1. Stem Cells and Regenerative Medicine
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CtBP impedes JNK- and Upd/STAT-driven cell fate misspecifications in regenerating Drosophila imaginal discs

  1. Melanie I Worley
  2. Larissa A Alexander
  3. Iswar K Hariharan  Is a corresponding author
  1. University of California, Berkeley, United States
Research Article
Cite this article as: eLife 2018;7:e30391 doi: 10.7554/eLife.30391
7 figures and 2 additional files

Figures

A system to study damage-induced cell-fate changes and a screen to identify genes that stabilize cell fates following damage.

(A) The rnts>eiger (rnts>egr) genetic system is used to induce temporally-controlled, tissue ablation in the wing pouch of larval wing imaginal discs. (B–C) Regeneration following ablation of the pouch typically results in viable adults with wings of relatively normal appearance. (D) Wing discs after 72 hr of recovery show regeneration of the pouch, shown here stained with antibodies to pouch marker Pdm2 and Wg. (E–F) In some genetic backgrounds, ectopic wings are observed following ablation. (E) An adult with ectopic wings on both sides. (F) The wing and the ectopic wing detached from one side. (G) Wing disc with an ectopic pouch after 72 hr of recovery. Ectopic wings and ectopic pouches are indicated with an asterisk (*). (H) Schematic of the screen of deletions encompassing most of the third chromosome for enhancers of ectopic wing formation. Each deletion was tested by crossing to rnts>egr. Density-controlled cultures were developmentally synchronized by collecting L1 larvae, shifted on day 7 from 18°C to 30°C for 40 hr. Adults were scored for ectopic wings. (I) Graph of frequency of adults with one or more ectopic wings for each deletion screened, arranged by relative location in the third chromosome (cytogenetic positions indicated). (J, K) Deletion screen data binned into classes for the entire screen (J) and subsets of different deletion collections (K).

https://doi.org/10.7554/eLife.30391.003
Figure 2 with 1 supplement
Loss-of-function mutations in C-terminal Binding Protein (CtBP) enhance the frequency of damage-induced ectopic wing formation.

(A) Frequency of ectopic wings (EW) following rnts>egr damage or mock ablation. EW scored in heterozygous genetic backgrounds for a wild-type Oregon R strain (wtOR), a deletion that spans CtBP (DEL = Df(3R)Exel8157), and two previously characterized alleles of CtBP, CtBP03463 and CtBPQ229*. Mock ablation controls 1) rn-GAL4, tub-GAL80ts (rnts>) and 2) tub-GAL80ts, UAS-egr (ts>egr), conducted in CtBPQ229*/+ genetic background. (B) An ectopic wing in CtBPQ229*/+ following ablation and regeneration. EW = ectopic wing, Sc = scutellum, W = wing. (C) A schematic of the two major protein isoforms of CtBP (short and long). The amino acid residues that correspond to codons at the CRISPR guide RNA target sites are indicated (148 and 334). (D) Frequency of EW following rnts>egr damage in a wild type isogeneic background (wtiso) and CRISPR-generated CtBP alleles (CtBPN148PY (N–to–PY), CtBP148Δ2 (Δ2)) in that background. (E) CtBPQ229*/+ wing imaginal disc following 72 hr of recovery stained with an antibody to Nubbin (Nub), a wing pouch marker (Ng et al., 1995). The ectopic pouch is marked with an asterisk (*). All bar graphs show averages between multiple experiments, error bars show standard deviations, and the numbers listed above the bars are the total adults scored.

https://doi.org/10.7554/eLife.30391.004
Figure 2—figure supplement 1
Ectopic wings occur most frequently when induced on day seven in CtBPQ229*/+.

