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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.HIGHLIGHTSIntegrated scRNA-seq and scATAC-seq of the adult zebrafish inner ear during hair cell regeneration.Support cells transition to an intermediate cell type that can differentiate to hair cells. Coordinated expression of Sox and Six transcription factors are key to hearing regeneration.Sox transcription factors trigger the regeneration response in the support cells while Sox and Six factors cooperate during hair cell differentiation.Deletion of an upstream enhancer that controls the timing of sox2 expression causes regeneration-specific defects in hearing regeneration.

Millions of Americans experience hearing or balance disorders due to loss of hair cells in the inner ear. The hair cells are mechanosensory receptors used in the auditory and vestibular organs of all vertebrates as well as the lateral line systems of aquatic vertebrates. In zebrafish and other non-mammalian vertebrates, hair cells turn over during homeostasis and regenerate completely after being destroyed or damaged by acoustic or chemical exposure. However in mammals, destroying or damaging hair cells results in permanent impairments to hearing or balance. We sought an improved method for studying hair cell damage and regeneration in adult aquatic vertebrates by generating a transgenic zebrafish with the capacity for targeted and inducible hair cell ablation in vivo. This model expresses the human diphtheria toxin receptor (hDTR) gene under the control of the myo6b promoter, resulting in hDTR expressed only in hair cells. Cell ablation is achieved by an intraperitoneal injection of diphtheria toxin (DT) in adult zebrafish or DT dissolved in the water for larvae. In the lateral line of 5 dpf zebrafish, ablation of hair cells by DT treatment occurred within 2 days in a dose-dependent manner. Similarly, in adult utricles and saccules, a single intraperitoneal injection of 0.05 ng DT caused complete loss of hair cells in the utricle and saccule by 5 days post-injection. Full hair cell regeneration was observed for the lateral line and the inner ear tissues. This study introduces a new method for efficient conditional hair cell ablation in adult zebrafish inner ear sensory epithelia (utricles and saccules) and demonstrates that zebrafish hair cells will regenerate in vivo after this treatment.

Mutations in the gene AK2 are responsible for reticular dysgenesis (RD), a rare and severe form of primary immunodeficiency in children. RD patients have a severely shortened life expectancy and without treatment die, generally from sepsis soon after birth. The only available therapeutic option for RD is hematopoietic stem cell transplantation (HSCT). To gain insight into the pathophysiology of RD, we previously created zebrafish models for Ak2 deficiencies. One of the clinical features of RD is hearing loss, but its pathophysiology and causes have not been determined. In adult mammals, sensory hair cells of the inner ear do not regenerate; however, their regeneration has been observed in several non-mammalian vertebrates, including zebrafish. Therefore, we used our RD zebrafish models to determine whether Ak2 deficiency affects sensory organ development and/or hair cell regeneration. Our studies indicated that Ak2 is required for the correct development, survival and regeneration of sensory hair cells. Interestingly, Ak2 deficiency induces the expression of several oxidative stress markers and it triggers an increased level of cell death in the hair cells. Finally, we show that glutathione treatment can partially rescue hair cell development in the sensory organs in our RD models, pointing to the potential use of antioxidants as a therapeutic treatment supplementing HSCT to prevent or ameliorate sensorineural hearing deficits in RD patients., Summary: A zebrafish model of reticular dysgenesis reveals hair cell developmental deficits that can be partially rescued by antioxidants, pointing to their potential use as a therapeutic treatment for reticular dysgenesis patients.

Cpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet, or even surpass, Cas9 targeting rates (approaching 100% targeting), but require higher concentrations of mRNA and guide. We also demonstrate that coinjecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.

Regenerative medicine holds great promise for both degenerative diseases and traumatic tissue injury which represent significant challenges to the health care system. Hearing loss, which affects hundreds of millions of people worldwide, is caused primarily by a permanent loss of the mechanosensory receptors of the inner ear known as hair cells. This failure to regenerate hair cells after loss is limited to mammals, while all other non-mammalian vertebrates tested were able to completely regenerate these mechanosensory receptors after injury. To understand the mechanism of hair cell regeneration and its association with regeneration of other tissues, we performed a guided mutagenesis screen using zebrafish lateral line hair cells as a screening platform to identify genes that are essential for hair cell regeneration, and further investigated how genes essential for hair cell regeneration were involved in the regeneration of other tissues. We created genetic mutations either by retroviral insertion or CRISPR/Cas9 approaches, and developed a high-throughput screening pipeline for analyzing hair cell development and regeneration. We screened 254 gene mutations and identified 7 genes specifically affecting hair cell regeneration. These hair cell regeneration genes fell into distinct and somewhat surprising functional categories. By examining the regeneration of caudal fin and liver, we found these hair cell regeneration genes often also affected other types of tissue regeneration. Therefore, our results demonstrate guided screening is an effective approach to discover regeneration candidates, and hair cell regeneration is associated with other tissue regeneration., Identifying regenerative genes in non-mammalian vertebrates A study on zebrafish has genetically screened 254 genes and identified 7 genes implicated in the development and regeneration of hair cells and other tissues. Humans and other mammals cannot regrow hair cells—inner-ear sensory receptors that enable hearing—whereas non-mammalian vertebrates, including zebrafish, can regrow these following injury. Researchers from the United States, led by the National Institutes of Health’s Shawn Burgess, screened adult zebrafish for genes active during the regeneration of inner-ear epithelium. The researchers then produced zebrafish without these genes to study their functions. The studies tested 254 genes known to respond during regeneration, and identified seven specifically impacting regeneration. Most of these seven genes also functioned in liver and fin tissue regeneration. Understanding the mechanisms of these genes may enable future research into regenerative therapies in humans.

Primary sex-determination "switches" evolve rapidly, but Doublesex (DSX)-related transcription factors (DMRTs) act downstream of these switches to control sexual development in most animal species. Drosophila dsx encodes female- and male-specific isoforms (DSX(F) and DSX(M)), but little is known about how dsx controls sexual development, whether DSX(F) and DSX(M) bind different targets, or how DSX proteins direct different outcomes in diverse tissues. We undertook genome-wide analyses to identify DSX targets using in vivo occupancy, binding site prediction, and evolutionary conservation. We find that DSX(F) and DSX(M) bind thousands of the same targets in multiple tissues in both sexes, yet these targets have sex- and tissue-specific functions. Interestingly, DSX targets show considerable overlap with targets identified for mouse DMRT1. DSX targets include transcription factors and signaling pathway components providing for direct and indirect regulation of sex-biased expression.

Cpf1 has emerged as an alternative to the Cas9 RNA-guided nuclease. Here we show that gene targeting rates in mice using Cpf1 can meet or even surpass Cas9 targeting rates (approaching 100% targeting) but require higher concentrations of mRNA and guide. We also demonstrate that co-injecting two guides with close targeting sites can result in synergistic genomic cutting, even if one of the guides has minimal cutting activity.

The creation of sexual dimorphism in the gonads is essential for producing the male and female gametes required for sexual reproduction. Sexual development of the gonads involves both somatic cells and germ cells, which often undergo sex determination by different mechanisms. While many sex-specific characteristics evolve rapidly and are very different between animal species, gonad function and the formation of sperm and eggs appear more similar and may be more conserved. Consistent with this, the doublesex/mab3 Related Transcription factors (DMRTs) are important for gonad sexual dimorphism in a wide range of animals, including flies, worms and mammals. Here we explore how sexual dimorphism is regulated in the Drosophila gonad, focusing on recent discoveries relating to testis development. We will discuss how sex determination in both the germline and the soma are utilized to create a testis, including the role of the key somatic sex determination factor doublesex.