We have achieved inducible, ectopic Atoh1-HA expression specifically in two subtypes of postnatal cochlear SCs, PCs and DCs, that reside directly underneath HCs within the organ of Corti. A fraction (6–11%) of neonatal and juvenile Atoh1-HA+ PCs and DCs were converted into HCs that first activated endogenous Atoh1 and subsequently expressed 11 HC and synaptic markers (myosin VI, myosin VIIa, calbindin, parvalbumin, calretinin, Lhx3, KCNQ4, α9 AChR, synaptogamin1, synaptophysin and CSP), migrated up into the HC layer, formed stereocilia, had MET channels, and survived for at least 2 months in vivo. However, newly generated HCs lacked oncomodulin and prestin expression, the morphology of mature OHCs (cylinder cell body shape and characteristic “V” shaped stereocilia), and likely remained at an intermediate or immature differentiation stage. In contrast, ectopic Atoh1-HA expression cannot convert adult PCs and DCs into immature HCs even after endogenous OHC damage. We also found that permanent Atoh1 expression did not block initiation of prestin expression in endogenous HCs but somehow subsequently resulted in cell loss of both IHCs and OHCs. Taken together, our data suggest that combination of Atoh1 expression with other factors is needed to convert PCs and DCs (especially at adult ages) into fully differentiated, functional HCs.
Because Cre activity is 100% specific to PCs and DCs within the organ of Corti in the Prox1CreER+; Atoh1-HA+ model, we could confirm, at single cell resolution, that these converted new HCs were derived from PCs and DCs, using Atoh1-HA expression as a lineage tracer. However, at juvenile ages, the Cre activity of Fgfr3iCreER is not 100% specific to PCs and DCs with 2% of Cre activity in OHCs (primarily in apical turns); therefore, it remains possible, albeit very low, that a few Atoh1-HA+/calbindin+ new HCs might be endogenous OHCs. We believe that the majority of Atoh1-HA+/calbindin+ new HCs are derived from PCs and DCs based on prestin expression and quantitative analysis. Specifically, new HCs derived from PCs and DCs never expressed oncomodulin or prestin ( and ), whereas endogenous OHCs with permanent Atoh1-HA expression expressed prestin (). Therefore, these Atoh1-HA+/calbindin+/prestin-negative HCs in OHC regions of Fgfr3iCreER+; Atoh1-HA+ mice are derived from PCs or DCs. In addition, quantitatively in each 160 μm cochlear length, the number (11 ± 2, n=4) of Atoh1-HA+/calbindin+ cells was much higher than that of OHCs (1 ± 1, n=3) traced by reporter gene EGFP in the Fgfr3iCreER+; CAG-loxP-stop-loxP-EGFP+ mice. Cre activity of Fgfr3iCreER+ was also detected in a very small fraction of Hensen’s and Claudius cells (). When we defined and quantified new HCs, Atoh1-HA+/calbindin+ cells in regions where Hensen’s and Claudius cells are located () were not included.
Although Atoh1 is required for HC fate commitment and possibly the differentiation process (Bermingham et al., 1999
; Pan et al., 2011
), not all Atoh1 expressing cochlear progenitors develop into HCs (Woods et al., 2004
; Matei et al., 2005
; Yang et al., 2010a
) and Atoh1-null HCs can be formed in the mosaic Atoh1 deletion model (Du et al., 2007
). Here, we also noticed heterogeneity among SCs which express Atoh1-HA, since the majority (89–94%) of Atoh1-HA+ SCs remained as SCs. Using the Atoh1-EGFP+ transgenic line, we showed that the Atoh1 enhancer region (or the endogenous Atoh1 gene) must be reactivated for a SC to become a HC. Within the organ of Corti, we saw that only those Atoh1-HA+ SCs which activated the Atoh1 enhancer became new HCs, while the rest maintained their original SC fate. In the future it will be interesting to investigate this heterogeneity at the single cell level. Such studies may provide the key for unraveling the long-standing mystery of how Atoh1 dictates cell fate determination (Pan et al., 2012
Atoh1 is believed to be maximally expressed at embryonic ages and declines rapidly within the first week after birth when HC maturation is far from completion (Woods et al., 2004
; Chow et al., 2006
), suggesting that Atoh1 is primarily required for HC fate commitment and/or initial differentiation of immature HCs. Such a notion is further supported by our result that conversion of SCs into HCs recapitulates normal HC development, but new HCs remain immature in our models. In addition, the new HCs derived from GER cells by Atoh1 ectopic expression seem immature (Zheng and Gao, 2000
). Does permanent ectopic Atoh1 expression cause the lack of prestin expression in our models? To test this, we permanently expressed Atoh1-HA in embryonic endogenous HCs in Gfi1Cre/+; Atoh1-HA+
mice and found that prestin was present in surviving OHCs at P14 (), which suggests that the absence of prestin in PC-and DC-derived new HCs was not caused by permanent Atoh1 expression. Instead, it is more likely that additional unknown transcription factors are needed for prestin expression and maturation of OHCs. Nonetheless, the significant HC loss in Gfi1Cre/+; Atoh1-HA+
mice suggests that permanent Atoh1-HA expression is detrimental to mature, adult HCs. Interestingly, PC- and DC-derived new HCs survived until adult ages, which is further evidence that these new HCs are immature since our data suggest that only adult, fully differentiated HCs are sensitive to permanent Atoh1 expression.
Converting one cell fate to another by overexpression of key transcriptional factors is a general and powerful approach for regenerative medicine (Cohen and Melton, 2011
). SCs in non-mammalian vertebrates are the source for regenerating HCs and Atoh1
is reactivated in the process of HC regeneration (Cafaro et al., 2007
). To endow mammals the capacity to regenerate HCs, similar to a recent study in the pancreas (Yang et al., 2011
), we tested the effects of ectopic expression of a single transcription factor, Atoh1, in postnatal PCs and DCs. Our data suggest that Atoh1 can convert neonatal and juvenile, but not adult, PCs and DCs to adopt a HC fate, thus demonstrating that Atoh1-mediated SC-to-HC conversion efficiency is age-dependent. Moreover, newly generated HCs recapitulated the normal HC developmental program, but failed to become fully differentiated. Thus, the combination of ectopic expression of Atoh1 and other factors that normally control HC terminal differentiation, especially those that can turn on prestin, may be valid approaches for the regeneration of fully differentiated, functional new HCs in vivo