Cellular reprogramming with ATOH1, GFI1, and POU4F3 implicate epigenetic changes and cell-cell signaling as obstacles to hair cell regeneration in mature mammals

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    Overall, this is an interesting paper that explains molecular underpinnings of hair cell reprogramming. This paper could have significant implications for our understanding of how different cellular programs can dictate phenotypic outcomes such as hearing.

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Reprogramming of the cochlea with hair-cell-specific transcription factors such as ATOH1 has been proposed as a potential therapeutic strategy for hearing loss. ATOH1 expression in the developing cochlea can efficiently induce hair cell regeneration but the efficiency of hair cell reprogramming declines rapidly as the cochlea matures. We developed Cre-inducible mice to compare hair cell reprogramming with ATOH1 alone or in combination with two other hair cell transcription factors, GFI1 and POU4F3. In newborn mice, all transcription factor combinations tested produced large numbers of cells with the morphology of hair cells and rudimentary mechanotransduction properties. However, 1 week later, only a combination of ATOH1, GFI1 and POU4F3 could reprogram non-sensory cells of the cochlea to a hair cell fate, and these new cells were less mature than cells generated by reprogramming 1 week earlier. We used scRNA-seq and combined scRNA-seq and ATAC-seq to suggest at least two impediments to hair cell reprogramming in older animals. First, hair cell gene loci become less epigenetically accessible in non-sensory cells of the cochlea with increasing age. Second, signaling from hair cells to supporting cells, including Notch signaling, can prevent reprogramming of many supporting cells to hair cells, even with three hair cell transcription factors. Our results shed light on the molecular barriers that must be overcome to promote hair cell regeneration in the adult cochlea.

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  1. eLife assessment

    Overall, this is an interesting paper that explains molecular underpinnings of hair cell reprogramming. This paper could have significant implications for our understanding of how different cellular programs can dictate phenotypic outcomes such as hearing.

  2. Reviewer #1 (Public Review):

    This manuscript addressed an important question regarding an obstacle to hair reprogramming in older mice that is not present in newborn mice. The conclusions were justified by experimental results. However, the scientific novelty is limited, and there is a lack of functional characterization of the newly formed hair cells.

    There are several strengths of this study: 1. It addressed a significant question as hearing loss is an important public health issue. 2. Well-designed genetic approaches. 3. Experiments were well designed and justified. 4. Experimental results are convincing. 5. Conclusions were well justified by experimental results. There are also several weaknesses:

    1. The scientific novelty is limited. It is known that overexpression of several transcription factors simultaneously can reprogram hair cells and non-hair cells with hair cell characteristics.

    2. Transcription factor Atoh1 and downstream GFI1and POU4F3 have been used to reprogram embryonic stem cells and chick otic epithelial cells in vitro to cells expressing several hair cell genes and displaying key hair cell features.

    3. There is no functional characterization of newly reprogramed hair cells in adult mice although FM 1-43 dye was used for characterizing reprogramed hair cells in neonatal mice.

    4. It is not understood why the changes in transduction channel protein expression were not highlighted in gene analysis.

    5. It will be nice if hair cell-like electrophysiological properties can be found in newly reprogramed hair cells.

  3. Reviewer #2 (Public Review):

    In this article, Iyer et al discuss the mechanics of reprogramming challenges encountered by supporting cells towards their potential pathway of dedifferentiating into hair cell types. Previous literature has shown the ability of ATOH-1, GFl-1, and POU4F3 to transform supporting cells into hair-like cell types. Here authors suggest that the combinatorial expression of these TFs can enhance the efficiency of the transcriptional remodeling of supporting cells to initiate the reprogramming toward hair-like cell lineage. It is a well-conducted study. Please see my comments/concerns below.

    1. In the representative images, the effect of GFl-1 seems to be less efficient or has no effect on reprogramming the lineage of supporting cells to hair cell-like cells in comparison to two other groups ATOH-1 alone or ATOH-1, GFl-1, and Pou4F3 combined (Figure 1, 1- S2, 2B, 4A) and even the single-cell RNA seq can be interpreted similarly (Figure 3C, 6C. According to authors and previous literature, GFl1 is supposed to be acting in concert to enhance the efficiency of this lineage conversion at least in older animals. The representative images and single-cell UMAPs show that either GFl-1 is not efficient or less efficient than ATOH-1 alone or ATOH-1, GFl-1, and Pou4F3 combined. Hence, why authors chose not to explore ATOH-1 and Pou4F3 without GFl-1.

    2. In Figure 3C, the authors find the most reduction in cell numbers in lateral GER during transcriptional reprogramming. Can authors comment on why the cells in this region are more susceptible to lineage reprogramming into hair cell-like cells?

    3. In figure 5A, how can the existing hair cells be distinguished from newly formed hair cell-like cells.

    4. Authors cited previous literature showing that existing hair cells can affect lineage reprogramming of supporting cells through Notch signaling. So would it not be a better experimental design when the hair cells were depleted prior to transcriptional reprogramming.

    5. Genetic mutations that lead to functional disruptions in supporting cells are also linked to hearing loss. Can authors predict how feasible would be the idea of in vivo conversion of one important cell type to another important cell type?

    6. Are reprogrammed hair cell-like cells transcriptionally similar to outer hair cells, inner hair cells, or none?