Abstract
Coordination of stem cell function by local and niche-derived signals is essential to preserve adult tissue homeostasis and organismal health. The vasculature is a prominent component of multiple stem cell niches. However, its role in adult intestinal homeostasis remains largely understudied. Here, we uncover a previously unrecognised crosstalk between adult intestinal stem cells (ISCs) in Drosophila and the vasculature-like tracheal system, which is essential for intestinal regeneration. Following damage to the intestinal epithelium, gut-derived reactive oxygen species (ROS) activate tracheal HIF-1α and bidirectional FGF/FGFR signaling, leading to reversible remodelling of gut-associated terminal tracheal cells and ISC proliferation following damage. Unexpectedly, ROS-induced adult tracheal plasticity involves downregulation of the tracheal specification factor trachealess (trh) and upregulation of IGF2 mRNA-binding protein (IGF2BP2/Imp). Our results reveal a novel intestine/vasculature interorgan communication program, which is essential to adapt stem cells response to the proliferative demands of the intestinal epithelium.
Main
Adult intestinal plasticity is largely owed to the action of stem cells, which must respond to constant signals from the intestinal epithelium and its microenvironment, to fulfil global tissue demands1–3. Surprisingly, little is known about the role of the vascular microenvironment in adult intestinal homeostasis.
The Drosophila tracheal system is an oxygen-delivering interconnected tubular network, functionally analogous to the mammalian vascular and respiratory systems4. Following specification from epidermal cells and the formation of a tracheal sac in the embryo, tracheal cells undergo extensive cell rearrangements and cell shape changes, leading to the formation of multicellular tubes that ramify into progressively thinner branches, culminating with a terminal tracheal cell5. Drosophila terminal tracheal cells (TTCs), analogous to mammalian vascular tip cells6, extend prominent cytoplasmic projections, which supply oxygen to their target tissues4, 5, 7, 8. While tracheal development and post-embryonic plasticity have been significantly studied in Drosophila5, 9, 10, there is scarce knowledge on the role and regulation of the adult tracheal system.
The adult Drosophila midgut shares remarkable homology with the mammalian intestine11. Critically, the midgut epithelium is maintained by intestinal stem cells (ISCs), which self-renew and replenish the differentiated intestinal lineage —secretory enteroendocrine cells and absorptive enterocytes— through the production of undifferentiated enteroblasts12, 13. As its mammalian counterpart, the Drosophila gastrointestinal tract is densely tracheated14. Beyond the requirement for tracheal derived Dpp/BMP to restrain ISC proliferation15, there is no knowledge on the role of the tracheal system in adult midgut biology.
Here, we combine genetics and image analysis with in vivo functional and molecular studies to characterise a novel inter-organ communication program between the adult Drosophila midgut and its closely associated tracheal tissue, which is essential to shape stem cell and tracheal plasticity during intestinal regeneration.
Results
Intestinal damage leads to dynamic and reversible remodelling of gut associated adult TTCs in Drosophila
The adult Drosophila melanogaster gut is extensively covered by TTCs, labelled with a GAL4 reporter driven under the control of the Drosophila homologue of Serum Response Factor (dSRF)16–18 (dSRF>GFP) (Fig. 1a and Extended data Fig. 1a). Transmission electron microscopy of adult posterior midguts denoted intimate contact between TTCs, enterocytes (ECs) (Fig. 1b) and ISCs (Fig. 1c). Oxygen and nutrient availability are two recognised determinants of TTC plasticity10, 19. We noticed that, damage to the adult Drosophila midgut epithelium caused by feeding animals with the pathogenic bacteria Pseudomonas entomophila (Pe)20–23, the DNA-damaging agent Bleomycin24–26, or the epithelial basement membrane disruptor Dextran Sulfate Sodium (DSS)25, 27, led to a significant increase in TTC coverage within the posterior midgut (Fig. 1d, e and Extended data Fig. 1b). Consideration of gut resizing upon Pe damage did not impact the overall increase in tracheal coverage (Extended data Fig. 1c-e). GFP labelled single terminal tracheal cell clones, unambiguously confirmed the increase in total number of cellular branches derived from individual TTCs in damaged (Pe) versus control (Sucrose) midguts (Fig. 1f, g). Interestingly, observation of single TTC clones within Pe treated midguts revealed direct correlation between the number of individual TTC branches and nearby PH3+ ISCs (Fig. 1h). Further quantification of tracheal phenotypes showed increase in primary, secondary and tertiary tracheal branches and total length of individual TTC extensions in damaged (Pe) versus control (Sucrose) midguts (Fig. 1i-k). Quantification of TTC numbers or assessment of potential co-localization between TTC bodies and the cell proliferation marker PH3 revealed no evidence of TTC proliferation following midgut damage (Extended data Fig. 1f, g). Collectively, these data suggest extensive cellular remodelling of TTCs in response to epithelial intestinal injury. A time course assessment of posterior midguts over a 16 hrs period of Pe infection (Damage phase) followed by 32 hrs on normal diet (Recovery phase) revealed direct correlation between tracheal coverage and ISC proliferation (Fig. 1l, m and Extended data Fig. 1h, i). These results strongly suggest that adult gut-associated-tracheal remodelling is a highly dynamic and reversible process, which accompanies changes in intestinal homeostasis.