Frequency of ectopic wings (EW) following rnts>egr induced damage initiated on different days of development in wild type and CtBPQ229*/+ genetic backgrounds. The day of the upshift AEL from 18o C to 30o C is indicated.

https://doi.org/10.7554/eLife.30391.005
Figure 3 with 5 supplements
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 rnts>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 CtBPQ229*/+ (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 rnts>egr damage at 0 hr recovery in wild type (G) and CtBPQ229*/+ (H) Boxed regions are shown at higher magnification in (G’–H’). (I) Frequency of ectopic wings (EW) in adults following rnts>egr damage with two (control) or one copy of the damage-dependent wg enhancer (wg1/+), in wild type and CtBPQ229*/+. (J) Frequency of EW in adults following overexpression of UAS-egr or UAS-wg alone, or UAS-egr and UAS-wg together driven by rnts> in wild type and CtBPQ229*/+. (K) Expression of dilp8-GFP (dilp8MI00727) in an undamaged disc. (L–N) Expression of dilp8-GFP in damaged discs after 0 hr of recovery for wild type (L) and CtBPQ229*/+ (M) and after 72 hr of recovery for CtBPQ229*/+ (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.

https://doi.org/10.7554/eLife.30391.006
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 ptcts> 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 CtBPQ229*/+ genetic backgrounds. (B–D) Wing discs after 48 hr of recovery with vgQE-RFP (pouch marker) and anti-Wg in control (B) and following ptcts>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.

https://doi.org/10.7554/eLife.30391.007
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 rnts>egr damaged (B–E) wing discs following 0 hr and 48 hr of recovery in wild type (B, D) and CtBPQ229*/+ (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 rnts>egr damaged (G–J) wing discs following 0 hr of recovery in wild type (G, I) and CtBPQ229*/+ (H, J) genetic backgrounds. The dilp8-GFP reporter (dilp8MI00727) 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.

https://doi.org/10.7554/eLife.30391.008
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 dilp8MI00727, CtBPQ229*/+ following rnts>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.

https://doi.org/10.7554/eLife.30391.009
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 dilp8MI00727, CtBPQ229*/+ 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.

https://doi.org/10.7554/eLife.30391.010
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 dilp8MI00727 /+ and dilp8MI00727 /MI00727 in a CtBPQ229*/+ 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.

https://doi.org/10.7554/eLife.30391.011
Figure 4 with 1 supplement
Characterization of the origin of cells that contribute to the ectopic pouch.

(A) rnts>egr, CtBPQ229*/+ wing disc with randomly-generated clones (two independent FLP-out constructs, Act<stop<lacZnls and Ubi<stop< GFPnls) generated with a 10 min heat-shock (37°C) at the start of the temperature shift from 18o C to 30o C and dissected 72 hr after the downshift. In (A’), there are three uniquely labeled clones (only Ubi-GFP, only Act-lacZ, or both), as indicated by arrowheads of matching color, in the ectopic pouch. (B–C) Lineage tracing following rnts>egr induced damage in wild type (B) and CtBPQ229*/+ (C) genetic backgrounds with R76B02-lexA, lexAOp-FLP, Ubi<stop<GFPnls after 72 hr of recovery. (D–F) rnts>egr, CtBPQ229*/+ wing discs with the enhancer trap mirr-lacZ stained with anti-Wg. (D) Undamaged. (E–F) Damaged discs, after 0 hr (E) and 72 hr (F) of recovery. N indicates notum, H indicates hinge. The asterisk indicates area of increased Wg expression in (E) and the ectopic wing pouch in (F). (G) Frequency of ectopic wings (EW) following rnts>egr damage in wild type or CtBP334Δ4/+ together with different mirr alleles, mirr1486/+ and mirr1825/+. Error bars show standard deviations. (H–J) rnts>egr, CtBPQ229*/+wing discs at different time points of recovery with dilp8-GFP, anti-Cubitus Interruptus (Ci) for marking the anterior compartment and anti-Engrailed (En) for marking the posterior compartment. Note that dilp8-GFP expression initiates in the posterior compartment and then spreads to the anterior compartment. (K) rnts>egr, CtBPQ229*/+ wing discs containing randomly generated marked clones (similar to A) that were induced on day 5 (48 hr before the temperature shift). Note the large GFP-positive clone in the posterior compartment of the ectopic pouch that abuts a significant portion of the anterior/posterior compartment boundary. Dotted boxes indicate areas that are enlarged in subsequent panels. The time of recovery (i.e. after downshift) is indicated in the bottom right corner of the relevant panel.

https://doi.org/10.7554/eLife.30391.012
Figure 4—figure supplement 1
Lineage tracing of cells from specific regions of the notum using lexA lines.

