DSS up-regulates Hh signaling through the JNK pathway. (A–B′) Expression of a JNK pathway reporter, puc-LacZ, in mock (Suc; A and A′) or DSS-treated (B and B′) midguts for 1 d. puc-LacZ expression was barely detectable in mock-treated guts but was elevated in Su(H)>GFP-positive cells in DSS-treated guts. Arrows indicate Su(H)>GFP⁺ cells with small nuclei. (C) Quantification of relative hh mRNA levels by RT-qPCR in control or Su(H)^ts>BSK^DN midguts treated with Suc or DSS for 1 d after adult flies were raised at 29°C for 10 d. Three independent experiments were performed and error bars are standard deviations. *, P < 0.05. (D) Quantification of PH3⁺ cells in control or Su(H)^ts>BSK^DN midguts treated with Suc or DSS for 1 d after adult flies were raised for 10 d at 29°C. Three independent experiments were performed and 20 guts were examined for each sample. Error bars are standard deviations. **, P < 0.01. (E) Quantification of hh and ptc mRNA levels by RT-qPCR in control and Su(H)^ts>Puc^RNAi guts grown at 29°C for 6 d. Three independent experiments were performed and error bars are standard deviations. Numbers indicate fold change over control guts. (F–I′) Expression of hh-lacZ (F–G′, red) or ptc-lacZ (H–I′, red) in control (F, F′, H, and H′) or esg^ts>Puc^RNAi guts grown at 29°C for 5 d. Arrows indicate elevated hh-lacZ (F-G′) and ptc-lacZ (H-I′) in precursor cells. (J–M) Adult midguts expressing esg^ts (Con; J), esg^ts>Puc^RNAi (K), esg^ts>Hh^RNAi (L), or esg^ts>Puc^RNAi + Hh^RNAi (M) for 7 d at 29°C were dissected out and immunostained for PH3 and Hoechst. (N) Quantification of PH3⁺ cells in adult midguts of the indicated genotypes. Three independent experiments were performed and 20 guts were examined for each sample. Error bars are standard deviations. **, P < 0.01. (O) A JNK–Hh–JAK–STAT signaling axis mediates DSS-stimulated ISC proliferation in adult midgut regeneration. BM, basement membrane; VM, visceral muscles. See text for details. Bars, 50 µm.