Low doses of whole body γ-irradiation in mice induce intestinal epithelial cell death, followed by a strong peak of crypt cell proliferation between 72- and 96 hrs after irradiation28. Staining with anti-CD31, showed an increase in vascular endothelial cells in regenerating (irradiated) versus control (non-irradiated) intestinal crypts (Fig. 1n). These results indicate a conserved phenomenology of vasculature/tracheal response to damage in the adult intestinal epithelium.
TTC remodelling is necessary for ISC proliferation following damage of the adult Drosophila midgut
An essential step in the intestinal regenerative response to damage, involves a robust increase in ISC proliferation1, 20,22, 25, 29 (Fig. 1d, h). To address the functional role of the tracheal system in adult intestinal regeneration, we severely reduced trachea by overexpressing the pro-apoptotic gene bax (UAS-bax) in adult TTCs using temperature sensitive dSRF-Gal4 (dSRFts>bax) (Fig. 2a, b). This caused a significant impairment of the regenerative response of the intestine to multiple damages, as evidenced by an approximate 50% decrease in damage induced ISC proliferation (Fig. 2c). TTCs are best known for their role facilitating gas exchange with their target tissues4. Thus, poor intestinal regeneration following TTC reduction might reflect the need of oxygen in this process. Exposing animals to high and prolonged hypoxic environmental conditions, induced activity of a reporter of Drosophila Hypoxia-inducible factor-1α (HIF-1α/Sima)30 and TTC remodelling in the adult midgut (Extended data Fig. 2a-c). However, while hypoxia clearly impaired damage induced ISC proliferation in the adult midgut (Fig. 2d), it did so to a lower extent than that observed upon TTC loss (dSRFts>bax) (Fig. 2c). This difference could be due to a compensatory effect of increased trachea upon hypoxia (Extended data Fig. 2a, c) or compromised ISC survival in dSRFts>bax midguts. We assessed apoptosis and ISC numbers in hypoxic and dSRFts>bax midguts through anti-caspase (Dcp-1) staining and the use of an ISC reporter (Delta-LacZ). Midguts overexpressing bax in adult ECs (NP1ts>bax), served as a ‘cell-death’ positive control (Extended data Fig. 2d, f). While we saw no evidence of cell death in hypoxic midguts, dSRFts>bax midguts showed significant apoptosis (Extended data Fig. 2e, g), which was restricted to ECs, distinguished by their large nuclei (Extended data Fig. 2e, lower panel, magnified view). Consistently, this cell death phenotype did not translate into defective ISC numbers (Extended data Fig. 2h, i). Therefore, impaired midgut regeneration following the hypoxia or TTC ablation regime used in our study is unlikely to be secondary to ISC loss. Alternatively, differences in the regenerative response of hypoxic versus dSRFts>bax midguts could be explained by the contribution of angiocrine factors to ISC proliferation, in addition to oxygen availability. Dpp/BMP ligand has been identified as an angiocrine factor in the adult midgut15. However, its action inhibits rather than induces ISC proliferation15. Therefore, a potential role of Dpp cannot directly explain our results (see also Discussion).
