Hh signaling is up-regulated by DSS-mediated tissue damage. (A) Quantification of hh and ptc mRNA levels by RT-qPCR in midguts treated with DSS or Suc for 1 d. Three independent experiments were performed and error bars are standard deviations. *, P < 0.05. (B–C′) Adult midguts expressing UAS-GFP under the control of hh-Gal4 (hh>GFP) were immunostained for GFP and DRAQ5 (B) or Pdm1 (C and C′). hh>GFP was mainly detected in Pdm1⁺ ECs. (D–E′) Adult midguts carrying two copies of hh-lacZ were immunostained for LacZ (D–E′) and Pdm1 (D and D′) or GFP under the control of esg-Gal4 (E and E′). (F–G″) Expression of one copy of hh-lacZ in midguts treated with Suc (F–F″) or DSS (G–G″) for 1 d. hh-lacZ was elevated in both ECs (large nuclei) and precursor cells (small nuclei) in response to DSS treatment. (H–I′) Expression of ptc-lacZ in control (Suc) and DSS-treated midguts for 1 d. ptc-lacZ was elevated in esg-GFP–positive cells after DSS treatment. (J and K) Quantification of hh mRNA levels by RT-qPCR (J) or PH3⁺ cells (K) in control, esg^ts>Hh^RNAi, or Myo1A^ts>Hh^RNAi guts treated with Suc or DSS after adult flies were raised for 10 d at 29°C. Three independent experiments were performed, and error bars are standard deviations. 20 guts were examined for each sample in K. **, P < 0.01; *, P < 0.05. Bars, 50 µm.