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. 2017 Dec 4:10:401.
doi: 10.3389/fnmol.2017.00401. eCollection 2017.

Loss of CIB2 Causes Profound Hearing Loss and Abolishes Mechanoelectrical Transduction in Mice

Yanfei Wang  1   2 , Jie Li  3 , Xuerui Yao  1   2 , Wei Li  1   2 , Haibo Du  1   2 , Mingliang Tang  4   5 , Wei Xiong  3 , Renjie Chai  4   5   6 , Zhigang Xu  1   2
Affiliations

Loss of CIB2 Causes Profound Hearing Loss and Abolishes Mechanoelectrical Transduction in Mice

Yanfei Wang et al. Front Mol Neurosci. .

Abstract

Calcium and integrin-binding protein 2 (CIB2) belongs to a protein family with four known members, CIB1 through CIB4, which are characterized by multiple calcium-binding EF-hand domains. Among the family members, the Cib1 and Cib2 genes are expressed in mouse cochlear hair cells, and mutations in the human CIB2 gene have been associated with nonsyndromic deafness DFNB48 and syndromic deafness USH1J. To further explore the function of CIB1 and CIB2 in hearing, we established Cib1 and Cib2 knockout mice using the clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease (CRISPR/Cas9) genome editing technique. We found that loss of CIB1 protein does not affect auditory function, whereas loss of CIB2 protein causes profound hearing loss in mice. Further investigation revealed that hair cell stereocilia development is affected in Cib2 knockout mice. Noticeably, loss of CIB2 abolishes mechanoelectrical transduction (MET) currents in auditory hair cells. In conclusion, we show here that although both CIB1 and CIB2 are readily detected in the cochlea, only loss of CIB2 results in profound hearing loss, and that CIB2 is essential for auditory hair cell MET.

Keywords: CIB2; Usher syndrome; hearing loss; knockout mice; mechanoelectrical transduction; stereocilia.

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Figures

Figure 1
Figure 1
Expression of Cib1, Cib2, Cib3 and Cib4 in different mouse tissues. (A) Amino acid sequences of Mus musculus calcium and integrin-binding protein 1 (CIB1), CIB2, CIB3 and CIB4 were aligned using the CLUSTALW multiple sequence alignment program. (B) The phylogenic tree of Mus musculus CIB1, CIB2, CIB3 and CIB4 was constructed using the rooted phylogenetic tree with branch length (UPGMA) method. (C) Expression of Cib1, Cib2, Cib3 and Cib4 in different tissues of 6-month-old mice was determined by RT-PCR. (D) Expression of Cib1, Cib2, Cib3 and Cib4 in the vestibule, basilar membrane and spiral ganglion cells of P2 mice was determined by RT-PCR. β-actin was included as the internal control.
Figure 2
Figure 2
Construction of Cib1 and Cib2 knockout mice. (A) The schematic drawing of the strategy for Cib1 gene disruption. The target sites of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 small guide RNAs (sgRNAs) in the Cib1 gene are indicated in red, and the deleted region in the Cib1 gene of knockout mice is indicated by dashes. The positions of RT-PCR primers are indicated by arrows. (B) The schematic drawing of the domain structure of CIB1 in wildtype and knockout mice. (C) The expression of Cib1 mRNA in the inner ear of P60 mice was determined by RT-PCR. β-actin was included as the internal control. (D) The schematic drawing of the strategy for Cib2 gene disruption. The target sites of CRISPR-Cas9 sgRNAs in the Cib2 gene are indicated in red, and the deleted regions in the Cib2 gene of knockout mice are indicated by dashes. The positions of RT-PCR primers are indicated by arrows. (E) The schematic drawing of the domain structure of CIB2 in wildtype and knockout mice. (F) The expression of Cib2 mRNA in the inner ear of P60 mice was determined by RT-PCR. β-actin was included as the internal control.
Figure 3
Figure 3
Hearing threshold is elevated in Cib2 knockout mice, but not in Cib1 knockout mice. (A) The auditory brainstem response (ABR) thresholds for click stimuli in Cib1+/− and Cib1−/− mice at different ages. (B) The ABR thresholds for pure tone stimuli in 1-month-old Cib1+/− and Cib1−/− mice. (C) The ABR thresholds for click stimuli in Cib2+/− and Cib2−/− mice at different ages. (D) The ABR thresholds for pure tone stimuli in 1-month-old Cib2+/− and Cib2−/− mice. (E) The distortion product otoacoustic emission (DPOAE) thresholds for pure tone stimuli in 1-month-old Cib2+/− and Cib2−/− mice. The numbers of animals for each group used in the experiments are indicated. The differences were evaluated by Student’s t-test (**p < 0.01; ***p < 0.001).
Figure 4
Figure 4
Whole-mount immunostaining in Cib2 knockout mice. Shown are single confocal sections. CIB2 immunoreactivity was visualized with FITC-conjugated secondary antibody, and F-actin was visualized with rhodamine-conjugated phalloidin. (A) P7 Cib2+/− mice. (B) P7 Cib2−/− mice. (C) P30 Cib2+/− mice. (D) P30 Cib2−/− mice. The images were taken at the middle turn of the cochlea. Stereocilia bundle fragmentation was indicated by arrows. Scale bars: 5 μm.
Figure 5
Figure 5
Stereocilia are disorganized in Cib2 knockout mice. (A–F) Low-magnification scanning electron microscopy (SEM) images of cochlear hair bundles from mice of different genotypes and ages as indicated. (A′–F′) High-magnification SEM images of outer hair cell (OHC) hair bundles from mice of different genotypes and ages. (A″–F″) High-magnification SEM images of inner hair cell (IHC) hair bundles from mice of different genotypes and ages. The images were taken at the apical-middle turn of the cochlea. Stereociliary bundle fragmentation is indicated by arrows, stereocilia loss is indicated by asterisks, and stereocilia fusion is indicated by triangles. Scale bars, 5 μm.
Figure 6
Figure 6
Hair bundle development is affected in Cib2 knockout mice. (A–F) High-magnification SEM images of cochlear hair bundles from mice of different genotypes and ages as indicated. The images were taken at the apical-middle turn of the cochlea. Three rows of stereocilia are indicated by numbers. Over-grown and retracted stereocilia are indicated by arrows up and down, respectively. The kinocilium that does not regress properly is indicated by asterisk. Scale bars, 1 μm.
Figure 7
Figure 7
Mechanosensitivity of hair cells is lost in Cib2 knockout mice. (A) Mechanoelectrical transduction (MET) current was examined in OHCs from control and Cib2 knockout mice. A fluid jet system that drives a sinusoidal deflection of hair bundles was used to evaluate the saturating MET current from hair cells. (B) The average peak current was 829.2 pA in control OHCs but absent in knockout OHCs. (C) Voltage-gated current was recorded from control and Cib2 knockout OHCs. The membrane potential was changed from −150 mV to +110 mV in 20 mV steps. (D) Current–voltage (I-V) curves were drawn from data similar to (C), indicating reduced membrane potassium currents in Cib2 knockout OHCs. (E) The average endocochlear potential (EP) was around 85 mV in control and 89 mV in Cib2 knockout mice. In all panels, data were collected from three control heterozygous mice (shown in black) and three Cib2 knockout mice (shown in red). The age of mice is P7 (A–D) or P30 (E). The numbers of cells used are shown in each panel. The differences were evaluated by Student’s t-test (*p < 0.05; **p < 0.01; ***p < 0.001).
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