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  1. Reviewer #3 (Public Review):

    In some species, supporting cells (SCs) of the cochlea can replace hair cells and thus restore hearing. In the mouse, neonatal SCs can also produce hair cells; however, this property is lost during early postnatal life. This study sought to test whether forced expression of two transcription factors normally associated with OHC development, Atoh1 and Ifzh2, can induce adult mammalian supporting cells to take OHC-like properties. Using Cre-dependent expression in mice, the authors showed that co-expression of Atoh1 and Izfh2 could induce a small number of adult SCs to express the OHC-specific gene, Prestin. This conversion was significantly enhanced when existing OHCs were ablated, in this case using a Prestin-DTR mouse model generated by the authors. A detailed phenotypic analysis combined with single cell RNA-sequencing (scRNA-seq) supports the idea that Atoh1/Izfh2 can partially convert adult SCs into OHC-like cells. However, the conversion is not complete, with immature bundles and a gene signature that resembles P1 OHCs (and sometimes E16 OHCs) more than P7/P30 OHCs or P60 SCs. Accordingly, the new OHCs are not sufficient to restore hearing in the Prestin-DTR mouse model. Together, these data encourage optimism that adult SCs can be steered along the OHC path, though clearly more manipulations will be needed to produce mature, functional OHCs.

    The main weakness of the study is the scRNA-seq analysis, which depends on very small sample sizes. Suggestions to improve upon the analysis are listed under Specific Recommendations.

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  2. Reviewer #2 (Public Review):

    The goal of this study is to devise a means of promoting adult mouse auditory sensory cell development from supporting cells (SCs), as occurs naturally in birds and fish following sensory cell death. Previous studies indicated that activating Atoh1, an early acting transcription factor that specifies sensory cell fate during embryogenesis, was not sufficient for such regeneration. The authors hypothesized that adding a second transcription factor, Ikzf2, which maintains outer hair cell (OHC) fate, would synergize with Atoh1 and push adult SCs to differentiate as OHCs. They tested this hypothesis by over-expressing both Atoh1 and Ikzf2 in supporting cells after killing the endogenous OHCs in adult cochleae. The authors showed that the induced cells first express the general HC marker, Myo6, and only later become Prestin-positive, much as occurs during normal development. Unfortunately, these induced OHC-like cells had abnormal stereocilia and did not restore auditory (ABR) thresholds. Moreover, there was a loss of IHCs (the primary auditory receptors) suggesting that much more is needed to induce a real OHC and to protect IHCs than simply inducing the two selected transcription factors. Single-cell RNAseq (scRNA-seq) results showed that the induced OHC-like cells are enriched for HC genes and depleted for SC genes, but overall are most similar to neonatal HCs as defined in published scRNA-seq data from other groups. Overall, the scRNA-seq data did not offer a clear path forward, other than to identify and test additional transcription factors that might push the induced cells to the next stage. Nevertheless, the extent of SC transformation is impressive and has not been seen in previous approaches. This is an important contribution to our understanding of the control of OHC gene expression and differentiation contributed by two important transcription factors.

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  3. Reviewer #1 (Public Review):

    Mature mammalian hair cells in the cochlea do not regenerate after damage. The outer hair cells of the cochlea, which function to amplify sound, are particularly susceptible to damage. Ectopic activation of two key transcription factors for outer hair cell formation, Atoh1 and Ikzf2, in damaged adult cochlea is sufficient to convert supporting cells into hair cells expressing Prestin, which is an essential protein mediating outer hair cell functions. Although there is no functional recovery in these transgenic mice based on auditory brainstem response, this study paves the way for future design of models for hearing recovery. The main concern is the identity of the OHC-like cells drawn from the small sample size in the scRNA-seq experiments.

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  4. Evaluation Summary:

    This manuscript demonstrated the effectiveness of combined activation of Atoh1 and Ikzf2 in converting adult supporting cells to outer hair cell (OHC)-like cells in a mouse model, in which the OHCs were selectively ablated with diphtheria toxin. The authors showed that while the number of regenerated hair cells was low and there was no functional recovery based on ABR, these OHC-like cells do express Prestin and exhibit a genetic profile that resembles nascent hair cells. This paper will be of great interest to researchers interested in hearing restoration, as well as regenerative biology.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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