Ethics declaration and approval for animal experiments.
All animal experiments and procedures were performed according to protocols approved by the Animal Care and Use Committee of the National Institute of Neurological Diseases and Stroke and the National Institute on Deafness and Other Communication Disorders, National Institutes of Health.
Generation of Kcnj10-Zsgreen BAC transgenic mice.
The bacterial artificial chromosome (BAC) clone RP23-157J4 from RPCI − 23 Female (C57BL/6J) Mouse BAC Library (GenBank: AC074311.28) was identified as containing the gene Kcnj10. This BAC (obtained from the BACPAC Resource Center located at Children’s Hospital Oakland Research Institute in Oakland, CA) contained 186 kb of mouse gDNA spanning from Chr1:172236604–172422466 (GRCm38/mm10). Kcnj10 mRNA (NM_001039484.1) spans from Chr1:172341210–172374085. This gene has 2 exons, with a coding sequence starting at the start of exon 2. The transgenic construct based on BAC RP23-157J4 was engineered with minor modifications using methods described in Lee and colleagues49 and Zeidler and colleagues50. Reporter mice were generated at the Transgenic Animal Model Core of the University of Michigan’s Biomedical Research Core Facilities.
The synthetic donor DNA was produced by PCR amplification of the recombineering plasmid R6K-PGK-ZsGreen. The PCR primers contained 80 nucleotides of homologous genomic sequences that matched the DNA 5’ and 3’ of the desired insertion in Kcnj10 genomic DNA. The BAC clone and the synthetic donor DNA were combined in DH10B competent bacteria. Introduction of the synthetic donor into the BAC resulted in kanamycin resistance of the bacteria clones containing the recombined BAC. The kanamycin cassette was then removed by induction of FLP recombinase expression, using plasmid pE-FLP (Addgene, #45978). DNA from kanamycin sensitive BACs were analyzed to identify correctly modified BACs by sequencing and enzyme restriction. A final recombination step replaced the BAC-backbone internal loxP site with ampicillin resistance cassette.
The modified BAC was sequenced to verify that the intended insertion of ZsGreen occurred, using 1350 bp amplicon obtained with primers located 5’ and 3’ of ZsGreen: 5’-CCACCACCTCCAACATGAAT-3’ and 5’-CTCTCTTTCCCCCAAGCTG-3’ and GoTaqGreen polymerase (Promega) with an annealing temperature of 55°C. The final BAC showed a silent (A > G) mutation in the amino acid Lysine at position 15 of ZsGreen. The absence of gross recombination inside the BAC was checked by restriction enzyme digestion followed by pulsed field gel.
This recombinant RP23-157J4 BAC was microinjected into fertilized eggs obtained by the mating of B6SJLF1/J female mice with B6SJLF1/J male mice (stock number #100012) obtained from the Jackson Laboratory (Bar Harbor, ME, USA) at the Transgenic Core Facility of the University of Michigan.
Four transgenic founder mice were obtained by random integration of the transgene in their genome. Genomic DNA prepared from tail or ear clips using the Maxwell 16 System (Promega, Madison, WI, USA) was used to identify the mice carrying ZsGreen (Table S1). Four independent transgenic lines were obtained. After maintaining these for 5 generations and noticing no differences in ZsGreen expression pattern between lines, two of them were cryopreserved and the two remaining, lines 850 and 858, were maintained alive and further backcrossed. Male mice hemizygous carrier of the ZsGreen transgene were mated with C57BL/6J wildtype female mice (stock number #000664) obtained from the Jackson Laboratory for over 10 generations leading to obtention of congenic mice carrier of the transgene. The mice studied here were from the 11th generation and were all hemizygous for the transgene. The resulting mouse strains was named B6.Cg-Tg(Kcnj10-ZsGreen)skMHa and B6.Cg-Tg(Kcnj10-ZsGreen)skMHb for lines 850 and 858, respectively. Location of the primers utilized to detect the Kcnj10-ZsGreen transgene in mice are shown in Fig. 2a. Primer sequences and PCR conditions used for genotyping are available in Table S10.