Given that tracheal remodelling and ISC proliferation show almost the exact same dynamics (Fig. 1l, m and Extended data Fig. 1 h, i), it is unclear whether these events are part of a feedforward mechanism or if one precedes the other. To address this, we assessed tracheal remodelling following damage while blocking ISC proliferation by overexpressing UAS-myc RNAi (myc-IR)31 using the stem/progenitor driver escargot-GAL4 (esgts>myc-IR) (Fig. 2e-g). As we needed to use the GAL4/UAS system to genetically manipulate gut cells, we established a scoring method for assessing tracheal coverage through the use of light microscopy, which was validated against our confocal microscopy tracheal quantification approach (Extended Data Fig. 3a-c). Gut-associated trachea remodelled normally in esgts>myc-IR midguts following Pe damage, in spite of the almost complete absence of ISC proliferation (Fig. 2e-g). Therefore, TTC remodelling precedes midgut ISCs proliferation following damage. We hypothesised that signals activated by damage upstream of ISC proliferation might induce gut-tracheal remodelling.
Gut-derived ROS initiates tracheal remodelling through activation of HIF-1α/FGFR signaling in TTCs
Pathogen-induced intestinal damage triggers a strong oxidative burst and the production of reactive oxygen species (ROS) from the intestinal epithelium32, 33. We therefore tested whether ROS could trigger tracheal remodelling in the regenerating intestine. Feeding animals with the antioxidant N-acetyl cysteine (NAC) or genetically blocking ROS production in ECs by overexpressing the enzyme catalase32 (NP1ts>catalase) significantly impaired damage-induced tracheal remodelling (Fig. 2h, i and Extended data Fig. 3d, e) in addition to the expected reduction in regenerative ISC proliferation (Fig. 2j and Extended data Fig. 3f)32. Conversely, driving adult intestinal epithelial cell death through bax overexpression in ECs (NP1ts>bax) was sufficient to induce TTC remodelling and ISC proliferation (Extended data Fig. 3g-i). Therefore, intestinal epithelial damage and ROS induce remodelling of gut associated trachea, which is in turn necessary to drive ISC proliferation during intestinal regeneration.
Exogenous H2O2 can stabilize HIF-1α —a key conserved driver of hypoxia-induced tracheal/vascular remodelling9, 34, 35— in normoxia36. The Sima/HIF-1α activity reporter ldh-lacZ was upregulated in gut-associated TTCs following midgut damage and in an ROS dependent manner (Fig. 2k-m). Furthermore, midguts from sima-/- whole mutant animals or upon adult specific sima knockdown within TTCs (dSRFts>sima-IR) showed significantly impaired tracheal remodelling and ISC proliferation following damage (Fig. 3a-f).
The Drosophila fibroblast growth factor receptor (FGFR), Breathless (Btl), is a well-known transcriptional target of HIF-1α during tracheal development and oxygen-driven tracheal remodelling19, 37. Consistently, a reporter of breathless (btl) expression (btl-lacZ) showed gene upregulation in TTCs following intestinal damage, which was abrogated by NAC (Fig. 3g-i). TTC knockdown of btl (dSRFts>btl-IR) inhibited tracheal remodelling and ISC proliferation following damage (Fig. 3j-l), without evidence of cell death or ISC loss (Extended data Fig. 2e, g-i). Therefore, ROS-dependent activation of HIF-1α/FGFR signaling within TTCs following gut damage induces tracheal remodelling and regenerative ISC proliferation in the adult midgut. Consistently, expression of the HIF-1α/FGFR target gene blistered (bs)/dSRF19, 38 was upregulated in damaged and hypoxic midguts (Extended data Fig. 4a-e) and knocking down bs in adult TTCs (dSRFts>bs-IR) impaired tracheal remodelling and ISC proliferation following damage (Extended data Fig. 4f-h).
ROS-dependent bidirectional FGF/FGFR signaling drives stem cell proliferation and TTC remodelling during intestinal regeneration
During development or hypoxia, the Drosophila FGF-like ligand Branchless (Bnl) is upregulated in target tissues and signals paracrinally to its receptor FGFR/Breathless (Btl) in the trachea to induce their remodelling16, 19, 39. Consistently, we observed upregulation of a bnl reporter (bnl-lacZ) in ISCs/EBs and ECs following intestinal damage, which was impaired by NAC (Fig. 4a-c). These results suggest that ROS induces Bnl activation within the intestinal epithelium following damage. Unexpectedly, using the same reporter, we observed that bnl was also upregulated in TTCs following intestinal damage, in an ROS dependent manner (Fig. 4d-e). Expression of bnl in TTCs was confirmed by the use of an independent reporter (Extended data Fig. 5a, b). Overexpressing bnl in adult TTCs (dSRFts>bnl) was sufficient to induce ISC proliferation without TTC remodelling (Extended data Fig. 5c, d).
