The integrity of cochlear hair cells is established and maintained through the localization of Dia1 at apical junctional complexes and stereocilia

摘要

a Lysates were obtained from the organ of Corti (OC) and the spiral ganglion (SG) of WT and DIA1TG/TG (TG) mice at P5. Expression of FLAG-tagged DIA1(R1204X) was confirmed by immunoblotting using FLAG and DIAPH1 antibodies. Comparable loading of proteins was confirmed using β3-tubulin and GAPDH antibodies. Uncropped images are shown in Fig. S1A. b Cochleae were obtained from WT and TG mice at P5, and immunostained using an mDia1 antibody followed by an Alexa568-conjugated secondary antibody and Alexa488-conjugated phalloidin. Arrowheads in the low-magnification view of the TG cochlea show the dense region of mDia1-positive cells, which was not detectable in WT mice. High magnification views of the OC from the boxed region in the upper panels are shown in lower panels (mDia1: red, phalloidin: green). Arrows and arrowheads show mDia1-positive inner pillar cells (IPCs) and outer hair cells (OHCs), respectively. Scale bars: 50 μm. Mid-modiolar-section images of the cochlea from TG mice at P8 are shown in Fig. S2A–C. c Statistical analysis of the number of mDia1-positive OHCs in the OC of WT and TG mice at P5 (n = 9). ***p = 0.0009 by unpaired Student’s t-test. d High magnification views of the OC from WT and TG mice at P6. Arrowheads, double-headed arrows, arrows, and asterisks show mDia1-positive OHCs, Deiters’ cells (DCs), IPCs, and inner hair cells (IHCs), respectively. Scale bars: 20 μm. Reconstructed lateral projections of the OC from TG mice at P5 are shown in Fig. S2D–F. e Whole-mount preparations of the OC dissected into three segments (Ap: apical turn, Md: middle turn, and Bs: basal turn) were obtained from WT and TG mice at P5, P7, and P14, and stained using an mDia1 antibody. Each panel shows a confocal microscopic image focused at an AJC plane containing both OHCs and IHCs. A basal-to-apical progressive increase in the immunoreactivity of mDia1 was observed, as seen with maturation of the OC. Scale bar: 10 μm. f Cryostat sections of cochleae at the SG plane from WT and TG mice were obtained at P14, and immunostained using DIAPH1 (red) and β3-tubulin (green) antibodies with DAPI (blue). DIAPH1 immunoreactivity was enhanced using tyramide signal amplification (TSA). Arrowheads indicate DIAPH1-highly positive cells. Scale bar: 50 μm.