Fig. 1: Loss of Pax triggers ISC proliferation in both homeostatic and stress conditions.

a Schematic diagram of the ISC lineage and intestinal epithelium in Drosophila adult midguts. ISC intestinal stem cell, EB enteroblast, EC absorptive enterocyte, Pre-EE Precursor enteroendocrine, EE enteroendocrine. Esg⁺ Dl⁺ cells are ISCs. Esg⁺ Su(H)⁺ cells are EBs. Esg⁺ Dl⁺ Pros⁺ cells are pre-EEs. Pdm1⁺ or Pros⁺ labels mature ECs or EEs, respectively. b–d’ Adult midguts from flies fed with either Glucose (b–b’) or DSS (c–c’) for 3 days, followed by 3 days of recovery (d–d’), were stained with Pax (red) and DAPI (nuclei, blue). Flies expressing EsgGal4; tubGal80^ts (Esg^ts>) were used. 5% Glucose solution with or without 3% DSS was fed to the flies. ISCs and EBs/pre-EEs were marked by EsgGal4-driven GFP expression. e–f” Adult midguts of Esg^ts>Ctrl (e–e”) and Esg^ts>Pax RNAi (V107789) (f–f”) flies were immunostained with p-H3 (gray), Dl (red) and DAPI (nuclei, blue). Phospho-Histone 3 (p-H3) marks mitotic cells derived from ISCs. ISCs and EBs/pre-EEs were marked by EsgGal4-driven GFP expression. White arrows indicate proliferative ISCs marked by p-H3. g Quantification of p-H3⁺ cells of adult midguts of the indicated genotypes of (e–f”) (n = 12,12). The counting of p-H3⁺ cells was conducted across the entire midgut. h Quantification of Dl⁺ cells of adult midguts of the indicated genotypes of (e–f”) (n = 12,12). i–j” Adult midguts of Ctrl (i–i”) and homozygous allele of Pax²⁰ (j–j”) were dissected and immunostained with Arm+Pros (green), p-H3 (red) and DAPI (nuclei, blue). Midguts were dissected 5 days after eclosion. k Quantification of p-H3⁺ cells of adult midguts of (i–j”⁾ (n = 12,12). The counting of p-H3⁺ cells was conducted across the entire midgut. l–o’ Adult midguts of Esg^ts>Ctrl (l–l’, n–n’) and Esg^ts > HA-Pax (m–m’, o–o’) were treated with Glucose or DSS for 3 days before gut dissection. Midguts were immunostained with p-H3 (red) and DAPI (nuclei, blue). A 5% Glucose solution with or without 3% DSS was fed to the flies. ISCs and EBs/pre-EEs were marked by EsgGal4-driven GFP expression. White arrows indicate proliferative ISCs marked by p-H3. p Quantification of p-H3⁺ cells of adult midguts of the indicated genotypes of (l–o’), n = 10,15,10,10. The counting of p-H3⁺ cells was conducted across the entire midgut. q–r” Adult midguts of MyoIA-Gal4; tubGal80^ts (MyoIA^ts)>Ctrl (q–q”) and MyoIA^ts >Pax RNAi (r–r”) were dissected and immunostained with Dl (red), p-H3 (gray) and DAPI (nuclei, blue). ECs are marked with MyoIA-GFP (green). White arrows indicate proliferative ISCs marked by p-H3. s Quantification of p-H3⁺ cells of adult midguts of the indicated genotypes of (q–r”) (n = 14, 14). The counting of p-H3⁺ was conducted across the entire midgut. t Quantification of the percentage of Dl⁺ cells in adult midguts of the indicated genotypes of (q–r”) (n = 11, 11). u–v^” Adult midguts containing MyoIA^ts>Ctrl (u–u”) and MyoIA^ts >Pax RNAi (v–v”) were immunostained with Phalloidin (red) and DAPI (nuclei, blue). ECs are marked with MyoIA-GFP (green). Three independent experiments were performed, and the error bars are mean ± SEM. In each box plot, the center line indicates the median, the edges of the box represent the first and third quartiles, and the whiskers extend to the minimum and maximum values. 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) were calculated by two-tailed Student’s t-test (g, h, k, s, t) and one-way ANOVA with Tukey’s test (p). Scale bars: 30 µm. Confocal images were taken from the boundary region between R4c and R5a of the posterior midgut.