We next assess the functional role of individual sources of FGF/Bnl in our system. Consistent with our reporter expression data (Fig. 4a-e), knocking down bnl from either TTCs (dSRFts>bnl-IR), ISCs/EBs (esgts>bnl-IR) or ECs (NP1ts>bnl-IR) restrained ISC proliferation following midgut damage but did not impair TTC remodelling (Fig. 4f-h and Extended data Fig. 5e-j). This is in line with the high sensitivity of the regenerative intestine to discrete fluctuations in individual signaling activity31, 40. Hence, small variations in Bnl levels, which are insufficient to affect tracheal remodelling are enough to impact ISC proliferation following damage. Instead, concomitant bnl knockdown from ECs and ISCs/EBs (NP1>, esgts>bnl-IR) significantly impaired TTC remodelling and subsequent ISC proliferation (Fig. 4i-k). Therefore, the combined action of gut-derived sources of Bnl, is necessary to induce tracheal remodelling following intestinal damage.
Given that multiple sources of Bnl—from the midgut and TTCs—can individually contribute to regenerative ISC proliferation independently of tracheal remodelling, we hypothesised this may be through a non-tracheal receptor. Consistently, knocking down btl from ISCs/EBs (esgts>btl-IR) significantly impaired ISC proliferation upon damage without affecting TTC remodelling (Extended data Fig. 6a-c). In the context of tracheal development, Bnl/Btl signals through the MAPK/ERK pathway5, 41, which is a key driver of ISC proliferation in the adult Drosophila midgut42–44. Knocking down btl from ISCs/EBs (esgts>btl-IR) impaired damage-induced MAPK/ERK activation in the midgut (Extended data Fig. 6d, e), suggesting that activation of Btl in the midgut regulates regenerative ISC proliferation through MAPK/ERK signaling.
Identification of novel TTC intrinsic mechanisms triggered during intestinal regeneration
We next used Targeted DamID (TaDa) for TTC in vivo profiling of RNA Pol II chromatin binding45 in control (Sucrose) and Pe treated midguts (Fig. 5a and Extended data Fig. 7a). TaDa is particularly advantageous in our system due to inherent difficulties to efficiently separate tracheal tissue from the midgut. We identified 1747 and 1712 genes significantly bound by RNA Pol II in TTCs from control (Sucrose) and Pe infected midguts, respectively (Supplementary Table 1). Gene ontology (GO) analysis of areas with significant RNA Pol II binding in control midguts revealed enrichment in components of the tracheal system, and genes previously involved in epithelial tube morphogenesis and respiratory/tracheal system development (Fig. 5b) (Supplementary Table 2 and 3). This validated the sensitivity of TaDa to reliably detect tracheal specific genes from combined gut and tracheal tissue samples.
Consistent with our reporter expression and functional data (Fig. 4d-h), TaDa analysis identified bnl/FGF as a gene with significant RNA Pol II binding in Pe treated midguts only (Supplementary Table 1) (Extended Data Fig. 7b). Unexpectedly, we were unable to detect significant RNA Pol II binding to btl in adult TTCs of Pe treated midguts (Supplementary Table 1). This is counterintuitive given our gene expression and functional data on btl (Fig. 3g-l).
Discrepancies between RNA pol II occupancy and mRNA transcript status are possible and could be due to pausing of the polymerase46, post-transcriptional mRNA regulation47 or temporally dynamic RNA pol II binding (e.g during intestinal damage), which may not be captured by a single time point assessment.
