Myo3a is expressed in cochlear hair cells and retinal cells and is responsible for human recessive hereditary nonsyndromic deafness (DFNB30). To investigate the mechanism of DFNB30-type deafness, we established a mouse model of Myo3a kinase domain Y137C mutation by using CRISPR/Cas9 system. No difference in hearing between 2-month-old Myo3a mutant mice and wild-type mice was observed. The hearing threshold of the ≥6-month-old mutant mice was significantly elevated compared with that of the wild-type mice. We observed degeneration in the inner ear hair cells of 6-month-old Myo3a mutant mice, and the degeneration became more severe at the age of 12 months. We also found structural abnormality in the cochlear hair cell stereocilia. Our results showed that Myo3a is essential for normal hearing by maintaining the intact structure of hair cell stereocilia, and the kinase domain plays a critical role in the normal functions of Myo3a. This mouse line is an excellent model for studying DFNB30-type deafness in humans.
Deafness presents the highest incidence among sensory defects. One-third of the global population suffer from hearing impairment. Approximately 300 million people currently possess hearing disabilities [
Our hearing depends on hair cells in the inner ear, which can convert the vibrations of sound into electrical signals. At the apical surface of each hair cell, three rows of stereocilium are arranged in high and low orders [
Based on different C-terminal cargo-binding domains, myosin superfamily members are classified into conventional myosins (class II) and unconventional myosins (classes I and III–XV) [
All animal experimental procedures were approved by the Ethics Committee of Shandong University. Animal management was performed strictly in accordance with the standards of the Animal Ethics of Shandong University.
Myo3a mutant mice were generated using the CRISPR-Cas9 genome-editing technology and maintained on the CBA/CaJ background. pX330 plasmid was obtained from Addgene (plasmid ID number 42230). The CRISPR-Cas9 genome-editing technology in mice was used as previously described [
CBA/CaJ female mice were superovulated and mated with CBA/CaJ male mice. The fertilized eggs were removed from the oviducts on the next day. pX330 plasmid (5 ng/
Genomic DNA was extracted from the tails of newborn pups. The genomic DNA fragment around the gRNA target site was amplified by PCR using the Myo3a forward primer 5
ABR was measured to determine the hearing thresholds of mice in a sound-isolated room as previously described [
The cochleae from Myo3a mutant and wild-type mice were removed, fixed with 4% formaldehyde in 10 mM phosphate buffer at 4°C overnight, decalcified in 10% EDTA in 10 mM phosphate-buffered saline (PBS) at room temperature for 2 d, dehydrated with 30% to 100% ethanol series, and treated with xylene for transparency. The cochlea was embedded in paraffin, and the specimen was sectioned at 7 mm thickness using a thin semiautomatic microtome. Sections were deparaffinized using xylene and 100% to 30% ethanol series, stained with H&E, and viewed under a light microscope (Nikon YS100).
Wild-type and Myo3a mutant mice were anesthetized with 0.007 g/mL pentobarbital sodium. The cochleae were removed from anesthetized mice, fixed in 4% formaldehyde in 10 mM PBS at 4°C overnight, and decalcified in 10% EDTA at room temperature for 2 days [
Myo3a mutant mice and wild-type mice were anesthetized using pentobarbital sodium and perfused with 4% PFA. The cochleae from Myo3a mutant and wild-type mice were removed, fixed with 2.5% glutaraldehyde in 0.1 M PBS at 4°C overnight, and decalcified in 10% EDTA [
FM1-43 staining experiments were performed as previously reported [
Nine 4.5-month-old Myo3a mutant mice and seven wild-type mice were anesthetized with 0.007 g/mL pentobarbital sodium by intraperitoneal injection (50 mg/kg body weight) and placed in a 24 cm × 24 cm × 18 cm stainless steel cage for full white noise handling. The loudspeaker was located just in front of the cage. Sound intensity was calibrated using a standard sound level meter prior to exposure to noise. The mice were continuously treated with 98 ± 2 dB SPL for 2 h to produce a temporary change in auditory threshold shifts.
