Figure 1

Cdk4 deletion in p19^−/− mice rescues hair cell (HC) survival and restores auditory function. (a) Whole-mount immunofluorescence of postnatal day 3 (P3) and 18 (P18) mouse cochlea using rabbit polyclonal antibody (sc-1063; Santa Cruz Biotechnology, Dallas, TX, USA) showing the early detection of p19 (red) at P3, and thereafter at P18 in all three rows of outer (OHCs) and one row of inner sensory HCs (IHC); actin is counterstained with phalloidin in green. Immunofluorescence experiments were performed as described.⁷ (b) Average thresholds of auditory brainstem recordings (ABRs) at pure tone frequencies 8, 16 and 32 kHz on P23-day-old p19^−/− (green; n=16) mice and their p19^−/−;Cdk4^−/− double knockout (brown; n=6) littermates, as well as their age-matched wild-type mice (blue; n=13) showing high thresholds at all frequencies in p19^−/− mice synonymous of a severe to profound hearing loss, and much lower thresholds for p19^−/−;Cdk4^−/− mice comparable to normal hearing wild-type mice. ABRs were performed as described.⁸ (c) Representative low- and high-magnification confocal micrographs of whole-mount immunofluorescence preparations of P3, P7, P12 and P23 cochlea from p19^−/− mice (green), and of P12 and P23 cochlea from p19^−/−;Cdk4^−/− mice (yellow). All preparations were stained with phalloidin to reveal actin, with the exception of one DAPI-stained P12 p19^−/−mouse cochlea panel (gray). In p19^−/− mice (green): (i) no HC death was detected at P3; (ii) sporadic HC death started to be detected at P7 around the mid-basal and basal turn of the cochlea; (iii) HC death and degeneration, and stereocilium bundle disorganization increased significantly at P12; and (iv) massive death of OHCs and IHCs, and damage of remaining HC stereocilium bundles is observable at P23. In p19^−/−;Cdk4^−/− mice (yellow), complete rows of OHCs and IHCs, and normal HC stereocilium bundles have been seen at all stages. Scale bars, 20 μm