Imp/IGF2BP is a novel regulator of adult tracheal remodelling and intestinal regeneration
Interestingly, we noticed that, within the genes showing significant RNA pol II binding in TTCs of Pe treated midguts only (Supplementary Table 1), there were several genes associated with neuronal function (Fig. 5c). Amongst them, was the highly conserved mRNA-binding protein Imp/IGF2BP (Fig. 5d) (Supplementary Table 1), which regulates axonal remodelling in Drosophila48, 49. We confirmed upregulation of Imp transcription by qRT-PCR (Fig. 5e) and Imp increase in TTCs through the use of a protein trap (Imp::GFP) (Fig. 5f, h). NAC treatment showed that Imp upregulation in TTCs following intestinal damage depends on ROS production (Fig. 5g, h). Importantly, adult specific knock down of Imp from TTCs (dSRFts>Imp-IR) significantly impaired tracheal remodelling and ISC proliferation following midgut epithelial damage (Fig. 5i-k). Next, we investigated two well-known post-transcriptional targets of Imp in our system: chickadee/profilin48 and myc49. Functional experiments on the role of chickadee led to inconclusive results (data not shown). However, Myc was upregulated in TTCs of Pe treated midguts and this was abrogated by Imp knockdown (dSRFts>imp-IR) (Fig. 6a, b). Moreover, adult specific knockdown of myc within TTCs using RNAi31 (dSRFts>imp-IR), impaired tracheal remodelling and ISC proliferation following gut damage (Fig. 6c-e). Altogether, these results establish Imp as a novel regulator of TTC remodelling and ISC proliferation during adult Drosophila midgut regeneration. This function of Imp is at least in part through tracheal intrinsic control of Myc.
Trachealess downregulation in TTCs is necessary for adult tracheal remodelling and damage induced ISC proliferation
While our TaDa analysis revealed significant binding of RNA Pol II to trachealess (trh) in TTCs of Sucrose treated midguts (Supplementary Table 1 and 3), this was not the case in the damaged tissues (Fig. 7a and Supplementary Table 1). This was surprising given that trh is known a master regulator of tracheal gene expression and it is present in all tracheal cells from the onset of embryonic development through adulthood50–52. Loss of trh during development impairs tracheal cell specification and tube morphogenesis51, 52. However, qRT-PCR confirmed downregulation of trh upon intestinal damage (Fig. 7b). Antibody staining (Fig. 7c, d) and a transgenic reporter (trh-lacZ) (Fig. 7e, f) further demonstrated protein and gene downregulation in TTCs upon gut damage, respectively. Remarkably, trh-lacZ signal was significantly restored upon Pe and NAC co-treatment or 32 hrs after removal of the damaging agent (Fig. 7e, f). This suggests that trh expression in adult TTCs is highly dynamic and its downregulation upon intestinal damage is dependent on ROS. Importantly, consistent with our gene and protein expression data, trh overexpression in adult TTCs (dSRFts>trh) significantly impaired tracheal remodelling and ISC proliferation (Fig. 7g-i), while trh knockdown (dSRFts>trh-IR) potentiates TTC remodelling and ISC proliferation following midgut damage (Fig. 7g-i). Altogether, these results suggest that ROS-induced trh downregulation in adult TTCs is necessary to allow gut associated TTC plasticity and robust regeneration of the intestine following damage.
Here, we reveal a novel inter-organ communication program in Drosophila, involving the adult tracheal system and the midgut, which drives reciprocal adaptation of both tissues to sustain a robust regenerative response of the intestine to injury (Fig. 7j). Our results may reflect vasculature/stem cell interactions in the mammalian intestine and other self-renewing tissues.
Discussion
The vasculature represents a prominent component of the gut microenvironment. However, its functional role in adult intestinal homeostasis remains largely unknown. Here, we report the cellular and molecular underpinnings of a novel inter-organ communication program between the adult Drosophila midgut and its closely associated vasculature-like tracheal tissue, which is fundamental to drive the regenerative response of ISCs following epithelial tissue damage.
ROS are key initiators of TTC remodelling and ISC proliferation, following intestinal damage by pathogenic infection (Fig. 7j). While our observations show a conserved phenomenology of TTCs/vasculature changes upon diverse intestinal insults and across species, it is highly conceivable that damage and species-specific molecular responses exist and that signals other than or in addition to ROS influence tracheal/vascular adaptations to intestinal damage.
HIF-1α/FGFR signaling drives tracheogenesis during development and in response to hypoxia following activation by FGF from target tissues9, 34, 35. As such, our findings suggest a repurposing of this developmental pathway during adult gut/tracheal crosstalk. However, the discovery of ROS-inducible angiocrine Bnl/FGF activating stem/progenitor cell FGFR signaling during adult intestinal regeneration is a novel finding from our study (Fig. 7j). FGF induces vascular endothelial cell differentiation in human intestinal organoids53 and acts as an angiocrine factor in various tumour settings54. This highlights the great degree of conservation between the Drosophila and mammalian system and raises the possibility of a conserved angiocrine role of FGF ligands in mammalian intestinal regeneration.
