1. Volume 3, Issue 3, Pages 919-930 (March 2013)
Homeostatic Epithelial Renewal in the Gut Is Required for Dampening a Fatal Systemic Wound Response in Drosophila 
Asuka Takeishi, Erina Kuranaga, Ayako Tonoki, Kazuyo Misaki, Shigenobu Yonemura, Hirotaka Kanuka, Masayuki Miura 
Cell Reports 
Volume 3, Issue 3, Pages (March 2013)
DOI: /j.celrep
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2. Cell Reports 2013 3, 919-930DOI: (10.1016/j.celrep.2013.02.022)
Copyright © 2013 The Authors Terms and Conditions

3. Figure 1 dpf-1K1 Has Defects in Gut Epithelial Turnover and Structure
(A) Morphological feature of the Drosophila midgut (left) and a schematic drawing for the self-renewal process of midgut cells at the adult stage (right). All of the immunohistochemical and EM analyses in this study were performed on the posterior midgut. ISCs proliferate to generate ISCs and EBs, which differentiate into ECs or EE cells (right). Markers for each cell type: Delta for ISC, Su(H) for EB, and Pros for EE (right). AMG, anterior midgut; PMG, posterior midgut; HG, hindgut; magenta, Hoechst (left). Scale bar, 100 μm.
(B) BrdU incorporation. Left: Magenta, Hoechst; green, BrdU labeling. Scale bars represent 10 μm. Right graph: The number of BrdU-incorporated cells in each gut. WT, wild-type.
(C and D) dpf-1K1 has defects in EC turnover at the adult stage.
(C) Experimental procedure (top) and EC turnover (bottom). GFP-negative cells with small nuclei (arrows) were EEs. Magenta, Hoechst; green, GFP. Scale bars represent 10 μm.
(D) Percentage of GFP-positive ECs 6 days after the temporary expression of CD8::PARP::Venus.
(E) BM visualization by collagen IV detection in sagittal sections of the posterior midgut by confocal microscopy. White, Hoechst; green, GFP (for collagen IV-GFP). Scale bars represent 10 μm.
(F) Electron micrographs of the gut. BMs are indicated with green dashed lines. Top: Lumen side. The CM and LM cells in dpf-1K1 were rounder and taller than those of the WT (asterisks), and the muscle cells between the ECs and body cavity were occasionally absent in dpf-1K1 (arrowhead). Scale bars represent 1 μm.
(G–I) Proportion of gut cell types. Percentage of (G) Dl-lacZ-positive cells (ISCs), (H) Su(H)-lacZ-positive cells (EBs), and (I) Pros-positive cells (EEs) in the posterior midgut. The number of cells that were positive for each marker was counted and calculated as a percentage of the Hoechst-positive cells.
Error bars in all graphs indicate SEM.
Cell Reports 2013 3, DOI: ( /j.celrep )
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4. Figure 2 dpf-1K1 Flies Are Sensitive to Wounding
(A–C) Survival rate of wounded flies. Flies 2 days after eclosion were pricked on the abdomen with a microinjection needle, or halteres were removed. Day 0 is the day of wounding or removing. Unwounded flies were used as a control. (A) Wounding. (B) Haltere removal. (C) Wounding of the dpf-1EX fly line.
(D) Number of BrdU-incorporating cells in the posterior midgut. PTEN was overexpressed in flies from 2 days after eclosion by shifting the temperature from 18°C to 29°C. BrdU was administered in the food for 3 days from 6 hr after the temperature shift to 29°C. Error bars in the graph indicate SEM.
(E) Top: Experimental procedure. PTEN expression was induced in the ISCs from 6 hr before wounding. Bottom: Survival rate after wounding.
See also Figures S2 and S3.
Cell Reports 2013 3, DOI: ( /j.celrep )
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5. Figure 3 Caspase Is Activated in ECs after Wounding
(A) Left: Imaging analysis with SCAT3 in the gut of a control fly or a fly 30 min after wounding. High FRET (Venus/ECFP) ratio (red) indicates low caspase activity, and low FRET ratio (blue) indicates strong caspase activity. Some ECs (arrowheads) showed strong caspase activity. Right: The FRET ratio of individual cells is represented in the graph. Scale bars, 10 μm.
