Hh signaling in EBs regulates ISC proliferation. (A–D′) 3- to 5-d-old female adult flies were heat shocked at 37°C for 1 h to induce MARCM clones for FRT40 (A, A′, B, and B′) or FRT40 smo³ (C, C′, D, and D′) and cultured at 18°C. 5 d ACI, the flies were treated with Suc (A, A′, C, and C′) or DSS (B, B′, D, and D′) for 2 d, followed by immunostaining for GFP (green), PH3 (red), and DRAQ5 (blue). (E) Clone size was quantified for ISC lineage clones in the posterior region of the midguts. Data are mean ± SEM. n = 150. ***, P < 0.001. (F) PH3⁺ cells within or outside clones were counted for individual guts. Three independent experiments were performed and 10 guts were examined for each sample. Error bars are standard deviations. (G–J) 3- to 5-d-old female adult flies were heat shocked at 37°C for 1 h to induce control, ptc^S2, or ptc^IIW clones and cultured at 18°C for 10 d ACI. Midguts were immunostained for GFP (green), PH3 (red), and DRAQ5 (blue) and quantified for PH3⁺ cell numbers within or outside clones per gut. Three independent experiments were performed and 10 guts were examined for each sample. Error bars are standard deviations. (K) Adult midguts by expressing two copies of UAS-Ptc^RNAi using Myo^ts or Dl^ts for 12 d were immunostained for PH3 (green), esg-lacZ (red), and DRAQ5 (blue). Quantification of PH3⁺ cells in each genotype is shown to the right. Three independent experiments were performed and 12 guts were examined for each sample. Error bars are standard deviations. (L–M′) Adult midguts expressing Su(H)^ts>GFP (L and L′) or Su(H)^ts>GFP + Ptc^RNAi (M and M′) for 10 d were immunostained for PH3 (red) and GFP (green). (N) Quantification of ISC proliferation (PH3⁺/gut) in midguts of the indicated genotypes cultured at 29°C for the indicated time. Three independent experiments were performed and 12 guts were examined for each sample. Error bars are standard deviations. (O–R) Adult midguts expressing Dl^ts>GFP (O), Su(H)^ts>GFP (P), Dl^ts>GFP + Ci^ΔN (Q), or Su(H)^ts>GFP + Ci^ΔN (R) for 6 d were immunostained for PH3 (red) and GFP (green). (S) Quantification of PH3⁺ cells in midguts of the indicated genotypes grown at 29°C for 6 d. Three independent experiments were performed and 12 guts were examined for each sample. Error bars are standard deviations. (T–W) Adult flies expressing Su(H)^ts>GFP or Su(H)^ts>GFP + Smo^RNAi for 10 d at 29°C were treated with Suc or DSS for 1 d before midguts were dissected out and immunostained for GFP (green) and PH3 (red). (X) Quantification of PH3⁺ cells in midguts of the indicated genotypes grown at 29°C for 10 d. Three independent experiments were performed and 20 guts were examined for each sample. Error bars are standard deviations. **, P < 0.01. Bars, 50 µm.