Fig 1.
Validation of γ-crystallin antibodies in HEK293 cells.
HEK293 transiently transfected with either Crygd-flag (HEK293Crygd) (A) or Crygn-flag (HEK293Crygn) (B) expression constructs were labeled with antibodies against Cryg (α-Cryg), Crygd/e (α-Crygd/e), or Crygn (α-Crygn). α-Cryg and α-Crygd/e both detect Crygd, but not Crygn in immunocytochemistry, whereas α-Crygn detects only Crygn. (C-E) Similar results were obtained in immunoblot analysis where α-Cryg and α-Crygd/e both detect Crygd, whereas α-Crygn detects Crygn. Scale bar is 100 μm.
Fig 2.
Cryg-immunreactivity in the rat, mouse, and gerbil SOC at P4.
All image series show Cryg-ir (α-Cryg) in the first, VGluT1-ir (α-VGluT1) in the second, and an overlay of both immunoreactivities in the third column. This order applies also to subsequent Figs 3 to 5. (A) Cryg-ir is clearly seen in the MNTB and in the ventral acoustic stria of rat. (B) The MSO and a subpopulation of the LSO show also prominent Cryg-ir. (C,D) The mouse displays a weaker labeling in the SOC. (E-F) MNTB, LSO and MSO of the gerbil show no Cryg-ir above background. MNTB, medial nucleus of the trapezoid body; MSO, medial superior olive; LSO; lateral superior olive. MNTB, medial nucleus of the trapezoid body, designated by a asterisk MSO, medial superior olive, designated by a star; () LSO; lateral superior olive, designated by a diamond. The abbreviations and symbols also apply to subsequent figures. Dorsal is up and medial to the left. n = 3, scale bar is 100 μm.
Fig 3.
Crygd/e-ir in the rat, mouse, and gerbil SOC at P4.
(A) Crygd/e labeling is observed in the rat MNTB and fibers of the acoustic stria. (B) LSO and MSO display moderate Cryge-ir. In the mouse, MNTB (C), LSO and MSO (D) are also labeled. (E-F) The gerbil MNTB, LSO and MSO show Crygd/e-ir similar to background. Dorsal is up and medial to the left. n = 3, scale bar is 100 μm.
Fig 4.
Crygn-ir in the rat, mouse, and gerbil SOC at P4.
Crygn clearly labels the MNTB (A), the LSO and the MSO (B) of rat. (C) The mouse MNTB shows a moderate labeling and the LSO and MSO a weak immunoreactivity (D). (E) Gerbils show a similar pattern of Crygn-ir as the mouse with the MNTB being strongest labeled. Dorsal is up and medial to the left. n = 3, scale bar is 100 μm.
Fig 5.
Cryg-ir in the mouse SOC at P25.
All three antibodies against crystallins, i.e. Cryg (A,B), Crygd/e (C,D) or Crygn (E,F) gave no signals above background in the MNTB, the LSO and the MSO of mice aged P25. Dorsal is up and medial to the left. n = 3, scale bar is 100 μm.
Fig 6.
Generation of a spatially restricted Crygn knockout mouse in the auditory hindbrain.
(A) Scheme of the knockout strategy, consisting of crossing a mouse line with a floxed allele of exon 2 of Crygn (Crygn:tm1a (EUCOMM)) and the Egr2::Cre driver line. Primers for probing recombination are depicted as black arrows. (B) Validation of the spatial ablation in the SOC. Left side: Genotyping of the floxed Crygn locus. In wt, a 524 bp long PCR product is amplified, whereas the mutant locus results in a 207 bp product. Right site: Confirmation of recombination in the SOC. Upon recombination, a 604 bp long product is amplified. The non-recombined locus is 3,411 bp in length and not amplified under the PCR conditions used. (C) RNA in situ hybridization analysis in the MNTB. An RNA probe complementary to exon 2 yields only signals in MNTB section of control mice whereas no signal is observed in CrygnEgr2 mice. In contrast, an RNA probe complementary to exons 2–4 still yields signals in the MNTB of CrygnEgr2 mice. This indicates transcription of the truncated Crygn gene. Scale bar is 200 μm.
