Journal: PLoS Biology

Article Title: Mouse screen reveals multiple new genes underlying mouse and human hearing loss

doi: 10.1371/journal.pbio.3000194

Figure Lengend Snippet: ABR thresholds of Klc2 mutants are close to those of controls at 2 weeks old, and show significant, progressive increases in thresholds from one month onwards, mainly at low frequencies (3–18 kHz), increasing to affect higher frequencies by 6 months old (A-E) (wild types 2 weeks n = 4, 1 month n = 13, 2 months n = 13, 3 months n = 9, 6 months n = 15; Klc2 homozygotes 2 weeks n = 6, 1 month n = 13, 2 months n = 13, 3 months n = 13, 6 months n = 18; Mann–Whitney U test, at 2 weeks old, p = 0.12 overall but p = 0.019 at 3 kHz and p = 0.01 at 6 kHz when testing stimuli separately; p < 0.001 at each later age shown by asterisks). F. DPOAEs at 6 months old show mutant amplitudes (red, n = 3) at the noise floor (grey) across all frequencies tested; wild types (green, n = 3) show normal emission amplitudes. Gi-vi. Scanning electron microscopy revealed extensive loss of OHC hair bundles at P28 in the cochlear regions, corresponding to the worst thresholds in mutants (12 kHz; Gi. heterozygote n = 4, Gii. homozygote n = 11), while there was little sign of hair cell loss at higher-frequency regions (42 kHz; Giii. heterozygote, Giv. homozygote). Remaining hair bundles had a normal appearance (Gv. heterozygote, Gvi. mutant). Scale bars, g-j, 10 μm; k-l, 1 μm. Hi-iv. Confocal imaging at P28 showed that many OHC nuclei were missing in the most affected regions (12 kHz; Hi. heterozygote n = 4, Hii. homozygote n = 5), but most hair cell nuclei were present at less affected regions (30 kHz; Hiii. heterozygote, Hiv. homozygote). Blue, DAPI-labelled nuclei; red, CtBP2-labelled ribbons and IHC nuclei; green, neurofilament-labelled unmyelinated dendrites. Scale bars, 10 μm. Hv. Quantification of OHC nuclei from confocal images demonstrated significant reduction in mutants (red) at best-frequency regions from 6 to 24 kHz and no significant difference with controls (green) at the 30-kHz region. Black line represents ABR threshold elevation in mutants compared with littermate controls. I. EPs in wild types (green) and homozygotes (red) show no significant difference in mutants (homozygotes 113.8 ± 11.5 mV, n = 10; wild-type littermates 116.0 ± 6.5 mV, n = 10; t = 0.518, df = 14, two-tailed p -value = 0.613). Maximum negative potentials during anoxia are significantly reduced in homozygotes (lower part of plot) (homozygotes −10.0 ± 5.39 mV, n = 6; wild-type littermates −33.7 ± 4.8 mV, n = 6; t = −8.050, df = 10, two-tailed p -value = 0.0000111). Ji-vi. Confocal imaging of IHCs at P28 showed no obvious abnormalities of GluR2-labelled postsynaptic densities (green), but less extensive Kcnma1-labelled patches (red) of IHCs in mutants compared with controls at the frequency regions showing the worst thresholds (6 and 12 kHz) ( n = 7 homozygotes, 5 littermate controls). Scale bars, 5 μm. Jvii. Counts of green-labelled GluR2 puncta per IHC show no difference between mutants and wild types (6 kHz, homozygotes n = 7, wild types n = 5, t test, p = 0.7422; 12 kHz, homozygotes n = 7, wild types n = 5, t test, p = 0.0737; 30 kHz, homozygotes n = 5, wild types n = 6, t test, p = 0.7089). Ki. Representation of the allele, with exons in grey, FRT sites in green, loxP sites in red, and lacZ and neo components of the inserted construct labelled. Kii-iii. Expression of Klc2 in the cochlear duct of a heterozygote ( n = 3) using the LacZ reporter system in the allele. Blue-labelled areas show expression in cells surrounding the cochlear duct and spiral ganglion. Kiii shows a higher magnification of the organ of Corti. Scale bar on Kii, 100 μm, on Kiii, 50 μm. Li-ii. Confocal images of the organ of Corti in a wild type (left) and homozygote (right) at P28 labelled with Myo7a antibody (false-coloured green) showing hair cell bodies and DAPI (false-coloured red) showing nuclei. Small images at left and right show the areas marked in white boxes rotated through 90° around the radial cochlear axis to show a mid-modiolar view of the hair cells. Nuclei appear in similar locations in mutants and controls (base of OHCs, middle of IHCs). Scale bars, 5 μm. M. qRT-PCR of Klc2 mRNA from brain at P28 showed complete knockdown of transcript in homozygotes (red, n = 3); heterozygotes (blue, n = 4) showed around half of the wild-type level (green, n = 2). All plots are means ± standard deviation. Plotted data points are given in . ABR, auditory brainstem response; DPOAE, distortion product otoacoustic emission; EP, endocochlear potential; FRT, flippase recombinase target; IHC, inner hair cell; lacZ , gene encoding β-galactosidase; loxP, locus of crossover in P1 bacteriophage; OHC, outer hair cell; P28, postnatal day 28; qRT-PCR, quantitative real-time PCR.

Article Snippet: The primary antibodies used overnight at RT were mouse anti-GluR2 (1:200, MAB397, Emd Millipore), rabbit anti-Kcnma1 (1:100, APC-021, Alomone), rabbit anti-Myo7a (1:200, PTS-25-6790-C050, Axxora), mouse anti-Ctbp2 (1:400, BD Transduction Laboratories 612044), and chicken anti-NF-H (1:800, Abcam ab4680).

Techniques: MANN-WHITNEY, Mutagenesis, Electron Microscopy, Imaging, Two Tailed Test, Construct, Expressing, Quantitative RT-PCR, Standard Deviation, Real-time Polymerase Chain Reaction