Fig. 2.

Generating “stalled” wing discs by compromising ecdysone (20E) production. (A and B) Stalled disc experimental paradigm. During normal development, 20E levels normally rise dramatically toward the end of the third larval instar, before declining abruptly at the onset of pupation, concomitant with the imaginal discs reaching full size (black, wing primordium shown in red) (28). Targeted knockdown of Fh in the larval prothoracic gland generated using either phm.Gal4+UAS.fh^RNAi or phm.LexA+LexOP.fh^RNAi causes an approximately three- to fourfold reduction in the late surge of 20E levels (A, dotted line, blue; quantified in F). Mutant larvae fail to pupate and continue to feed and grow (B). (C) Growth trajectories of stalled discs (phm.Gal4 UAS.fh^RNAi; blue, open circles, dashed line) versus control discs (phm.Gal4; black, closed squares, solid line). In wing discs destined to stall, growth slows down during the second half of the third larval instar and ceases ∼1 to 2 d later than normal, after which the disc remains quiescent for extended periods of > 2 to 3 wk, until larvae cease feeding and die (disc size quantified by pixel number; Experimental Methods; n = 10 discs for each time point; error bars indicate SEM). (D and E) Proliferative growth in wild-type (D) versus stalled discs (E), as monitored by both the number of cells that incorporate EdU (and hence are in S phase) as well as the intensity of the signal (which reflects the rate of synthesis and declines as proliferative growth slows during the stall; SI Appendix; EdU, green; DNA, blue). (F) ELISA measurement of 20E concentration in wild-type larvae (late third instar, 120 h AEL), compared to phm.Gal4/UAS.fh^RNAi larvae in which the discs have begun to stall or have completed the stall (120 and 168 h AEL), as well as with adult female abdomens. Error bars are SEM; n = 3 biological replicates for each treatment, each replicate contained 10 animals. (G and H) Expression of the protein product of the 20E target gene Broad (Br) provides an independent corroboration of reduced 20E transduction in stalled discs: Wild-type third instar imaginal discs express high levels of Br (G, magenta), in contrast to stalled discs from phm.LexA+LexOP.fh^RNAi larvae, which show only low-level expression (H). Wild-type and stalled discs were treated identically but distinguished by ap.Gal4 driven expression of green fluorescent protein (GFP) in the wild-type discs (red; DNA, blue; EdU incorporation, green; SI Appendix).