The pathogenesis of common Gjb2 mutations associated with human hereditary deafness in mice

Gjb2 35delG homozygous mice show complete hearing loss at P35

To determine the phenotypes and functional effects caused by complete loss of the GJB2 protein, we tried to obtain mouse models carrying homozygous 35delG or 235delC by mating heterozygotes. No homozygotes were observed and the proportion of heterozygotes did not follow Mendel’s laws (Supplemental Fig. S3E), and this might also be caused by embryonic lethality due to placental defects similar to what was seen for Gjb2 knock-out mice in previous work [22]. Tetraploid embryo complementation is the gold standard for demonstrating and rescuing placental defects and can result in live births [30–32]. Thus, we first derived embryonic stem cell (ESC) lines carrying homozygous 35delG in Gjb2 from the inner cell mass of preimplantation blastocysts, which were obtained from in vitro fertilization (IVF) (Supplemental Fig. S4A–C). Previous studies showed that the efficiency of producing ESC-derived mice via injection into 4–8-cell tetraploid embryos is greater than that obtained via tetraploid blastocyst complementation [20, 21]. Thus, we attempted to obtain homozygous mutant mice by injecting about ten Gjb2^{35delG/35delG} ESCs into one 4–8-cell tetraploid embryo using a piezo manipulator (Fig. 2A). In total, we successfully obtained 26 live pups carrying the homozygous 35delG mutation out of 1719 reconstructed blastocysts in 18 independent experiments (Fig. 2B, Supplemental Fig. S5A and Table S1). Taken together, these results demonstrated that GJB2 plays an indispensable role in mouse placenta development.

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Fig. 2

The generation of Gjb2 35delG homozygous mice with profound deafness. A Schematic diagram of the generation of Gjb2 35delG homozygous mice via tetraploid embryo complementation (created in BioRender.com). B Representative Sanger sequencing results of wild-type and Gjb2^{35delG/35delG} males. C ABR waveforms were recorded at 8 kHz in the wild-type and Gjb2^{35delG/35delG} mice at P35. The blue and red traces indicate the thresholds of wild-type and Gjb2^{35delG/35delG} males, respectively. The scale bar for all traces is shown. D The average ABR thresholds of the P35 wild-type and Gjb2^{35delG/35delG} mice at 4, 8, 16, 24, and 32 kHz. Upward arrows indicate that there were no ABR responses at 90 dB SPL for the corresponding frequencies, and the hearing threshold was recorded as 90 dB SPL. n represents the number of tested mice. E Representative confocal microscopy images of Myo7a and Sox2 immunofluorescence in the apical, middle, and basal turns of the cochleae from wild-type and Gjb2^{35delG/35delG} mice at P35. White arrows indicate the loss of hair cells. Scale bar, 20 μm. F Quantification of surviving IHCs, OHCs, supporting cells of Deiters’ cells and outer/inner pillar cells (DCs and PCs) in the wild-type and Gjb2^{35delG/35delG} males at P35, respectively. Individual values are shown. Data are shown as the mean ± s.e.m of the indicated biological replicates. n.s, no significance, **P < 0.01, ***P < 0.001 by Student’s unpaired two-sided t-test