(B and C) Immunohistochemistry of the gut with CD8::PARP::Venus 24 hr after wounding. The cPARP-positive cells (magenta) were negative for (B, left) Delta (ISCs; green, arrow) and (C, left) Pros (EEs; green, arrow), although CD8::PARP::Venus was expressed in all of the cells (right panels of C and B for CD8 staining). Blue: Hoechst. Scale bars represent 10 μm.
(D) Immunohistochemistry of guts expressing CD8::PARP::Venus in the ECs. Left: top, white: Hoechst; middle, white: cPARP; bottom, green: CD8. Scale bars represent 100 μm. Right: Graph of the number of cPARP-positive cells in each gut after wounding.
(E) Number of cPARP-positive cells in each posterior midgut of flies 24 hr after wounding. NAC was administered in the food at 100 mM.
(F) Number of cPARP-positive cells in the posterior midgut of Hayan mutant flies or transgenic flies with NP1-Gal4-driven SOD1 overexpression (o/e) or NOX1 knockdown (IR) 24 hr after wounding.
(G) Confocal microscopy of the sagittal section of a control gut (24 hr after wounding). Magenta, arrow: cPARP-positive EC in the gut cell layer. Magenta, arrowheads: cPARP-positive ECs outside the cell layer. Blue, Hoechst; green, phalloidin. L, lumen. Scale bars represent 10 μm.
(H) EM of the WT gut 24 hr after wounding. Arrowhead: a dying EC. Scale bar represents 10 μm.
(I) Location and nuclear Hoechst staining of cPARP-positive ECs in a control fly 6 hr and 24 hr after wounding. Inside/outside of the gut cell layer: nuclear Hoechst positive/negative. The number of observed gut samples is shown on top of each column (n = 85, 81).
Error bars in all graphs indicate SEM. See also Figures S4 and S5.
Cell Reports 2013 3, DOI: ( /j.celrep )
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6. Figure 4 dpf-1K1 Hemolymph Contains a Lethal Factor after Wounding
(A) Experimental procedure for hemolymph injection. The hemolymph of 20 flies was collected into 10 μl of PBS, and 65 nL was injected into the abdomen of each recipient fly.
(B) Survival rate of WT flies after injection of PBS or hemolymph from wounded WT or unwounded or wounded dpf-1K1flies.
(C) Survival rate of dpf-1K1 flies after injection of PBS or hemolymph from wounded WT flies.
(D) Survival rate of WT flies after injection of dpf-1K1 hemolymph collected at various time points after wounding. The lethal factor appeared within 3 hr after wounding.
(E) Caspase activity observed in a fly expressing CD8::PARP::Venus in the ECs 24 hr after injection of hemolymph from wounded dpf-1K1 flies. Green, Hoechst; magenta, cPARP; white, CD8. Scale bars represent 100 μm.
Cell Reports 2013 3, DOI: ( /j.celrep )
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7. Figure 5
Caspase Activity in ECs Is Required for the Systemic Wound Response
(A–E) Survival rate of wounded flies.
(A) Flies expressing GFP or p35 in the gut in ECs.
(B) The wound-induced lethal phenotype of dpf-1K1 (control) was rescued by the overexpression of dark in ECs (NP1 > dark).
(C) Experimental procedure for starting p35 overexpression in adulthood before wounding (top). Beginning 6 hr before wounding, p35 expression in ECs was induced by a temperature shift from 18°C to 29°C. Survival rate after wounding (bottom).
(D) Flies expressing GFP or p35 in the hemocytes (blood cells) using the Pxn-Gal4 driver.
(E) Survival rate of WT flies after injection of hemolymph from wounded control flies or wounded flies expressing p35 in the ECs (NP1 > p35). Hemolymph was taken 48 hr after wounding.
