Fig. 7: JAG1-peptide treatment boosts HC formation in cochlear explants in a stage and mTOR-dependent manner.
From: The Notch ligand Jagged1 plays a dual role in cochlear hair cell regeneration
a Experimental scheme for isolating Lfng-GFP(+) cochlear SCs used in (b). b RT-qPCR analysis of Jag1 mRNA expression in stage P1, P5, and P13 FACS-purified cochlear SCs (n = 2 for P1, n = 3 for P5 and n = 2 for P13. N = independent biological replicates that were obtained from at least 2 independent experiments). c Experimental scheme for (d–g). Cochlear explants from P2 or P5 Atoh1-nGFP tg mice were cultured with JAG1-Fc peptide or without JAG1-Fc peptide (control) in the presence of CHIR99021 and LY411575 for 4 days. EdU was added on day 1. d Representative confocal images of the HC layer taken at the cochlear mid-apical turn following culture. Newly formed HCs (white asterisks) are labeled by MYO7A (magenta) and SOX2 (blue) immuno-staining and Atoh1-nGFP (green) expression. EdU (red) labels actively and previously dividing cells. Scale bars = 25 µm. e–g Quantification of SC proliferation (EdU+SOX2+) (e), mitotic HC formation (EdU+ MYO7A+) (f), and the total number of newly formed HCs (MYO7A+SOX2+Atoh1-nGFP+) (g) for (d) (n = 5 animals per group, two independent experiments). h Experimental scheme for (i, j). Cochlear explants from stage P4 Atoh1-nGFP tg mice were cultured with JAG1-Fc peptide or without JAG1-Fc peptide (control) in the presence of rapamycin (4 ng/mL) or DMSO (0.05%, vehicle control) for 5 days. CHIR99021 and LY411575 were added on day 1 to induce SC-to-HC conversion. i Representative confocal images of the HC layer taken at the cochlear mid-apical turn following culture. Newly formed HCs are marked by MYO7A (magenta) and SOX2 (blue) immuno-staining and Atoh1-nGFP (green) expression. Scale bars = 25 µm. j Quantification of newly formed HCs in (i) (n = 4 animals per group, two independent experiments). Graphed are individual data points and mean ± standard deviation. Two-way ANOVA with Tukey’s correction was used to calculate exact p-values. Source data are provided as a Source Data file. N.s. not significant.
