Animals
Sox10 missense mutant(c.325A>T) Bama-minipigs were described previously (Hai et al., 2017)(Hao et al., 2018).All experiments involving animals or embryos were performed by guidelines of the Institutional Animal Care and Use Committee at the Third Military Medical University and Chinese PLA Medical School.
Auditory Brainstem Response (ABR) tests
Miniature pigs were anesthetized with xylazine (0.1 mg/kg, i.m.) and ketamine (15 mg/kg, i.m.) prior to auditory brainstem response (ABR). Smart-EP software (Intelligent Hearing System, Miami, USA) was used to test the ABR and cVEMP. For ABR recording, four needle electrodes were inserted into the skin of both pinnas, the base of the tail and vertex, sound stimuli earphone at the testing ear. ABR was evoked with click stimuli. The repetition rate was 12.1/s, and 1250 sweeps were averaged.
Miniature pigs were laid on baoding hammock without anesthesia to test cervical vestibular evoked myogenic potential(cVEMP). cVEMP was recorded in miniature pigs by using methods established in guinea pigs(Yang and Young, 2005), insteading of fixing pigs’ heads, we used feeding stimulation to keep heads elevated and neck hyperextended during recording, each pig was trained many times before the test. Two needle electrodes were placed into the skin of the base of the tail for grounding and vertex for reference, two needle electrodes were inserted into both neck extensor muscles (Corneil and Camp, 2018), and electromyography (EMG) was monitored throughout the test. Acoustic stimuli consisted of tone-burst at 1kHz, the EMG signal was amplified with a band-pass filter between 10 and 1000 Hz. 40 sweeps were averaged for each run. All miniature pigs underwent a serial cVEMP testing, starting with the 130 dB sound pressure level (SPL) and then lowering the 10dB step until there were no waveforms, so the threshold was determined.
Immunofluorescence Phalloidin staining of vestibular hair cells and hair cell quantification
Miniature pigs of postnatal 1 day were euthanized by urethane(1.5g/Kg, i.m.)/CO2 gas overdose, temporal bones were obtained and fixed immediately in 4% paraformaldehyde at 4 °C overnight. The vestibular sensory epithelia were subsequently micro-dissected in 0.1 M phosphate buffer, then permeabilized in 1% Triton X-100 in PBS for 30 min. F-actins to visualize stereocilia bundles and cell nuclei were stained by Alexa Fluor 555-Phalloidin (1:1000 dilution in PBS, 1h, room temperature. Sigma, 1:800) and DAPI (Invitrogen D3571, 1:1000) respectively.
Counting and data analysis
For confocal microscopy analysis, we used Zess 880 Confocal Microscope (Olympus, Center Valley, PA). Photographs taken with these microscopes were cropped and labeled with Adobe Photoshop and Illustrator software (Adobe System, San Jose, CA). For each utricle, three areas (36.9 μm × 36.9 μm) each of the striolar and extrastriolar regions were imaged and averaged as one data point for each region (striola and extrastriola).
Scanning electron microscopy (SEM)
Miniature pigs of postnatal 1 day and embryos at E75 were euthanized, and inner ears were harvested as described above. Tissues were fixed with 2.5% glutaraldehyde in 0.1 M PB at 4 °C overnight. The vestibular sensory epithelia were collected. They were treated with 1% osmium in 0.1 M PB for 1h, after dehydration via an ethanol gradient of increasing concentrations, they were dehydrated within a critical point dryer. then mounted on aluminum stubs and coated with 10 nm gold layer, and viewed under a scanning electron microscope.
Vestibular end organ were fixed with 2.5% (vol/vol) glutaraldehyde with Phosphate Buffer (PB) (0.1 M, pH 7.4), washed four times in PB. Then were first immersed in 1% (wt/vol) OsO4 and 1.5% (wt/vol) potassium ferricyanide aqueous solution at 4℃ for 1 h. After washing, the tissues were incubated in filtered 1% thiocarbohydrazide aqueous solution (Sigma-Aldrich) at room temperature for 30 min, 1% unbuffered OsO4 aqueous solution at 4℃ for 1h and 1% UA aqueous solution at 4℃ overnight following four rinses in ddH2O for 5 min each between each step. Then were dehydrated through graded alcohol (30,50,70,80,90,100%, 100%, 5min each) into pure acetone (2×5min). Samples were infiltrated in a graded mixtures (3:1, 1:1, 1:3) of acetone and SPI-PON812 resin (19.6 ml SPI-PON812, 6.6ml DDSA and 13.8ml NMA), then changed pure resin. Finally, samples were embedded in pure resin with 1.5% BDMA and polymerized for 12h at 45°C, 48 h at 60°C. The ultrathin sections (70nm thick) were sectioned with microtome (Leica EM UC6), and imaged by a Scanning Electron Microscope (FEI Helios Nanolab 600i dual-beam SEM) with its an immersion high magnification mode (CBS detector, 2kV, 0.34nA).
