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. 2019 Nov 22;13:1255.
doi: 10.3389/fnins.2019.01255. eCollection 2019.

A Novel Mouse Model of MYO7A USH1B Reveals Auditory and Visual System Haploinsufficiencies

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Free PMC article

A Novel Mouse Model of MYO7A USH1B Reveals Auditory and Visual System Haploinsufficiencies

Kaitlyn R Calabro et al. Front Neurosci. .
Free PMC article

Abstract

Usher's syndrome is the most common combined blindness-deafness disorder with USH1B, caused by mutations in MYO7A, resulting in the most severe phenotype. The existence of numerous, naturally occurring shaker1 mice harboring variable MYO7A mutations on different genetic backgrounds has complicated the characterization of MYO7A knockout (KO) and heterozygote mice. We generated a novel MYO7A KO mouse (Myo7a - / -) that is easily genotyped, maintained, and confirmed to be null for MYO7A in both the eye and inner ear. Like USH1B patients, Myo7a - / - mice are profoundly deaf, and display near complete loss of inner and outer cochlear hair cells (HCs). No gross structural changes were observed in vestibular HCs. Myo7a - / - mice exhibited modest declines in retinal function but, unlike patients, no loss of retinal structure. We attribute the latter to differential expression of MYO7A in mouse vs. primate retina. Interestingly, heterozygous Myo7a + / - mice had reduced numbers of cochlear HCs and concomitant reductions in auditory function relative to Myo7a +/+ controls. Notably, this is the first report that loss of a single Myo7a allele significantly alters auditory structure and function and suggests that audiological characterization of USH1B carriers is warranted. Maintenance of vestibular HCs in Myo7a - / - mice suggests that gene replacement could be used to correct the vestibular dysfunction in USH1B patients. While Myo7a - / - mice do not exhibit sufficiently robust retinal phenotypes to be used as a therapeutic outcome measure, they can be used to assess expression of vectored MYO7A on a null background and generate valuable pre-clinical data toward the treatment of USH1B.

Keywords: Usher syndrome; cochlea; hearing; myosin-7A; retina; vision.