To determine whether Gjb2^{35delG/35delG} mice can mimic human hereditary deafness, we performed ABR tests at frequencies of 4, 8, 16, 24, and 32 kHz on homozygous and wild-type mice at 5 weeks of age. The results showed that Gjb2^{35delG/35delG} mice had significantly elevated ABR thresholds across all frequencies tested and had completely lost their hearing (Fig. 2C D), which is consistent with patients carrying homozygous 35delG mutations in the GJB2 gene [33]. Meanwhile, we found that the homozygous 35delG mutation also had no effect on the growth of the mice (Supplemental Fig. S5A, B). The organ of Corti is the epithelium of the cochlea and contains one row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs), which can convert the pressure fluctuations of sound into electrical signals through mechanoreceptors [34]. Supporting cells, such as Deiters’ cells and pillar cells surrounding sensory hair cells, play an essential role in the physiology of the cochlea and help convey auditory inputs to the brain [35, 36]. Immunofluorescence staining showed that OHCs, but not IHCs, were partially lost in 35delG homozygotes from the apical turn to the basal turn of the cochlea, which damaged frequency selectivity and signal amplification (Fig. 2E, F). However, supporting cells did not show a significant change in the cochlea of the Gjb2^{35delG/35delG} mice at P35 (Fig. 2E, F). Taken together, hearing loss in the Gjb2^{35delG/35delG} mice appears to be caused by the degeneration of sensory hair cells, which is similar to what is seen in the R75W transgene and Gjb2 P0 knockdown mouse models [19, 37].

GJB2 absence disrupts the GJCs formation of the cochlea

In previous studies, Gjb2^loxP/loxP;Rosa26^CreER mice were injected with 4-hydroxytamoxifen to knockdown Gjb2 at different time points after birth, including P0, P5, P6, P8, P10, P12, P15, and P20, and this showed that Gjb2 knock-down before P8 caused profound hearing loss, failure of the tunnel of Corti to open up, and massive hair cell degeneration [37–39], indicating that GJB2 plays important roles in the postnatal maturation of the cochlea. Immunostaining showed that GJB2 was localized in the cochlea from embryonic day 16 (E16) [40]. To reveal the function of GJB2 in the cochlea during embryonic development, we first analyzed the supporting cells and hair cells at E17.5 and P0 using immunofluorescence. The results showed that the number and distribution of supporting cells and hair cells were unaffected in the apical, middle, and basal turns of the cochlea at E17.5 and P0 when Gjb2 was disrupted (Supplemental Fig. S6A, B). Moreover, scanning electron microscopy (SEM) showed that the structures of hair cell stereocilia in Gjb2^{35delG/35delG} mice were also normal compared to wild-type mice during embryonic development (Supplemental Fig. S6C, D). These results indicated that Gjb2 is dispensable for the prenatal development of hair cells and supporting cells in the cochlea.

The mouse cochlea is not fully developed at birth and it continues to mature until P14, at which point hearing becomes active, so we tested the ABR of Gjb2^{35delG/35delG} mice at P14. As expected, the homozygous mice were completely deaf at P14 at frequencies of 4, 8, 16, 24, and 32 kHz (Fig. 3A, B). Subsequently, we performed distortion product otoacoustic emission (DPOAE) reflecting the active cochlear amplification to assess fine changes in OHCs function. We found that the DPOAEs could not be induced in the 35delG homozygous mice with profound deafness (Fig. 3C–E), indicating that the function of OHCs is damaged. Besides, Gjb2^{35delG/35delG} mice exhibited the delayed apoptosis of greater epithelial ridge (GER) cells and GER retention (Supplemental Fig. S7A), which is similar to the previous study [41]. To our surprise, the hair cells and supporting cells around the hair cells were also intact in the homozygous mice (Fig. F, G), indicating that the degeneration of hair cells at P35 in the cochlea was not the direct cause for hearing loss in 35delG homozygous mice.

