Fig. 4: AAV8-GFAP-ATOH1 induced hair cell regeneration in the adult mouse utricle in vivo.

a 4-10-week-old mice were treated with IDPN (4 or 5 mg/g) at day 1, AAV8-GFAP-ATOH1 (1.71 × 10¹³ vg/mL) were injected at day 2 or 15. Organs were examined at 2 weeks and 1 month after injection. b, c” Treated utricles had more Myosin7a+ (red) hair cells compared to contralateral ear. d, d’ High magnification images of injected and contralateral ears (from b and b”). GFP+ hair cells (arrowhead) were observed after GFAP-ATOH1 injection. e, f” Crista ampullaris from injected ear has more Myosin7a+ (red) hair cells compared to contralateral ear. g, h GFAP-ATOH1 significantly increased Myosin7a+ (red) hair cells compared to contralateral ear. i, j’ Utricles from injected ear had more hair bundles (phalloidin, green) than contralateral ear. i’, j’ High magnification pictures in boxes. k Phalloidin-labeled bundles increased significantly in utricles from injected ear compared to contralateral controls. l–n Representative images of bundles less than 6 µm, between 6 µm and 12 µm, and more than 12 µm. o Measurements of bundle height. p–r Expression of Ctbp2+ puncta (gray) on the basolateral surfaces of utricular hair cells from naive, injected ear and contralateral ear. Insets are the 3D view of hair cells at 1 month. s Quantification of Ctbp2+ puncta in Myosin7a+ (green) hair cells. Number of Ctbp2+ puncta per hair cell increased significantly in contralateral ear compared to naive and injected ear. Data shown as mean ± S.D, compared using two-tailed paired Student’s t tests and ordinary one-way ANOVA. p = 0.0033 and 0.005 in extrastriola and striola, respectively in (g), p < 0.0001 and =0.0003 in extrastriola and striola, respectively in (h), p = 0.0110 in (k), p = 0.002 between naive and injected ear in striola and p < 0.0001 for all the others in (s). n = 8 at 2 weeks and =10 at 1 month in (g) and (h). n = 4 in (k). n = 114 and 79 bundles from injected and contralateral ears in (o). n = 36 cells from 3 naive mice ear, 72 cells from 6 injected mice ear and 72 cells from 6 contralateral mice ear in (s). Scale bars = 100 μm in (b–f”, i, j), 20 μm in (d, d’, p–r), 10 μm in (i’, j’), 2 μm in (l–n), 5 μm in (p–r) inserts. Source data are provided as a Source Data file.