Post-natal day 1 (P1) C57BL/6 mice (n=480; average weight 1.0 g) were obtained from the Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). The study protocol was approved by the Institution Review Board of Sun Yat-sen University (Guangzhou, China). All animal experiments were performed within 2–3 h of the arrival of the mice and in compliance with the guidelines of the Animal Care and Use Committee of the National Institutes of Health of USA for experimental use of laboratory animals.

Hank's balanced salt solution (HBSS, pH 7.4), supplements N2 (100×) and B27 (50×), Dulbecco's modified Eagle's medium/F12 (DMEM/F12) were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Collagen-coated cover slides, penicillin G, heparin sulfate, and bromodeoxyuridine (BrdU) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). C57BL/6 mice were euthanized at postnatal day 1 and cochlear sensory epithelium was collected and dissected in HBSS. The stria vascularis, Reissner's membrane and the tectorial membrane were removed prior to transfer onto the collagen-coated cover slides. One group of organ samples from 20 mice were incubated in serum-free DMEM/F12 media supplemented with N2, B27 and 100 U/ml penicillin G. Culture medium was changed every other day. Following 8 days culture the incubated cochleae were then fixed with 4% paraformaldehyde at room temperature for 30 min.

The inner ear sensory epithelial sheets were isolated from the saccule and utricle of C57BL/6 mice. The otolith was carefully dissected under a stereoscopic microscope in a separate dish with ice-cold HBSS. The isolated inner ear sensory epithelial sheets were transferred into Eppendorf tubes, digested in 500 µl of 0.125% trypsin in phosphate-buffered saline (PBS; Gibco; Themo Fisher Scientific, Inc.) at 37°C for 15 min. The cells were carefully triturated with plastic 200 µl pipette tips, centrifuged (3,000 × g, 5 min at room temperature) and suspended in 2 ml DMEM/F12 medium with N2 and B27 supplements, epidermal growth factor (EGF; 20 ng/ml; Invitrogen; Thermo Fisher Scientific, Inc.), insulin-like growth factor 1 (IGF-1, 20 ng/ml, PeproTech, Rocky Hill, NJ, USA), basic fibroblast growth factor (bFGF; 20 ng/ml, R&D Systems, Minneapolis, MN, USA). The dissociated cells were passed through a 70 µm cell filter (BD Biosciences, Franklin Lakes, NJ, USA) to remove cell clumps. Half of the medium was exchanged every other day. The solid spheres were collected after 5 days of culture, transferred into chamber slides coated with Matrigel™ (BD Biosciences), and allowed to cultivated up to 11 days in the same medium without growth factors. The inner ear sensory precursor cells were fixed with 4% paraformaldehyde at room temperature for 30 min.

In order to induce injury in hair cells, the isolated organs were incubated with 150 µM gentamicin (Shanghai DingGuo Biotech Co., Ltd., Shanghai, China) for 14 h. DAPT (5 µM, D5942; Sigma-Aldrich; Merck KGaA) or dimethyl sulfoxide (DMSO; 15 µM; Invitrogen; Thermo Fisher Scientific, Inc.; negative control) were subsequently added to substitute for gentamincin. The cochleae were fixed after 3.5 days for sex determining region Y-box 2 (Sox2; SAB2104660; 1:250; Sigma-Aldrich; Merck KGaA), anti-active Notch1 (1:200; ab8925), 4′6-diamidino-2-phenylindole (DAPI; 1:1,000; ab104139; both Abcam, Cambridge, UK) and myosin VI (1:200; 25-6791; rabbit polyclonal, Proteus Biosciences Inc., Ramona, CA, USA) staining. Organs were cultured for 8 days for myosin VI, active Notch1, anti-bromodeoxyuridine (anti-BrdU; 1:200; Sigma-Aldrich; Merck KGaA) Rhodamine-phalloidin (1:200; Invitrogen; Thermo Fisher Scientific, Inc.), and DAPI staining. For proliferation analysis, BrdU was added at a final concentration of 10 µM following washes to remove gentamycin. The organs with BrdU and the control group were cultured for 3.5 days following the washing out of the gentamicin prior to staining. Inner ear precursor cells were incubated with 50 µM DAPT as a negative control reagent, with or without small interfering RNA (siRNA) transfection of the miR-183 family (miR-183, miR-182, miR-96) by electroporation 5 days post-culture. The inner ear precursor cells continued to be cultured for 5 days.

