to those that reside in the inner ear are also located on the skin in fish and amphibians in small structures called “neuromasts,” which reside in an organ normally referred to as the “lateral line.”2,3 Although lateral line HCs differ in morphology compared with the adult inner ear, the accessibility of the lateral line HCs on the skin surface has made them a popular in vivo model. However, more effort is justified in studying inner ear HC regeneration in fishes with the goal of restoring lost hearing in mammals.
Key genes in inner ear development can also have important roles in regeneration.5 However regeneration is distinct from embryonic development6 and recent genome-wide analyses suggest that, while regeneration programs may target many of the same genes, they may do so through distinct regulatory sequences:7,8 reviewed in Rodriguez et al.9 and Yang et al.10
Enhancer elements are critical in the control of development,11 and several groups have made connections between enhancer regulation and tissue regeneration programs. Injury-responsive or regeneration-associated enhancers that direct gene expression in injured tissues have been identified in the regenerating heart and fin of zebrafish.7,9,12, 13, 14, 15, 16, 17, 18, 19, 20 Comparative epigenomic profiling and single-cell genomics experiments have revealed species-specific and evolutionarily conserved genomic responses to regeneration in fish termed tissue regeneration enhancer elements (or TREEs).7,13,21 However, ablation of enhancers in these studies showed that, even though these enhancers respond to injury, they are generally not essential for normal regeneration.
Here, we profiled changes in chromatin accessibility (scATAC-seq) and gene expression single-cell 3′ RNA sequencing (scRNA-seq) during regeneration of the zebrafish inner ear at single-cell resolution. We showed the support cells (SCs) potentially transitioned into a “progenitor-like” state that differentiated into new HCs. We also identified a key regulator of sox2 expression that, when deleted, the HCs developed normally, but HC regeneration after injury was significantly disrupted….
