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. 2018 Jul 5:2018:4372913.
doi: 10.1155/2018/4372913. eCollection 2018.

Knock-In Mice with Myo3a Y137C Mutation Displayed Progressive Hearing Loss and Hair Cell Degeneration in the Inner Ear

Peipei Li  1 , Zongzhuang Wen  1 , Guangkai Zhang  1 , Aizhen Zhang  1 , Xiaolong Fu  1 , Jiangang Gao  1
Affiliations

Knock-In Mice with Myo3a Y137C Mutation Displayed Progressive Hearing Loss and Hair Cell Degeneration in the Inner Ear

Peipei Li et al. Neural Plast. .

Abstract

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.

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Figures

Figure 1
Figure 1
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 μm.
Figure 2
Figure 2
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 t-test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. Error bars indicate SEM. n > 5 for control and mutant mice for each experiment.
Figure 3
Figure 3
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 μm. The stereocilium degeneration and the loss of hair cells can be seen in the Myo3a mutant mice from 6 months old, and this phenomenon becomes more serious in 12-month-old Myo3a mutant mice.
Figure 4
Figure 4
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 μm.
Figure 5
Figure 5
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 μm for (a) and 2 μm for (b, c, and d).
Figure 6
Figure 6
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 μm.
Figure 7
Figure 7
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 (n≧7); error bars indicate SEM.
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