The DPP/BMP ligand has been reported as an angiocrine factor required to restrain stem cell proliferation in the adult Drosophila midgut15. However, knocking down dpp in adult TTCs did not affect basal stem cell proliferation as reported upon global ligand downregulation in the adult trachea15 (data not shown). Ligand compensation from main tracheal branches or the intestinal epithelium55 may explain discrepancies between our results and the published study.
In addition to the well-established role of oxygen, nutrition regulates TTC remodelling in the larval and adult Drosophila midgut10. In this context, a defined subset of enteric neurons influence TTC remodelling through the delivery of Insulin- and Vasoactive Intestinal Peptide-like neuropeptides10. Nutrient induced-tracheal remodelling involves activation of Insulin Receptor (InR) signaling within TTCs. We observed no requirement for InR or enteric neuronal activity in the plastic response of adult TTCs to intestinal damage (data not shown). Reciprocally, FGF/FGFR signaling does not appear to mediate nutrient dependent TTC remodelling10. These results suggest that molecular events driving tracheal tissue plasticity are diverse and highly dependent on the biological context and/or stimuli.
We identified two novel tracheal-intrinsic molecular mechanisms triggered in response to intestinal epithelial damage and necessary to induce TTC remodelling and ISC proliferation (Fig. 7j). One, involving upregulation of DrosophilaIGF2 mRNA-binding protein (Imp) and its downstream target Myc (Fig. 7j). The other, requiring downregulation of the tracheal cell specification factor trh (Fig. 7j). Known functions of Imp had been restricted to the induction of neuronal remodelling and growth48, 49. Its mammalian orthologue, IGF2BP2, has been studied for its involvement in metabolic disease56 and its potential role in the vasculature remains to be addressed. Trh, homologous to mammalian NPAS357, has been exclusively known for its requirement in the specification of tracheal cells from undifferentiated progenitors in the developing embryo50–52. Here, we present the first report for a role of Trh in terminally differentiated adult tracheal cells, which involves its unexpected downregulation. Emerging evidence suggests that adult tissues and cells, such as the intestine and neurons, lose differentiation markers and acquire ‘naïve’ or ‘foetal-like properties’ during the process of tissue regeneration58–60. Our work suggests the exciting possibility that this may also be the case for the adult vasculature.
The vasculature is a largely uncharacterized component of the adult intestinal niche. Vascularization of in vitro organ culture systems has been notoriously difficult, representing a major roadblock in the field of tissue engineering. As such, our in vivo findings may be of broad interest and impact to the vascular and intestinal research fields.
Methods
Fly stocks and rearing
A complete list of fly lines and full genotypes used in this study can be found in Supplementary Tables 5 and 6. In experiments using the GAL4/GAL80ts system, flies were crossed, F1 progenies reared, and adults aged for 5 days after eclosion at 18°C. Animals were then transferred to 29°C for 5-7 days to allow activation of most transgenes prior to phenotypic analysis. The exception was bax, which was overexpressed for only 3 days. If not carrying temperature sensitive transgenes, crosses and offspring were kept at 25°C. Overall, experimental animals were used 10-12 days following adult eclosion. Animals for experiments were maintained in food vials at low densities (10-15 flies per vial) and were transferred to fresh food every 2 days. Only adult posterior midguts from mated females were analysed in this study.
Damage induced intestinal regeneration
Oral administration of Pe, Bleomycin or DSS was performed as previously described21, 25, 61 with minor modifications. Briefly, 10 day-old females of the desired genotypes were starved in empty vials for 2hrs followed by feeding with a 5% sucrose solution only (Sucrose), or Sucrose containing either Pe at OD 100, 25μg/ml Bleomycin (Sigma-Aldrich, Cat#B2434), or 3% DSS (Sigma-Aldrich, Cat#42867) applied on filter paper discs (Whatman). Pe infection was carried out for 16hrs, Bleomycin feeding was done for 1-day and DSS feeding lasted for 2-days, with fresh media applied each day.
