Fig 1.
ROS produced after physical injury and after cell death.
(A) Cut disc cultured ex vivo (white wedge indicates cut edges) and thermal LUT of CellROX Green. (B) Cut disc cultured ex vivo, imaged at just after cut (0–5’) and 30’ later. Thermal scale indicates pixel intensity. (C) Sketch of wing imaginal discs with the area (black square) shown in A, B and E. (D) Fixed disc stained for nuclei to show disc contour (TP-3: TO-PRO-3) and caspase-3 after ptc>rpr activation for 11h at 29°C. (E) ptc>rpr disc cultured ex vivo; basal images at the bottom, apical at the top. Left, cell death (TO-PRO-3). Right, thermal LUT taken from the ROS channel (CellROX Green) of the same preparation. Note that most dead cells (TO-PRO-3 positive) show high ROS (red in thermal image) whereas living cells (TO-PRO-3 negative) had low ROS (green-cyan in thermal image). (F) Mean pixel intensity (grey value) of the indicated zones in control discs without cell death (ptc>rpr OFF) and discs with cell death (ptc>rpr ON). The pixel intensity in the ptc domain in the absence of cell death (ptc>rpr OFF) was 1.76 ± 0.55 (SD; from 48 regions of interest [ROI] in n = 5 discs). The mean pixel intensity for the apoptotic region (basal; ptc>rpr ON) was 33.14±8.18 (SD), measured in 27 ROI on confocal images taken from n = 6 discs. Living cells adjacent to the apoptotic zone showed a mean grey value of 8.51±2.12 (SD; 15 ROI from 6 discs taken from cells near the ptc domain). White rectangles in E: example ROI for Basal Apoptotic Zone (1) and Apical Living Zone (2). The ROI’s for the ptc Zone, in discs in which ptc>rpr is OFF, were placed as (1). ***P<0.001. Thermal scale indicates sample values from raw images.
Fig 2.
ROS are required for tissue repair.
(A) Design for chemical antioxidant intake and cell death induction. At 17°C and 24 h before cell death induction, larvae were transferred to a vial with standard food supplemented with antioxidant. Cell death (salE/Pv>rpr ON) was induced by shifting temperature to 29°C for 11 h (blue stripes in the disc). Larvae were transferred to 17°C where they regenerated and emerged into adults, in which wings were scored. Blue color in the wing: area emerged from salE/Pv. Controls salE/Pv>rpr OFF were kept at 17°C to avoid cell death. (B) Percentage of regenerated wings after cell death (salE/Pv>rpr ON) in the absence of antioxidant (Std Food), or in the presence of antioxidants (NAC, Trolox or Vitamin C). (C) Examples of salE/Pv>rpr ON wings with the indicated food supplement. In controls without antioxidants (Std Food), the complete wing recovered. For each antioxidant an example of incomplete regeneration after cell death induction is shown. (D) Mitosis number in ptc>rpr discs from larvae fed with and without NAC and with or without rpr-ablation (ON versus OFF). Ptc>rpr OFF: 41.86 ± 9.84 (S.D.); NAC ptc>rpr OFF: 39.9 ± 4.68 (S.D.); ptc>rpr ON: 49.73 ± 8.18 (S.D.); NAC ptc>rpr ON: 29.52 ± 9.41 (S.D.) (E) Design for ectopic expression of enzymatic antioxidant transgenes and simultaneous cell death induction when shifted to 29°C for 11 h. The Gal4/UAS (red) activate Cat, Sod or Cat+Sod transgenes. Blue striped area: salE/Pv-LHG lexO-rpr. Adult wings were scored for complete regeneration of the missing zone. Red coloration indicates zone influenced by the enzymatic antioxidant; purple: zone influenced by enzymatic antioxidant and cell death. salE/Pv-LHG and nub-Gal4 are under the control of tubGal80TS. (F) Percentage of regenerated wings in Cat, Sod or Cat and Sod ectopically expressed transgenes. (G) Wings from individuals after cell death and transgene activation (ON). For Cat, Sod or Sod:Cat and example of incomplete regeneration is shown. TP-3: TO-PRO-3. ***P<0.001
Fig 3.
(A) Test of JNK reporters. All images in A correspond to the same disc after ptc>rpr induction. Top row: apical sections. Bottom row: basal sections. Note that puc is more abundant in apical than basal sections, particularly in the notum (n; arrowhead) and wing pouch (wp; arrow). Cell death (TUNEL) and high TRE-red are more abundant in basal sections. (B) Zoom of a digital cross section of the zone marked with a white line in A. Endogenous puc-lacZ is found in the outer layer of peripodial membrane cells (pm). Puc-lacZ cells in the disc columnar epithelium are apical (white), most apoptotic cells are basal (red), and TRE-red positive cells are apical and basal (blue). (C) Three digital cross section in an apical puc-lacZ zone of the wing pouch (wp) and notum (n1, n2). Each example contains three to four cells with co-localization of ß-galactosidase and EdU. (D) TRE-red reporter in ptc>rpr discs of larvae fed with standard food or NAC-supplemented food (NAC). TP-3: TO-PRO-3. (E) Mean pixel intensities of TRE-red reporter in ptc>rpr discs with standard or NAC food. The pixel intensity for standard food was 26.06 ± 7.22 (S.D.; n = 15) and for NAC 18.12 ± 8.32 (S.D.; n = 25). (F) TRE-red reporter expression in physically injured discs, cultured for 7 h ex vivo in Schneider’s culture medium with or without NAC. Outline: disc contour. Wedges: cut. (G) Mean pixel intensities of TRE-red reporter in ex vivo cultured discs with or without NAC. The pixel intensity for standard culture was 88.98 ± 22.25 (S.D.; n = 6) and for NAC 23.98 ± 10.26 (S.D.; n = 16). **P<0.01, ***P<0.001.
