Remodelling of oxygen-transporting tracheoles drives intestinal regeneration and tumorigenesis in Drosophila

Abstract

The Drosophila trachea, as the functional equivalent of mammalian blood vessels, senses hypoxia and oxygenates the body. Here, we show that the adult intestinal tracheae are dynamic and respond to enteric infection, oxidative agents and tumours with increased terminal branching. Increased tracheation is necessary for efficient damage-induced intestinal stem cell (ISC)-mediated regeneration and is sufficient to drive ISC proliferation in undamaged intestines. Gut damage or tumours induce HIF-1α (Sima in Drosophila), which stimulates tracheole branching via the FGF (Branchless (Bnl))-FGFR (Breathless (Btl)) signalling cascade. Bnl-Btl signalling is required in the intestinal epithelium and the trachea for efficient damage-induced tracheal remodelling and ISC proliferation. Chemical or Pseudomonas-generated reactive oxygen species directly affect the trachea and are necessary for branching and intestinal regeneration. Similarly, tracheole branching and the resulting increase in oxygenation are essential for intestinal tumour growth. We have identified a mechanism of tracheal-intestinal tissue communication, whereby damage and tumours induce neo-tracheogenesis in Drosophila, a process reminiscent of cancer-induced neoangiogenesis in mammals.

Extended Data Fig. 1. Infection and oxidative damage increase esg>GFP+ cells in the midgut and associate with increased TTC branching

a. Adult midgut intestinal progenitors labeled with esg^NP5130-Gal4>UAS-srcGFP were imaged in unchallenged conditions (4% sucrose) and upon oral P.a. infection (48hrs), and feeding with H₂O₂ (48hrs) and PQ (24hrs). DAPI (blue) in the upper panels stains all midgut nuclei. The bottom panels show the GFP-labeled progenitors separately. P.a. and PQ expanded the intestinal progenitors with a posterior midgut bias, whereas H₂O₂ exhibited an anterior midgut bias. b-c. Quantification of midgut mitosis (b, n=10 each) and TTC branching (c, n=7,6) in PQ-treated flies. d-e. Posterior midgut (R4) images of btl-Gal4>UAS-srcGFP flies in baseline conditions (sucrose) and upon PQ feeding. DAPI (blue) staining all the nuclei. Single channel images of the GFP are shown in d’-e’. f-g. Posterior midgut images of QF6>QUAS-mtdTomato flies in baseline conditions exhibit tracheal expression of the reporter. Midgut epithelial ECs with low expression of the reporter are visible is zoomed image (g). Single channel images of the Tomato are shown in f’-g’. Scale bars: 300 μm in a, 75 μm in d-g. Data are presented as mean values ± SD. Statistical significance (t-tested, two-sided for b, and U-tested for c): ns, not significant, * 0.01<p≤0.05, ** 0.001<p≤0.01 and *** p≤0.001.

Extended Data Fig. 2. The FGFR/Btl is necessary and sufficient for midgut TTC branching and ISC mitosis

a-b. Brightfield images of the tracheae of P.a. infected R5 regions of the midgut in trh-Gal4 control (a) and trh-Gal4>UAS-btl^DN (b). c-d. Brightfield images of the tracheae of uninfected R5 regions of the midgut in trh-Gal4 control (c) and trh-Gal4>UAS-λbtl (d). e-g. Quantification of TTCs (e, n=10,11,10,9,10), TTC branching (f, n=10,11,10,8,10), and midgut mitosis (g, n=8,6,10,9,6) upon trh-Gal4-driven btl manipulation with or without P.a. infection. h-i. Brightfield images of the tracheae of P.a.-infected R5 regions of the midgut in dSRF-Gal4 control (h) and dSRF-Gal4>UAS-btl^DN (i). j-k. Brightfield images of the tracheae of uninfected R5 regions of the midgut in dSRF-Gal4 control (j) and dSRF-Gal4>UAS-λbtl (k). l-n. Quantification of TTCs (l, n=10,10,8,8,11), TTC branching (m, n=10,10,8,8,11), and midgut mitosis (n, n=11,9,12,12,9) upon dSRF-Gal4-driven btl manipulation with or without P.a. infection. All scale bars: 75 μm. Data are presented as mean values ± SD. Statistical significance (t-tested, two-sided): ns, not significant, * 0.01<p≤0.05, ** 0.001<p≤0.01 and *** p≤0.001.

