Combinatorial Atoh1 and Gfi1 induction enhances hair cell regeneration in the adult cochlea

Abstract

Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits. Attempts to induce HC regeneration in adult mammals have used over-expression of the HC-specific transcription factor Atoh1, but to date this approach has yielded low and variable efficiency of HC production. Gfi1 is a transcription factor important for HC development and survival. We evaluated the combinatorial effects of Atoh1 and Gfi1 over-expression on HC regeneration using gene transfer methods in neonatal cochlear explants, and in vivo in adult mice. Adenoviral over-expression of Atoh1 and Gfi1 in cultured neonatal cochlear explants resulted in numerous ectopic HC-like cells (HCLCs), with significantly more cells in Atoh1 + Gfi1 cultures than Atoh1 alone. In vitro, ectopic HCLCs emerged in regions medial to inner HCs as well as in the stria vascularis. In vivo experiments were performed in mature Pou4f3^DTR mice in which HCs were completely and specifically ablated by administration of diphtheria toxin. Adenoviral expression of Atoh1 or Atoh1 + Gfi1 in cochlear supporting cells induced appearance of HCLCs, with Atoh1 + Gfi1 expression leading to 6.2-fold increase of new HCLCs after 4 weeks compared to Atoh1 alone. New HCLCs were detected throughout the cochlea, exhibited immature stereocilia and survived for at least 8 weeks. Combinatorial Atoh1 and Gfi1 induction is thus a promising strategy to promote HC regeneration in the mature mammalian cochlea.

Figure 1

Gfi1 enhances Atoh1-induced ectopic hair cell generation in neonatal cochlear explant cultures. Whole mounts of cultured explants stained for myosin VIIa photographed with epi-fluorescence showing the OC and adjacent areas (a,b) and lateral wall (a′,b′). Ectopic HCs (bracket) were induced on the medial side of the inner HC area by Atoh1 treatment (a). Larger numbers of ectopic HCs were observed after Atoh1 + Gfi1 treatment (b). Ectopic HC induction in the lateral area was evident in Atoh1 (a′) and Atoh1 + Gfi1 treatment (b′). Statistical analysis showed significantly more ectopic Myosin VIIa positive cells in the region medial to the OC after Atoh1 + Gfi1 treatment than after Atoh1 alone (c). I inner HC area, O outer HC area, Brackets ectopic HC. Scale bars represent 30 μm. Error bars are ± SD. Differences between groups were assessed via Student’s t test; (*) indicates p < 0.05.

Figure 2

Efficient HC ablation after DT injection and adenovirus scala media inoculation. Images show cochleae of adult mice 10 days after adenoviral inoculation of scala media with or without HC ablation. (a,a′,b,b′) Ad.Atoh1 adenovirus inoculation of wild-type (WT) mouse without DT-induced HC ablation. (c,c′,d,d′) Ad.Atoh1 adenovirus inoculation of Pou4F3^DTR mouse with simultaneous DT injection. (e,e′,f,f′) adenovirus Ad.Gfi1.Atoh1 inoculation of Pou4F3^DTR mouse with simultaneous DT injection. Dotted lines in a, b indicate where OHCs would be found in normal mice. In animals receiving only the injection surgery, most outer HCs are damaged and only a few OHCs remain in the most apical region, but inner HCs are largely intact. In contrast, all HCs both outer HCs and inner HCs, are ablated by simultaneous DT injection in both the Ad.Atoh1 group (c,d) and the Ad.Gfi1.Atoh1 group (e,f). All groups robustly expressed the tdTomato reporter (a′–f′). Myosin VIIa, Myosin VIIa. tdTom, tdTomato. Representative figures from 5 biological replicates for each group. Scale bar = 100 μm.

Figure 3

Gfi1 enhances Atoh1-induced hair cell regeneration in the adult cochlea. Images show cochleae of adult mice 4 weeks after DT-induced HC (HC) ablation and adenovirus injection into scala media. The tdTomato (tdTom) reporter is robustly expressed in OC epithelial cells in both cochlear turns (a). Mice that received Ad.Atoh1 had fewer Myosin VIIa positive cells in both apical and basal turn than mice that received Ad.Gfi1.Atoh1. Higher magnification images (b) show that Myosin VIIa-positive cells are large, variable in shape but usually round, and all co-express tdTom. Linear regression analysis (c) shows that the proportion of tdTom-positive cells that were also Myosin VIIa-positive were similar in the apex and base for both treatments and differed between treatments (see text for details). Scale bar = 100 μm.

Figure 4

Long term survival of hair cell-like cells. 8 weeks after transgene delivery, there were still numerous Myosin VIIa-positive/tdTomato-positive HCLCs in both treatment groups (a). Scatter plots showing counts of Myosin VIIa-positive and tdTomato-positive cells (b) demonstrate patterns similar to those seen at 4 weeks; namely, similar proportions tdTomato-positive cells that are Myosin VIIa-positive in the apex and base of each group, and a higher proportion of Myosin VIIa-positive/tdTomato-positive cells in the Ad.Gfi1.Atoh1 group than in the Ad.Atoh1 group. The total number of new Myosin VIIa-positive cells was significantly higher in the Ad.Gfi1.Atoh1 group than in the Atoh1-only group (c); although difference were not significant due to small sample size at the 8-week time point. Scale bar = 100 μm.

Figure 5

SEM of the organ of Corti in control and Ad.Gfi1.Atoh1 treated ears. SEM images taken 8 weeks after Ad.Gfi1.Atoh1 surgery and DT injection in control (contralateral) ear (a) or experimental ears (b–e). Contralateral ear showed no remaining inner and outer hair cell stereocilia. Microvilli on surface delineate rhomboidal shaped cell borders of Deiters cells in the reorganized reticular lamina (a). Ad.Gfi1.Atoh1 injected ear exhibits several round shaped cells bulging from the surface of the reticular lamina and displaying microvilli resembling stereocilia, in a cauliflower appearance (b). Some cells are displaying multiple thick kinocilia-like projections arranged in bundles (c). Cells with stereocilia throughout the surface with multiple long kinocilia (d). Some cells had stereocilia in a clustered form (e). Representative images from 3 biological replicates. Scale bar = 10 μm for (a–c), 5 μm for (d,e).

Figure 6

Nerve fibers near regenerated HCLCs. Samples collected at 4 weeks after adenovirus (Ad) and DT-induced HC (HC) ablation. Spiral ganglion neuron (SGN) fibers were visualized by Neurofilament 200 (NF200) staining. Myosin VIIa (MyoVIIa) and tdTomato (tdTom) double-positive cells were associated with nerve fibers along the cell body. Both groups—adenovirus with Atoh1 (Ad.Atoh1) and with Atoh1 + Gfi1 (Ad.Gfi.Atoh1) showed similar association with SGNs. Representative images are shown from 4 biological replicates for each group. Scale bar = 100 μm.