Immunostaining of the cultured organs of Corti were performed as previously described (12) using the following primary antibodies: Myosin VI, mouse monoclonal anti-BrdU, mouse monoclonal anti-active Notch1 and rabbit polyclonal anti-Sox2 (SAB2104660; 1:250; Sigma-Aldrich; Merck KGaA; all overnight at 4°C). The following secondary antibodies were used: Alexa Fluor^® 488-conjugated and Alexa Fluor^® 594-conjugated donkey anti-rabbit, Alexa Fluor^® 488-conjugated and Alexa Fluor^® 594-conjugated donkey anti-mouse (1:400; R37118, R37119, R37114, R37115 respectively; Invitrogen; Thermo Fisher Scientific, Inc.). Following the fixation of the cultured organs, they were permeabilized in 0.1% Triton X-100 in PBS for 15 min and washed three times for 5 min in PBS. The organs were blocked in 5% bovine serum albumin (AR1006; Boster Biological Technology, Ltd., Wuhan, China)/0.05% Triton X-100 in PBS for 1 h prior to incubation with the primary antibodies overnight at 4°C. Incubation with the secondary antibodies (1:400) was performed at 4°C overnight or at room temperature for 2 h. Rhodamine-phalloidin was used to stain the stereocilium and reticular laminae of the cochlear hair cells for 30 min in room temperature. DAPI was used to stain the nucleus for 15 min at room temperature. Specimens were mounted and observed under a fluorescence microscope (Olympus IX71 Inverted Microscope; Olympus Corporation, Tokyo, Japan) and a FluoView FV1000 confocal microscope (Olympus Inc., Guangzhou, Guangdong, China). Specimens were examined at ×20 or ×40 magnification. Images were processed using Image-Pro Plus software version 6.0 (Media Cybernetics, Inc., Rockville, MD, USA). Cultured precursor sensory epithelia were prepared for staining and fixed in 4% PFA/PBS for 20 min at room temperature and then washed in PBS three times. Subsequently, permeabilization, blocking and antibody incubation were performed as described for organ samples.

For organ culture, the number of hair cells was determined by calculating the number of myosin VI-positive cells per 200 µm in the organ of Corti. For cultured precursor sensory epithelia, the percentage of hair cells was determined by calculating the number of myosin VI-positive cells over the total number of cells in the whole sphere, multiplied by 100. Cells positive for DAPI or myosin VI were counted in one representative optical section. Cells were only counted when their nucleus comprised >50% of the cell area. Quantification was obtained from at least 6 random areas over the length of an entire cochlea. At least 5 or 6 inner ear precursor spheres were counted for each experiment.

The cultured organs (at day 8) were carefully dissected and collected in Eppendorf tubes. The total RNA was extracted and column-purified using RNeasy purification kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol. Total RNA was treated with RNase-free DNase (Roche Diagnostics, Indianapolis, IN, USA). Reverse transcription was performed using SuperScript III one-step RT-PCR system with Platinum Taq High Fidelity DNA polymerase (1274-030; Invitrogen; Thermo Fisher Scientific, Inc.) according to the protocol provided by manufacturer. The PCR sequential steps are as follows: Denaturation at 94°C for 2 min, annealing at 60°C for 30 sec, and extension at 72°C for 45 sec: 35 cycles were performed and the last amplification was followed by a final 10-min elongation step at 72°C. The primers (Invitrogen; Thermo Fisher Scientific, Inc.) used for PCR are presented in Table I. The PCR products were resolved by 1% agarose gel electrophoresis containing 0.4 µg/ml of ethidium bromide. The level of gene expression was semi-quantified by grey scale ratio of Atoh1, Notch1, Jagged1, Delta1, Hes1, P27 vs. GAPDH by Quantity One (version 4.6.2; Bio-Rad Laboratories, Inc. Hercules, CA, USA). Each experiment was performed (containing at least four different cochleae) at least three times from each group.

RT-qPCR was performed based on a stem-loop Rtprimer design, as previously described (13). The cDNA was synthesized from extracted total RNA from cultured organs as mentioned above. Quantitative PCR (qPCR) was performed using 2 µl of SYBR-Green/ROX qPCR Master Mix (Suzhou GenePharma Co., Ltd., Suzhou, China). All procedures were subjected to a TaqMan miRNA assay (Applied Biosystem 7500) according to the manufacturer's instructions. U6 was employed as an endogenous control to normalize data using the 2^−ΔΔCq (14) method.

