TSK-knockout (KO) mice
TSK-KO C57BL/6 J mice (Mus musculus domesticus) were generated by inserting a LacZ/Neo cassette into the TSK-coding exon, as described previously . Wild-type littermates by genotyping were used as CONT. Heterozygous littermates by genotyping were used for LacZ expression experiments. Mice were housed in an air-conditioned room, maintained at about 25 °C and 50% humidity. Standard commercial pellet diet and water were available ad libitum. The day at which vaginal plug was observed was designated as E0.5 at noon.
Embryo heads were dissected and fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 12 h at 4 °C. For postnatal assessments, mice were anesthetized by intraperitoneal injection of 4 mg/kg xylazine (Bayer, Shawnee Mission, KS, USA) and 120 mg/kg ketamine-HCl (Daiichi Sankyo, Tokyo, Japan) in 0.9% NaCl, and fixed by cardiac perfusion of 4% PFA in PBS. Subsequently, inner ears were dissected from temporal bones and decalcified in 0.5 M EDTA (Wako, Osaka, Japan) for 3 days at 25 °C. Cochleae were embedded in OCT (Sakura Finetek Japan, Tokyo, Japan) and serially sectioned at 12 μm thickness by a cryostat.
TSK expression was investigated throughout the developmental stages of the inner ear by staining for LacZ in mice heterozygous for LacZ insertion in TSK. In particular, the inner ear was examined at embryonic stages E9.5, E11.5, E13.5, and E15.5, as well as at P0, P10, and P30 (each stage, n = 5). Slides were dried for 30 min at 25 °C, stained with 400 mg/mL X-gal (Genlantis, San Diego, CA, USA) for 18 h at 37 °C, and imaged at 1360 × 1024 pixels using a BZ-9000 microscope (Keyence, Osaka, Japan) using uniform photographic exposure parameters.
Sections were blocked with 10% (v/v) normal goat serum in PBS containing 0.1% Triton X-100 (IBI Scientific, Peosta, IA, USA) for 10 min at 25 °C and labeled using rabbit antibody against acetylated α-tubulin (Tuj1; 1:500, COVANCE, Princeton, NJ, USA) and mouse antibody against Sox2 (1:200, Cell Signaling, CA, USA) in a humidified chamber for 1 h at 25 °C. Subsequently, sections were washed and incubated with goat anti-mouse or anti-rabbit IgG conjugated to Alexa 594 (1:500, Thermo Fisher Scientific, Rockford, IL, USA) for 1 h at 25 °C. After washing with PBS, tissue sections were counterstained with Hoechst 33342 (Molecular Probes, OR, USA), mounted, covered with coverslips, and imaged at 1360 × 1024 pixels by using a BZ-9000 fluorescence microscope (Keyence) with uniform photographic exposure parameters. Sections representative of at least five mice were presented.
Enumeration of hair cells
HCs were counted as described previously . In brief, cochleae were removed from temporal bones at P0, P6, and P30 and fixed for 18 h at 4 °C. Bony capsules and lateral walls were then removed. After blocking with 0.3% Triton X-100 in PBS for 10 min, the organ of Corti (OC) was labeled using rabbit antibody against Myo7a (1:500, Thermo Scientific Pierce, PA1–936) and goat anti-mouse or anti-rabbit IgG conjugated to Alexa 488 (1:500, Thermo Fisher Scientific). Tissue sections were counterstained with Hoechst 33342 (Molecular Probes), mounted, and covered with coverslips for P30 cochleae, while the OC was stained by Texas Red-X phalloidin (Molecular Probes) for 30 min for P0, P6, and P30 cochleae (P0, each n = 2; P6, each n = 2; P30, each n = 5). Between steps, tissue sections were washed with PBS thrice for 5 min each. Surface images of the OCs were captured, and the inner and outer HCs were counted after Myo7a staining over 300 μm2 of tissue. The viability of HCs was also assessed (CONT and TSK-KO, each n = 5 at P30).