Fig 4.
ROS stimulate p38 phosphorylation.
(A) P-p38 staining of intact (uncut, controls) and cut discs cultured for the indicated times after injury. White lines: wound edges; white arrowhead: small incision. (B) Discs cultured with or without NAC, cut and stained for P-p38. (C) Mean pixel intensities of P-p38 fluorescence from cut discs cultured with standard medium (95.29 ± 17.52; S.D.) or NAC-supplemented (22.45±2.56; S.D.). (D) Apical and basal images of P-p38 after ptc>rpr induction. (E) Apical and basal images of ptc>rpr after NAC supplementation showing reduction of P-p38 localization. (F) Mean pixel intensities of P-p38 fluorescent labeling from ptc>rpr discs fed with standard (52.17±19.96; S.D.) or NAC-supplemented food (7,85 ± 2,42; S.D.). (G) Genetic ROS scavenging using ci>Sod:Cat, activated in the anterior compartment (ci, black in the sketch). SalEPv>rpr cell death (blue in the sketch) in the same disc results in inhibition of P-p38 only in anterior compartment. TP-3: TO-PRO-3 nuclei staining. Outlined white in D, E and G: apoptotic zone. ***P<0.001.
Fig 5.
p38 inhibition impairs tissue repair.
(A) Adult wing parameters in p38 signaling mutant backgrounds after genetic ablation. Left: ratios of the wing areas between experimental groups (rpr induction salE/Pv>rpr ON) and control (no rpr induction salE/Pv>rpr OFF). Right: percentage of fully regenerated wings. Far right: examples of wings with full regeneration (control) and incomplete regeneration (indicated genotypes) after salE/Pv>rpr. (B) Percentage of fully regenerated wings after SB202190 intake in salE/Pv>rpr flies. Right: wing fully regenerated (top) and examples of incomplete regeneration for each SB202190 concentration.
Fig 6.
p38 and JNK are activated independently.
(A) Hepr75 hemizygous disc cut (wedge) and stained with P-p38. Sketch of wing discs with square indicate location of images. (B) Hepr75 hemizygous disc after ptc>rpr induction and stained for P-p38. Dead domain is outlined white. TP-3; TO-PRO-3. (C) Wild type and p38a1-/- discs, cultured for 7h showing TRE-red activation close to the cut edges. (D) Mean pixel intensity for TRE-red measured in discs with physical injury in wild type (88.24 ± 22.58; S.D.) and p38a1-/- (70.80 ± 19.14; S.D.). P = 0.33 n.s.
Fig 7.
Cytokine signaling is controlled by ROS and JNK.
(A, B) In situ hybridization of upd (A) and upd3 (B) in wild type (wt) discs and JNKK hepr75 hemizygotes (C) after injury. (D) hepr75 hemizygote stained with anti-Upd after injury. (E) Mean pixel intensities of upd reporter (upd>myrtom Std food: 16.68 ± 3.22 and NAC: 9.01 ± 3.41, S.D.) and STAT92E reporter (10xSTAT92E-GFP Std food: 18.24 ± 2.73 and NAC: 8.63 ± 4.07, S.D.) after standard or NAC-supplemented food. White wedges indicate zone of injury. (F, G) Upd (anti-Upd) is mainly expressed in living cells and not in dead cells after ptc>rpr or sal>rpr. (H) Upd expression declines after NAC intake. TP-3: TP-PRO-3 nuclei staining. (I) Mean pixel intensities of Upd stained ptc>rpr discs with or without NAC feeding (150.29 ± 7.11 and 96.69 ± 18.97, S.D.). (J) Inhibition of the JAK/STAT signaling within domeDN impairs wing regeneration. Genetic design (J) using double transactivator system (as in Fig 2) to induce death (blue) and activate domeDN (red). (K) Percentage of regenerated wings for controls (rpr or domeDN expression only) and experimental (rpr and domeDN dual expression). Note that domeDN wings were not able to regenerate (rpr and domeDN dual expression), whereas domeDN wings in the absence of cell death are normal. Examples of wings (left) of controls and experimental. (L) Experimental design for testing the rescue of NAC effects by ectopic activation of upd. (M) NAC effect on repair ability was rescued by upd overexpression. Quantification of the percentage of wings that regenerate after NAC feeding for the indicated genotypes. (N) Examples of wings from NAC-feeding with rpr-ablation defects (upper) and with rescue after rpr-ablation and upd activation (lower). *P<0.05 **P<0.01.
Fig 8.
(A) In situ hybridization of upd in wild type (wt) and p38a1-/- cut discs. (B) In situ hybridization of upd3 in wild type (wt) and p38a1-/- cut discs. (C) Immunostaining with anti-Upd in wild type (wt) and p38a1-/- cut discs. White lines and wedges indicate the position of the cut (D) Experimental design for testing the rescue of SB202190 effects by ectopic activation of upd. (E) SB202190 effect on repair ability was rescued by upd overexpression. Quantification of the percentage of wings that regenerate after SB202190 feeding for the indicated genotypes. (F) Examples of wings from SB202190-feeding with rpr-ablation defects (upper) and with rescue after rpr-ablation and upd activation (lower).
Fig 9.
Cell protection module activated by injury or cell death.
Oxidative stress in dying cells is likely of mitochondrial origin and results in highly toxic JNK. However, low levels of ROS propagate to adjacent surviving cells (arrows). Non-deleterious ROS will activate moderate levels of JNK and p38 only in surviving cells. P38 and JNK are required for cytokine activation and tissue repair.