Probe and primers specific for miR-183, miR-182, miR-96, or for U6 RNA internal control were purchased from LC Sciences (Hangzhou, China). RT-qPCR analyses of at least 3 independent cultures were performed.

The qPCR reaction was performed using an ABI 7500 sequence detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) and used the following PCR protocol: 95°C for 10 min followed by 40 cycles of 95°C for 10 sec and 55°C for 30 sec.

In situ hybridization procedure was performed as previously described (15). The cochleae explants were cultured in vitro for 8 days and subsequently fixed in fresh 4% paraformaldehyde at room temperature for 1–2 h. Then the slides of cochleae were washed for 3×5 min in 1XPBS at room temperature. Followed proteinase K treatment, the slides were rinsed in 1XPBS at room temperature and placed horizontally in the hybridization chamber with 70 µl of hybridization buffer. Cochleae were prehybridized by incubating for 4–8 h at room temperature. Next, the cochleae were carefully pipetted with probed hybridization buffer and hybridized at 50–60°C overnight. The next day, the slides were soaked in prewarmed 60°C 5XSSC and then incubated in 0.2XSSC at 60°C for 1 h. Finally, the slides were incubated in B1 solution (0.1 M Tris pH 7.5/0.15 M NaCl) at room temperature for 10 min and then blocked in blocking solution (2 ml of fetal calf serum, 18 ml of B1 and 0.1% Tween-20) for 1 h at room temperature. The slides were carefully pipetted with blocking solution containing anti-DIG-alkaline phosphatase antibody (diluted 1:1,000), followed by incubation at 4°C overnight. The next day, the slides were washed three times for 5 min each time in B1 solution at room temperature. Then, cochleae were equilibrated for 10 min in B3 solution (0.1 M Tris pH 9.5/0.1 M NaCl/50 mM MgCl₂). Finally, the developer solution (3.4 µl of 100 mg/ml NBT, 3.5 µl of 50 mg/ml BCIP, 2.4 µl of 24 mg/ml levamisole, 5 µl of 10% Tween and 986 µl of B3) was carefully added to the slides, which were kept in a humidified chamber. The reaction was developed at room temperature in the dark for times ranging from 1–3 days, depending on the microRNA expression levels. A light microscope (Leica Microsystems GmbH, Wetzlar, Germany) was used to monitor the reaction, which was terminated when a strong blue stain was observed. Washing the slides for 3×10 min in 1XPBT terminated the color reaction. The slides were mounted in fluorescent mounting media (S3023; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for further observation.

The siRNA oligoribonucleotides for the miR183 family (miR-183, miR-182, miR-96) were purchased from Shanghai GenePharma Co., Ltd., (Shanghai, China), cultured inner ear precursor cells were dissociated into a single-cell suspension with 0.25% trypsin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C for 3 min. Then, 1×10⁶ cells/well were transfected by electroporation with 5 µg miR-183 siRNA oligoribonucleotides, according to the manufacturer's protocol. An enhanced green fluorescent protein RNA oligoribonucleotide (Shanghai GenePharma Co., Ltd.) was used to monitor the efficiency of the electroporation. The transfected inner ear precursor cells were plated on cover glasses coated with poly-L-lysine and laminin (R&D Systems, Inc.) and fixed with 4% paraformaldehyde at room temperature for 30 min following an additional 5 days culture. The inner ear precursor spheres with the average diameter of 55 µm were selected for statistical evaluation.

Data are expressed as the mean ± standard deviation and analyzed using SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). One-way analysis of variance and Tukey's Honest Significant Difference was used as a post-hoc test in order to determine significant differences in multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To investigate the expression of activated Notch in hair and supporting cells, Notch1 expression was determined in control and gentamycin-damaged neonatal cochlear explants. An activated signal was not detected in control cochlear hair cells and supporting cells (Fig. 1A). However, fluorescence was detected in the nucleus of the supporting cells in the gentamicin-damaged cochleae, indicating an activated Notch signal (Fig. 1B).

