Fig. 4: Calcium waves in supporting cells in live mice.

a Diagram showing a cross-section of an immature Organ of Corti. b Top left: average intensity projection displaying GCaMP6f expression in the cochlear epithelium from a P4 GCaMP6f^fl/flPax2-Cre^+/- mouse. Bottom left: maximal intensity projection, highlighting Ca²⁺ activity in the supporting cells during a 10-minute recording. Right: semi-automatic ROI segmentation. c Fluorescence trace from a square ROI drawn across the entire GER. d Histogram of the full duration at half maximum of Ca²⁺ waves (1145 Ca²⁺ waves, 20 mice). e Average frequency of Ca²⁺ waves during pre-hearing stages of development (Ca²⁺ waves/min). Each data point represents one recording from 33 mice (number of recordings from left to right: 1,10,3,7,6,8,7,18,21). Mean Ca²⁺ wave expansion (f) and contraction (g) speed as a function of age. Number of Ca²⁺ waves from left to right for each panel: 47, 151, 86, 108, 41, 399, 77 from 20 mice. Top panels h and j: average intensity projection displaying GCaMP6f signals in the cochlear epithelium from GCaMP6f^fl/flPax2-Cre^+/- mice at P8 (h, broken seal, orange: in vivo but with the exposed cochlear duct following the rupture of the membrane sealing the cochlear partition Fig. 1d) and P7 (j, ex vivo, green: cochlear explant). Bottom panels i and k: corresponding fluorescence traces from the ROI drawn across the GER in panels h and j, respectively. l, m Comparison of the maximum area (l) and full duration at half maximum (m) of Ca²⁺ waves recorded from P7-P9 cochleae under in vivo (blue: 621 Ca²⁺ waves (l), 614 Ca²⁺ waves (m), 10 mice), in vivo but with the broken membrane (orange, 329 Ca²⁺ waves (l), 324 Ca²⁺ waves (m), 6 mice) and ex vivo (green: 135 Ca²⁺ waves (l), 120 Ca²⁺ waves (m), 3 mice) conditions. Average data are shown as mean ± SD (median: red lines). Note that the different number of Ca²⁺ waves between panel (l) and (m) is because some Ca²⁺ waves start or end outside the recording time and their duration could not be mesured. Statistical tests in panels l, m are from Kruskal-Wallis with Dunn’s post-test.