(A–A′, C–E′) High magnification views of wild type adult midguts immunostained for pMad (red in A–A′, D–E′), dad-lacZ (red in C), Dl-lacZ (blue in A′, E′), Su(H)-GFP (green in A′, C, E′) or PH3 (green in D). (B–B′, F–F′) Sagittal views of wild type adult midguts immunostained for pMad (red), Su(H)-GFP (green in B′), Dl-lacZ (green in F′), and Phalliodin (blue). Arrows and arrowheads indicate ISCs and EBs, respectively. Asterisks indicate the pMad signals in ECs. (G–O) Adult midguts expressing esgts>GFP (G, J, M), esgts>GFP + TkvQ235D (H, K, N), or esgts>GFP + Dpp + Gbb (I, L, O) at 29°C for the indicated time periods were immunostained for Dl (red in G–L), PH3 (red in M–O), GFP (green), and Su(H)-lacZ or DRAQ5 (blue). (P) Scheme for twin-spot experiments involving TkvQ235D overexpression. 3–5-day-old control or esgts>TkvQ235D adult flies were grown at 29°C for 3 days before heat shock (hs) to induce clones. After 1-day recovery at 29°C, the flies were raised at 18°C for 4 days prior to analysis. (Q–R) Representative twin-spot clones from control and esgts>TkvQ235D guts. (S) Quantification of twin spots of different classes from control and esgts>TkvQ235D guts: Con (n = 160, ISC/EB: 83%, ISC/ISC: 9%, EB/EB: 8%), TkvQ235D (n = 190, ISC/EB: 23%, ISC/ISC: 77%, EB/EB: 0%).