All data were expressed as mean ± SD, and all experiments were repeated at least thrice to ensure the data accuracy and repeatability. Statistical analyses were implemented using Microsoft Excel, and charts were constructed using GraphPad Prism 5 software. Two-tailed, unpaired Student’s
To mimic Myo3a Y129C mutation in human, we introduced the A410G (Y137C) mutation in mice by using CRISPR/Cas9 technology (Figure
The generation of Myo3a mutant mice using CRISPR/Cas9. (a) Schematic diagram of targeting the mouse myosin IIIA gene. sgRNA was at exon 4 (indicated by the blue rectangles). The point mutation is in red. (b) The comparison of DNA sequences between Myo3a mutant mice (now referred to as Myo3a KI/KI mice) and wild-type mice. (c) Sequence of wild-type mice, heterozygous mice, and homozygous Myo3a KI/KI mutant mice. TAT was changed to TGT, demonstrating the missense mutation at mouse Y137C. (d) Gross morphology of Myo3a KI/KI and wild-type mice at the age of two months. There was no obvious difference. (e) Cochlea morphology is normal in Myo3a mutant mice. Hematoxylin and eosin (HE) staining showed no prominent difference between Myo3a mutant and wide-type mice cochlear at the age of two months. Scale bar = 20
The sgRNA containing the 20 bp target sequence complexed with Cas9 protease can introduce DSB into the target sequence near a protospacer-adjacent motif (PAM) sequence. On the basis of this principle, we designed a specific sgRNA targeting the sequence near the A410 and cloned the sgRNA into the Px330 plasmid containing the Cas9 gene sequence. Thereafter, we designed a repair template with the mutation of interest based on the location of the DSB. The two synonymous mutations on the repair template aim to prevent a secondary targeting of sgRNA. The Px330 plasmid (5 ng/
Myo3a mutant mice were viable and fertile with no apparent abnormalities in their gross morphology (Figure
In humans, Myo3a mutation can cause nonsyndrome-type deafness. Thus, we wanted to test whether Myo3a mutant mice show the same symptoms. To determine whether Myo3a mutant mice demonstrate age-related deafness, we tested the hearing threshold of 2-, 6-, and 12-month-old Myo3a mutant and wild-type mice by ABR measurement. There results indicated no significant difference in the hearing threshold between 2-month-old wild-type (
ABR analysis in Myo3a mutant mice (red) and wild-type mice (black) at two months, six months, and twelve months. (a) ABR measurements for broadband click. (b) Frequency-specific pure tone stimulation of Myo3a KI/KI mice and wild-type mice at two months old (b), six months old (c), and twelve months old (d). In contrast to wild-type mice, Myo3a KI/KI mutant mice showed progressive hearing loss. Compared with WT threshold at the corresponding frequency as determined by Student’s
We investigated the mechanism of senile deafness in Myo3a mutant mice and examined cochlear changes. We dissected the cochleae of 2-, 6-, and 12-month-old wild-type and mutant mice and used phalloidin, DAPI, and Myo7a to stain hair cell stereocilia, hair cell nucleus, and hair cells, respectively. Confocal images of the basilar membrane showed that the stereocilia and hair cells of the wild-type and Myo3a mutant mice were intact at the age of 2 months; by contrast, the stereocilia of the Myo3a mutant mice started to degenerate, and the hair cells began to disappear at 6 months. By the age of 12 months, stereocilium degeneration and hair cell loss became incrementally serious (Figure
The degeneration of stereocilia and the hair cell loss in the Myo3a mutant mice showed by confocal images. Confocal images of the stereocilia, hair cells, and nucleus in Myo3a mutant and WT mice at two months old, six months old, and 12 months old. Images were taken from the middle turn of the cochlea. Scale bar = 20
To further verify the accuracy and reliability of the above result, we used SEM to observe the hair cells of mutant and wild-type mice in detail. Consistent with the above results, stereocilium degeneration and hair cell loss occurred in 6-month-old Myo3a mutant mice, especially in the stereocilia of inner hair cells (Figure
The degeneration of stereocilia in the Myo3a mutant mice showed by SEM. SEM images of the hair cells in Myo3a mutant and WT mice at two months old (a, d), six months old (b, e), and 12 months old (c, f). The inner hair cell stereocilium loss was found in 6-month-old Myo3a mutant mice (e), and this phenomenon becomes more serious in 12-month-old Myo3a mutant mice (f). Scale bar = 20
SEM images showed the abnormal structure of the stereocilia in Myo3a mutant mice. (a) The outer hair cell stereocilium loss was serious in 6-month-old Myo3a mutant mice. (b) Fusion phenomenon was observed in some stereocilia of mutant mice. (c) The stereocilia of some outer hair cells were found to be shorter, and the degeneration started from the innermost line of the stereocilia. (d) The stereocilia of inner hair cells become sharp in Myo3a mutant mice at the age of 2 months, 6 months, and 12 months. Scale bar = 10
