Figure 3. The formation of ectopic pouches is marked by the activation of Wg and JNK at a secondary location.

(A–F) Wg and apoptosis visualized in wing discs following rn^ts>egr ablation of the pouch at different time points during recovery and regeneration (0 hr, 24 hr, and 48 hr after the downshift) for wild type (A–C) and CtBP^Q229*/+ (D–F). Apoptotic cells and their debris stain with an antibody to cleaved Drosophila caspase-1 (DCP-1). (A’–F’) Higher magnification images of the notum region. The site of secondary pouch formation is indicated with an asterisk (*). The arrow in (D’) points to a region where the normal stripe of Wg expression in the notum is disrupted. (G–H) Damage-dependent wg enhancer, BRV-B-GFP, following rn^ts>egr damage at 0 hr recovery in wild type (G) and CtBP^Q229*/+ (H) Boxed regions are shown at higher magnification in (G’–H’). (I) Frequency of ectopic wings (EW) in adults following rn^ts>egr damage with two (control) or one copy of the damage-dependent wg enhancer (wg^1/+), in wild type and CtBP^Q229*/+. (J) Frequency of EW in adults following overexpression of UAS-egr or UAS-wg alone, or UAS-egr and UAS-wg together driven by rn^ts> in wild type and CtBP^Q229*/+. (K) Expression of dilp8-GFP (dilp8^MI00727) in an undamaged disc. (L–N) Expression of dilp8-GFP in damaged discs after 0 hr of recovery for wild type (L) and CtBP^Q229*/+ (M) and after 72 hr of recovery for CtBP^Q229*/+ (N). (O) Graph of frequency of discs with a spot of dilp8-GFP expression at different time points of recovery (0 hr, 24 hr, 48 hr, 72 hr), along with the frequency of ectopic wings per side (or heminotum) in adults.

Figure 3—figure supplement 1. wingless overexpression is not sufficient to induce ectopic wings on day 7.

(A) Frequency of ectopic wings following >wg driven by ptc^ts> shifted from 18°C to 30°C for 40 hr beginning on either day 5, day 6, or day 7 AEL in wild type or CtBP^Q229*/+ genetic backgrounds. (B–D) Wing discs after 48 hr of recovery with vgQE-RFP (pouch marker) and anti-Wg in control (B) and following ptc^ts>wg overexpression initiated on day 5 (C) or day 7 (D) AEL. (B’, C’, D’) Close-up of the notum or ectopic pouch as indicated by the dotted yellow box.

Figure 3—figure supplement 2. JNK activity at secondary location in the notum and in the ectopic pouch.

(A–E) AP-1-RFP reporter and anti-Wg in undamaged (A) and rn^ts>egr damaged (B–E) wing discs following 0 hr and 48 hr of recovery in wild type (B, D) and CtBP^Q229*/+ (C, E) genetic backgrounds. AP-1-RFP reporter is shown in green and anti-Wg is in magenta. (F–J) JNK target MMP-1 in undamaged (F) and rn^ts>egr damaged (G–J) wing discs following 0 hr of recovery in wild type (G, I) and CtBP^Q229*/+ (H, J) genetic backgrounds. The dilp8-GFP reporter (dilp8^MI00727) expression largely overlaps with the area of MMP-1 expression in the regenerating wing pouch (I, J) and at the secondary location in the notum (J’). Regions of the notum are enlarged in (F’–J’) and asterisks mark areas of increased JNK activity in the notum and ectopic pouch.

Figure 3—figure supplement 3. Timing of dilp8-GFP spot appearance in the notum relative to Tsh downregulation and onset of Nub expression.

(A–F) Close-up of the notum/ectopic pouch in dilp8^MI00727, CtBP^Q229*/+ following rn^ts>egr induced damage on day 7 for 40 hr. The transcription factor Teashirt (Tsh) is downregulated in the wing pouch during normal development and Nubbin (Nub) is expressed in the wing pouch (Zirin and Mann, 2007). Wing discs with dilp8-GFP and either anti-Tsh (A–C) or anti-Nub (D–F). Note that dilp8-GFP expression partially overlaps the Nub-positive domain, but there are regions that express only dilp8-GFP or Nub. (G) Model of timeline of marker expression during the formation of the ectopic pouch: dilp8-GFP is initially co-expressed with Tsh in the notum. Tsh is progressively downregulated as the pouch forms and Nub starts to be expressed.

Figure 3—figure supplement 4. Bilateral ectopic wings slightly more frequent than expected for completely independent events.

The observed (A) and expected (B) frequency of dilp8^MI00727, CtBP^Q229*/+ adults with zero, one or two ectopic wings, following damage and regeneration. The expected frequency is calculated from the observed frequency of ectopic wings per heminotum, assuming that the events in one wing disc are completely independent from the other.

Figure 3—figure supplement 5. dilp8 is needed for EW in adults and additional dilp8 increases the frequency.

(A) Frequency of ectopic wings (EW) in dilp8^{MI00727 /+} and dilp8^{MI00727 /MI00727} in a CtBP^Q229*/+ genetic background. (B) Frequency of EW with UAS-dilp8 alone UAS-egr alone and both together. Numbers above graph represent the total number of adults scored.