Suppression of Dpp signaling in the PG does not cause accelerated pupariation except overexpression of brk which induces L2 arrest. Suppression of Dpp signaling in the PG does not cause accelerated pupariation except overexpression of brk which induces L2 arrest. (A) Expression of tkvRNAi or MadRNAi does not significantly accelerate pupariation compared with phm>. All UAS-RNAi transgenes were tested in a separate assay and found to be effective in disrupting Dpp signaling by causing patterning disruptions. (B) Expression of both MadRNAi and MedRNAi has at most a very small effect on accelerating pupariation compared with phm> outcrossed to w− and both UAS transgenes outcrossed to the same w−. Average pupariation times are phm> 135.8 ± 1.1 h, >MadRNAi;MedeaRNAi/+ 121.3 ± 0.6 h, phm>MadRNAi;MedeaRNAi 127.8 ± 2.3 h. (C) Expression of tkvD95K or dad does not significantly accelerate pupariation compared with phm> outcrossed to w− or either UAS- transgene outcrossed to the same w− stock. (D) Expression of dppRNAi in dpp-Gal4–expressing cells does not significantly accelerate pupariation. Note that larvae develop without any imaginal discs and reach the L3 stage before pupariating. (E) Developmental timing of larvae with UAS-brk overexpression in the PG which do not pupariate (n = 375 for phm>brk represents the number of L1 larvae that were raised under conditions of controlled density). (F–Q) Temporal comparison of phm> (F–I), phm>tkvQD (J–M), and phm>brk (N–Q) larvae. Images of stage- and density-controlled animals raised at 25°C. On day 2 AEL, (F) control phm> animals, (J) phm>tkvQD, and (N) phm>brk animals are at the second instar stage (L2). phm> and phm>tkvQD animals progress to the early third instar (eL3) on day 3 (G, K) and wandering third instar (wL3) on day 5 (H, L). phm> animals pupariate by day 6 (I), whereas phm>tkvQD animals remain in wL3 (L) until a much delayed pupariation on day 8 or day 9. phm>brk animals do not progress to eL3 on day 3 (O) and remain small and in second instar through days 5 and 6 (P, Q). However, although still in L2, they exhibit wandering behavior for multiple days followed by lethality without pupariation. Wandering behavior in L2 is reminiscent of the early pupariation phenotype observed with expression of a constitutively active form of the Activin receptor, Baboon. Data information: Scale bars = 1 mm. Linda Setiawan et al. LSA 2018;1:e201800216 © 2018 Hariharan et al.