(A) Undamaged wing disc with R76B02-lexA driving the expression of lexAOp-GFP. (B–D) Wing discs with R81E08-lexA driver in undamaged disc (B) with lexAOp-RFP (shown in green) and following rnts>egr induced damage with lineage tracing in wild type (C) and CtBPQ229*/+ (D) genetic backgrounds. (E–G) Wing discs with R76B06-lexA driver in undamaged disc (E) with lexAOp-GFP and following rnts>egr induced damage with lineage tracing in wild type (F) and CtBPQ229*/+ (G) genetic backgrounds. Ectopic wing pouches are indicated with an asterisk (*). Lineage tracing is with lexAOp-FLP, Ubi<stop<GFPnls and the damaged discs are shown after 72 hr of recovery and stained with anti-Wg (magenta).

https://doi.org/10.7554/eLife.30391.013
Figure 5 with 7 supplements
Egr activation of the JNK pathway is required for the formation of ectopic wings.

(A) Frequency of ectopic wings (EW) following rnts>egr damage in wild type or CtBP334Δ4/+ (CtBP334Δ4 was generated via CRISPR/Cas9 on the same chromosome that contains rnts>egr) in listed genotypes: (1) hepr75/+, (2) co-expression of UAS-JNKDN and (3) co-expression of UAS-FosDN. (B) Frequency of ectopic wings (EW) following rnts> expression of listed transgenes (UAS-rpr, UAS-hepwt, UAS-hepCA, UAS-grindICD, UAS-ecto-egr) in wild type and CtBP-/+ genetic background. Temperature shifts for experiments shown in (A) and (B) were done on day 7 AEL for 40 hr. (C–H) Wing discs with homozygous mutant clones of FRT82B CtBPQ229* generated by mitotic recombination with the transcriptional reporters for AP-1-GFP (C, D), MMP1-lacZ (E, F) and dilp8-GFP (G, H).

https://doi.org/10.7554/eLife.30391.014
Figure 5—figure supplement 1
Co-expression of a dominant-negative Fos decreases the amount of tissue damage caused by rnts>egr.

Wing imaginal discs following rnts>egr damage alone (A) or with UAS-FosDN (B). Note that in (B), the wing pouch (WP) has less apoptotic debris (DCP-1) and less Wg pattern disruption (compare B’) to A’). WP = wing pouch, N = notum, hal = haltere disc, leg = leg disc.

https://doi.org/10.7554/eLife.30391.015
Figure 5—figure supplement 2
Shorter ablation period does not increase the frequency of ectopic wings.

The frequency of ectopic wings (EW) following a 24 hr temperature shift on day 7 AEL in wild type and CtBPQ229*/+ genetic backgrounds. Damage was induced by rnts> driven expression of UAS-egr, UAS-hepCA, UAS-rpr, or UAS-ecto-egr alone, or by the co-expression of both UAS-rpr and UAS-ecto-egr. Compare EW frequencies to ones from 40 hr temperature shift, shown in Figure 2 and Figure 5. Both UAS-egr and UAS-ecto-egr show a decrease in EW frequency when shifted for a shorter period.

https://doi.org/10.7554/eLife.30391.016
Figure 5—figure supplement 3
Expression of eiger in the wing pouch, but not in the myoblasts or notum epithelium, is sufficient to induce ectopic wings.

