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

Abstract

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.

Defective mechanosensory transduction of the new inner hair cells prevents hearing recover in the damaged cochlea

“…How might IHC damage enhance the reprogramming efficiency of Tbx2 and Atoh1? We speculate that, one, certain unknown components released from the dying IHCs might stimulate the response of IBCs/IPhs to Tbx2 and Atoh1 manipulation; or two, intercellular signaling, such as Notch signaling (21) (34).…”

“…Notably, both OHCs and IHCs are critical for sound detection and their damage causes permanent deafness in mammals, which have lost HC regeneration capacity (11–13). Intriguingly, the b-HLH transcription factor Atoh1 is necessary for cochlear pan-HC development: whereas all HCs disappear in Atoh1 -/- mice (14–16), Atoh1 overexpression induces supernumerary new HCs (17–21). Furthermore, Insm1 and Ikzf2 play crucial roles in specifying or stabilizing the OHC fate (22, 23), and our group and two other groups have independently shown that Tbx2 is required in IHC fate specification, differentiation, and maintenance (24–26).…”

Future Pharmacotherapy for Sensorineural Hearing Loss by Protection and Regeneration of Auditory Hair Cells

“…Chen et al (2021) reported the generation of mature and functional HCs by co-expression of GFI1, POU4F3, and ATOH1 in postnatal mouse cochlea [ 78 ], suggesting that co-transfection of GFI1 and POU4F3 contributes to the maturation of converted HCs. More recently, Iyer et al (2022) also demonstrated a higher efficiency of co-transfection of GFI1, POU4F3, and ATOH1 for SC-to-HC conversion than that of co-transfection of GFI1and ATOH1 or transfection of ATOH1 alone [ 79 ]. Walters et al (2017) showed that a combination of ATOH1, Gata3, and POU4F3 improved the efficiency of HC regeneration in adult mice [ 56 ].…”

“…These findings suggest the potential of POU4F3 as a pioneer factor in the induction of SC-to-HC conversion in adult mammalian cochleae. However, the efficiency of co-transfection with GFI1, POU4F3, and ATOH1 was also decreased by aging [ 79 ]. One of the major causes of this is a decrease in the accessibility of HC loci in mature SCs [ 79 ].…”

“…However, the efficiency of co-transfection with GFI1, POU4F3, and ATOH1 was also decreased by aging [ 79 ]. One of the major causes of this is a decrease in the accessibility of HC loci in mature SCs [ 79 ].…”