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Fig. 3

The influence of GJB2 in the cochlea at P14. A ABR waveforms were recorded at 8 kHz in the wild-type and Gjb2^{35delG/35delG} males at P14. The blue and red traces indicate the thresholds of wild-type and Gjb2^{35delG/35delG} mice, respectively. The scale bar for all traces is shown. B The average ABR thresholds of the P14 wild-type and Gjb2^{35delG/35delG} males at 4, 8, 16, 24, and 32 kHz. Upward arrows indicate that there were no ABR responses at 90 dB SPL for the corresponding frequencies, and the hearing threshold was recorded as 90 dB SPL. n represents the number of tested mice. Data are shown as the mean ± s.e.m. *** P < 0.001 by Student’s unpaired two-sided t-test. C Spectra of DPOAE were recorded from the wild-type (blue trace) and Gjb2^{35delG/35delG} (red trace) males at P14. f₀ = 16 kHz, f2/f1 = 1.2, L₁/L₂ = 60/60 dB SPL. The peak of DPOAE (2f₁–f₂) in Gjb2^{35delG/35delG} mice was not available but f1 and f2 peaks remained the same as those in wild-type mice. D The average DPOAE thresholds were measured on the same animals performed with ABR recording at 4, 8, 16, 24, and 32 kHz. Upward arrows indicate that there were no DPOAE responses at the stimulus level of 80 dB SPL for the corresponding frequencies, and the DPOAE threshold was recorded as 80 dB SPL. n represents the number of tested mice. Data are shown as the mean ± s.e.m. **** P < 0.0001 by Student’s unpaired two-sided t-test. E Input/output function of the DPOAE (2f₁–f₂) amplitudes of wild-type and Gjb2^{35delG/35delG} males at 16 kHz frequency at P14. Data are shown as the mean ± s.e.m. The dotted line is the noise level. F Representative confocal microscopy images of Myo7a and Sox2 immunofluorescence in the apical, middle, and basal turns of the cochleae from wild-type and Gjb2^{35delG/35delG} males at P14. Scale bar, 20 μm. G Quantification of surviving inner hair cells (IHCs), outer hair cells (OHCs), supporting cells of Deiters’ cells and outer/inner pillar cells (DCs and PCs) in the wild-type and Gjb2^{35delG/35delG} males at P14, respectively. Data are shown as the mean ± s.e.m of the indicated biological replicates. n.s., no significance by Student’s unpaired two-sided t-test

GJB2 and GJB6 form the intercellular gap junction channels (GJCs) of non-sensory cells in the cochlea [13, 42]. Gjb6 knock-out mice show profound hearing loss with a reduction of GJB2 protein, which disrupts the formation of GJCs [12, 43]. Thus, we first determined the integrity of the GJCs by immunofluorescence of GJB2 and GJB6 in the whole cochlea at P14. As expected, GJB2 was not observed in 35delG homozygous mice (Fig. 4A). However, to our surprise, GJB6 protein was significantly down-regulated in the supporting cells of 35delG homozygous mice resulting in the total disruption of GJCs, suggesting that the macromolecular complex had been degraded (Fig. 4A and Supplemental Fig. S8A). In a detailed analysis of the GJCs structure in the inner sulcus cells (ISCs), we observed that in wild-type cochleae, the GJCs were extensive and flat at the cell periphery, arranged in neat pentagonal or hexagonal shapes, while in 35delG homozygous cochleae, the GJCs were considerably broken down and appeared as tiny, vesicle-like structures (Fig. 4B, C). We observed that GJC disruption was already present in P0 mice (Supplemental Fig. S8B), indicating the impairment of the cochlea prior to the onset of hearing. We further analyzed the protein expression of GJB2 and GJB6 in different tissues (cerebellum, liver, bladder, tail, and cochlea) between wild-type and Gjb2^{35delG/35delG} mice at P14 by western blot. The result showed that GJB6 was comparable to wild-type bladder and tail in 35delG homozygous mice with a low expression, but was reduced in cerebellum and cochlea with a high expression (Fig. 4D). To answer the question of whether the loss of GJB6 protein is due to transcriptional regulation, the qPCR results showed that the expression of Gjb2 and Gjb6 in 35delG homozygotes was also reduced in cerebellum and cochlea (Fig. 4E, F). Taken together, these results suggested that GJB2 and GJB6 expression is interrelated at the transcription and translation levels in the cerebellum and cochlea, which may be regulated through the NF-κB pathway and contribute to deafness [12].

