Dual expression of Atoh1 and Ikzf2 promotes transformation of adult cochlear supporting cells into outer hair cells

Abstract

Mammalian cochlear outer hair cells (OHCs) are essential for hearing. OHC degeneration causes severe hearing impairment. Previous attempts of regenerating new OHCs from cochlear supporting cells (SCs) had yielded cells lacking Prestin, a key motor protein for OHC function. Thus, regeneration of Prestin+ OHCs remains a challenge for repairing OHC damage in vivo. Here, we reported that successful in vivo conversion of adult cochlear SCs into Prestin+ OHC-like cells could be achieved by simultaneous expression of Atoh1 and Ikzf2, two key transcriptional factors necessary for OHC development. New OHC-like cells exhibited upregulation of hundreds of OHC genes and downregulation of SC genes. Single cell transcriptomic analysis demonstrated that the differentiation status of these OHC-like cells was much more advanced than previously achieved. Thus, we have established an efficient approach to promote regeneration of Prestin+ OHCs and paved the way for repairing damaged cochlea in vivo via transdifferentiation of SCs.

In vivo CRISPR‐Cas9‐mediated DNA chop identifies a cochlear outer hair cell‐specific enhancer

“…In contrast, Ikzf2 is specifically and permanently expressed in OHCs, and expression patterns of Ikzf2 and Prestin have perfect overlap. Moreover, it has been shown that ectopic Ikzf2 in IHCs can derepress Slc26a5 50,52 …”

“…Historically, besides cochlear OHCs, Prestin has been considered to be expressed in vestibular HCs, 47,48 but this is not supported by analyzing two knockin mouse strains Slc26a5 YFP /+ and Slc26a5 CreER /+ 23,24 . Recently, Prestin has been reported to be additionally expressed in cardiomyocytes, 49 but paradoxically, Slc26a5 DTR /+ mice treated with diphtheria toxin, survived 50 . Although such controversies are not yet completely resolved, we speculated that Slc26a5 is exclusively turned on in cochlear OHCs at perinatal ages, then permanently maintained afterward while being repressed or expressed at very low levels in other tissues.…”

“…23,24 Recently, Prestin has been reported to be additionally expressed in cardiomyocytes, 49 but paradoxically, Slc26a5 DTR/+ mice treated with diphtheria toxin, survived. 50 Although such controversies are not yet completely resolved, we speculated that Slc26a5 is exclusively turned on in cochlear OHCs at perinatal ages, then permanently maintained afterward while being repressed or expressed at very low levels in other tissues. This raises two key questions: (1) what factors are necessary to turn on Slc26a5 specifically in cochlear OHCs, on time?…”

“…In contrast, Ikzf2 is specifically and permanently expressed in OHCs, and expression patterns of Ikzf2 and Prestin have perfect overlap. Moreover, it has been shown that ectopic Ikzf2 in IHCs can derepress Slc26a5 50,52. Thus, it is more likely that Ikzf2 is involved in maintaining specific Prestin expression in cochlear OHCs, although direct biochemical evidence is needed to prove binding of Ikzf2 to Slc26a5 cis-regulatory elements, including 1.4 kbp enhancer or other unknown enhancers.…”

Fate‐mapping analysis of cochlear cells expressing Atoh1mRNA via a new Atoh13*HA‐P2A‐Cre knockin mouse strain

“…Atoh1 has been the focus of studies on HC development and regeneration since the importance of this gene was discovered 16,27,37,38,44 ; however, which cochlear cell lineages express Atoh1 has remained widely debated because distinct assays or mouse models have yielded divergent or contradictory results. The transgenic Atoh1Cre+ model supports that Atoh1 is also expressed in SGNs, 31 whereas Atoh1 is not detected in SGNs of Atoh1 Cre/+ knockin strains 45 .…”