Differential regulation of hair cell actin cytoskeleton mediated by SRF and MRTFB
- Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, China
- Ear Institute, Shanghai Jiao Tong University School of Medicine, China
- Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, China
- Department of Neuroscience, University of Virginia, United States
Figures
Figure 1 with 1 supplement
Srf cKO mice have defects in the cuticular plate.
(A) Reduced phalloidin staining in the cuticular plates of Srf cKO hair cells (HCs) at P10. (B) Quantification of phalloidin reactivity in the cuticular plates of HCs in control and Srf cKO mice at …
Figure 1—figure supplement 1
Conditional knockout of Srf in mice caused growth defects.
(A) Representative examples of P10 control and Srf cKO pups. (B) Body mass measurements of control and Srf cKO mice at P10. Analyzed numbers (animals): control (7), Srf cKO (8). Error bars indicate …
Figure 2 with 1 supplement
The defects of stereocilia dimensions in Srf cKO mice.
(A) Phalloidin-stained inner hair cell (IHC) bundles of control and Srf cKO at different ages. En face views (scale bar, 5 μm) are on the left, and side views (scale bar, 1 μm) are on the right. (B) …
Figure 2—figure supplement 1
Srf cKO mice have defects in hair bundles.
(A) Phalloidin staining of control and Srf cKO outer hair cells (OHCs) hair bundles at various ages. (B) Phalloidin staining of control and Srf cKO hair bundles in P15 utricle. Upper, bundles of …
Figure 3
Altered distribution of tip proteins and actin crosslinker in Srf cKO mice.
(A–C) Immunofluorescence localization of ESPN1, EPS8, and GNAI3 in inner hair cells (IHCs) of control and Srf cKO at P4 and P10. Arrows indicate proteins staining on row 2 tips. Left panels are …
Figure 4 with 2 supplements
Mrtfb cKO mice have defects in the cuticular plates.
(A) Reduced phalloidin staining in the cuticular plates of Mrtfb cKO HCs at P10. (B) Quantification of phalloidin reactivity in the cuticular plates of inner hair cells (IHCs) and outer hair cells …
Figure 4—figure supplement 1
No obvious defects in HCs and hearing function of Mrtfa cKO mice.
(A) RNAscope in situ hybridization of Mrtfa in E16 control cochleae. (B) Genomic structures of mouse Mrtfa gene, CRISPR-mediated targeting allele, and mutated allele after Cre-mediated …
Figure 4—figure supplement 2
Conditional knockout of Mrtfb in mice.
(A) RNAscope in situ hybridization of Mrtfb in E16 control cochleae. (B) Genomic structures of mouse Mrtfb gene, CRISPR-mediated targeting allele, and mutated allele after Cre-mediated …
Figure 5 with 1 supplement
The defects of stereocilia dimensions in Mrtfb cKO mice.
(A) Phalloidin staining of inner hair cell (IHC) bundles at different ages. En face views (scale bar, 5 μm) are on the left, and side views (scale bar, 1 μm) are on the right. (B) Fluorescence-based …
Figure 5—figure supplement 1
Mrtfb cKO mice have stereocilia impairments.
(A) Phalloidin staining of outer hair cell (OHC) bundles in control and Mrtfb cKO at different ages. (B) Phalloidin staining of hair bundles in P60 control and Mrtfb cKO utricle. Upper, bundles of …
Figure 6
Altered distribution of tip proteins and actin crosslinker in Mrtfb cKO mice.
(A–C) Immunofluorescence localization of ESPN1, EPS8, and GNAI3 in inner hair cells (IHCs) of control and Mrtfb cKO mice at different ages. Left panels are proteins staining separately from the …
Figure 7 with 1 supplement
Early-onset and progressive hearing loss in Mrtfb cKO mice.
(A) Auditory brainstem response (ABR) analysis demonstrates Mrtfb cKO mice exhibited early-onset and progressive hearing loss. (B) Distortion product otoacoustic emission (DPOAE) thresholds in Mrtfb …
Figure 7—figure supplement 1
Hair cell numbers were not affected in Mrtfb cKO mice.
(A, B) The hair cell numbers were not affected in Mrtfb cKO mice at P26. Analyzed numbers (animals): P26, control (4), Mrtfb cKO (4). P42, control (3), Mrtfb cKO (3). Scale bar, 5 μm. Error bars …
Figure 8 with 1 supplement
Srf cKO and Mrtfb cKO hair cell transcriptome analysis.
(A) Diagram of fluorescence-activated cell sorting (FACS) purification strategy to isolate hair cells. The genotypes of mice used for cell sorting are on the right of the mouse cartoon. (B) The …
Figure 8—figure supplement 1
Validation of RNA-sequencing results.
(A) Comparison of Srf cKO or Mrtfb cKO’s regulated genes which are known downstream of MRTF–SRF pathway. Dotted lines on the plot represent the cutoff to annotate the differentially expressed genes …
Figure 9 with 1 supplement
The injection of Anc80L65-Cnn2 partially restores the cuticular plate integrity and stereocilia morphology in Srf cKO mice.
(A, B) CNN2 immunostaining in the stereocilia and cuticular plates of P6 inner hair cells (IHCs). (C) Quantitation of CNN2 reactivity in row 1 IHC stereocilia. Analyzed numbers (stereocilia, cells, …
Figure 9—figure supplement 1
The injection of AAV-Anc80L65-Cnn2 partially restores outer hair cell (OHC) stereocilia morphology in Srf cKO mice.
(A) RNAscope in situ hybridization of Cnn2 in E16 control cochlea. Inset indicates hair cells. (B) Immunohistochemical analysis of CNN2 expression in control inner hair cells (IHCs) at different …
Figure 10
An illustration model of differential regulation of actin cytoskeleton mediated by SRF and MRTFB in hair cells.
Additional files
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Supplementary file 1
Primer sequences used for PCR and RT-qPCR.
- https://cdn.elifesciences.org/articles/90155/elife-90155-supp1-v3.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/90155/elife-90155-mdarchecklist1-v3.pdf