Extended Data Fig. 3. Infection and oxidative damage induce FGF/bnl in the midgut epithelium

Adult midgut bnl-expressing cells labeled with the reporter bnl-Gal4>UAS-srcGFP were imaged in unchallenged conditions (4% sucrose) and upon oral P.a. infection (48hrs), feeding with H₂O₂ (48hrs) and PQ (24hrs). DAPI (blue) in the upper panels stained all midgut nuclei. The bottom panels show the GFP-labeled bnl expressing cells separately. P.a. and PQ induced the reporter throughout the midgut, whereas H₂O₂ exhibited an anterior midgut bias. Scale bar: 300 μm.

Extended Data Fig. 4. Btl/Bnl signaling in the epithelial cells is necessary for efficient tracheal remodeling and mitosis in response to infection

a-b. Quantification of TTC branching upon progenitor- (a) and EC-specific (b) silencing of bnl (bnl^RNAi3) and btl (btl^RNAi) (a, n=10,8,5,10,7,7 and b, n=10,7,9,10,9,8). c-d. Quantification of midgut mitosis upon progenitor- (c) and EC-specific (d) silencing of bnl (bnl^RNAi3) (c, n=6,8,12,12 and d, n=9,9,11,13). e. Quantification of esg+ progenitors as a percent of total number of cells in the posterior regions of the midgut upon progenitor-specific knockdown of btl (btl^RNAi) and bnl (bnl^RNAi3) (n=12,15,15). f-g. Quantification of midgut mitosis upon progenitor- (f) and EC-specific (g) silencing of btl (btl^RNAi) (f, n=8,13,11,13 and g, n=12,11,11,16). h. Quantification of esg+ progenitor cells/total number of cells in the posterior midgut upon progenitor-specific knockdown of btl (btl^DN, n=9,9). Data are presented as mean values ± SD. Statistical significance (t-tested, two-sided): ns, not significant, * 0.01<p≤0.05, ** 0.001<p≤0.01 and *** p≤0.001.

Extended Data Fig. 5. Infection and oxidative damage activate Hif-1α/Sima in the midgut epithelium and the visceral TTCs

Hif-1α/Sima activation was monitored via the ldh-Gal4>UAS-nlsGFP reporter expression in the adult midgut epithelium and the intestinal trachea of the R5 region in unchallenged flies (sucrose) and upon P.a. and PQ treatment (a, c, e), and of the R2 region in unchallenged flies (sucrose) and upon H₂O₂ feeding (b, d). Epithelial sections (a-d) and trachea surface sections (a’-d’) of the same midguts were imaged. DAPI (blue) in a-d and a’-d’ stains all the nuclei. a”-d” and a”’-d”’ correspond to separated channels for reporter expression in the epithelium and the intestinal trachea, respectively. The ldh-Gal4>UAS-nlsGFP reporter was expressed in cells of the midgut epithelium and in the midgut TTCs in baseline conditions in the anterior (R2 in b, b’) and posterior (R5 in a, a’) midgut. P.a. (c, c’), H₂O₂ (d, d’) and PQ (e, e’) induced the reporter in the epithelium and the trachea at varying degrees. All images were acquired at the same confocal settings as their respective controls. Scale bar: 75 μm.