(A, B) A 3D-projection of a late L3 wing disc of rn>G-TRACE, which shows current (UAS-RFPnls) and past (UAS-FLP, Ubi<stop<GFPnls) expression of rn-GAL4. There is strong rn> expression in the wing pouch (WP) and much weaker expression in a subset of the myoblasts (MB). (B) The same disc with MB marker Twist and a close-up to highlight the three cell layers in the notum: the peripodial epithelium (PE), the disc proper (DP) and the myoblasts (MB). (C–F) Close-up of the notum. (C) In addition to the MB, past expression (GFP) is detected in a few scattered cells in the DP of the notum (arrows). (D–F) Current expression (RFP) in the notum is limited to a subset of the MB, all of which express Twist. (G) nub>G-TRACE shows that current and past expression is mostly limited to the wing pouch (WP). (H) R15B03>G-TRACE shows expression is limited to the MB (anti-Cut identifies myoblasts and the stripe at the dorsoventral boundary of the DP). (I) R76A01>G-TRACE shows current and past expression is limited to the notum of the DP. (J) dpp>G-TRACE shows expression in the DP of the notum, the hinge, the WP and PE. (K) Frequency of EWs in adults following expression of >egr with the listed GAL4 drivers, in wild type or CtBPQ229*/+ genetic backgrounds. All cultures were shifted from 18°C to 30°C on day 7 AEL for 40 hr. Error bars show standard deviations.

https://doi.org/10.7554/eLife.30391.017
Figure 5—figure supplement 4
Manipulations of apoptosis levels during the damage period.

(A) Frequency of ectopic wings (EW) following rnts>egr damage in wild type or CtBP334Δ4/+ in listed genotypes: (1) Df(3L)XR38/+ and (2) co-expression of UAS-DroncDN. Same controls as shown in Figure 5A. (B) Frequency of EW following rnts>egr damage in wild type or CtBPQ229*/+ with the co-expression of UAS-p35, by transgenes inserted on chromosome 2 and 3. Same controls as shown in Figure 3J. (C, D) Wing imaginal discs following rnts>egr damage with co-expression of UAS-p35. Note that there is still a high level of apoptosis (DCP-1), and Wg pattern disruption (C) and JNK activity visualized by anti-MMP-1 (D). (E) Frequency of EW following rnts>egr damage with co-expression of UAS-rpr and UAS-ecto-egr. Shown same data as Figure 5B to compare to UAS-ecto-egr alone.

https://doi.org/10.7554/eLife.30391.018
Figure 5—figure supplement 5
The regenerating wing pouch and ectopic pouch are derived from cells that express rn-GAL4 during the ablation period.

(A) Diagram of the system used to lineage label cells that express rn-GAL4 during the ablation period. When shifted to the higher temperature (30°C), rn-GAL4 drives the expression of both UAS-egr and UAS-FLP. There are two independent FLP-out constructs (Ubi<stop<GFP and Act<stop<lacZ). There will be several possible classes of cells: (1) cells that do not express rnts>FLP and remain unlabeled, (2) cells that express low levels of rnts>FLP during the temperature shift and stochastically activate one construct (either express GFP or β-GAL alone), and (3) cells that express high levels of rnts>FLP and activate both flip-out constructs (express GFP and β-GAL). (B–I) Wing discs following 72 hr of recovery from rnts>egr ablation with an ectopic pouch (B–E) and without an ectopic pouch (F–I), as shown by pouch marker Pdm2. The wing pouch (WP) is largely regenerated from cells that expressed rnts>egr and UAS-FLP during the temperature shift. The ectopic pouch (EP) and notum (N), as highlighted in dotted rectangle in (D, H) are enlarged in (E, I) to show the classes of cells present. Green arrow = GFP only, Blue arrow = β-GAL only, White arrow = GFP and β-GAL and Red arrows are unlabeled regions that express Pdm2.

https://doi.org/10.7554/eLife.30391.019
Figure 5—figure supplement 6
CtBP acts outside of the rn-GAL4 domain to prevent damage-induced ectopic wings.

(A) Frequency of ectopic wings (EW) following rnts>egr damage as calculated by the fraction of adults without (no EW) and those with one or two ectopic wings (EW) from multiple biological replicates in listed genetic backgrounds. gCtBP is a genomic rescue construct that is located at attp40 (Zhang and Arnosti, 2011). Last column is rnts> control for >CtBPRNAi. Error bars show standard deviations. Numbers above bars represent the total number of adults scored for each genotype. (B–C) Wing imaginal discs stained with antibodies against CtBP and Patched (Ptc) in control (B) and with dpp-GAL4 driving the expression of UAS-CtBPRNAi (BL:32889) (C). CtBP immunoreactivity is lost in cells that currently express dpp-GAL4, which significantly overlaps with Ptc expression domain, as well as more anterior cells that expressed it earlier in development (likely due to RNAi persistence [Bosch et al., 2016]). (D) Wing disc following rnts>egr damage in CtBPQ229*/+ genetic background with AP-1-RFP reporter showing activation in the notum, stained with anti-CtBP. Note relatively uniform expression of CtBP (D’).

https://doi.org/10.7554/eLife.30391.020
Figure 5—figure supplement 7
CtBP-/- mutant clones upregulate AP-1-GFP expression without evidence of apoptosis.