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Fig. 4

The mutation of GJB2 disrupts the formation of GJCs by reducing the GJB6 protein. A The panoramic view of representative confocal images of apical turns from wild-type and 35delG homozygous mice at P14, by staining GJB2, GJB6, and Sox2. The regions containing different supporting cells were delineated in the figure. Scale bar, 20 μm. B The diagram of the organ of Corti. C Representative confocal microscopy images of GJB2 and GJB6 immunofluorescence in the apical, middle, and basal turns of the cochleae from wild-type and Gjb2^{35delG/35delG} mice at P14. The inner sulcus areas were shown. Three independent mice were analyzed for each group. Scale bar, 20 μm. The bottom row shows high magnification views of GJB6. D Western blots showing GJB2 and GJB6 protein expression levels in the different tissues of wild-type and Gjb2^{35delG/35delG} mice, including cerebellum, liver, bladder, tail, and cochlea. Results obtained with GADPH are shown as controls for the amount of protein loading in each lane. Two biological repeats for each group. E and F Transcriptional analysis of Gjb2 (E) and Gjb6 (F) genes in different tissues of wild-type and Gjb2^{35delG/35delG} mice by qPCR, including cerebellum, liver, bladder, tail, and cochlea. Three biological repeats for each group. The expression values were normalized to that of Gapdh. Data are shown as the average mean ± s.e.m. *P < 0.05; n.s, no significant difference

Plasmid construction

For the construction of CRISPR-Cas9 plasmids, sgRNAs targeting Gjb2 (termed pX330-mCherry-35delG and pX330-mCherry-235delC, respectively) were designed, synthesized, annealed, and ligated to the pX330-mCherry plasmid [61], which was digested with Bpi I (Thermo Scientific, Cat# FD1014). For the construction of donor plasmids (termed 19T-Gjb2-35delG and 19T-Gjb2-235delC), the sequences of the left and right homologous arms with 35delG and 235delC were amplified from the genome using Phanta Max Super-Fidelity DNA Polymerase (Vazyme, Cat# P505). The left/right arms of 35delG and 235delC were obtained by overlap PCR with mixed templates and then ligated to the pMD19T vector. All plasmids were collected using the EndoFree Maxi Plasmid Kit (TIANGEN, Cat#DP117) and were confirmed by Sanger sequencing. The oligonucleotide sequences used for plasmid construction in this study are listed in Supplemental Table S2.

Cell culture and transfection

Mouse DKO-AG-haESCs (IG^ΔDMR-H19^ΔDMR-AGH-2 cells or O48 cells) [24] were cultured in ESC medium containing 15% FBS (Excell Bio), penicillin–streptomycin, non-essential amino acids, Nucleosides, l-glutamine, β-mercaptoethanol, 1000 U/ml Lif, 1 μM PD03259010 (Selleck), and 3 μM CHIR99021 (Selleck) in DMEM (Gibco) at 37 ℃ and 5% CO₂. As previously described [23, 24], DKO-AG-haESCs were passaged every 2–3 days by trypsinization, and haploid cells were enriched every 2–3 weeks by fluorescence-activated cell sorting (FACS). One million AG-haESCs were passaged into a well of a six-well plate and then transfected with 2 μg CRISPR-Cas9 plasmids (pX330-mCherry-35delG or pX330-mCherry-235delC) and 1 μg pMD19T vectors (19 T-Gjb2-35delG or 19 T-Gjb2-235delC, respectively) using Lipofectamine 3000 (Life Technologies) following the manufacturer’s instructions. After 18–24 h, mCherry-positive haploid cells were enriched by FACS and then 6000–10,000 cells were seeded into a new well of a six-well plate. After 6–8 days, single clones derived from a single cell were picked and genotyped using PCR and Sanger sequencing.