Single-cell and single-nucleus RNA-sequencing dataset analysis
Adult spiral ganglion neuron and satellite glia cells and P7 organ of Corti single cell RNA-Seq datasets and adult SV single-nucleus RNA-Seq datasets from the mouse were analyzed for expression of Kcnj103,18,23. Violin plots of expression among cell types in the SV (marginal, intermediate, basal and spindle cells), cell types in the P7 organ of Corti (inner and outer hair cells, pillar cells, and Deiters’ cells) and in the spiral ganglion region (Type IA, IB, IC and type II spiral ganglion neurons, satellite glial cells) were constructed as we have previously described3,4.
Reporter transgene copy number assessment.
To assess the number of transgene copies integrated in the gDNA of the transgenic mice, digital droplet PCR (ddPCR, Bio-Rad, Hercules, CA, USA) was performed. Seven animals (4 males and 3 females) from each of the two founder lines (850 and 858) were tested in Kcnj10-ZsGreen group. Five wildtype littermates were included as control. gDNA was isolated from tail snip using Maxwell 16 DNA purification system (Promega, RRID:SCR_020254), DNA concentration was measured using Thermo Scientific NanoDrop One/OneC Microvolume UV Vis Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA, RRID:SCR_023005) and was adjusted to 50 ng/uL. The autosomal gene Actb was used as reference. In ddPCR reaction, each well contained 1 uL of ZsGreen probe (FAM labeled)/primers mix, 1 uL of the probe recognizing the gene Actb (HEX labeled)/primers mix, 1uL of gDNA (50 ng), 10 uL Bio-Rad ddPCR Supermix (Bio-Rad, Hercules, CA, USA, Cat#1863024) for Probes and 7 uL molecular grade water. The ddPCR Droplets were generated using the QX200 AutoDG Droplet Digital (Bio-Rad, Cat#1864101). PCR was performed as described in the QX200 ddPCR EvaGreen Supermix instructions. Droplets were read with a QX200 Droplet Reader (Bio-Rad, Cat#1864003) and analyzed with QuantaSoft software (Bio-Rad, Cat#1864011). Sequences of the primers and probes used for this experiment are presented in Table S11.
Tissue preparation for immunofluorescence labeling and in situ hybridization.
Hemizygous transgenic mice and their wildtype littermates were studied. Unless indicated otherwise mice were studied at P56 to P65 (summarized here at P60) for all experiments. Inner ears from these mice were dissected and placed in 4% paraformaldehyde (PFA in 1 x PBS solution) overnight at 4°C. Fixed adult mouse inner ears were then decalcified in 0.25 M EDTA for 3 days in 4°C on orbital shaker. If the tissue was designated for immunostaining or hybridization as whole mounts, it was washed in PBS, dissected and stored in PBS in 4°C for further use. If the tissue was designated for cryosections, it was washed in PBS, transferred to 30% sucrose in PBS at 4°C overnight, followed by immersion in 50/50 mix of 30% sucrose in PBS and finally in super cryo-embedding medium (SCEM) (C-EM001, Section-Lab Co, Ltd.; Hiroshima, Japan). Tissue was flash-frozen in liquid nitrogen after the transfer and 2-hour incubation in fresh 100% SCEM in a cryomold biopsy square.
Fluorescent immunohistochemistry was performed as follows. Cryosections or whole mount tissue were washed in PBS then permeabilized and blocked (2 hours at room temperature (RT) for cryosections, overnight at 4°C for whole-mounts) in PBS with 0.2% Triton X-100 (PBS-T) with 10% fetal bovine serum (A3840001, ThermoFisher Scientific, Waltham, MA, USA). Samples were then incubated in the appropriate primary antibodies in PBS-T with 10% fetal bovine serum for 24 hours in 4°C, followed by two 10-minute washes in PBS-T and labelling for 2 hours at RT with AlexaFluor 488, 555 and/or 647-conjugated secondary antibodies made in donkey and directed against appropriate species (Life Technologies, Waltham, MA) diluted at 1:250 in PBS-T. DAPI (4,6-diamidino-2-phenylindole,1:10,000, Life Technologies, Waltham, MA) was included with the secondary antibodies to detect nuclei. Alexa Fluor 647 phalloidin was used to label F-actin in a subset of experiments. Samples were washed in PBS four times for five minutes and mounted in SlowFade Gold (S36937, Invitrogen, ThermoFisher). Specimens were imaged using Zeiss LSM880 confocal microscopes (Zeiss, Oberkochen, Germany) using 40x, 1.4 numerical aperture and 63x, 1.4 numerical aperture objectives. Primary antibodies utilized are detailed in Table S12.