Figures

FIGURE 1
FIGURE 1
Generation and confirmation of MYO7A knockout in Myo7a–/– mice. (A) Schematic of how Myo7a–/– mice were generated using Cre-lox recombination. Exons 10 and 11 of Myo7a were deleted by breeding Myo7aTM1a(EUCOMM)Wtsi mice with a Sox2-Cre deleter line, creating the Myo7aTM1b(EUCOMM)Wtsi or knockout allele. Immunoblotting was performed to compare differences in MYO7A expression in 4-month-old Myo7a+/+, Myo7a+/–, and Myo7a–/– (B) eyes (n = 3 per genotype) and (C) cochlea (n = 2–4 per genotype). Protein levels were quantified and compared using Fiji. MYO7A expression was normalized to vinculin and analyzed relative to MYO7A expression in Myo7a+/+ mice. FRT, flippase recognition target; Kan, kanamycin; kDa, kilodaltons; VCL, vinculin.
FIGURE 2
FIGURE 2
Characterization of cochlear hair cell structure and function in Myo7a+/+, Myo7a+/–, and Myo7a–/– mice at 5 months of age. (A,B) Assessment of auditory brainstem response (ABR) thresholds in male (A) and female (B) mice were measured with a tone burst stimulus at 8, 16, and 32 kHz in (n = 5–6). Two-way ANOVA with Bonferroni’s multiple comparisons tests were used. Data are shown as means ± SEM. p < 0.05 between Myo7a/ vs. Myo7a+/+; +p < 0.05 between Myo7a+/– vs. Myo7a+/+. (C–E) Cochleograms were recorded in Myo7a+/+ (C), Myo7a+/– (D), and Myo7a/ (E) mice at 5 months of age. Graphs show percent loss of inner hair cells (IHC, solid line) and outer hair cells (OHC, dotted line) as a function of percent distance from the apex of the cochlea. Lower x-axes show the frequency-place map for mouse cochlea. (F–H) HC morphology in the middle turn of cochlear basilar membranes of Myo7a+/+ (F), Myo7a+/– (G), and Myo7a–/– (H) mice at 5 months of age.
FIGURE 3
FIGURE 3
Assessment of basal–cochlear and utricle structure. Organ of Corti (A), spiral ganglion neurons (B), stria vascularis (C), and vestibular hair cells (D) in 5-month-old Myo7a+/+, Myo7a+/–, and Myo7a–/– mice. (A–C) Representative H&E-stained sections from the basal-cochlear region, imaged at 40× magnification. (D) Low magnification photomicrograph of representative hematoxylin-stained surface preparations of the macular utricle showing a dense array of vestibular hair cells covering the sensory epithelium (scale bar: 50 μm). Inset panels show a higher magnification view of densely packed vestibular hair cells with darkly stained nuclei (arrows, scale bar: 15 μm). SGN, spiral ganglion neuron; SV, stria vascularis. Scale bars for organ of Corti, SGN, and SV = 20 μm.
FIGURE 4
FIGURE 4
Characterization of retinal function and structure in Myo7a+/+, Myo7a+/–, and Myo7a–/– mice (n = 11–12 mice/genotype) over 6 months. Average scotopic b-wave (A), scotopic a-wave (B), and photopic b-wave (C) amplitudes. Equal numbers of males and females were used. Representative scotopic (D) and photopic (E) ERG waveforms at 2-, 4-, and 6-months of age. Scotopic and photopic values in panels (A–D) were recorded at 1.0 and 25.0 cd.s/m2, respectively. Average outer nuclear layer (ONL) thickness (F) in Myo7a+/+, Myo7a+/–, and Myo7a–/– mice over 6 months (n = 11–12 mice/genotype). Equal numbers of males and females were used. No gross changes in ONL thickness were observed. p < 0.5 between Myo7a–/– vs. Myo7a+/+; +p < 0.5 between Myo7a+/– vs. Myo7a+/+.
FIGURE 5
FIGURE 5
Characterization of retinal structure and function in Myo7a+/+:RHOGFP+/–, Myo7a+/–:RHOGFP+/–, Myo7a–/–:RHOGFP+/–, and P23H+:RHOGFP+/– mice (n = 11–12 mice/genotype) over 6 months. (A) Retinal cross sections (n = 4 mice/genotype) were co-stained with DAPI (left). Endogenous GFP expression from RHOGFP in the same section (without DAPI) is shown (right). Endogenous GFP expression on the right is overexposed for optimal visualization of RHOGFP localization in photoreceptor inner segments and ONL. All images taken at 60× magnification with identical exposure settings. Scale = 50 μm. Average (B) scotopic b-wave, (C) scotopic a-wave, and (D) photopic b-wave amplitudes in Myo7a+/+:RHOGFP+/–, Myo7a+/–:RHOGFP+/–, Myo7a–/–:RHOGFP+/–, and P23H+:RHOGFP+/– mice (n = 10–12 mice/genotype) over 6 months. Equal numbers of males and females were used. Scotopic and photopic averages were recorded at 1.0 and 25.0 cd.s/m2, respectively. (E) Average outer nuclear layer (ONL) thickness in Myo7a+/+:RHOGFP+/–, Myo7a+/–:RHOGFP+/–, Myo7a–/–:RHOGFP+/–, and P23H+:RHOGFP+/– mice (n = 10–12 mice/genotype) over 6 months. Equal numbers of males and females were used. No P23H+:RHOGFP+/– ONL data are included at 6 months because retinas were too thin for accurate measurement. p < 0.5 between Myo7a–/–:RHOGFP+/– vs. Myo7a+/+:RHOGFP+/–; +p < 0.5 between Myo7a+/–:RHOGFP+/– vs. Myo7a+/+:RHOGFP+/–; ∗∗p < 0.5 between P23H+:RHOGFP+/– vs. Myo7a+/+:RHOGFP+/–.
FIGURE 6
FIGURE 6
Expression of MYO7A in non-human primate vs. mouse. Neural retina and RPE from a 5-year-old male macaque and 5-month-old WT mice were manually separated. Protein from each compartment was immunoblotted with an antibody raised against MYO7A. The majority of MYO7A expression in macaque was found in neural retina (left). The majority of MYO7A expression in mouse was found in RPE (right). Protein levels were quantified and compared using Fiji. MYO7A expression was normalized to beta-actin. WT mouse = C57BL/6J.

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