ICAHCI and embryo transfer

To generate semi-cloned mice, Gjb2^35delG and Gjb2^235delC haploid cells were arrested at M phase by culturing in ESC medium containing 0.05 μg/ml demecolcine for 12 h before ICAHCI (intracytoplasmic AG-haESC injection). Arrested haploid cells were trypsinized and suspended in H-CZB medium for ICAHCI. A single haploid cell was then injected into the cytoplasm of MII oocytes, which were collected from superovulated B6D2F1 females, using a Piezo-drill micromanipulator. The reconstructed zygotes were cultured in CZB medium for 30 min and then activated for 5–6 h in Ca²⁺-free CZB with SrCl₂. After activation, the reconstructed zygotes were cultured in AA-KSOM (Merk) medium at 37 °C and 5% CO₂. After 18 h, the semi-cloned embryos reached the two-cell stage and then were transferred into each oviduct (18–23 embryos) of 0.5 day post-coitum (dpc) pseudo-pregnant ICR females.

Derivation of ESCs

To generate blastocysts to derive Gjb2^{35delG/35delG} ESCs, zygotes were obtained through IVF. Cumulus-oocyte complexes and sperms were obtained from Gjb2^+/35delG females and males, respectively, and incubated in a drop of HTF (Millipore, Cat#MR-070-D) containing reduced L-glutathione (170 µg/µL) (Sigma, Cat#G4251) for 3 h at 37 °C and 5% CO₂. The zygotes from IVF cultured in AA-KSOM developed into two-cell embryos after 1 day and formed blastocysts after 4 days. The zona pellucida of the blastocysts was removed using acidic Tyrode’s solution. Each blastocyst was transferred into one well of a 96-well plate seeded with ICR embryonic fibroblast feeders in ESC medium. After 7–10 days of culture, the ESC colonies derived from the inner cell mass of blastocysts were trypsinized and transferred to a 48-well plate without a feeder layer in fresh ESC medium. Clonal expansion of the ESCs proceeded from 48-well plates to 6-well plates without feeder cells and then in 6-well plates for routine culture and genotyping. All derived ESC lines were male.

Generation of tetraploid embryos

Eight-week-old B6D2F1 females were superovulated with 6 international units of pregnant mare’s serum gonadotropin (PMSG) for 48 h and then injected with human chorionic gonadotropin (hCG), then mated to B6D2F1 males for 12 h. Zygotes were harvested from the oviducts of B6D2F1 females with a vaginal plug after 24 h post-hCG injection using hyaluronidase (Sigma, Cat# H3884). After 12 h, the zygotes developed into two-cell embryos and were electrofused to produce one-cell tetraploid embryos. Two-cell embryos were aligned using an alternating current in 0.3 M mannitol solution, and a single direct current pulse of 1000 V/cm was applied for 20 µs. After electrofusion, the tetraploid embryos were returned to AA-KSOM medium and cultured for one day to reach the 4–8-cell stage.

Tetraploid embryo injection and blastocyst transfer

To generate Gjb2^{35delG/35delG} mice, male Gjb2^{35delG/35delG} ESCs were trypsinized and suspended in KnockOut DMEM (Gibco, Cat# 10,829,018) containing 15% KSR for tetraploid embryo injection. A total of 10–15 ESCs were then injected between the blastomere under the zona pellucida of 4–8-cell tetraploid embryos in a droplet of 15% KSR medium using a Piezo-drill micromanipulator. The reconstructed embryos were cultured in AA-KSOM medium for 24 h at 37 °C and 5% CO₂ and then transferred into each uterine horn (10–13 embryos) of 2.5 dpc pseudo-pregnant ICR females following standard procedures. On day 19.5 of gestation, the recipients were subjected to cesarean section, and live pups were nursed by lactating ICR females.

Genotyping and sanger sequencing

The tails and toes of heterozygotes (Gjb2^+/35delG and Gjb2^+/235delC mutant mice) and homozygotes (Gjb2^{35delG/35delG} mutant mice) were directly lysed by 50 μL lysis buffer from the Mouse Direct PCR Kit (Bimake), incubated at 55 °C for 90 min and 95 °C for 5 min. The lysate (about 2 μL) was used as a PCR template to amplify the mutant site in Gjb2 using Phanta Flash Master Mix (Vazyme). Products of PCR were purified by gel electrophoresis using Universal DNA Purification Kit (TIANGEN) and then performed Sanger sequencing. The primer sequences are listed in Table S2.