Enumeration of the spiral ganglion cells
SGCs were counted as described previously . Briefly, after immunostaining for Tuj1 and counterstaining with Hoechst, we delineated the Rosenthal’s canal as the area that contains clusters of doubly stained cells and extends laterally to the modiolus. SGCs in the Rosenthal’s canal, which were positive for both Tuj1 and Hoechst stain, were then marked and counted visually using ImageJ (NIH, Bethesda, MD, USA) at the apical, middle, and basal turns using three randomly selected sections per cochlea (CONT and TSK-KO, each n = 5). A second researcher reviewed the results for accuracy and calculated the average.
Measurement of the thickness of the stria vascularis
The average thickness of the stria vascularis was measured by analyzing images of the sections containing the midmodiolar region by using ImageJ (NIH) (CONT and TSK-KO, each n = 3).
In situ hybridization
Cochleae collected at E13.5, E15.5, and P0 were examined by in situ hybridization according to Moorman et al. , by using antisense riboprobes for BMP4 and Sox2 that were labeled with digoxigenin using DIG RNA Labeling Kit (Roche, Indianapolis, IN, USA). The probes were produced using the corresponding DNA constructs. Detailed protocols are available upon request. A minimum of five samples were prepared for each time point (CONT and TSK-KO, each n = 5).
Scanning electron microscopy
Each mouse was anesthetized as described above and briefly perfused with 0.9% NaCl, followed by 20 mL of 2.5% glutaraldehyde and 2 mM CaCl2 in 0.1 M sodium cacodylate (pH 7.4), through the ascending aorta. The inner ear was removed by dissection. The top of the cochlea was punctured using a fine-tipped pair of forceps, and semicircular canals were sliced open. The inner ear was then gently flushed through these openings using 0.3 mL of 2.5% glutaraldehyde and subsequently fixed in the same solution for 18 h at 4 °C. The membranous labyrinth containing the cochlea was removed by dissection. Cochlear specimens were prepared by removing the stria vascularis, Reissner’s membrane, and tectorial membrane. The cochlear spiral was sectioned into basal, middle, and apical segments and stained with 1% OsO4 thrice for 1 h, based on the technique by Hunter-Duvar . Samples were incubated with saturated thiocarbohydrazide for 20 min after the first and second OsO4 treatments. Specimens were then dehydrated in a graded series of ethanol and critical-point-dried using liquid CO2 as transitional fluid. Uncoated specimens were mounted on a Hitachi specimen stub, using silver electroconductive paint, and imaged using a Hitachi S-4800 field-emission scanning electron microscope operated at 5 kV. Five mice from each group (CONT and TSK-KO) were studied.
Quantification of stereocilia
Stereociliary dimensions were measured by using ImageJ (NIH) as described previously . Ten inner HCs per mouse were imaged at 20,000× magnification at basal, middle, and apical turns to measure the average length of the four tallest stereocilia in each HC (CONT and TSK-KO, each n = 5).
At E13.5, E15.5, and P0, whole heads were dissected and incubated in 4% PFA for 1 h at 25 °C. Samples were then embedded in OCT, sectioned at 10 μm in the plane of the long axis of the cochlear modiolus, mounted on uncharged slides (Leica Microsystems, Wetzlar, Germany), and dried at 25 °C. Slides were incubated in 95% acetone at − 20 °C and dried at 25 °C immediately before laser microdissection using an LMD7 system (Leica Microsystems), as described by Pagedar et al. . Cell samples were obtained from cochlear epithelium or SGCs. Each slide contained multiple adjacent sections, and all cells in each category were pooled from individual slides onto a single cap (CONT and TSK-KO, each n = 5).
Quantitative reverse-transcription polymerase chain reaction
Using a microRNA extraction kit (QIAGEN, Valencia, CA, USA), total RNA was extracted from each sample obtained by laser microdissection, quantified using a GeneQuant100 (GE Healthcare, Amersham, UK), and diluted as needed to achieve uniform concentrations. cDNA was then synthesized using a One-Step PrimeScript RT-PCR Kit (Takara Bio, Otsu, Japan) according to the manufacturer’s instructions, using primers for BMP4, Sox2, and GAPDH (Applied Bionics, Foster City, CA, USA). Targets were amplified using a Takara Dice TP960 over 40 cycles of denaturation at 95 °C for 15 s and annealing at 60 °C for 1 min. Relative gene expression was calculated by generating a standard curve and normalized to GAPDH signal (CONT and TSK-KO, each n = 7).