(A–B) Wing disc with homozygous CtBP-/- mutant clones, which are RFP negative with the AP-1-GFP transcriptional reporter. (A) Disc stained for apoptosis (DCP-1). (B) Disc stained for MMP1. Note that there is clear AP-1-GFP reporter activation in CtBP-/- mutant clones, but without detectable apoptosis or MMP1 protein (arrows).

https://doi.org/10.7554/eLife.30391.021
Figure 6 with 4 supplements
JAK/STAT signaling is important for the formation of the ectopic wings.

(A) Frequency of ectopic wings (EW) following rnts> driven expression of >upd1, >egr or >upd1 together with >egr in wild type and CtBPQ229*/+discs. Data were compared using ANOVA followed by Tukey test for significance (*p<0.05, **p<0.01, ***p<0.001) (B) Wing disc following rnts> driven co-expression of >egr and >upd1 after 72 hr of recovery. Pouch identity is shown by anti-Nub and an asterisk marks the ectopic pouch. (C–D) Early L3 (C) and late L3 (D) wing discs stained with anti-Wg and anti-GFP to detect the fast turnover STAT-DGFP reporter. (E–F) Wing discs with STAT-DGFP reporter following rnts>egr damage at 0 hr of recovery in wild type (E) and CtBPQ229*/+ (F) stained with anti-MMP1. Note expression of MMP1 in a subset of cells expressing STAT-DGFP in the area of ectopic pouch formation (asterisk); see (Figure 6—figure supplement 1). (G–I) Wing imaginal discs with both upd3-lacZ and STAT-DGFP reporters in undamaged (G) and damaged wild type (H) and CtBPQ229*/+ (I) discs at 0 hr of recovery. Note area in the notum that expresses STAT-DGFP but does not have elevated levels of upd3-lacZ. (J) Frequency of EW following rnts>egr damage when crossed to 1) FRT82B, 2) FRT82B Stat92E85C9, 3) FRT82B CtBPQ229*, and 4) FRT82B CtBPQ229* Stat92E85C9. Data were compared using ANOVA followed by Tukey test for significance (*p<0.05, **p<0.01, ***p<0.001). (K) Frequency of EW following rnts>egr damage in wild type and CtBP334Δ4/+ in the listed genotypes (1) +/+, (2) upd1YM55/+, (3) upd2Δ/+, (4) upd3Δ/+, and (5) upd2Δupd3Δ/+. In addition, co-expression of >upd1 with rnts>egr in (1) CtBP334Δ4/+ and (2) upd2Δupd3Δ/+;; CtBP334Δ4/+ genetic backgrounds. Note the rescue of EW induction in upd2Δupd3Δ/+ background by the co-expression of >upd1.

https://doi.org/10.7554/eLife.30391.022
Figure 6—figure supplement 1
STAT-DGFP expression in the notum of damaged wing discs is independent of MMP1 expression.

(A–D) Multiple examples of discs with variable amounts of STAT-DGFP and MMP1 expression in the notum. The green arrowheads (A–A’’’) point to a region in the notum with STAT-DGFP expression without detectable MMP1 protein. The white arrowheads (B–C’’’) show an example of a large patch of STAT-DGFP expression with only small region of MMP1 expression. The red arrowheads (D–D’’’) show a high level of MMP1 expression within a region of STAT-DGFP expression. Note that the STAT-DGFP expression appears lower where it overlaps with MMP1 expression.

https://doi.org/10.7554/eLife.30391.023
Figure 6—figure supplement 2
Upd1 overexpressed in the myoblasts can act on the disc proper epithelium and disrupt the notum Wg stripe.