Extended Data Fig. 6. Hif-1a/Sima is necessary in the intestinal epithelium and the trachea for TTC branching

a-b. Brightfield images of the midgut TTCs (R5 region) upon trachea-specific (via btl-Gal4) sima knockdown in the background of heterozygous simaKG in baseline conditions. c-d. Bright-field images of the midgut TTCs (R5 region) upon EC-specific (via Myo1A-Gal4) sima knockdown in the background of heterozygous simaKG in baseline conditions. e-f. Bright-field images of the midgut TTCs (R5 region) upon trachea-specific (via btl-Gal4) sima knockdown in the background of heterozygous sima^KG in P.a.-infected conditions. g-h. Bright-field images of the midgut TTCs (R5 region) upon EC-specific (via Myo1A-Gal4) sima knockdown in the background of heterozygous sima^KG in P.a.-infected conditions. The images correspond to examples of those quantified for Fig. 4e,i. Scale bar: 75 μm.

Extended Data Fig. 7. Time-course analysis of Notch^DN progenitor-derived midgut tumors

a-d. The R4a region of control (reared for 4 days at 18°C) (a) and tumorous midguts (reared for 4, 7 and 10 days at 29°C) (b-d) of the esg-Gal4 UAS-eGFP tub-Gal80^ts>UAS-Notch^DN genotype with concomitant expression of QF6>QUAS-mtdTomato (red) to label the trachea. DAPI (blue in a-d) is used to label all midgut nuclei and Prospero (a”-d”) labels the EEs. a’-d’, a”-d” and a”’-d”’ correspond to the individual channels for eGFP, Prospero and Tomato-labeled trachea, respectively. Scale bars: 75 μm. e-g. Quantification of TTC branching in the R4a of control (Notch^DN uninduced) and Notch^DN-expressing midguts (e, n=4,8,4,6,4,7), in the Notch^DN tumor-region vs. neighboring non-tumor area on the same image (f, n=6,6,4,4,6,6), and midgut mitosis of control (Notch^DN uninduced) and Notch^DN-expressing midguts (g, n=20 each) during a time-course analysis at 4, 7, and 10 days post-tumor induction. Scale bar: 75 μm. Data are presented as mean values ± SD. Statistical significance (t-tested, two-sided): ** 0.001<p≤0.01 and *** p≤0.001.

Extended Data Fig. 8. Time-course analysis of Ras^V12 progenitor-derived midgut tumors

a-d. The R5 region of control esg-Gal4 UAS-eGFP tub-Gal80^ts (reared for 1 day at 29°^C) and esg-Gal4 UAS-eGFP tub-Gal80^ts>UAS-Ras^V12-tumor bearing midguts (reared for 1, 3 and 5 days at 29°C) with concomitant expression of QF6>QUAS-mtdTomato (red) to label the trachea. DAPI (blue in a-d) was used to label all midgut nuclei. a’-d’ and a”-d” correspond to the individual channels for the eGFP and the Tomato-labeled trachea, respectively. Scale bar: 75 μm.

Figure 1.. Remodelling of visceral TTCs covering the midgut is associated with ISC proliferation.

a-f. The adult intestinal trachea is increased upon P.a. infection (a, b), administration of H₂O₂ (c, d) and P.e. infection (e, f). The intestinal TTCs were labeled with btl-Gal4>UAS-srcGFP in a-d and with QF6>QUAS-mtdTomato in e-f, and DAPI (blue) labeled all nuclei in a-f. The R5 midgut region was imaged in a-b, the R2 region in c-d and the R4-R5 region in e-f. Single channel images of the trachea are shown in a’-f’. g-l. Quantification of TTC branching and mitosis upon P.a. infection (g, n=10,9 and h, n=15,18), administration of H₂O₂ (i, n=10,9 and j, n=15,11) and P.e. infection (k, n=14,15 and l, n=7,15). m. Quantification of TTCs upon trachea-specific btl manipulation with or without P.a. infection (n=7 each). n. Tracheae (green) and cleaved Caspase-3 (red) upon trachea-specific btl knockdown. o. Quantification of mira-GFP-positive ISCs upon trachea-specific btl knockdown via trh-Gal4 (n=10,12). p-q. Quantification of TTC branching (p, n=7,9,10,10,10) and mitosis (q, n=35,12,8,10,10) upon trachea-specific btl manipulation with or without P.a. infection. r-u. Tracheae (green) of P.a.-infected R5 regions of the midgut in btl-Gal4>UAS-srcGFP control (r) and btl-Gal4>UAS-srcGFP, UAS-btl^DN-expressing (s), as well as control (t) versus btl-Gal4>UAS-srcGFP, UAS-λbtl-expressing flies (u). v. Quantification of midgut mitosis of flies heterozygous for btl pathway mutations with or without P. e. infection compared to controls (n=9,11,11,13,11,13,10,14,9,12). Scale bars: 75 μm in a-d, 60 μm in e-f, 37.5 μm in n, r-u. Data are presented as mean values ± SD. Statistical significance (t-tested, two-sided): ns, not significant, * 0.01<p≤0.05, ** 0.001<p≤0.01 and *** p≤0.001.