Micro-CT scanning
In order to assess the 3D structure and integrity of the inner ear, samples from WT and heterozygous pigs at postnatal day 1 were fixed with 4% paraformaldehyde and scanned by a micro-CT system (Quantum GX, Perkin Elmer) using the following parameters: 90 kV, 160 mA, 24 mm of field of view, and time 4.5 min. The structure of the inner ear was analyzed and reconstructed using IMARIS 9 (Bitplane, AG) software.
RNA Isolation From Vestibular Tissue
Vestibular end organs were obtained from 3 wild-type and 3 mutant embryos at E75, respectively. Total RNA was extracted using the Total RNA Extractor(Trizol)kit (B511311, Sangon, China) according to the manufacturer’s protocol, and treated with RNase-free DNase I to remove genomic DNA contamination. RNA integrity was evaluated with a 1.0% agarose gel. Thereafter, the quality and quantity of RNA were assessed using a NanoPhotometer ® spectrophotometer (IMPLEN, CA, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). The high quality RNA samples were subsequently submitted to the Sangon Biotech (Shanghai) Co., Ltd. for library preparation and sequencing.
Library Construction and Sequencing
The VAHTSTM mRNA-seq V2 Library Prep Kit for Illumina was used for the RNA-seq library preparation, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The libraries were then quantified and pooled. Paired-end sequencing of the library was performed on the HiSeq XTen sequencers (Illumina, San Diego, CA).
Data assessment and quality control
FastQC (version 0.11.2) was used for evaluating the quality of sequenced data. Raw reads were filtered by Trimmomatic (version 0.36). And the remaining clean data was used for further analysis.
Alignment with reference genome
Clean reads were mapped to the reference genome by HISAT2 (version 2.0) with default parameters. RSeQC (version 2.6.1) was used to statistics the alignment results. The homogeneity distribution and the genome structure were checked by Qualimap (version 2.2.1). BEDTools (version 2.26.0) was used to statistical analysis the gene coverage ratio.
Expression analysis
Gene expression values of the transcripts were computed by StringTie (version 1.3.3b). Principal Component Analysis (PCA) and Principal co-ordinates analysis (PCoA) were performed to reflect the distance and difference between samples. The TPM (Transcripts Per Million), eliminates the influence of gene lengths and sequencing discrepancies to enable direct comparison of gene expression between samples. DESeq2 (version 1.12.4) was used to determine differentially expressed genes (DEGs) between two samples. Genes were considered as significant differentially expressed if q-value <0.001 and |FoldChange| >2. When the normalized expression of a gene was zero between two samples, its expression value was adjusted to 0.01 (as 0 cannot be plotted on a log plot). If the normalized expression of a certain gene in two libraries was all lower than 1, further differential expression analysis was conducted without this gene. Gene expression differences were visualized by scatter plot, MA plot and volcano plot.
Gene Ontology (GO) and Pathway Enrichment Analysis of DEGs
Functional enrichment analyses including Gene Ontology (GO) and KEGG was performed to identify which DEGs were significantly enriched in GO terms or metabolic pathways. Gene Ontology (GO) is an international standard classification system for gene function. DEGs are mapped to the GO terms (biological functions) in the database, the number of genes in every term is calculated, and a hypergeometric test is performed to identify significantly enriched GO terms in the gene list out of the background of the reference gene list. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database is a public database of pathway data, KEGG pathway analysis identifies significantly enriched metabolic pathways or signal transduction pathways enriched in DEGs compared to a reference gene background, using the hypergeometric test. GO terms and KEGG pathway with false discovery rate (q-value) < 0.05 were considered as significantly altered.
Real-time PCR (RT-PCR)
Total RNA was extracted from vestibular end organs using TRIzol reagent (Invitrogen, USA). Random hexamers were used for synthesizing the first cDNA strand synthesis with 1 μg of total RNA (Molecular Biology). The thermal cycling profile was 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s, 58 °C for 10 s, and extension at 95 °C for 5 s, 65 °C to 95 °C, and 0.5 °C for 5 s. The PCR primers are listed in Table 1. The expression of each target gene in WT and mutant littermates was standardized against that of actin mRNA using the 2-ΔΔCt method.
Western blot analysis
Vestibular end organs were obtained from 3 wild-type and 6 mutant embryos at E75, respectively. The proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes(Bio-Rad, CA). After three 5-min washes in TBST (Tris-buffered-saline with Tween). The membranes were blocked overnight in TBST containing 5% dried milk for 1 h at room temperature, and then were incubated overnight at 4∘C with either SOX-10 anti- body (1:100 dilution in blocking buffer) (sc-365692, Santa) or Anti-S100 antibody (1:2500 dilution in blocking buffer) (ab52642, Abcam) or Anti-TRP2/DCT antibody (1:1000 dilution in blocking buffer) (ab74073, Abcam). The membranes were then washed three times for 5 min each in TBST and incubated for 1 h with the secondary antibody (1:4000 dilution )(Zhongshan Golden Bridge Biotechnology Co.Ltd. China) at room temperature. The membranes were washed three times for 10 min withTBST. and then visualized with the Super ECL Plus Detection Reagent (Applygen, China). The membranes were wrapped in plastic wrap then exposed to X-ray film.