During hearing formation, transformation from mechanical energy to electric energy is crucial, and ion channel plays a critical role in this process. The ion channel is located at the tip of the stereocilia where Myo3a is expressed. Thus, we investigated whether the function of the MET activity was affected in Myo3a mutant mice. We used the FM1-43 dye to stain the hair cells of wild-type and Myo3a mutant mice to determine whether the function of the MET activity was affected. The results are shown in Figure
MET activity is not affected in Myo3a mutant mice. FM1-43 staining showed that the MET activity is normal in Myo3a mutant mice. Scale bar = 20
Presbyacusis is highly similar to noise-induced deafness in clinical pathology. Numerous senile deaf individuals are extremely sensitive to noise. To verify whether the noise resistance in the Myo3a mutant mice was affected, we performed continuous white-noise experiment for 2 h in 4.5-month-old wild-type and Myo3a mutant mice. Hearing test was conducted in wild-type and mutant mice before, at 4 h after, and at 1 week after noise treatment. The experimental results are shown in Figure
ABR analysis in Myo3a mutant and wide-type mice after noise exposure. ABR threshold was tested for broadband click (a) and frequency-specific pure tone (b, c, d, e) on Myo3a mutant and wide-type mice before (control), 4 h after, and 1 week after noise exposure (
Using CRISPR/Cas9 technology, we generated a model of Myo3a Y137C mice consisting of a mutation in the kinase domain similar to that observed in the human Myo3a Y129C mutation. Through ABR hearing detection in mutant mice and wild-type mice, we found that the mutant mice exhibited progressive hearing loss. In 6 months, the hearing threshold of the mutant mice increased relative to that of the wild-type mice, whereas the hair cells of the 6-month-old mice started to degenerate. At 12 months of age, mutant mice exhibited significantly different hearing threshold and a more severe hair cell degeneration in the inner ear compared with that in the wild-type mice. The increased hearing threshold coincided with the loss of hair cells. This finding shows that kinase activity is crucial for the function of Myo3a and inner hair cells. Progressive hearing loss in Myo3a mutant mice was similar to that observed in human Myo3a mutants. Thus, the Myo3a mutant mouse model that we constructed was not only a good model for presbyacusis but also a satisfactory human disease model.
As a member of the myosin family, Myo3a transports cargo Espin1 to the top of the stereocilia [
Myo3a retains its motility by using the motor domain and the C-terminal THDII domain in combination with actin. In the wild-type mice, the THDI domain specifically binds to cargo Espin1, ensuring that Myo3a can transport Espin1 to the tip of the stereocilia to stabilize its structure [
Myo3a kinase activity may be disrupted in our mutant mouse model. Myo3a autophosphorylation cannot occur even at high Myo3a concentration at the top of the stereocilia so that the motor domain cannot be phosphorylated. The active motor domain tightly binds with actin and cannot be detached from actin. Thus, Myo3a that is located at the tip of the stereocilia cannot return to the cytoplasm and continue to transport Espin1. Thus, the structure of the top of the stereocilia is abnormal, thereby affecting the normal stereocilia functioning and causing progressive hearing loss.
We observed normal hearing threshold and normal inner ear hair cell development in the 2-month-old Myo3a mutant mice. The elevated hearing threshold and hair cell degeneration of mutant mice presented at ≥6 months of age. The explanation why mutant mice exhibit progressive rather than profound hearing loss at a young age is unknown. We speculate that Myo3b compensates for the loss of Myo3a.
Compensatory effects in higher organisms are highly common. Myo3b localizes at cochlear hair cell stereocilium tips, similar with the localization of Espin1 and Myo3a [
In Myo3a mutant mice, Myo3b can compensate for the loss of function of Myo3a because Myo3b is located at the stereocilium tip of the inner ear hair cells. Thus, we observed normal development of the inner ear hair cells. Myo3a and Myo3b double knock-out mice are profoundly deaf, demonstrating that class III myosins play redundant roles in hearing function [
Unlike a previous Myo3a knock-out mouse model [
The knock-in mice with Myo3a kinase domain mutation displayed progressive hearing loss and stereocilium degeneration in inner ear hair cells. Our mouse model of Myo3a point mutation made by CRISPR/Cas9 technology can simulate human diseases well and provide a good mouse model for the study of senile deafness.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare no conflict of interests regarding the publication of this paper.
Jiangang Gao and Peipei Li conceived and designed the experiments. Peipei Li and Zongzhuang Wen performed the experiments. Peipei Li analyzed the data. Jiangang Gao contributed reagents/materials/analysis tools. Peipei Li, Zongzhuang Wen, and Jiangang Gao wrote the paper.
This work was supported by grants from the Natural Science Foundation of China (31171194), the National 973 Basic Research Program of China (2014CB541703), and Shandong Provincial Science and Technology Key Program (2009GG10002039).