Mice were anesthetized at P30 as described above, and electrodes were placed beneath the pinna of the test ear and at the vertex just below the surface of the skin. The ground electrode was placed under the contralateral ear. Auditory thresholds were measured at 4, 12, and 20 kHz by recording the ABR (15 ms duration, 1 ms rise/fall time, and tone burst) on a System 3 (Tucker-Davis Technologies, Alachua, FL, USA). For each recording, 1024 sweeps were averaged. Stimulus levels near the threshold were changed in 10-dB steps, and the threshold was defined as the lowest level at which waves in the ABR were clearly detectable by visual inspection (CONT and TSK-KO, each n = 5).
Five mice from each group were anesthetized as described above, and pinnae were removed. An ER10B+ probe microphone/speaker system with two speaker ports (Etymotic Research, Inc., Elk Grove Village, IL, USA) was fitted tightly into the ear canal and linked to two closed-field EC-1 speakers (Tucker-Davis Technologies). Two primary tones were generated (1 s duration with 20 ms rise/fall cosine ramp; f 2/f 1 = 1.22, f 2 varied at a one-fourth octave step from 4 to 29 kHz) and routed separately to the two EC-1 speakers at SPL1 = 75 dB and SPL2 = 65 dB. The SPL was calibrated in a 0.1-mL coupler  using a Brüel and Kjær 1/4″ pressure field microphone (model 4938), which has a flat frequency response from 4 Hz to 70 kHz. The calibration was conducted for primary tones and all the components of the DPOAE. The DPOAE response from the ER10B+ microphone was amplified by 20 dB and digitized at 150 kHz using a PCI-MIO-16E-1 A/D converter (National Instruments, Austin, TX, USA). Data were acquired and analyzed using customized software written in Matlab (The Mathworks, Natick, MA, USA). Recordings were repeated 10 times at 20-s intervals and averaged as a function of time. The noise was estimated by averaging three adjacent frequency bins that were above and below the DPOAE frequency .
Endocochlear potentials were recorded at P30 under general anesthesia . The cochlea was firstly exposed ventrally. The bone over the spiral ligament was thinned, and a small opening was generated with a pick to access the endolymphatic compartment (scala media) of the basal turn. A heat-pulled micropipette electrode filled with 150 mM KCl was inserted into this compartment until a stable potential was recorded, at which point potentials no longer depended on the electrode depth. The signal was amplified through an MEZ-7200 amplifier (Nihonkoden, Tokyo, Japan), and direct current potentials were recorded using a USB-6216 A/D converter (National Instruments) (CONT and TSK-KO, each n = 3).
Contextual and cued fear-conditioning test
To assess fear-associated learning and memory, each mouse was placed in a test chamber (33 × 25 × 28 cm) with a stainless-steel grid floor (0.2 cm diameter, spaced 0.5 cm apart; O’HARA & CO., Tokyo, Japan) in a sound-attenuating chamber to control environmental conditions and allowed to explore for 2 min . Subsequently, a conditioned stimulus (CS; 55 dB white noise) was presented for 30 s, followed by a mild foot shock (2 s, 0.3 mA), which served as the unconditioned stimulus (UCS). Two more CS–UCS pairs were presented at 2-min interstimulus intervals. Context test was conducted 1 day after conditioning in the same chamber for 300 s on each mouse. A cued test with an altered context was then conducted in a triangular chamber (33 × 29 × 32 cm) made of white opaque plastic, placed in a different room. Tone stimulus for the cued test (55 dB white noise) was applied for 180 s. In each test, freezing percentage was calculated automatically using the ImageFZ software based on the public domain Image J program and developed and modified by Tsuyoshi Miyakawa (available through O’HARA & CO.) (CONT: n = 15, TSK-KO: n = 17).
Experimental design and statistical analyses
Data are reported as mean ± s.d. Two groups were compared using Mann–Whitney U test. For comparisons of more than two groups, one-way or two-way repeated-measures ANOVA was performed, followed by Bonferroni’s post-hoc test of pairwise group differences. Statistical calculations were performed using Microsoft Excel (Redmond, WA, USA). Results with a p value < 0.05 were considered statistically significant.