Figure 2.. The FGF/Bnl is expressed in the midgut epithelium and controls ISC proliferation and TTC remodelling.

a-b. Control (a) and P.a.-infected (b) bnl-Gal4>UAS-srcGFP midguts (R5 region) with esg-lacZ-labeled (red) ISCs and EBs, and DAPI (blue) staining all the nuclei. c-d. Control (c) and H₂O₂-fed (d) bnl-Gal4>UAS-srcGFP midguts (R2 region) with Prospero-labeled (red) EEs, and DAPI (blue) staining all the nuclei. Yellow arrowheads indicate bnl-positive ECs (big cells) and white arrowheads indicate bnl-positive esg-positive progenitors (a-b) and EEs (c-d). e. RT-qPCR for bnl mRNA expression in w¹¹¹⁸ whole adult midguts upon different stresses. The average and standard deviation of n=3 biological expreriments are plotted. f-g. Quantification of TTC branching (f, n=12,12,10,10,10,10) and mitosis (g, n=15,11,12,12,13,15) upon progenitor-specific bnl knockdown with or without P.a infection. h-i. Quantification of TTC branching (h, n=6 each) and mitosis (i, n=12,14) upon progenitor-specific bnl overexpression. j-k. Quantification of TTC branching (j, n=9,10,9,9,10,10) and mitosis (k, n=15,12,13,13,7,10) upon EC-specific bnl knockdown with or without P.a infection. l-m. Quantification of TTC branching (l, n=6 each) and mitosis (m, n=9,11) upon EC-specific bnl overexpression. n-p. Midguts (R5 region) of control (n) and esg^ts-Gal4 progenitor-specific bnl knockdown (o-p), stained for ISCs+EBs (green), cleaved Caspase-3 (red) and Topro-3 (blue). q. Quantification of esg-positive ISCs+EBs in control and progenitor-specific bnl knockdown (n=9,10,9). Scale bars: 37.5 μm in a-d, 75 μm in n-p. Data are presented as mean values ± SD. Statistical significance (t-tested, two-sided): ns, not significant, * 0.01<p≤0.05, ** 0.001<p≤0.01 and *** p≤0.001.

Figure 3.. Damage-induced TTC remodelling and ISC mitosis require epithelial FGFR/Btl and tracheal FGF/Bnl.

