Fig. 5

aLacZ reporter is specifically expressed in HCs in the Gpr156^del/+ auditory epithelium at P4 and P21 (right panels: cochlear base). b GPR156 immunolabeling shows polarized protein enrichment at the medial HC junction in control (left, arrowhead) but not Gpr156 mutant cochlear HCs (right). Peanut agglutinin (PNA) labels hair bundles in OHCs. c GPR156 enrichment per cochlear HC type at P0 (base) at the medial (M) and lateral (L) junction. GPR156 is expressed as ratio of ZO1 signal (mean ± SD; n, HC numbers in 3 animals; Mann-Whitney test (two-tailed), ****p < 0.0001). d P0 auditory epithelium at 3 positions along the cochlea (base, 15%; mid, 50%; apex, 75%). PCNT and phalloidin labeling respectively reveal HC orientation by the position of the off-center basal body and the hair bundle shape. Arrows indicate OHC1 orientation and the arrowhead indicates a rare misoriented IHC. Magnified insets: less mature OHCs at the apex are already reversed in orientation (see also Supplementary Fig. 5c-d). e, f Circular histograms of P4 HC orientation by row. Histograms show frequency distribution at 50% cochlea position (10° bins in a referential where 90° (top) is lateral and 0° (right) is towards the cochlear base; n indicates HC number in 5–7 animals; Watson U2 test of homogeneity; normalized difference (ND) value indicates how many standard deviations separate the circular means of each distribution). In e, PTXa indicates the Cre-inducible R26-LSL-PTXa allele. Littermate controls for Atoh1-Cre; PTXa are Cre-negative PTXa animals. Scale bars are 100 µm (a, left), 50 µm (a, right), 5 µm (b), 10 µm (d).