Age-dependent in vivo conversion of mouse cochlear pillar and Deiters' cells to immature hair cells by Atoh1 ectopic expression

Abstract

Unlike nonmammalian vertebrates, mammals cannot convert inner ear cochlear supporting cells (SCs) into sensory hair cells (HCs) after damage, thus causing permanent deafness. Here, we achieved in vivo conversion of two SC subtypes, pillar cells (PCs) and Deiters' cells (DCs), into HCs by inducing targeted expression of Atoh1 at neonatal and juvenile ages using novel mouse models. The conversion only occurred in ∼10% of PCs and DCs with ectopic Atoh1 expression and started with reactivation of endogenous Atoh1 followed by expression of 11 HC and synaptic markers, a process that took approximately 3 weeks in vivo. These new HCs resided in the outer HC region, formed stereocilia, contained mechanoelectrical transduction channels, and survived for >2 months in vivo; however, they surprisingly lacked prestin and oncomodulin expression and mature HC morphology. In contrast, adult PCs and DCs no longer responded to ectopic Atoh1 expression, even after outer HC damage. Finally, permanent Atoh1 expression in endogenous HCs did not affect prestin expression but caused cell loss of mature HCs. Together, our results demonstrate that in vivo conversion of PCs and DCs into immature HCs by Atoh1 is age dependent and resembles normal HC development. Therefore, combined expression of Atoh1 with additional factors holds therapeutic promise to convert PCs and DCs into functional HCs in vivo for regenerative purposes.

Figure 1.

Reprogramming of neonatal PCs and DCs into HCs by ectopic Atoh1-HA expression in Prox1^CreER/+;Atoh1-HA+ mice. A, Cross-section of a P6 wild-type mouse cochlea. HCs are labeled by myosin VI (red), SCs by Sox2 (green), and nuclei by Hoechst (blue). B, Illustration of the strategy to induce ectopic Atoh1-HA expression. C, D, Mice of the indicated genotypes received tamoxifen (TMX) at P0 and P1 and were analyzed at P22. Triple staining of HA (green), prestin (red), and myosin VI (pink) at P22 in control (Ctrl) (C) and experimental mice (D). Arrowheads in D and its inset are the same two newly generated HCs. Hen, Hensen's cells; Clau, Claudius cells. Scale bars: 20 μm.

Figure 2.

Reactivation of multiple HC markers in newly generated HCs. A, B, Mice with the indicated genotypes received tamoxifen (TMX) at P0 and P1 and were analyzed at P22. Double labeling of HA (white) and parvalbumin (red) in control (A) and experimental mice (B). Arrowheads in B are two new HCs. Inset in B shows Atoh1-HA+/parvalbumin-negative PCs/DCs that remained in the lower SC nuclear layer. Double labeling of HA (white) and calbindin (red) at P22 in control (C) and experimental mice (D) is shown. Arrowheads in D are two newly generated HCs. Arrow in D points to the regions where endogenous OHCs are missing. E, Quantification of the new HCs at P22 and P60 in the entire cochlea (*p < 0.05 as determined by a Student's t test). Scale bars, 20 μm.

Figure 3.

Expression of additional HC and synaptic markers. Mice of the indicated genotypes received tamoxifen (TMX) at P0 and P1 and were analyzed at P22. A, B, Double staining of HA (white) and calretinin (red) in control (A) and experimental (B, B′) mice at P22. B is calretinin alone and B′ is the merged image. C–F′, Triple staining of HA (white), calbindin (green), and KCNQ4 (red) in control (C, D) and experimental (E–F′) mice at P22. E is KCNQ4 alone and E′ is the merged image. F is KCNQ4 alone and F′ is the merged image. E–F′ show the same cell visualized at confocal Y–Z plane (E, E′) and X–Y plane (F, F′) to better show the KCNQ expression pattern. G–L′, Costaining of α9 AChR-EGFP (green) and HA (white) in control (G) and experimental (H, H′) mice at P22. H is α9 AChR-EGFP alone and H′ is the merged image. Triple staining of HA (white), α9 AChR-EGFP (green), and CSP (red) in control (I) and experimental (J, J′) mice at P22. J is CSP alone and J′ is the merged image. Triple staining of HA (white), α9 AChR-EGFP (green), and synaptophysin (red) in control (K) and experimental (L, L′) mice at P22 is shown. L is synaptophysin alone and L′ is the merged image. All images were visualized in the confocal Y–Z plane, except D, F, and F′, which were imaged in the X–Y plane. Scale bars: 20 μm (A, C, G, I, K), 10 μm (B′, E′, H′, J′, L′), and 5 μm (D).