NAC treatment
Flies were placed in empty vials with a filter paper soaked with a 5% sucrose solution containing 20mM NAC (Sigma-Aldrich, Cat#A7250) for 24 hrs. Animals were then fed with either 5% sucrose + NAC or 5% sucrose + NAC + Pe (OD 100) for an additional period of 24 hrs.
Mouse intestinal regeneration and IHC
C57BL/6 mice were subject to whole body 10 Gy gamma-irradiation and the intestines were analysed 72 hrs post-irradiation, which represents the proliferative phase of the regenerative response to damage in the mouse small intestine28. Small intestines were isolated and flushed with tap water. 10× 1cm portions of small intestine were bound together with surgical tape and fixed in 4% neutral buffered formalin. Intestines from 3 mice per condition were used. 4 μm sections of formalin–fixed paraffin–embedded (FFPE) tissues were cut, mounted onto adhesive slides and incubated at 60°C overnight. Prior to staining, sections were dewaxed for 5 minutes in xylene followed by rehydration through decreasing concentrations of alcohol and final washing with H2O for 5 minutes. FFPE sections underwent heat–induced epitope retrieval in a Dako pre-treatment module. Sections were heated in Target Retrieval Solution High pH (Dako, K8004) for 20 minutes at 97°C before cooling to 65°C. Slides were removed and washed in Tris Buffered Saline with Tween (TbT) (Dako, K8000) and loaded onto a Dako autostainer link48 platform where they were stained with anti-CD31 antibody 1:75 (Abcam, ab28364) following standard IHC procedures. All animal work has been approved by a University of Glasgow internal ethics committee and performed in accordance with institutional guidelines under personal and project licenses granted by the UK Home Office to J.B.C (PPL PCD3046BA).
Single terminal tracheal cell clones
To generate single cell clones of terminal tracheal cells, parental lines were allowed to mate for 2-3 days, after which adults were moved into new vials and F1 progenies where heat shocked in a water bath for 1 hr at 37°C. Adults of the correct genotype, emerging from the heat shocked animals, were selected and aged at 25°C for 10 days followed by feeding with Sucrose (control) or Pe for 16 hrs to cause intestinal damage and induce regeneration. Tissues were then dissected and processed for immunofluorescence staining and confocal imaging.
Hypoxia treatment
Adult flies were aged at 25°C or 29°C in 21% O2 (Normoxia) at a density of 15-20 flies per vial. Then, animals were transferred overnight to 3% O2 (Hypoxia) in a Whitley Scientific H35 hypoxystation incubator.
Drosophila immunohistochemistry
Immunohistochemistry was carried out as described previously31. The following antibodies were used: chicken anti-GFP 1:200 (Abcam, ab13970), mouse anti-PH3 1:100 (Cell Signaling, 9706), rabbit anti-Dcp1 1:100 (Cell Signaling, #9578S), rabbit anti-βgal 1:1000 (MP Biochemicals #559761), rabbit anti-DsRed 1:1000 (Clontech, #632496), mouse anti-Arm 1:3 (Hybridoma Bank, N2 7A1), mouse anti-Dlg 1:100 (Hybridoma Bank, 4F3), rabbit anti-p-Erk 1:100 (Cell Signaling, #9101), guinea pig anti-Myc 1:100 (gift from Gines Morata) and rabbit anti-Trh 1:100 (gift from M. Llimargas). Chitin Binding Protein (CBP 1:100; gift from M. Llimargas) was used to visualise all tracheal tissue. Alexa Fluor 488, 546 and 647 (Invitrogen) were used as secondary antibodies labels at 1:200 and 1:100 respectively. Guts were mounted in Vectashield anti-fade mounting medium for fluorescence with DAPI (Vector Laboratories, Inc) to visualize all nuclei.