Notch inhibition by DAPT (5–20 µM) increased the number of myosin VI-positive cells in cochlear explants (Figs. 2 and 3) in a dose-dependent manner, with the most evident increase observed in the 20 µM DAPT group (P<0.05; Fig. 2E). The majority of the increase in hair cell number occurred at the apex turn, with limited changes in the basal turn (Fig. 2E). The ability of DAPT stimulate new hair cell growth in damaged postnatal cochlea was also examined. The results revealed that gentamicin treatment reduced the number of myosin VI-positive hair cells (Fig. 3C, G and J) compared with the controls (Fig. 3A, E and I). This reduction was mostly confined to the basal and middle turn of the cochleae, with virtually no hair cell loss occurring in the apex turn. Co-treatment of gentamicin and DAPT resulted in a significant increase in the number of myosin VI positive cells (P<0.05; Fig. 3D, H and L) compared with gentamicin treatment alone. No hair cell loss was observed in the gentamicin-treated apex turn of the cochleae. DAPT and gentamicin treatment markedly increased the number of hair cells compared with gentamicin treatment alone (Fig. 3M; P<0.05). Additionally, DAPT treatment and gentamincin and DAPT co-treatment resulted in the blurring of the Corti's tunnel between inner and outer hair cells and crowding in the apex and middle turns due to new hair cell growth (Fig. 3B, D, F and H). Hair cells in the sensory epithelium region displayed stereocilia with varying degrees of disarrangement and random orientation (Fig. 3D, H and L).

Activated Notch was revealed to be confined to the supporting cells of gentamicin-damaged cochleae (Fig. 1B). Thus, it was investigated whether hair cell proliferation was induced in response to DAPT treatment. The majority of BrdU-positive nuclei were observed in the area near outer hair cell and thus may be Hensen cells, according to the inner ear anatomy of mice. No BrdU and myosin VI double-positive cells were identified under any treatment conditions (Fig. 4A-D). Sox2, a marker of supporting cells, and myosin VI co-expressing cells were observed within the sensory epithelium in the DAPT and gentamicin co-treatment group (Fig. 4E). This suggested that the newly generated hair cells were derived from the differentiated, but not proliferating, supporting cells.

RT-sqPCR results revealed that gentamicin-induced injury was associated with the upregulation of Notch1, Jagged1 and Hes1 expression and downregulation of Atoh1 expression (Fig. 5A). The expression of Jagged1 and Hes1 was downregulated in cochlear explants treated with DAPT. No apparent changes were observed in Delta1 or p27 expression, regardless of gentamicin or DAPT treatment. These results indicate that DAPT efficiently downregulated Notch signaling.

Previous studies revealed that the miR-183 family (miR-183, miR-182, miR-96) is expressed in differentiated hair cells in spatially exclusive pattern with Notch1 (9–11). To investigate the association between the miR183 cluster and hair cell differentiation and regeneration in damaged cochlea, the expression of the miR183 cluster was examined in cochlea following gentamicin-induced injury. The same pattern of miR-183 cluster expression was observed in all groups (Fig. 5B). miR-183 cluster expression was significantly downregulated (P<0.05) in gentamicin-treated cochleae, whereas DAPT treatment significantly increased miR-183 cluster expression alone and in the presence of gentamicin (Fig. 5B; P<0.05). Furthermore, in situ hybridization with LNA probes revealed similar results to the RT-qPCR analysis, demonstrating that DAPT treatment markedly increased miR-183 cluster expression, with or without gentamicin co-treatment (Fig. 5C-F). These findings suggest that the miR-183 cluster may play a role in DAPT-induced hair cell differentiation and regeneration.

Electroporation transfection efficiency was detected by eGFP protein with fluorescence microscopy 24 h after eGFP transfection (Fig. 6A). The cultured organs of Corti were repeatedly electroporated with miR-183 cluster inhibitors and eGFP fluorescence was not observed 24 h post-transfection. Therefore, inner ear precursor cells from neonatal mice were cultured. Cultured inner ear precursor cells were subsequently transfected with miR-183 cluster siRNA oligoribonucleotides. Transfection efficiency was determined to be 40–50% by eGFP. After 5 days of DAPT treatment, the number of myosin VI-positive cells was significantly increased (Fig. 6C) compared with controls (P<0.05; Fig. 6B). However, the number of myosin VI-positive cells observed in the inner ear precursor cells pretreated with DAPT and miR-183 siRNA (Fig. 6D) was below that of the controls (Fig. 6B; P<0.05), indicating that the DAPT-induced increase in myosin VI-positive cell numbers was abolished by miR-183 inhibition (Fig. 6, P<0.05). These results suggest that miR-183 is required for hair cell differentiation in Notch signaling-prohibited inner ear precursor cells.