Fig. 1: Loss of Jag1 reduces cell cycle re-entry and proliferation of perinatal cochlear SCs and KCs in organoid culture.

a Schematic of early postnatal murine cochlear sensory epithelium. SC-subtypes (He, Hensen cells; DC1-3 Deiter’s cells 1–3, OP, outer pillar cell; IP, inner pillar cell; IPh, inner phalangeal cells) highly express SOX2 (bright green nuclei). KCs express lower levels of SOX2 (faint green nuclei). Inner HC (IHC) and three outer HCs (OHC) are labeled red. b Experimental scheme (c–j). Cochlear epithelial cells from stage P2 mice were used to establish control and Jag1 cKO organoid cultures, which were analyzed on day 7 of expansion. c Representative confocal images of control and Jag1 cKO organoids stained for JAG1 protein (red). Hoechst labels nuclei (blue). Scale bars = 50 µm. d RT-qPCR of relative Jag1 mRNA expression in control (Ctrl, blue bar) and Jag1 cKO (red bar) organoids (n = 3 animals per group, three independent experiments). e Bright field (BF) images of control and Jag1 cKO organoids. Scale bars = 200 µm. f, g Organoid forming efficiency (f) and organoid diameter (g) for (e) (n = 8 animals per group, three independent experiments). h Cell proliferation in control and Jag1 cKO organoids on day 7. Hoechst labels cell nuclei (blue). Scale bars = 50 µm. i Percentage of EdU⁺ cells in (h) (n = 6 animals per group, three independent experiments). j RT-qPCR of Notch target Hes1, SC markers (Fgf20, Sox2, S100a1, Lfng) and HC markers (Atoh1, Pou4f3, Gfi1, Myo7a) in control and Jag1 cKO organoids (n = 3 animals per group). k Experimental scheme (l, m). FACS-purified p27-GFP⁽⁺⁾ SCs and p27-GFP⁽⁻⁾ KCs from stage P2 mice were used to establish control and Jag1 cKO organoid cultures, which were analyzed on day 7 of expansion. l BF images of control and Jag1 cKO organoid cultures established from GFP^(-) KCs and GFP⁽⁺⁾ SCs. Scale bars = 200 µm. m, n Colony (organoid) forming efficiency (m) and organoid diameter (n) for (e) (n = 3 independent biological replicates per group, two independent experiments). Graphed are individual data points and mean ± standard deviation. A two-tailed, unpaired Student’s t test was used to calculate exact p-values. Source data are provided as a Source Data file. N.s. not significant. Created in BioRender. Doetzlhofer, A. (2025) https://BioRender.com/vo09ioe.