Fig. 2: Loss of Jag1 enhances the rate of HC formation by perinatal cochlear SCs and KCs in organoid culture.

a Experimental scheme (b–d). Cochlear epithelial cells from stage P2 mice were used to establish control and Jag1 cKO organoid cultures. HC formation was analyzed using RT-qPCR and immuno-staining after 5 days of differentiation. b RT-qPCR was used to analyze relative mRNA expression of early HC genes (Atoh1, Pou4f3, Myo7a) in control and Jag1 cKO organoids (n = 3 animals per group). c Confocal images of MYO7A (magenta) and SOX2 (green) stained control and Jag1 cKO organoids. Note MYO7A and SOX2 co-staining labels new HCs. Control organoids only contain a few scattered new HCs, whereas Jag1 cKO organoids contain clusters of new HCs. Scale bars = 50 µm. d Quantification of the percentage of HCs per control and Jag1 cKO organoid in (c) (n = 3 animals per group, two independent experiments). e Experimental scheme (f–i). FACS-purified P27-GFP⁽⁺⁾ SCs and p27-GFP⁽⁻⁾ KCs from stage P2 mice were used to establish control and Jag1 cKO organoid cultures. HC formation was analyzed by RT-qPCR and immuno-staining after 5 days of differentiation. f RT-qPCR was used to analyze relative mRNA expression of early HC-specific transcription factors (Atoh1, Gfi1, Pou4f3) in control and Jag1 cKO organoids (n = 3 independent biological replicates per group, two independent experiments). g Confocal images of KC-derived (GFP⁽⁻⁾) and SC-derived (GFP⁽⁺⁾) control and Jag1 cKO organoids stained for MYO7A (magenta) and SOX2 (green) Scale bars = 100 µm. h Percentage of HC-containing organoids in control and Jag1 cKO organoid cultures (n = 4-6 independent biological replicates per group, two independent experiments). i Percentage of HCs in HC-containing organoids in control and Jag1 cKO organoid cultures (n = 12 independent biological replicates for control and n = 8 independent biological replicates for Jag1 cKO groups). 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.