Slowly progressive hearing and hair cell loss in Atoh1–Cre;Cdc42^flox/flox mice. (A) Age-related click ABR thresholds (dB SPL) in Cdc42^flox/flox (cont, blue) and Atoh1–Cre;Cdc42^flox/flox (Cdc42-KO, red) mice. n≥4 animals; *P<0.05. (B) Age-related click and tone-burst (8, 16, 24 and 32 kHz) ABR thresholds in Cdc42^flox/flox and Atoh1–Cre;Cdc42^flox/flox mice. Atoh1–Cre;Cdc42^flox/flox mice show a progressive and high-frequency sound-dominant hearing loss. n≥4; *P<0.05. (C) Age-related DPOAE (f2 frequency at 8, 12, 16 and 20 kHz) thresholds in Cdc42^flox/flox and Atoh1–Cre;Cdc42^flox/flox mice. Progressive and high-frequency sound-dominant hearing loss is detected. n≥4; *P<0.05. (D) Representative (n≥4) Alexa-Fluor-488–phalloidin staining shows hair cell loss (white and red circles) in the middle turn of cochleae from Cdc42^flox/flox and Atoh1–Cre;Cdc42^flox/flox mice at the age of 2, 4 and 8 weeks. (E) The percentages of remaining IHCs and OHCs in each turn in Cdc42^flox/flox and Atoh1–Cre;Cdc42^flox/flox mice at 8 weeks. Note the IHC- and basal turn (Bs)-dominant hair cell loss. n≥3; *P<0.05. Ap, apical; Md, middle.

Cdc42 localization and function at the hair cell stereociliary membranes and AJCs. (A,B) Dissected organ of Corti from P1 wild-type mice cultured for 16 h and infected with the indicated adenoviruses (green). The top panels of A show membrane localization of GFP–Cdc42 covering an individual stereocilum (arrows) and at apical cell junctions in a IHC (arrowhead). Scale bar: 2 µm. The lower panels of A and B show reconstructed lateral view (in the xz axis) images of cochlear OHCs; GFP–Cdc42, but not GFP–Cdc42(17N;4A), is localized at the stereociliary membranes (arrow). Scale bars: 10 µm (C) Schematic representation of the intramolecular Cdc42 FRET biosensor: YPet and Turquoise-GL are variants of YFP and CFP, respectively. (D–G) Dissected organ of Corti from P2 Cdc42-FRET mice were observed under a two-photon excitation microscope. D, F and G are obtained from different organs of Corti. Scale bars: 10 µm. (D) The FRET/CFP ratio is highest at the stereocilia (arrowheads) and is higher at the apicolateral membranes (large arrow) than at the basolateral membranes (small arrow). Note that the image plane is oblique to the apical surface to show cross-sections of various depths, from the level of stereocilia to that of the basolateral membrane (see the illustration above it). A 3D movie is available in supplementary material Movie 1. (E) Schematic drawing showing an overview of IHCs and OHCs1–3 in an organ of Corti. (F) Composite image showing the FRET/CFP ratio of a series of five OHCs in the row of OHC3 obtained in serial sections from the base to top of stereocilia. Note that the five OHCs are obliquely aligned in vertical direction (see the illustration). The FRET/CFP ratio is higher in upper portions than in basal portions of stereocilia in all OHCs. (G) Representative FRET/CFP ratio images (n≥3) showing the effect of the Cdc42 inhibitor ML141 (before and 4 min after 500 µM ML141 treatment). The right-hand panel shows a quantification of these results (mean ± s.e.m.) showing that FRET/CFP ratios at the stereocilia and AJCs are significantly decreased by ML141. *P<0.01.

Degeneration of cochlear stereocilia in Atoh1–Cre;Cdc42^flox/flox mice. SEM images of the organ of Corti at the middle turn (A–J) and the apical turn (K–M) obtained from Cdc42^flox/flox (cont; A,C,G) and Atoh1–Cre;Cdc42^flox/flox (B,D–F,H–M) mice at the age of P8 (E,F), 2 (H), 4 (I), 6 (J), and 8 weeks (A–D,G,K–M). (A) Both IHCs and OHCs are regularly aligned in a plane in Cdc42^flox/flox mice at 8 weeks. (B) In Atoh1–Cre;Cdc42^flox/flox mice at 8 weeks, IHCs mostly disappeared, whereas OHCs are partially depleted and have scattered stereocilia. (C) In Cdc42^flox/flox OHCs at 8 weeks, stereocilia have the characteristic W-profile. (D) In Atoh1–Cre;Cdc42^flox/flox OHCs at 8 weeks, stereocilia are fewer in number and have lost their characteristic W-profile and precise staircase pattern with consistent length of stereocilia in each row. (E,F) The morphology of IHC (E) and OHC (F) in Atoh1–Cre;Cdc42^flox/flox mice at P8 is identical to that in Cdc42^flox/flox mice (not shown). (G) The regular array of stereocilia in IHCs of Cdc42^flox/flox mice at 8 weeks. (H) In the middle turn of cochlea in Atoh1–Cre;Cdc42^flox/flox mice, stereocilia fusion (arrowheads) is first observed at 2 weeks. (I–M) The stereoilia fusion (arrowheads) and IHC loss (asterisks) increase in number through 4 (I) to 6 weeks (J). The apical turn of cochlea at 8 weeks retains some IHCs, which have frequently fused stereocilia (K–M, arrowheads), and long (L, arrows) and short stereocilia (M, double arrow). Scale bars: 5 µm (A,B); 2 µm (C–L).