RNA extraction and quantitative real-time PCR (qPCR)

Total RNA was isolated from the different tissues (cerebellum, liver, bladder, tail, and cochlea), which were cut with scissors, of wild-type and Gjb2^{35delG/35delG} mice at P14 using TRIzol reagent (Invitrogen). The cDNA was obtained from about 1 µg RNA with reverse transcription reaction by the HiScript III 1st Strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme). Quantitative real-time PCR reactions were performed on a qTOWER³ (Analytik Jena) using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) in triplicate. All gene expression was calculated based on the 2^−∆∆Ct method after normalization to the transcript level of the internal standard gene, Gapdh. The primer sequences are listed in Table S2.

Western blot

The different tissues (cerebellum, liver, bladder, tail, and cochlea), which were homogenized by a homogenizer, of wild-type and Gjb2^{35delG/35delG} mice at P14, were lysed in 1 × RIPA buffer [50 mM Tris–Cl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.5% sodium pyrophosphate, 0.1% sodium dodecyl sulfate] (Cell Signaling Technology) containing protease inhibitor cocktail (Sigma) on ice for 2 h. Before gel electrophoresis, protein lysate was boiled with 5 × loading buffer at 60 °C for 10 min. Proteins were separated by 15% SDS–PAGE and then blotted on a PVDF (polyvinylidene difluoride) membrane, blocked with blocking solution (10% non-fat dry milk in TBST buffer) for a few hours and incubated with the appropriate primary antibody (rabbit anti-CX26, 1:1000 dilution, Invitrogen, Cat#512800; mouse anti-CX30, 1:1000 dilution, SANTA CRUZ, Cat#sc-514847; and mouse anti-GAPDH, 1:5000, Proteintech, Cat#60004-1-lg) in TBST buffer overnight at 4 °C. The membranes were washed three times for 10 min each with TBST and incubated with the appropriate secondary antibody (Goat anti-rabbit IgG, 1:5000 dilution, BBI, Cat#D110058-0001 and goat anti-mouse IgG, 1:10,000 dilution, Proteintech, Cat#SA00001-1) in TBST for 1 h at room temperature. Chemiluminescence detection was performed using an ECL reagent solution kit from Share-Bio.

Auditory testing

ABR and DPOAE measurements were recorded using an RZ6 acoustic system (Tucker-Davis Technologies, Alachua, FL, USA). Mice were anesthetized with xylazine (10 mg/kg) and ketamine (100 mg/kg) via intraperitoneal injection. For ABR measurements, three needle electrodes were inserted separately into the subcutaneous tissue of the cranial vault between the two ears (the reference electrode), the mastoid portion behind the left pinna (the recording electrode), and the dorsal rump of the animal (the grounding electrode). Each animal was stimulated with 5-ms tone pips to induce ABR potentials, followed by amplification (10,000 responses), filtering (300 Hz–3 kHz passband), and averaging (512 responses) at each sound pressure level (SPL). Tone burst acoustic stimuli were given at frequencies of 4, 8, 16, 24, and 32 kHz. The ABR threshold was determined as the lowest SPL at which an ABR wave peak could be visually detected and repeated compared to background noise. For DPOAE measurements, the f₁ and f₂ primary tones (f₂/f₁ = 1.2) were presented with f₂ at 4, 8, 16, 24, and 32 kHz and L₁ − L₂ = 0 dB sound pressure level (dB SPL). For each f₂/f₁ primary pair, L₂ was swept in 5 dB increments from 80 to 20 dB SPL. Waveform and spectral averaging were used at each level to increase the signal-to-noise ratio of the recorded ear-canal sound pressure. DPOAE threshold was defined from the average spectra as the 2f₁–f₂ level producing a DPOAE of magnitude 5 dB SPL above the noise floor. The mean noise floor level was under 0 dB across all frequencies. In general, ABR and DPOAE thresholds and amplitudes were defined by two independent observers.