Single molecule fluorescent in situ hybridization (smFISH).
In situ hybridization on PFA fixed, frozen tissue was performed with RNAscope® Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, Hayward, CA, USA) with substantial modification of pretreatment process was performed as previously described3,4,35. Briefly, frozen sections on Superfrost Plus microscope slides were removed from − 80°C freezer, placed on heating block (37°C) for 30 minutes to thaw and dry, then moved to RT. Hydrogen peroxide solution was applied on the section and incubated for 10 minutes at RT. Slides were washed 2 times in deionized water, dried initially on heating block for 10 minutes at 37°C, then placed on heating block and baked for 30 minutes at 60°C. After the baking, slides were moved to RT and hydrophobic barrier was applied around the specimen and left to dry. After 10 minutes, Protease PLUS was applied on the sample and slide was moved to hybridization oven set to 40°C and incubated for 25 minutes. Then slides were washed twice in deionized water, appropriate probe was applied to cover the sample and slides were incubated in hybridization oven for 2 hours at 40°C. Next steps of the protocol directly followed RNAscope® Multiplex Fluorescent Reagent Kit v2 User Manual (Doc. No. 323100-USM).
Light sheet microscopy imaging of Kcnj10-ZsGreen adult mouse cochlea.
Sample processing
Mouse cochlea fixed in 4% paraformaldehyde was washed in PBS at RT for half a day and then transferred into 50 mL of decalcification solution (20% EDTA in 1X PBS, pH = 9) for 7–10 days at 40°C. Fresh decalcification solution was supplied every 3 days. Finally, the decalcified cochlea was washed in PBS for 1 day at 4°C. To clear the tissue, decalcified mouse cochlea was dehydrated in a step gradient of tetrahydrofuran/water solutions (20%, 40%, 60%, 80%, 100% tetrahydrofuran) at 4°C. The duration of each step was 12 h and the cochlea was incubated in 100% tetrahydrofuran one extra time at 4°C overnight. Dehydrated cochlea was incubated in 100% dibenzyl ether at 4°C until the sample sank and was incubated one more time in fresh 100% dibenzyl ether at 4°C overnight before imaging.
Cleared tissue DISPIM
The dual-view inverted selective plane illumination microscope optimized for cleared tissue (CT-DISPIM; Applied Scientific Instrumentation, Eugene, OR, USA), and the associated data processing pipeline have been described in detail previously51.
We used a pair of 0.7 N.A. multi-immersion objectives (Special Optics; Denville, NJ, USA) to acquire images of a cochlea that had been cleared using iDISCO and mounted in dibenzyl ether (DBE). The sample was excited by a digitally scanned OBIS laser (Coherent; Santa Clara, CA, USA) light sheet. Fluorescence was filtered through an emission bandpass filter before being recorded on a Hamamatsu Flash 4 v3 sCMOS camera (Hamamatsu Photonics; Shizuoka, Japan). Kcnj10-ZsGreen was imaged using 488 nm excitation and a 525/50 bandpass emission filter. Autofluorescence was imaged using 637 nm excitation and a 676/37 bandpass emission filter (both filters from Semrock; West Henrietta, NY, USA). Image volumes were acquired in single view mode as stage-scanned tiles at full frame (2048 × 2048 pixels, FOV 520 µm, 0.254 µm per pixel) with 1 µm perpendicular inter-plane distance and 10% overlap between adjacent tiles. Image tiles were then stitched and deskewed on the NIH Biowulf supercomputer. Three-dimensional rendering was done in Imaris (version 9.9, Oxford Instruments).