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8. Figure 6 Model for the Caspase-Mediated Systemic Wound Response
Caspase activation is required to overcome wounding. ROS in ECs activate caspases. The fat body and hemocytes may also contribute to the systemic wound response, for example, by mediating signaling from the wound site to the gut or by generating cytokines as positive feedback. In wounded WT flies, caspase is activated to induce EC death (1) and ISC proliferation (2), resulting in gut epithelial turnover. In flies in which caspase is inhibited in ECs, gut homeostasis—i.e., EC death (1), gut cell turnover (2), gut cell repopulation, and gut structure—is impaired. Although these flies can survive under normal culture conditions, they are sensitive to wounding; the mechanism involves a lethal factor introduced into the hemolymph as a result of the wound. In other words, the wound changes the fly’s condition from a latent phase to a crisis phase (3).
Cell Reports 2013 3, DOI: ( /j.celrep )
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9. Figure S1
Expression Pattern of NP1-Gal4 and esg-Gal4 (Adult Expression), Related to Results
(A–D) GFP or RedStinger was expressed by elav-Gal4 (2nd panels), NP1-Gal4 (3rd panels), or esg-Gal4 (adult expression, bottom panels). After antibody staining, the expression pattern of the fluorescence was observed in the brain (A, magenta: nc82 labeling; green: GFP or RedStinger fluorescence, scale bar, 10 μm), epithelium and fat body (B, green: GFP or RedStinger fluorescence), muscle in thorax (C, red: Phalloidin, green: GFP or RedStinger fluorescence), and gut (D, green: GFP or RedStinger fluorescence; inset blue: Hoechst; inset magenta: anti-Delta; inset scale bar, 10 μm).
UAS-GFP (top panels), elav-Gal4/UAS-GFP (2nd panels), NP1-Gal4 UAS-GFP (3rd panels), esg-Gal4/+;UAS-RedStinger/tub-Gal80ts7 (bottom panels).
(E) GFP expression was driven by NP1-Gal4 (NP1-Gal4/UAS-GFP). Top panels: ISC, magenta for anti-Delta staining. Bottom panels: EE, magenta for anti-Pros staining. Scale bars, 10 μm.
(F) ECs 6 days after the temporary expression of CD8::PARP::Venus (dpf-1K1 NP1-Gal4/+; UAS-CD8::PARP::Venus/tub-Gal80ts7). The staining patterns of GFP antibody and CD8 antibody were identical. White: Hoechst. Green: CD8. Magenta: GFP. Scale bars, 10 μm.
(G) ECs 24 hr after injury. The CD8 staining pattern of cPARP positive cell (arrowheads) was same as that of surrounding cells that were cPARP negative (n (number of observed cPARP positive cells) ≥ 50). Green: CD8. Magenta: cPARP. Scale bars, 10 μm.
Cell Reports 2013 3, DOI: ( /j.celrep )
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10. Figure S2
dpf-1K1 Flies Have No Obvious Defects under Normal Culture Conditions, Related to Figure 2
(A) Body weight of the adult fly (bar graph represents mean ± s.e.m. of three independent experiments).
(B) Food intake was estimated with blue-colored food (normal cornmeal food containing 1% Brilliant Blue FCF, Wako); 0 hr was the time that flies were moved from colored to normal food. Anterior: left; posterior: right.
(C) Scab formation proceeded immediately, within 20 min, at the wounded site (asterisks) in both wild-type and dpf-1K1flies. White line denotes the scab edge. Scale bars, 10 μm.
Cell Reports 2013 3, DOI: ( /j.celrep )
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11. Figure S3
dpf-1K1 Is Sensitive to Noninfectious Stresses, Related to Figure 2
(A) Survival rate of flies. Flies 2 days after eclosion were cultured on 1% agar containing 5% sucrose and 3% DSS. Day 0 is the starting day of DSS treatment. Flies cultured on agar containing sucrose were used as a control. p < 0.01 for DSS-treated wild-type versus DSS-treated dpf-1K1. P-value was calculated by the log-rank test.
(B) Flies were sprinkled with 70% ethanol and pricked with a needle soaked in 70% ethanol. dpf-1K1 flies showed a decreased survival rate even under the cuticle-sterilized condition. p < for lethality of wounded dpf-1K1 versus wounded wild-type or unwounded dpf-1K1. P-value was calculated by the log-rank test.