a-d. Quantification of TTC branching (a, n=10 each, and c, n=10,10,10,9) and mitosis (b, n=31,27,25,30 and d, n=29,23,26,23) upon progenitor-specific (a-b) and EC-specific (c-d) btl knockdown with or without P.a. infection. e-f. Quantification of TTC branching (e, n=10 each) and mitosis (f, n=32,23,27,24,28,27) upon trachea-specific btl knockdown via btl^ts-Gal4 with or without P.a. infection. g-h. Quantification of TTC branching (g, n=7,9,7,10) and mitosis (h, n=10,10,9,8) upon trachea-specific btl knockdown via dSRF^ts-Gal4 with or without P.e. infection. i-j. Quantification of TTC branching (i, n=12,11) and mitosis (j, n=8,11) upon progenitor-specific λbtl overexpression. k-l. Quantification of TTC branching (k, n=12,11) and mitosis (l, n=12,8) upon EC-specific λbtl overexpression. m-n. Quantification of TTC branching (m, n=4,6) and mitosis (n, n=28,24) upon trachea-specific λbtl overexpression via btl^ts-Gal4. The flies were uninfected in i-n. Data are presented as mean values ± SD. Statistical significance (t-tested, two-sided): ns, not significant, * 0.01<p≤0.05, ** 0.001<p≤0.01 and *** p≤0.001.

Figure 4.. The Hif-1α/Sima is expressed in the midgut epithelium and the visceral trachea, and is necessary and sufficient for ISC proliferation and TTC remodelling.

a-b. Uninfected (a) and P.a.-infected (b) ldh-Gal4>UAS-srcGFP midguts (R5 region) with Prospero-labeled (red) EEs. c-d. Control (c) and H₂O₂-fed (d) midguts (R2 region) with Su(H)-lacZ-labeled (red) EBs. Purple arrowheads indicate ldh-positive Su(H)-positive EBs and white arrowheads indicate ldh-negative Prospero-positive EEs. e-f. Quantification of TTC branching (e, n=5,7,6,6) and mitosis (f, n=14,13,18,14) with or without P.a. infection in trachea-specific sima knockdown in the background of heterozygous sima^KG. g-h. Quantification of TTC branching (g, n=15,16) and mitosis (h, n=10,8) upon trachea-specific sima overexpression in baseline conditions. i-j. Quantification of TTC branching (i, n=18,18,8,7) and mitosis (j, n=24,22,11,11) with or without P.a. infection upon EC-specific sima knockdown in the background of heterozygous sima^KG. k-l. Quantification of TTC branching (k, n=8,10) and mitosis (l, n=9,7) upon trachea-specific sima overexpression in baseline conditions. m. RT-qPCR for ldh mRNA expression in w¹¹¹⁸ whole adult midguts upon different stresses. n=3 biological experiments. n. RT-qPCR analysis of btl, bnl and ldh upon EC-specific sima overexpression. n=3 biological experiments. o. RT-qPCR analysis of Dl, wg, upd, upd3, spi, vn, krn, and egr upon EC-specific sima overexpression. n=3 biological experiments. The average and standard deviation are plotted in m-o. Scale bars: 37.5 μm in a-d. Data are presented as mean values ± SD. Statistical significance (t-tested, two-sided in f-k, m-o and U-tested in e,l): ns, not significant, * 0.01<p≤0.05, ** 0.001<p≤0.01 and *** p≤0.001.

Figure 5.. Hypoxia induces TTC branching, but not ISC proliferation.

