Figure 1.

Adgrv1 Y6236fsX1 mutation leads to hearing loss and stereocilia disorganization in mice. a) Schematic diagrams showing the sequence features of wide‐type (WT) human ADGRV1 and Y6244fsX1 mutant. The ADGRV1 Y6244fsX1 mutation leads to a premature translational termination, which results in the truncation of the C‐terminal 63 residues. b) Schematic representation of the generation of Adgrv1 Y6236fsX1 mice using the CRISPR/Cas9 strategy. A deletion of 19 bp was introduced into exon 89 of the mouse Adgrv1 gene, resulting in a frameshift and premature translational termination. c) Representative blotting showing endogenous ADGRV1 in the cochlea of WT or Adgrv1 Y6236fsX1 mice. A band with > 200 kDa molecular weight corresponding to the intact ADGRV1 (or an endogenous isoform) and a 45‐kDa band corresponding to the ADGRV1‐CTF subunit were detected by both antibodies in the WT mice. In the Y6236fsX1 mutant, bands corresponding to truncated ADGRV1 were detected by the antibody recognizing the sequence (residue 6150–6200) before the truncating site but not the other antibody recognizing the C‐terminus (residue 6250–6300). Representative blotting from three independent experiments were shown. d) Cochlear whole mounts from P1 mice of indicated genotypes were stained with an anti‐ADGRV1 antibody specifically recognizing its N‐terminal extracellular fragment. TRITC‐phalloidin was used to visualize the F‐actin core of stereocilia. ADGRV1 immunoreactivity in the stereocilia of WT mice is indicated by arrows; ADGRV1 immunoreactivity in the apical bare zone of hair cells in the mutant mice is indicated by arrowheads; absence of ADGRV1 immunoreactivity at the corresponding position in the knockout mice is indicated by asterisks. All images were taken from the basal turn of the basilar membrane using a confocal microscope. Scale bar: 10 µm. Representative images from three independent experiments was shown. e) ABR thresholds of 2‐week‐old mice for click or pure tone stimuli. The number of animals used in each group was indicated in the brackets. f) DPOAE thresholds for each representative f2 frequency of P60 mice of different genotypes as indicated. The number of animals used in each group was indicated in the brackets. g,h) Representative SEM images showing hair bundle morphology of Y6236fsX1 heterozygous or homozygous mice at g) P8 or h) P30. Images were taken from the middle turn of the auditory sensory epithelia. Asterisks indicate complete loss of hair bundles. IHC, inner hair cell; OHC, outer hair cell. Scale bar: 10 µm (in low magnification images) or 2 µm (in high magnification images). Representative images from three independent experiments were shown. Data information: e–f) ***p < 0.001; ns, no significant difference; Adgrv1/del7TM or Y6236fsX1 mutant mice compared with WT mice. The bars indicate the mean ± SEM values. Data were statistically analyzed using one‐way ANOVA with Dunnett's post hoc test.