Cell Reports 2013 3, DOI: ( /j.celrep )
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12. Figure S4
Wounding Does Not Induce Caspase Activation in the Fat Body, Related to Figure 3
SCAT3 imaging analysis (FRET ratio imaging) of the fat body of unwounded and wounded flies 30 min after epithelial wounding (tubP-LexA::GAD; lexAop-SCAT3/MKRS). Graph: FRET ratios of individual cells (n (number of calculated cells in each time) > 150; n.s., not significant, unpaired Student’s t test). Scale bars, 10 μm. Error bars in the graph indicate SEM.
Cell Reports 2013 3, DOI: ( /j.celrep )
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13. Figure S5
Immunohistochemistry with CD8::PARP::Venus to Detect Caspase Activation after Wounding, Related to Figure 3
(A) Schematic drawing of CD8::PARP::Venus. CD8::PARP::Venus possesses a caspase cleavage site, and the cleaved PARP fragment (cPARP) is detected by an anti-cPARP antibody to visualize caspase-activated cells. CD8::PARP::Venus can be detected by an anti-CD8 antibody.
(B) Anti-cPARP-positive cells were not observed in the fat body, epithelium (left panels, arrowheads) (da-Gal4/+; UAS-CD8::PARP::Venus/+), or nervous system (right panels) (act-Gal4/+; UAS-CD8::PARP::Venus/+) of control flies 24 hr after wounding. Green: anti-CD8 staining. Scale bars, 100 μm.
(C) Caspase activation 24 hr after cuticle wounding was investigated in the gut overexpressing PTEN. PTEN expression was induced from 2 days after eclosion by temperature shift, and flies were pricked 6 hr after PTEN induction. Control: NP1-Gal4/UAS-PTEN; tub-Gal80ts7/UAS-CD8::PARP::Venus, esg > PTEN: esg-Gal4 NP1-Gal4/UAS-PTEN; tub-Gal80ts7/UAS-CD8::PARP::Venus). n (number of flies in each column) ≥ 4, ∗p < 0.05, unpaired Student’s t test. Error bars in the graph indicate SEM.
(D) Reconstructed 3D images of the gut 24 hr after wounding (NP1-Gal4/+; UAS-CD8::PARP::Venus/+), showing cPARP-positive cells (magenta) outside the gut cell layer; cells were visualized by nuclear Hoechst (blue) and Phalloidin (green) staining. Scale bars, 10 μm (1 unit). BM: basement membrane; L: lumen.
Cell Reports 2013 3, DOI: ( /j.celrep )
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14. Figure S6
Upd3 Was Expressed at the Wound Site and in the Midgut in Both Wild-Type and dpf-1K1, Related to Results
(A) X-gal staining of upd3 expression in the epidermis. upd3 expression was observed around the wound site (asterisks) of both wild-type and dpf-1K1 flies within 24 hr after wounding (control: upd3-Gal4 UAS-GFP/UAS-lacZ 4.1.2), dpf-1K1: dpf-1K1 upd3-Gal4 UAS-GFP/dpf-1K1 UAS-lacZ 4.1.2).
(B) upd3 expression was observed within 24 hr after wounding in the gut of both control and dpf-1K1 flies (control: upd3-Gal4 UAS-GFP/CyO, dpf-1K1: dpf-1K1 upd3-Gal4 UAS-GFP/dpf-1K1). (Top and middle panels: low magnification; bottom panel: Z section of the high-magnification image of a wild-type gut 24 hr after wounding; lumen is at the top.) Green: anti-GFP staining. Red: Phalloidin staining. White: Hoechst staining. Scale bars, 100 μm (top and middle panels), 10 μm (bottom panel).
(C) upd3 expression level 24 hr after wounding was evaluated by the percentage of GFP-positive cells in the posterior midgut: 0%–30% (level 1), 30%–60% (level 2), and 60%–100% (level 3) (control: upd3-Gal4 UAS-GFP/CyO, dpf-1K1: dpf-1K1 upd3-Gal4 UAS-GFP/dpf-1K1). N (number of flies examined) = 15 (unwound control), 12 (wound control), 8 (unwound dpf-1K1), 11(wound dpf-1K1). Magenta: Hoechst staining. Green: anti-GFP staining. Scale bars, 100 μm.
Cell Reports 2013 3, DOI: ( /j.celrep )
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