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Role of Dach1 Revealed Using a Novel Inner Ear-Specific Dach1-knockdown Mouse Model

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Role of Dach1 Revealed Using a Novel Inner Ear-Specific Dach1-knockdown Mouse Model

Toru Miwa et al. Biol Open.

Abstract

The Dach1 gene is expressed in the inner ear of normal mouse embryos in the area that differentiates into the cochlear stria vascularis (SV). We hypothesised that Dach1 downregulation in the inner ear would lead to SV dysplasia. However, because Dach1 knockout is embryonic lethal in mice, the role of Dach1 in the inner ear is unclear. Here, we established inner ear-specific Dach1-knockdown mice and showed that Dach1 downregulation resulted in hearing loss, reduced endocochlear potential and secondary outer hair cell loss. There were no abnormalities in marginal cells and basal cells in the SV or spiral ligament in inner ear-specific Dach1-knockdown mature mice. However, intermediate cell dysplasia and thinning of the SV were observed. Moreover, dynamic changes in the expression of key genes related to the epithelial-mesenchymal transition were observed in the lateral wall of the cochlear epithelium, which differentiated into the SV in inner ear-specific Dach1-knockdown mice at embryonic stages. In summary, suppression of Dach1 expression in the inner ear caused the epithelial-mesenchymal transition in the lateral wall of cochlear epithelium, resulting in loss of intermediate cells in the SV and SV dysplasia.This article has an associated First Person interview with the first author of the paper.

Keywords: Dach1; Epithelial-mesenchymal transition; Intermediate cell; Knockdown mouse; Stria vascularis.

Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Electroporation-mediated transuterine gene transfer into otocysts (EUGO) in mice at E11.5. (A) The arrowhead indicates an E11.5 embryo otocyst. Fast Green dye was microinjected into the otocysts, and the uterine wall was then removed. (B) Development of cochlea. (C) A cross-sectional image of a typical adult cochlea. The adult mammalian cochlea is divided into three compartments: the scala vestibuli, scala tympani and scala media. Depicted here is a cross-section of the scala media, which contains the organ of Corti (OC). The OC contains three types of cell populations: inner hair cells (IHCs), outer hair cells (OHCs) and supporting cells (SCs). The two types of auditory hair cells play critical roles in hearing as mechanoelectrical transducers. The auditory hair cells are overlaid by the tectorial membrane (TM). The SV, in the lateral wall of the scala media, is responsible for the secretion of K+ into the endolymph and for endocochlear potential production. SL, spiral limbus; SLi, spiral ligament; SG, spiral ganglion. (D) Scheme of the cellular structure of the SV. Cell types are as indicated in the figure. SV integrity relies on a three-layered tissue architecture of marginal, intermediate and basal cells that enclose a dense capillary network. The formation of long cell processes by all three cell types and a high degree of interdigitation characterises strial architecture. (E) EUGO was performed in mice at E11.5. Embryos were delivered via C-section at E18.5, and the pups that underwent gene transfer at E11.5 were passed to surrogate dams.
Fig. 2.
Fig. 2.
The shRNA-Dach1 plasmid was expressed in the treated developing cochlear duct. (A) After injecting the GFP-containing shRNA-Dach1 plasmid and the GFP-containing shRNA-scramble plasmid into the inner ear of a wild-type (WT) mouse embryo, GFP signals were observed in the precursor neuroepithelium, medial wall, lateral wall and the spiral ganglion cells in the inner ear at E15.5. (B) No difference was observed in the expression of GFP between groups transfected with shRNA-scramble and shRNA-Dach1 (P=0.44). Scale bar: 50 μm. N.S., not significant.
Fig. 3.
Fig. 3.
The Dach1 signal was lost in the treated cochleae. After transferring the shRNA-Dach1 plasmid, Dach1 mRNA expression was lost in the lateral wall of the cochlear duct at E15.5 (asterisks), and the cochlear duct was observed to balloon slightly. The images shown in the lower panels are higher magnification images of the lateral wall. All images were taken in the middle turns of the cochlear duct.
Fig. 4.
Fig. 4.
Intermediate cells were absent from the SV of the cochlea of inner ear-specific Dach1-KD mice. (A,B) Expression of the potassium channel KCNQ1, a marginal cell marker (A), and Claudin11, a basal cell marker (B), in mature inner ear-specific Dach1-KD mice and control mice. No difference was observed in the expression of KNCQ1 and Claudin11 between control mice and KD mice. (C) Thickness of the SV of KD mice was significantly decreased compared to control mice (****P<0.0001). (D,E) Expression of Kir4.1, a marker of intermediate cells in KD mice was significantly decreased, as confirmed by western blotting (*P=0.01). (F) No difference was observed in the expression of Connexin26, a marker of the spiral ligament between KD mice and control mice. Square brackets show the thickness of SV.
Fig. 5.
Fig. 5.
The EMT in the lateral wall of the treated developing cochlear duct. (A–F) Protein and mRNA expression in the lateral wall of the cochlear epithelium, utilising immunohistochemistry and qRT-PCR analyses in inner ear-specific Dach1-KD mouse embryos and control embryos at E15.5. (A,B) Vimentin (*P=0.03) (C,D) E-cadherin (*P=0.04). (E,F) TGFβR2 (*P=0.04).
Fig. 6.
Fig. 6.
Loss of Dach1 signalling in the cochlea caused decreased EP and deafness. (A) EP in mature inner ear-specific Dach1-KD mice and control mice (****P<0.0001). (B) Hearing thresholds in mature inner ear-specific Dach1-KD mice and control mice (4 kHz, **P=0.003; 8 kHz, **P=0.004; 12 kHz, ***P=0.0009; 20 kHz, *P=0.01; 32 kHz, ***P=0.0005). (C,D) Auditory outer hair cells, but not inner hair cells, were malformed and degenerated in the cochleae of adult inner ear-specific Dach1-KD mice in the absence of Dach1 signalling. White arrowheads show the loss of hair cells. ****P<0.0001; N.S., not significant.

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