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.
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The authors would like to thank the BDSC, the VDRC, the Kyoto Stock Center and the TRiP for fly stocks; A. Bardin, S. Hou, J. Casanova, T. Kornberg, C. Potter, P. Wappner and A. Ignatiou for fly stocks; E. Snyder for use of the Nikon Ti microscope; and the DSHB for antibodies. This project was supported by FP7-PEOPLE-2011-CIG-303727, the Fondation Santé and the Cyprus Research and Innovation Foundation EXCELLENCE/0918/0082 to C.P., and by ERC AdG 268515, DFG SFB873, the Huntsman Cancer Foundation and NIH GM124434 to B.A.E.
The authors declare no competing interests.
Peer review information Nature Cell Biology thanks Rongwen Xi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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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 labelled with esgNP5130-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-btlDN (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-btlDN (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 remodelling and mitosis in response to infection.
a-b, Quantification of TTC branching upon progenitor- (a) and EC-specific (b) silencing of bnl (bnlRNAi3) and btl (btlRNAi) (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 (bnlRNAi3) (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 (btlRNAi) and bnl (bnlRNAi3) (n = 12,15,15). f-g, Quantification of midgut mitosis upon progenitor- (f) and EC-specific (g) silencing of btl (btlRNAi) (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 (btlDN, 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 simaKG 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 simaKG 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 NotchDN 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-Gal80ts > UAS-NotchDN 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 (NotchDN uninduced) and NotchDN-expressing midguts (e, n = 4,8,4,6,4,7), in the NotchDN tumor-region vs. neighboring non-tumor area on the same image (f, n = 6,6,4,4,6,6), and midgut mitosis of control (NotchDN uninduced) and NotchDN-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 RasV12 progenitor-derived midgut tumors.
a-d, The R5 region of control esg-Gal4 UAS-eGFP tub-Gal80ts (reared for 1 day at 29 °C) and esg-Gal4 UAS-eGFP tub-Gal80ts > UAS-RasV12-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.
Supplementary Table 1: Drosophila and Pseudomonas strains used in this study. Supplementary Table 2: reagents and antibodies used in this study. Supplementary Table 3: primer sequences used in this study.
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Tamamouna, V., Rahman, M.M., Petersson, M. et al. Remodelling of oxygen-transporting tracheoles drives intestinal regeneration and tumorigenesis in Drosophila. Nat Cell Biol 23, 497–510 (2021). https://doi.org/10.1038/s41556-021-00674-1
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