Immunofluorescence

Cochleae from E17.5, P0, 2-week-old, and 5-week-old mice were harvested and fixed with 4% paraformaldehyde at room temperature for 2 h. The cochleae of 2-week-old and 5-week-old mice were decalcified in 10% ethylenediamine tetraacetic acid (EDTA) at room temperature for 4–6 h. The entire basilar membranes from neonatal mice and adult mice were divided into the apical, middle, and basal turns according to the sensitivity to different sound frequencies. The tissue was permeabilized and then infiltrated with 1% Triton X-100 and blocked with 10% bovine serum for 12–16 h at 4 °C before primary antibody incubation. To observe the degeneration of hair cells and supporting cells, rabbit anti-Myosin VII (1:800 dilution, Proteus BioSciences, Cat#25-6790) and goat anti-human/mouse/rat Sox2 (1:500 dilution, R&D Systems, Cat#AF2018), respectively, were incubated at 4 °C overnight. Donkey-anti-rabbit Cy3 (1:500 dilution, Jackson ImmunoResearch, Cat#711-165-152) and donkey anti-goat Alexa Fluor 647 (1:500 dilution, Jackson ImmunoResearch, Cat#705-605-147) secondary antibodies were incubated in the dark at room temperature for 2 h after rinsing three times with 1 × PBS. To observe the expression of gap junction channel proteins, rabbit anti-connexin 26 (1:200 dilution, Invitrogen, Cat#512800) and mouse IgG2a anti-connexin 30 (1:400 dilution, SANTA CRUZ, Cat#sc-514847) were incubated at 4 °C overnight. Donkey-anti-rabbit Cy3 (1:500 dilution, Jackson ImmunoResearch, Cat#711-165-152) and donkey anti-mouse Alexa Fluor 488 (1:500 dilution, Jackson ImmunoResearch, Cat#AB_2340846) secondary antibodies were incubated in the dark at room temperature for 2 h after rinsing three times with 1 × PBS. To observe apoptosis, rabbit cleaved caspase-3 antibody (1:500 dilution, Cell Signaling Technology, Cat#9664S) was incubated at 4 °C overnight, and donkey-anti-rabbit Cy3 secondary antibody (1:500 dilution, Jackson ImmunoResearch, Cat#711-165-152) was incubated in the dark at room temperature for 2 h after rinsing three times with 1 × PBS. DAPI was used as a nuclear counterstain (1:1000 dilution, Sigma, Cat#D9542). Fluorescent Z-stack images were collected using a Leica TCS SP8 laser scanning confocal microscope and a 40 × 1.30 or 63 × 1.40 glycerol immersion objective. The maximum intensity projections are shown in the figures.

Cell counting

For cell counting, the numbers of Myo7a⁺ hair cells, Sox2⁺ supporting cells, including three rows of Deiters’ cells and one row of outer/inner pillar cells [62], were manually counted in every 100-μm region of the apical, middle, and basal turns of the cochlea using Leica TCS SP8 software. The results are presented as the mean ± s.e.m.

Scanning electron microscopy (SEM)

Cochleae from E17.5 and P0 mice were harvested and fixed in 2.5% glutaraldehyde at 4 °C overnight, then decalcified in 10% ethylenediamine tetraacetic acid (EDTA) at room temperature for 4–6 h and dissected. The dissected tissues were fixed with 1% osmium tetraoxide (OsO₄) at 4 °C for 2 h and dehydrated in an increasing ethanol series and pentyl acetate, and then dried in an HCP-2 critical point desiccator for 2 h. SEM images were obtained with a high vacuum field emission scanning electron microscope (Hitachi SU-8010) at 3.5 kV (low magnification) or 10.0 kV (high magnification).