Wing discs with STAT-GFP reporter in wild type (A–B) and CtBPQ229*/+ (C–D) genetic backgrounds. (A, C) Control discs. (B, D) Discs with myoblast driver, R15B03-GAL4, expressing UAS-upd1. Note the disruption of the normal notum Wg stripe (arrows) and the expression of STAT-GFP reporter in the notum epithelium. (WP >RFP = R15C03-lexA, lexAOp-RFP is used to visualize the wing pouch). (E) Adult R15B03>upd1 flies. Note the disruption of the thorax including the loss of many bristles and macrochaetes (arrow).

https://doi.org/10.7554/eLife.30391.024
Figure 6—figure supplement 3
CtBP-/- mutant clones do not alter expression of upd3-lacZ.

(A) The upd3-lacZ reporter in CtBP-/- mutant clones, which is marked by the absence of RFP (shown in magenta), in a wing disc. Note that upd3-lacZ is not expressed in the late L3 wing disc during normal development and that no expression was detected in CtBP-/- mutant clones.

https://doi.org/10.7554/eLife.30391.025
Figure 6—figure supplement 4
Effect of reducing CtBP function on STAT activity.

Wing discs with homozygous mutant clones of CtBPQ229* with the STAT-DGFP reporter. CtBP-/- mutant clones are marked by the absence of RFP. Areas in the white dotted box in (A) and (C) are enlarged in the subsequent panels (B–B”) and (D–D”).

https://doi.org/10.7554/eLife.30391.026
Figure 7 with 1 supplement
‘Weak point’ in the wing imaginal disc is responsive to Upd1 ligand and model for notum to wing transdetermination.

(A, B) Wing discs with dilp8-GFP and vgQE-RFP reporters: control (A), and dpp-GAL4, UAS-upd1 (B). Note the specific expression of dilp8-GFP along the posterior edge of the notum and in the wing pouch (arrows). (C) Overexpression of UAS-upd1 by heat-shock induced flip-out clones (hs-FLP, Act<stop<GAL4) marked with UAS-RFP. Note the specific area in the notum that responds to Upd1 by expressing dilp8-GFP (arrows). (D) Model for how JAK/STAT and JNK/AP-1 signaling work together to trigger a fate change and the growth of an ectopic pouch. Damage to the normal wing pouch generates both Egr and Upd ligands. The weak point, an area near the anterior/posterior compartment boundary in the notum responds to Upd ligands and the resulting JAK/STAT signaling disrupts the normal notum identity (as observed with the disruption of the notum Wg stripe). JNK activity within a field of JAK/STAT activity leads to the expression of Wg, which triggers cells to adopt the new wing pouch fate. Then, as in the regenerating wing pouch, additional cells are recruited to generate a new pouch. CtBP functions, either directly or indirectly, to antagonize these pathways.

https://doi.org/10.7554/eLife.30391.027
Figure 7—figure supplement 1
The notum and wing pouch respond to the overexpression of UAS-upd1 by dpp-GAL4.

(A–B) JAK/STAT activity as shown by the STAT-DGFP reporter in late L3 wing discs in the control (A) and dpp>upd1 (B). Note the specific activation of STAT-DGFP in the notum and wing pouch but not in-between. (C–D) Apoptosis (DCP-1) in control (C) and dpp>upd1 (D) wing discs, with the dilp8-GFP reporter and a wing pouch reporter (vestigial quadrant enhancer, vgQE-RFP). Note the increase in DCP-1 staining following the overexpression of UAS-upd1, which is mostly adjacent to the areas of dilp8-GFP expression (arrows). (E) The adult phenotype of dpp>upd1. Note the disruption of the thorax including the loss of many bristles and macrochaetes.

https://doi.org/10.7554/eLife.30391.028

Additional files

Supplementary file 1

Screen summary for third chromosome deletions.

The frequency of ectopic wings (EW) following rnts>egr damage for third-chromosome deletions screened. The deletions are ordered based on the cytogenetic position along the third chromosome.

https://doi.org/10.7554/eLife.30391.029
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https://doi.org/10.7554/eLife.30391.030

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