Disturbed ultrastructure of stereocilia and AJCs in Atoh1–Cre;Cdc42^flox/flox mice. TEM images of IHCs at the middle turn of cochlea at 6 weeks of age. (A) In Cdc42^flox/flox (cont) mice, IHCs and supporting cells (SC) are alternately aligned (left) and there is a thick actin-rich cuticular plate bordered by the arcuate-shaped AJCs with the circumferential actin belt (arrowheads) observed as a dense layer beneath the plasma membrane (right, magnified view). Stereocilia are located on the apical surface and each has a single rootlet inserted into the cuticular plate (arrow). (B) Upper panels: Atoh1–Cre;Cdc42^flox/flox (Cdc42-KO) mice are missing some IHCs, which were displaced by supporting cells, characterized by the apical microvilli. Lower panels: stereocilia in the remaining IHCs are often fused at the base (black arrows) and they contain several actin cores (black arrowheads) with rootlets (white arrows). The right two panels are close-up views of supporting cells with microvilli and neighboring hair cells (upper) and fused stereocilia base (lower). (C) Close-up views of the AJCs in Cdc42^flox/flox (upper panels) and Atoh1–Cre;Cdc42^flox/flox mice (lower panels). Each panel shows a cross section of a supporting cell in the middle, flanked by two hair cells (asterisks). The AJCs display smooth and vertical arcs in controls but shows a range of ruffling in Atoh1–Cre;Cdc42^flox/flox mice. (D) Close-up views of the AJCs and perijunctional actin belts. Hair cells (asterisks) are on the right side. The actin belt (outlined by white dots) is thinner in Atoh1–Cre;Cdc42^flox/flox hair cells. Scale bars: 1 µm (A,B); 500 nm (C); 200 nm (D).

Reduced number and shape-abnormalities of microvilli in MDCK^Cdc42-KD cells. (A) Confocal images of MDCK^GFP-Cdc42 cells cultured in Matrigel and counterstained with Alexa-Fluor-568–phalloidin. Note the strong and weak GFP–Cdc42 accumulation at the apical (arrow) and lateral surfaces of cyst-cells (arrowheads), respectively. Scale bar: 10 µm. (B,C) Immunoblot showing the effects of three different shRNA-plasmids targeting canine Cdc42 (sh29, sh197, sh333) in MDCK cells. Using the most effective shRNA (sh197), two different clones (MDCK^Cdc42-KDa and MDCK^Cdc42-KDg) were established. (D) SEM images showing the apical surface of MDCK cells grown on a filter insert. Both MDCK^Cdc42-KDa and MDCK^Cdc42-KDg cell lines had substantially fewer microvilli compared with cells expressing a control shRNA (MDCK^cont). Scale bar: 5 µm. (E) SEM images of MDCK^cont and MDCK^Cdc42-KDg cells grown on a filter insert, and infected by the indicated adenoviruses. Colored insets show the GFP signal. Note the localization of GFP–Cdc42 at cell–cell junctions (arrowheads). Scale bars: 10 µm. (F) Recovery of reduced microvilli in MDCK^Cdc42-KDg cells by the expression of adenovirus-encoded GFP–Cdc42 but not GFP–Cdc42(17N;4A). *P<0.01. (G) High-power images obtained by scanning helium-ion microscope (SHIM) showing the range of morphological abnormalities in microvilli in MDCK^Cdc42-KDg cells. Black arrow, black arrowhead, red arrows, and red arrowhead showing shortened, lengthened, ragged and fused microvilli, respectively. Scale bar: 500 nm.

Disruption of TJs in MDCK^Cdc42-KD cells. (A,B) Confocal images of ZO1 immunostaining in confluent MDCK^cont (A) and MDCK^Cdc42-KDg (B) cells. Note the absence of ZO1 at cell–cell junctions in MDCK^Cdc42-KDg cells. (C,D) Confocal images of ZO1 immunostaining in MDCK^Cdc42-KDg cells infected by indicated adenoviruses. Note the presence of ZO1 immunostaining at cell–cell junctions in cells expressing GFP–Cdc42 (C) but not GFP–Cdc42(17N;4A) (D). Insets, DAPI staining. Scale bars: 20 µm.