Resin sections and transmission electron microscopy (TEM)

Cochleae from 2-week-old mice were harvested and fixed in 2.5% glutaraldehyde at 4 °C overnight, and decalcified in 10% ethylenediamine tetraacetic acid (EDTA) at room temperature for 4–6 h. After decalcification, the cochleae were placed with 2.5% glutaraldehyde and fixed with 1% osmium tetraoxide (OsO₄) at 4 °C for 2 h. Then, the tissues were dehydrated in an increasing ethanol series and acetone prior to incubation in a dilution of liquid epoxy resin and acetone at a ratio of 1/2 at room temperature for 3 h. Then, a mixture of 67% epoxy resin and 33% acetone replaced the liquid at room temperature overnight. Subsequently, tissue samples were incubated in pure epoxy resin at 37 °C for 2–3 h. The specimens were subsequently transferred to an embedding mold and labeled followed by incubation in a drying oven at 37 °C, 45 °C, and 60 °C for overnight, 12 h, and 24 h, respectively. The samples were sectioned (0.8 μm in thickness) on a LEICA EM TRIM2 and stained with Toluidine Blue (89,640-5G; Sigma-Aldrich, USA) at 60 °C for light microscopy observation with a magnification of 100 × and 400 ×. For TEM analysis, specimens were then trimmed and ultrathin sections (70 nm in thickness) were acquired on a Leica EM UC7 and stained with 3% uranium acetate-lead citrate. TEM images were obtained with transmission electron microscopy (PHILIPS CM-120) at 460 × and 1400 × magnification.

RNA-seq and data analysis

The cochleae of wild-type and Gjb2^{35delG/35delG} mice were carefully collected and surgically divided into three equal sections, including the apical, middle, and basal turns. The total RNA of these sections was collected using a miRNeasy Micro Kit (QIAGEN, Cat#217084) following the manufacturer’s instructions. All collected RNA was used for library construction with the VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme, Cat# NR605). RNA-seq libraries were subjected to deep sequencing on an Illumina NovaSeq6000 platform following the manufacturer’s instructions (Illumina) to produce 150 bp paired-end reads by Genergy Biotechnology Co. Ltd. (Shanghai, China). Raw RNA-seq reads were cleaned using fastp (v0.20.0) and aligned to mm10 using STAR (v2.7.3a). Only unique mapped reads were kept and counted by featureCounts (v2.0.0), and FPKM (Fragments Per Kilobase of transcript per Million mapped fragments) and TPM (Transcripts Per Kilobase Million) were calculated by stringtie (v2.0). Genome coverage was counted by bamCoverage (v3.3.1). Differential gene expression was determined using DESeq2 (v1.30.0), and clusterProfiler (v3.18.1) was used for GO and KEGG analysis in R.

Endocochlear potential (EP)

EP was recorded in mice at P14 and P35. Mice were anesthetized with xylazine (10 mg/kg) and ketamine (100 mg/kg) via intraperitoneal injection and placed on a heating pad to maintain body temperature. Hair on the neck and anterior chest of anesthetized mice was removed. The trachea was dissected to maintain a normal airway, and soft tissues including muscle, fat, and vessels were ligated and separated away from the bulla. Some of the bulla wall was removed to allow clear visualization of the cochlea, and a small opening was made over the stria vascularis in the middle turn of the cochlea. A glass micropipette filled with 150 mM KCl was connected to a capacity-compensated direct-current preamplifier, and a ground electrode was inserted into the chest muscle. The micropipette was attached to a micro-manipulator and slowly advanced through the lateral wall into the scala media. Amplified electrical signals were recorded through an EPC10/2 amplifier (HEKA, Lambrecht/Pfalz, Germany) driven by a PC running Patchmaster (HEKA). The EP was calculated as the maximum change in voltage as the micropipette tip passed from the lateral wall tissue into the endolymph of the scale media [56].

We are grateful to Min Yao and Yu Han for helping with experiments of this manuscript.