Figure 4.

New Atoh1-HA+ HCs have stereocilia bundles and MET channels. Prox1^CreER/+;Atoh1-HA+ mice received tamoxifen (TMX) at P0 and P1 and were analyzed at P60. A–A″, Triple staining of phalloidin (actin, green), HA (white), and parvalbumin (red) in experimental mice at P60. A was visualized in the confocal X–Y plane, while A′ and A″ are in the X–Z plane. Arrow in A labels the new HC imaged at high magnification in A′ without obvious actin signal. Arrowhead in A labels the new HC imaged at high magnification in A″ with intensive actin signal. B–B⁗, Triple staining of FM4-64 FX (red), HA (green), and parvalbumin (white) in experimental mice at P60. Arrowheads label one new HC that had taken up FM4-64 FX dye, which suggests the presence of MET channels. Nuclei are labeled by Hoechst (blue). C, Summarized reprogramming efficiency of Atoh1-HA in PCs/DCs. Scale bars: 10 μm (A′) and 20 μm in (A, B″).

Figure 5.

Ectopic Atoh1-HA expression reprogrammed juvenile PCs and DCs into HCs. Mice of the indicated genotypes received tamoxifen (TMX) at P12 and P13 and were analyzed at P21 (A–E) or P42 (F–H⁗). A–D, Double staining of EGFP (green) and calbindin (red) at HC layer (A), DC layer (B), outer PC layer (OPC) (C), and inner PC layer (IPC) (D) at P21. Nuclei are labeled by Hoechst (blue). E, Quantification of EGFP+ cells among different cell types. Data are presented as mean ± SEM. F–G⁗, Triple staining of HA (green), calbindin (blue), and prestin (red) in control (F--F⁗) and experimental (G–G⁗) mice at P42. Arrows in G–G⁗ point to one new HC. Inset in G shows Atoh1-HA+ PCs and DCs that maintain the SC fate and reside at the SC layer. Many endogenous prestin+ OHCs are missing in experimental mice, while IHCs were normal (G–G′). H–H⁗, Triple staining of HA (red), Lhx3 (green), and parvalbumin (blue) in experimental mice at P42. Arrows in H–H⁗ point to one new HC. Arrowheads in H′ and H⁗ point to one endogenous Lhx3+ HC. Note that the endogenous HCs have parvalbumin expression, but it is expressed at a much lower level than in new HCs. Scale bars, 20 μm.

Figure 6.

Ectopic Atoh1-HA expression cannot convert adult PCs and DCs into immature HCs in Fgfr3^iCreER+;Atoh1-HA+ mice. A–E, Fgfr3^iCreER+;Rosa26-CAG-tdTomato^Loxp/+ mice received tamoxifen (TMX) at P30 and were analyzed at P39. Double staining of tdTomato (endogenous epifluorescence; red) and myosin VIIa (green) at the HC layer (A), DC layer (B), outer PC layer (OPC) (C), and inner PC (IPC) layer (D) at P39. E, Quantification of tdTomato+ cells among different cell types. F–H″, Fgfr3^iCreER+;Atoh1-HA+ mice were injected with tamoxifen (TMX) at P30 and analyzed at P60. F, Low-magnification cochlear whole mount image with triple staining of HA (green), calbindin (pink), and Sox2 (red) in experimental mice at P60. High-magnification images of the square area in (F) at the HC layer (G) and SC nuclear layer (H, H″) are shown. Neither OHC loss nor new HCs were found in the HC layer (G). Using inner PCs as an example, all Atoh1-HA+ cells expressed Sox2 and remained in the SC layer (H–H″). I–K, Fgfr3^iCreER+;Atoh1-HA+ mice were injected with kanamycin at P30 and TMX at P33 and analyzed at P63. I, Low-magnification cochlear whole mount image double stained of HA (green) and calbindin (pink) after OHC damage in experimental mice at P63. High-magnification images of the square area in I taken at the HC layer (J) and the SC nuclear layer (K). Arrow in J labels a single OHC that remained. All Atoh1-HA+ PCs and DCs were found in the SC layer (K) and did not express calbindin. Scale bars: 200 μm (A, D); 20 μm (B, C″, E, F).