Auditory brainstem responses and distortion product otoacoustic emissions.
Auditory brainstem responses (ABRs) were detected in both ears of anesthetized mouse at age P60-P65. Wildtype littermates lacking the transgene were used as a control group. Mice were anesthetized with an intraperitoneal injection of ketamine (56 mg/kg) and dexdomitor (0.375 mg/kg) and placed on a heating pad connected to a temperature controller (TC-2000, World Precision Instruments, Sarasota, FL, USA) inside a sound-attenuated booth (Acoustic Systems, ETS-Lindgren, Austin, TX, USA) to maintain animal body temperature at 37°C. Recordings were obtained using Tucker-Davis Technologies (Alachua, FL, USA) hardware (RZ6 Processor) and software (BioSigRZ, version 5.7.5). For ABR testing, subdermal electrodes (Rhythmlink, Columbia, SC, USA) were placed at the vertex, under the test ear, and under the contralateral ear (ground). Blackman-gated tone burst stimuli (3 ms, 29.9/s, alternating polarity) were presented to the test ear at 8, 16, 32, and 40 kHz via a closed-field Tucker-Davis Technologies MF-1 speaker. Responses were amplified (20×), filtered (0.3-3 kHz), and digitized (25 kHz) with 512–1024 artifact-free responses per waveform. For each frequency, testing began at 80 dB SPL and decreased in 10 dB steps until the ABR waveform was no longer discernable. Once the response was lost, testing continued in 5 dB steps with a minimum of two waveforms per stimulus level to verify repeatability of ABR waves. ABR thresholds were determined by visual inspection of stacked waveforms for the lowest stimulus level that yielded repeatable waves.
Distortion-product otoacoustic emissions (DPOAEs) were measured in both ears using Tucker-Davis Technologies hardware (RZ6 Multi I/O processor, MF-1 speakers) and software (BioSigRz, version 5.7.5) in conjunction with an Etymotic ER-10B + microphone. Two tones were presented simultaneously at levels of f1 = 65 dB SPL and f2 = 55 dB SPL with the higher frequency tone (f2) set between 4-44.8 kHz (5 points per octave) and f2/f1 = 1.25. Mean noise floors were calculated from levels at six frequencies surrounding the 2f1-f2 DPOAE frequency.
Endocochlear potential measurement.
Methods for EP measurement have been described previously52–55. Briefly, P63-P68 mice were anesthetized with 2,2,2-tribromoethanol (T4842, Sigma-Aldrich, St. Louis, MO, USA) at a dose of 0.35 mg/g body weight. EP measurements were made using glass microelectrodes inserted into the round window and through the basilar membrane of the first turn of the cochlea. Induction of anoxia, allowing measurement of anoxic-state EP, was accomplished by intramuscular injection of succinylcholine chloride (0.1 µg/g, NDC-0409-6629-02, Pfizer, New York, NY, USA) after establishment of deep anesthesia followed by additional injection of 2,2,2-tribromoethanol. Anoxic-state EP provides an indicator of the lowest EP and sensory hair cell function. In the presence of functional hair cells, the anoxic-state EP is negative, whereas the EP is zero if the hair cells are not functional. Data were recorded digitally (Digidata 1440A and AxoScope 10; Axon Instruments) and analyzed using Clampfit10 (RRID: SCR_011323, Molecular Devices, San Jose, CA, USA). Eight Kcnj10-ZsGreen transgenic mice and eight wildtype C57BL/6J littermates were evaluated.
Statistical Analysis
Results of quantifications are presented as mean ± standard deviation (SD). For pairwise comparisons of ABR and DPOAE data between Kcnj10-ZsGreen mice and wildtype littermates, two-way ANOVA was conducted. For pairwise comparison of EP between Kcnj10-ZsGreen mice and wildtype littermates, an unpaired t-test with Welch’s correlation was used. All statistical analysis were performed using GraphPad Prism version 8.4.3 (GraphPad Software, San Diego, CA, USA).