In Vivo Interplay between p27Kip1, GATA3, ATOH1, and POU4F3 Converts Non-sensory Cells to Hair Cells in Adult Mice

Abstract

Hearing loss is widespread and persistent because mature mammalian auditory hair cells (HCs) are nonregenerative. In mice, the ability to regenerate HCs from surrounding supporting cells (SCs) declines abruptly after postnatal maturation. We find that combining p27^Kip1 deletion with ectopic ATOH1 expression surmounts this age-related decline, leading to conversion of SCs to HCs in mature mouse cochleae and after noise damage. p27^Kip1 deletion, independent of canonical effects on Rb-family proteins, upregulated GATA3, a co-factor for ATOH1 that is lost from SCs with age. Co-activation of GATA3 or POU4F3 and ATOH1 promoted conversion of SCs to HCs in adult mice. Activation of POU4F3 alone also converted mature SCs to HCs in vivo. These data illuminate a genetic pathway that initiates auditory HC regeneration and suggest p27^Kip1, GATA3, and POU4F3 as additional therapeutic targets for ATOH1-mediated HC regeneration.

Keywords: aging; cancer; cochlea; development; differentiation; hearing; proliferation; regeneration; sensory.

Figure 1

Co-manipulation of p27 and ATOH1 converts SCs to HCs. Little to no co-expression of HA and POU4F3 was observed in Fgfr3iCreER+;Atoh1HA+ (Atoh1HA+) samples (A), while numerous HA+ cells were POU4F3+ in CAP27 mice (B). Some HA-negative SCs were also seen to upregulate POU4F3 (arrowheads). Little to no co-expression of HA and MYO6 was observed in Atoh1HA+ samples (C), but many HA+ cells were MYO6+ (arrows) in CAP27 samples (D). Little to no co-expression of HA and PVALB was observed in Atoh1HA+ samples (E), while several HA+ cells were PVALB+ (arrows) in CAP27 samples (F). Little to no co-expression of HA and Calb was observed in Fgfr3iCreER+;Atoh1HA+ samples (G), but HA and Calb double-positive cells (arrows), as well as Calb-negative cells (asterisk) were readily observed in CAP27 samples (H). Scale bars = 20 μm

Figure 2

Converted cells represent different stages of HC maturation, but fail to terminally differentiate. Significantly more POU4F3+ PCs & DCs were observed in CAP27 mice as compared to Fgfr3iCreER+;Atoh1HA+ (Atoh1HA+) littermates (A). Significantly more MYO6+ PCs & DCs were observed in CAP27 mice than in Atoh1HA+ littermates (B). Significantly more PVALB+ PCs & DCs were observed in CAP27 mice than in Atoh1HA+ littermates (C). Significantly more Calb+ PCs & DCs were observed in CAP27 mice than in Atoh1HA+ littermates (D). Co-expression of HA and the mature HC marker prestin was not observed in any of the CAP27 mice, not even in cells that were co-labeled for ATOH1HA and MYO7A (arrow). Data are presented as mean ± 1 S.E.M. ***p ≤ 0.001, *p < 0.05, scale bar = 20 μm.

Figure 3

Converted cells have hair bundles and recruit neuronal fibers. Co-labeling of HA, MYO6, and phalloidin (Phall) demonstrates a converted SC that has actin bundles (A, A′). The first panel provides an orthogonal view (Ortho), while the second and third panels show a top view. A′ is an enlarged image of the white square in the third panel of A. SEM images reveal ectopic hair bundles (artificially colored magenta) with atypical morphologies (B–F). Some cells exhibited one or even two kinocilia (arrows) (F and B &D, respectively). Some HCs appeared to be dying and possibly extruded from the membrane (asterisks, C) though whether these were converted cells or existing HCs was indeterminate. (G) Tuj1+ terminals were readily observed underneath endogenous HCs (arrows), and seen to contact HA & MYO6 double positive cells (arrowhead). (H) A partial projection image revealed a Tuj1+ nerve fiber stretching across the tunnel of Corti to contact an HA and MYO7A double-positive cell. (I) An HA+ cell that is also PVALB+ is contacted by a NF-H+ neurite. Scale bars = 5 μm

Figure 4

GATA3 expression positively correlates with SC responsiveness to ATOH1. GATA3 is widely distributed in neonatal cochlear SCs (A & B), but is selectively lost from PCs & DCs by P30 (C & D). IPh/BCs and Hensen cells remain positive for GATA3 at P30 (D). Ectopic ATOH1 in Fgfr3iCreER+;Atoh1+ (Atoh1+) mice does not alter GATA3 expression (E). p27CKO, either in conjunction with ATOH1HA (F), or independently (G), causes a significant increase in GATA3+ PCs & DCs as compared to ectopic ATOH1HA alone (H). Data are presented as mean ± 1 S.E.M. *** p < 0.01. Scale bars = 20 μm

Figure 5

GATA3 promotes ATOH1-mediated conversion of SCs to HCs. A diagram of the typical pattern of GATA3 expression in the mature organ of Corti (A). IPC = inner pillar cell, OPC = outer pillar cell. A representative image of GLAST-CreER+;R26tdTomato+ activity in IPh/IB cells one week after Tamox induction at P28 (B). Ectopic ATOH1HA results in the upregulation of POU4F3 (C) and MYO7A (D) in IPh/IB cells (arrows). POU4F3 (E), MYO6 (G), and PVALB (I) are upregulated in PCs & DCs (arrows) of Fgfr3iCreER+;Atoh1HA+;GATA3+ mice. Co-manipulation of GATA3 and ATOH1HA (GATA3+) resulted in significantly more POU4F3+ (F), MYO6+ (H), and PVALB+ (J) SCs than ectopic ATOH1HA alone (GATA3−). Data are presented as mean ± 1 S.E.M, *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars = 20 μm

Figure 6

Ectopic hPOU4F3 causes upregulation of HC-specific markers in mature PCs & DCs. Tamox induction at P12–P13 did not result in any mCherry or hPOU4F3 expression in PCs or DCs of control mice (Fgfr3iCreER+) at P35 (A). Fgfr3iCreER+;POU4F3-mCherry mice exhibited robust expression of mCherry and POU4F3 in PCs & DCs (B). When Fgfr3iCreER+;POU4F3-mCherry mice were induced with Tamox at P28, several mCherry+ cells upregulated the HC markers MYO7A (C) and PVALB (D) by P49. Combined POU4F3-mCherry and ATOH1HA expression also resulted in MYO7A (E,F) and PVALB (G,H) expression in a number of PCs & DCs. The number of MYO7A and mCherry double-positive cells per cochlea was significantly higher in Fgfr3iCreER+;Atoh1HA+;POU4F3-mCherry+ samples than in Fgfr3iCreER+;POU4F3− mCherry+ samples ***p = 0.001 (F). The number of PVALB and mCherry double positive cells per cochlea was significantly higher in Fgfr3iCreER+;Atoh1HA+;POU4F3-mCherry+ samples than in Fgfr3iCreER+;POU4F3mCherry+ samples, **p < 0.01 (H). Data are presented as mean ± 1 S.E.M. Scale bars = 20 μm