Fig 7.
Immunohistochemical and anatomical analysis of the SOC in CrygnEgr2 mice.
(A) Normal gross anatomy of the SOC in CrygnEgr2 mice. GlyT2 and VGluT1 immunoreactivity in coronal brainstem sections of P25 CrygnEgr2 mice and control litter mates indicate normal gross morphology of SOC nuclei. (B) Structural alterations in nuclei of the SOC. In CrygnEgr2 mice, the volumes of the LSO and MNTB were significantly decreased. The MNTB of CrygnEgr2 mice displayed also lower cells number but normal cross sectional area of neurons. At P4, MNTB cell number was not affected by the genotype. Volume and cell number were analysed in Nissl-stained serial sections of the respective nucleus (6 nuclei from 3 animals/genotype). As a test for statistical significance, a two-tailed student’s t-test was used. Color coding (black: control control litter mice; red: CrygnEgr2 mice). Dorsal is up and medial to the left. * p ≤ 0.01; ***p ≤ 0.001.
Fig 8.
Altered auditory brainstem responses (ABR) in CrygnEgr2 mice.
(A) Auditory brainstem response (ABR) thresholds for click, noise burst, and pure tone stimuli for control (white) and CrygnEgr2 mice (red) (n = 4 and 5 mice for controls and CrygnEgr2, respectively). No difference was observed for ABR thresholds in response to click stimuli (left) or noise burst stimuli (middle panel, n.s., not significant, p > 0.05). Threshold for pure tone stimuli were slightly but significantly better in CrygnEgr2 mice (p = 0.0279, 2-way ANOVA comparing the genotype). (B) Outer hair cell function measured by distortion product otoacoustic emission (DPOAE) growth function (left), maximal signal strength (middle, max. amplitude) and threshold for the 2f1-f2 distortion product (right). CrygnEgr2 mice had slightly improved 2f1-f2 distortion products indicated by increased amplitudes at 45 dB SPL stimulation (left, p = 0.1048, 2-way ANOVA) and small though non-significant improvements of DPOAE thresholds (right, p = 0.1013, 2-way ANOVA). This indicated intact outer hair cells and cochlear amplification in both genotypes. Arrows in left panel illustrate how threshold and amplitude were determined from the individual growth functions. (C) Processing of fast temporal modulation was measured by auditory steady state responses (ASSR) to amplitude modulated stimuli of increasing modulation speed (left, modulation frequency), as function of the modulation index (middle, modulation depth in %,) and for increasing level of the carrier (right, -20 to 60 dB hearing level, HL). For both genotypes, responses dropped for stimulation speeds above 1,024 Hz and were lost for faster modulation (2,048 Hz). Detection thresholds for modulated stimuli were at ca. 3–4% modulation, and response strength increased with carrier level. There was a tendency for CrygnEgr2 signals to level off at lower signal strength than the control (right panel, 55–60 dB hearing level), though this was not statistically significant. Insets schematically illustrate the used stimuli. (D) Changes of average peak amplitude for ABR wave I to IV (defined as peak to peak amplitudes, illustrated in the inset showing an example of an ABR recording with marked negative (n) and positive (p) peaks of either wave. The growth function shows significantly increased ABR amplitudes at wave IV at higher stimulus levels (> 50 dB, p < 0.001, 2-way ANOVA). Growth functions for ABR wave I and II amplitudes also were significantly changed, corroborating the results from the slightly improved ABR thresholds and DPOAE functions in CrygnEgr2 mice. Data for controls are shown as open bars, symbols and black lines, data for CrygnEgr2 mice are shown in red. Data represent mean and standard deviation (A,B) or standard error of the mean (C,D). The number of measured ears is indicated in each panel. n.s., not significant, *: p < 0.05, ***: p < 0.001, ****: p < 0.0001 in Holm-Sidak's multiple comparisons test. (*) indicates statistical results from uncorrected single comparison with p < 0.05 in 2-sided t-test.