a-b. Brightfield images of the midgut TTCs (R5 region) of wild-type (w¹¹¹⁸) uninfected flies reared in normoxia (21% O₂) vs. hypoxia (5% O₂). c-d. Quantification of TTC branching (c, n=10 each) and mitosis (d, n=11,13) in normoxia vs. hypoxia. e-f. esg-Gal4>UAS-srcGFP (ISCs+EBs, green) midguts (R5 region) of flies reared in normoxia (e) and hypoxia (f) stained for cleaved Caspase-3 (red) and Topro3 (blue) staining all nuclei. g-h. Quantification of TTCs (g, n=18, 14) and esg-lacZ+ progenitors (h, n=12,13) in normoxia and hypoxia. i-j. Fluorescence images showing normoxia- (i) and hypoxia-reared (j) bnl-Gal4>UAS-srcGFP (Bnl reporter, green) midguts with Dl-lacZ (red) labeling the ISCs and DAPI (blue) staining all nuclei. Yellow arrowheads indicate bnl-positive and Dl-positive progenitors and white arrowheads indicate bnl-negative and Dl-positive progenitors. k-l. Fluorescence images showing normoxia- (k) and hypoxia-reared (l) ldh-Gal4>UAS-srcGFP (Sima reporter, green) midguts with esg-lacZ (red) labeling the ISCs and EBs and DAPI (blue) staining all nuclei. Yellow arrowheads indicate bnl-positive and esg-positive progenitors and white arrowheads indicate bnl-negative and esg-positive progenitors. m-n. Brightfield images of the midgut TTCs (R5 region) of wild-type (w¹¹¹⁸) P.a.-infected flies reared in normoxia (21% O₂) vs. hypoxia (5% O₂). o-p. Quantification of TTC branching (o, n=6,11,6,8) and mitosis (p, n=19,14,11,11) of uninfected and P.a.-infected flies in normoxia and hypoxia. q. RT-qPCR analysis of btl, bnl and ldh in normoxia vs. hypoxia. The average and standard deviation of n=3 biological experiments are shown. r-s. Quantification of TTC branching (r, n=12,10,10,17) and mitosis (s, n=13,10,14,17) of untreated vs. H₂O₂-fed flies in normoxia and hypoxia. All scale bars: 75 μm. Data are presented as mean values ± SD. Statistical significance (t-tested, two-sided, and U-tested for o): ns, not significant, * 0.01<p≤0.05, ** 0.001<p≤0.01 and *** p≤0.001.

Figure 6.. Stress-responsive increased TTC remodelling is ROS dependent.

a-e. Brightfield images of the midgut TTCs (R5 region) of wild-type (w¹¹¹⁸) flies fed on LB (a, negative control) and P.a. culture supernatants of the virulent wild-type PA14 strain (b, positive control), the quorum-sensing mutant MvfR (c), and the phenazine mutants PhzS (d) and PhzM (e). f. Schematic of the P.a. quorum-sensing and phenazine production pathway. g-h. Quantification of TTC branching (g, n=10 each) and midgut mitosis (h, n=12,11,11,11,10) upon P.a. culture supernatant feeding. i-j. Quantification of TTC branching (i, n=10,10,8,10) and midgut mitosis (j, n=22,23,10,21) upon EC-specific Duox knockdown with or without P.a. infection. k-l. Quantification of TTC branching (k, n=10,10,8,10) and midgut mitosis (l, n=42,32,36,36) upon trachea-specific (via btl^ts-Gal4) Duox knockdown with or without P.a. infection. m-o. Quantification of midgut mitosis (m, n=37,30), TTC number (n, n=10 each) and TTC branching (o, n=7,10) upon trachea-specific (via dSRF^ts-Gal4) Cat knockdown in uninfected flies. p. Quantification of midgut mitosis upon trachea-specific (via dSRF^ts-Gal4) Cat and Sod1 overexpression in P.e.-infected flies (n= 12,13,15). q-r. Quantification of TTC branching (q, n=7,9,10,10) and midgut mitosis (r, n=15,11,8,11) upon trachea-specific (via btl^ts-Gal4) Cat and Sod1 overexpression with or without P.a. infection. All scale bars: 75 μm. Data are presented as mean values ± SD. Statistical significance (t-tested, two-sided): ns, not significant, * 0.01<p≤0.05, ** 0.001<p≤0.01 and *** p≤0.001.

Figure 7.. Midgut ISC-derived tumors induce TTC branching required for tumor growth.

