(a) A scheme to genetically remove dpp from the second or third instar larval stages using Gal80ts system. At 17°C, Gal4 activity is blocked by Gal80ts. At 29°C, Gal80ts is inactivated and Gal4 starts to induce FLP expression in the anterior stripe of cells. After embryo collection for 2–4 hr at room temperature, the embryos were incubated at 17°C until temperature shift. Temperature was shifted after 4 days (second instar) or 6 days (third instar) at 17°C, and late third instar wing discs were dissected after 53 hr or 43 hr later respectively. (b–e) Removal of dpp using ptc-Gal4 during second instar larval stages (b, c) or third instar larval stages (d, e). (b, d) anti-pMad staining and anti-β-gal staining (lineage tracing) in a control male wing disc (dppFO, ptc-Gal4/CyO; tub-Gal80ts/UAS-FLP, act5C(-FRT)lacZ) (left), and a male wing disc removing dpp during the specified time point (dppFO, ptc-Gal4/dppFO; tub-Gal80ts/UAS-FLP, act5C(-FRT)lacZ) (right). (c, e) anti-Dpp staining in a control male wing disc (dppFO, ptc-Gal4/+; tub-Gal80ts/ +) (left), and a male wing disc removing dpp during the specified time point (dppFO, ptc-Gal4/dppFO; tub-Gal80ts/UAS-FLP) (right). (b’–e’) Quantification of the wing disc size of (b–e). Mean ± s.d. p<0.001 by two sided Student’s t-test. Scale bars 100 μm.