Figure 7.

Reactivation of endogenous Atoh1 in Atoh1-HA+ cells. A–B′, Mice of the indicated genotypes received tamoxifen (TMX) at P12 and P13 and were analyzed at P15. Images taken from cochlear samples stained with EGFP (green), HA (pink), and calbindin (red) in control (A–A⁗) and experimental (B–B⁗) mice at P15 are visualized in the confocal Y–Z plane. Arrowheads (B–B⁗) label the same Atoh1-HA+/EGFP+ PC that was calbindin negative, and its nucleus resided at the bottom of the cell body. Arrows in A and B are IPHs with EGFP expression in both the control and experimental group. The endogenous HCs were also EGFP+. Scale bar, 20 μm.

Figure 8.

Reactivation of endogenous Atoh1 precedes differentiation of new HCs in Fgfr3^iCreER+;Atoh1-HA+;Atoh1-EGFP+ mice. A–D⁗, Fgfr3^iCreER+;Atoh1-HA+;Atoh1-EGFP+ mice received tamoxifen (TMX) at P12 and P13 and were analyzed at P19 (A–B⁗) and P42 (C–D⁗). Triple staining of HA (white), EGFP (green), and calbindin (pink). B–B⁗, Images were visualized at the confocal Y–Z plane through the vertical yellow dashed line in A. Yellow arrows point to an Atoh1-HA+/EGFP+ PC that did not turn on calbindin, and its nucleus was migrating into the HC layer. White arrows pointed to endogenous IPH cells. D–D⁗, Images were visualized at the confocal Y–Z plane through the vertical yellow dashed line in C. Arrowheads point to an Atoh1-HA+/EGFP+/calbindin+ new HC. Arrows point to an Atoh1-HA+/EGFP-negative/calbindin-negative cell (with low level of Atoh1-HA expression). Scale bars: 200 μm (A, C); 20 μm (B–B⁗, D–D⁗).

Figure 9.

Effects of permanent Atoh1 expression in endogenous differentiating HCs of Gfi1^Cre/+;Atoh1-HA+ mice. A–A⁗, Double labeling of HA (green) and myosin VI (red) in cochlear samples from Gfi1^Cre/+;Atoh1-HA+ mice at P3. All Atoh1-HA+ HCs appeared normal. B–E⁗, Triple labeling of HA (green), prestin (red), and calbindin (blue or pink) in the basal (B–C⁗) and middle/apical (D–E⁗) turns of the cochlea at P14. B and D are images showing calbindin channel alone. C″ and E″ are images showing calbindin in pseudo blue color. Many HCs are lost in the basal turn. a, Apical turn; m, middle turn; b, basal turn. Scale bars, 200 μm (A″, B, D), 20 μm (A⁗, C⁗, E⁗).

Figure 10.

Loss of mature HCs in Gfi1^Cre/+;Atoh1-HA+ mice. A, B, Calbindin (pink) and HA (green) double staining in cochlear samples from control (A) and experimental (B) mice at P35 is shown. C, Auditory brainstem response test comparing Gfi1^Cre/+;Atoh1-HA+ (blue line) and Gfi1^Cre/+ control (black line) mice. **p < 0.01 for all frequencies as determined by a two-way ANOVA followed by a Student's t test with a Bonferroni correction. Scale bar, 20 μm.