a. Midgut Ras^V12 MARCM clone (green) and corresponding brightfield image (a’) of the trachea. b. Quantification of TTC branching in tumorous Ras^V12 clone-bearing midguts vs control clone-bearing midguts (n=26,14). c. Schematic of the transplant experimental process. d. Fluorescently-labeled midgut tumor pieces from a donor fly are transplanted to a recipient fly with fluorescently-labeled trachea. Images showing GFP+ green tumors (first two images) recruiting CD8-mCherry marked tracheae from the recipient. The third image shows RFP+ (red) tumor mass overlayed with CD8-GFP+ (green) recipient trachea. e. Table showing comparison of the growth ability and survival of transplanted tumors in wild-type or trachea-defective (ppk-Gal4>UAS-btl^RNAi) recipients. f-g. The R5 region of control esg-Gal4 UAS-eGFP tub-Gal80^ts (f) and esg-Gal4 UAS-eGFP tub-Gal80^ts>UAS-Ras^V12-tumor (g) bearing midguts with concomitant expression of QF6>QUAS-mtdTomato (red) to label the trachea reared for 1 day at 29°C. DAPI (blue) was used to label all midgut nuclei. f’- g’, and f”-g” show the individual channels for the eGFP and the Tomato-labeled trachea, respectively. h-i. Quantification of TTC branching (h, n=10,11,9,9,10,10) and midgut mitosis (i, n=20 each) in control and Ras^V12-tumor bearing flies during a time-course analysis at 1, 3, and 5 days post-tumor induction. j. qRT-PCR analysis of btl, bnl, ldh, puc and upd3 upon Ras^V12 tumor progression. The average and standard deviation of n=3 biological experiments are plotted. k-l. Control genotype and Ras^V12 tumor reversal (3 days at 29°C followed by 7 days at 18°C). k’-k” and l’-ll” show the individual channels for eGFP and the Tomato-labeled trachea, respectively. m-n. Ras^Q13 tumor mitosis in the presence or knockdown of sima (m, n=10 each), and bnl (n, n=10,8,7). o-p. Quantification of TTC branching (o, n=10,10,10,10,10,11) and midgut mitosis (p, n=20 each) in control and Ras^V12 following reversal of the tumors after 1, 3, and 5 days post tumor initiation. Data are presented as mean values ± SD. All scale bars: 75 μm in f-g, k-l. Statistical significance (t-tested, two-sided): ns, not significant, * 0.01<p≤0.05, ** 0.001<p≤0.01 and *** p≤0.001.

Figure 8.. Tumor-induced increased tracheal coverage, oxygen supply, and ROS are necessary for tumor growth.

a-d. Brightfield images of the TTCs (a) and fluorescent images of the GFP-positive (b) progenitor or progenitor derived tumor cells in control midguts, midguts bearing esg-Gal4 UAS-eGFP tub-Gal80^ts>UAS-Ras^V12 UAS-Notch^RNAi tumors, and esg-Gal4 UAS-eGFP tub-Gal80^ts>UAS-Ras^V12 UAS-Notch^RNAi co-expressing UAS-bnl^RNAi. Quantification of TTC branching (c, n=7,10,11) and mitosis (d, n=7,13,10) in control, Ras^V12 Notch^RNAi and Ras^V12 Notch^RNAi bnl^RNAi tumor-bearing flies. e. Percent survival of control, Ras^V12 Notch^RNAi and Ras^V12 Notch^RNAi bnl^RNAi tumor-bearing flies (n=160,78,120). f-h. Images showing growth of Ras^V12 Notch^RNAi tumors (f), quantification of TTC branching (g, n=8,9,9,9) and midgut mitosis (h, n=13,8,7,12) in control and Ras^V12 Notch^RNAi tumor-bearing flies in normoxia vs. hypoxia. i-k. Images showing tumorous midguts (R5 region) with progenitor derived Ras^Q13 tumors (green) with Cat and Sod1 overexpression or btl knockdown. Quantification of TTC branching (j, n=11,10,10) and midgut mitosis (k, n=15,13,13) in the same genotypes. Scale bars: 200 μm in a, 100 μm in b and 75 μm in f and i. l. Model of the intestinal trachea-midgut communication during damage-induced regeneration. Data are presented as mean values ± SD. The Kaplan-Meier method was used to test significance in e. For all others, a two-sided t-test was used: ns, not significant, * 0.01<p≤0.05, ** 0.001<p≤0.01 and *** p≤0.001.