Figure 6. Loss of apical stability of RhoA-depleted outer hair cells and its effect on planar cell polarity, microtubule network, and junctional stability. Recombination was induced in RhoAfl/fl;Fgfr3-iCre-ERT2 mice at E13–14, and analysis was performed at E18.5. A, B, Cochlear ducts of control and mutant mice viewed under dark-field illumination show comparable lengths. C, D, Compared with the control specimen, basal turn of the mutant cochlea shows distinct abnormalities in the apical domain of OHCs, revealed by phalloidin labeling. E–G, Shown at a higher magnification and compared with the control specimen (E), medial turn of the mutant cochlea shows rather mild expansion of the OHC apical surface area. Hair bundles appear relatively normal as well (F). In contrast, the OHC apical area displays distinct expansion in the basal turn. Extrusion of OHC nuclei is evident (G). H, Quantification of the width of the apical surface area of hair cells in the basal turn of control and mutant cochleas. Differences are statistically significant in the case of recombined OHCs, as opposed to non-recombined inner hair cells. I, In controls, Vangl2 is expressed in the contact sites between the OHC’s medial wall and Deiters cells (arrows). J, K, In mutants, Vangl2 expression is maintained until OHCs are extruded (red arrow), based on phalloidin and Vangl2 double-labeling. K′, K″, An OHC with expanded apical surface is contacted by Vangl2-positive domains, shown at a high magnification. L, Acetylated tubulin-positive microtubules radiate from the kinocilia in OHCs of the control specimen. M, N, In mutant OHCs with a slightly expanded apical surface area, microtubules appear to be largely intact. Microtubules become disorganized along with further expansion of the OHC apex (asterisk) and cell extrusion (two asterisks). N′, N″, In a mutant OHC, acetylated tubulin and phalloidin double-labeling shows an intact appearance of microtubules around the kinocilium (arrowhead). Microtubules become less organized along radiation toward the cortex. O–Q, Similar to control OHCs (O), ZO-1 localizes evenly to tight junctions of mutant OHCs with an expanded apical surface area (asterisks; P, Q). This junctional expression is also seen in extruding OHCs (two asterisks; P, Q). Q′, Q″, A higher-magnification view of ZO-1–labeled tight junctions of an expanded OHC, shown together with phalloidin labeling. Images in I–Q″ are from the medial turn of the cochlea. Abbreviations: Ac. tub, acetylated tubulin; TM, tamoxifen. Scale bar (in Q″): A, B, 0.3 mm; C, D, 8 µm; E–G, I–K, L–N, O–Q, 5.5 µm; K′, K″, N′, N″, Q′, Q″, 3.5 µm.