Fig. 8: Pax regulates midgut proliferation and regeneration by promoting Yki cytoplasmic localization, thereby restricting its activity.

a–d’ Adult midguts containing GFP-positive MARCM clones of FRT82 (Ctrl) (a–a’), wts^x1 clones (b–b’), overexpressing Pax clones (c–c’) and Pax overexpression plus wts^x1 clones (d–d’) were immunostained with Yki (red), HA (blue), and DAPI (nuclei, gray). Midguts were dissected 4 days after clone induction at 25 ˚C. Dotted lines mark the edges of the clone region. Yellow arrows mark the GFP⁺ clone region, and white arrows mark the GFP^- region. e Quantification of the percentage of nucleocytoplasmic distributions in panels (a–d’) (n = 3 for each group, counting 123, 125, 112 Yki⁺ cells). f–i’ Adult midguts of Esg^ts>Ctrl (f–f’, h–h’) and Esg^ts > HA-Pax (g–g’, i–i’) flies treated with Glucose (Glu) or DSS for 3 days before dissection. Midguts were immunostained with Yki (red), DAPI (nuclei, gray), and HA (blue). ISC and EB/pre-EE were marked by EsgGal4-driven GFP expression. Arrows indicate HA⁺ and Yki⁺ cells. 5% Glucose solution with or without 3% DSS was fed to the flies. j Quantification of the percentage of nucleocytoplasmic distributions in panels (f–i’) (n = 3 for each group, counting 100, 125, 112 Yki⁺ cells). k Proposed model illustrating how Pax regulates Hippo and Notch signaling to suppress ISC proliferation and promote its differentiation in adult Drosophila midgut. Three independent experiments were performed, and the error bars are mean ± SEM. P values of significance (indicated with asterisks, NS no significance P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****p < 0.0001) was calculated by one-way ANOVA with Tukey’s test (e, j). Scale bars: 20 µm. Confocal images were taken from the boundary region between R4c and R5a of the posterior midgut.