Image acquisition
Transmission Electron Microscopy
Guts were dissected under Schneider’s insect medium and were subsequently fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1hr at room temperature. Samples were rinsed repeatedly in 0.1M sodium cacodylate buffer, before fixation treatment in 1% Osmium Tetroxide/buffer for 1hr, followed by washing with dH20 for 30mins. Samples were then stained in 0.5% Uranyl Acetate/dH20 for 1hr (in the dark) and dehydrated by incubation in a graded series of Ethanol. Samples were subject to three subsequent incubations in Propylene Oxide followed by Epon 812 resin/Propylene Oxide (50:50) mix and left on a rotator overnight, followed by several incubations in pure Epon resin. Samples were then embedded into blocks and oven incubated at 60°C for 48hrs. Ultrathin sections (50-70nm thickness) were cut using a Leica Ultracut UCT. The sections produced were collected on Formvar coated 100 mesh copper grids and subsequently contrast stained in 2% Methanolic Uranyl Acetate for 5mins followed by Reynolds Lead Citrate for 5mins. Gut samples were viewed using a JEOL 1200 EX TEM and Images captured using a Cantega 2K x 2K camera and Olympus ITEM Software.
Confocal microscopy (Zeiss LSM 780)
Each image represents half-volume of the full posterior midgut (area comprised between hindgut and Copper Cell Region) and were acquired with 20x, 40x or 63x lenses using identical acquisition conditions for all samples from a given experiment. Images represent maximal intensity projection of a stable number of Z-Stacks and were processed with ImageJ and Carl Zeiss Zen 3.0 to adjust brightness and contrast.
Light microscopy (Axio observer Zeiss)
Adult guts were dissected in PBS, mounted in 100% glycerol and imaged immediately. Up to three pictures per posterior midgut were taken to cover most of the area. Images were taken at their most apical plane to best detect TTCs with a 20x lens. Images from mouse intestinal samples (Fig. 1n) were acquired under a 20x lens using this microscope and were processed with ImageJ.
Quantifications in the adult posterior midgut
Quantification of ISC proliferation
Antibodies against Phosphorylated Histone H3 (PH3) were used to detect ISC proliferation in the adult midgut. The total number of PH3+ve cells per posterior midgut was quantified manually upon visual inspection using an Olympus BX51 microscope. The number of midguts analysed (n) for each experiment are indicated in the figures.
Quantification of tracheal coverage from immunofluorescence images
Unless otherwise noted, confocal images of dSRF>GFP expressing midguts were used and tracheal values provided were obtained from quantification of tile scan images of the entire R4-R5 posterior midgut regions, acquired with a 20x lens. Values of tracheal coverage represent pixel per area and were obtained from maximum intensity Z-projections. Pictures were individually processed on ImageJ as follows: 1) maximum intensity projection from Z stacks were produced; 2) area of interest was cropped to eliminate Malpighian tubules, hindgut and copper cell region; 3) “threshold” was adjusted to ensure the detection of most of terminal tracheal branches; 4) function “skeletonize” was applied to generate a skeleton of the tracheal network; 5) maximum intensity of this skeleton was measured (Extended data Fig. 1a). The number of posterior midguts analysed (n) for each experiment are indicated in the figures.
Quantification of tracheal branching form light-microscopy images
Acquired images were blindly scored using a 1 to 5 scoring system (Extended Data Fig. 3a, c). The custom ImageJ macro used for blind tracheal scoring is available at “Blind_scoring.ijm”. Number of midguts analysed (n) for each experiment are indicated in the figures.
Quantification of total branches per TTC and TTC ramifications
Maximum intensity projections from confocal Z stacks were used. The number of primary, secondary and tertiary branches derived from individual TTCs was assessed (Fig. 1i). Due to the intrinsic complexity of the tracheal, it is difficult to unambiguously assign cellular extensions/branches to a single TTC. To circumvent this issue, we counted tracheal branches starting from a TTC body and defined the end of a TTC extension when it touched the body of another TTC. Additionally, the generation of single TTC clones allowed us to unambiguously quantify the total number of branches from individual TTC, which we did manually (Fig. 1f, h). These two approaches led to same outcome. Number of TTC (n) and midguts analysed for each condition are indicated in the corresponding figures and figure legends.
Quantification of total tracheal length
We used the plugin “NeuronJ” from ImageJ to quantify the total length of all branches emerging from a TTC (Fig. 1k). Number of TTC (n) and midguts analysed for each condition are indicated in the corresponding figures and figure legends.
Quantification of TTC nuclei
Confocal images of posterior midguts from animals expressing dSRFts>RedStinger to label TTC nuclei were used. The custom ImageJ macro used for quantifying the number of TTC nuclei is available at “dSrf_pH3_overlap_for Jessica.ijm”. This macro was also used to quantify the number of TTC nuclei positive for PH3+ staining (Extended data Fig. 1g). Number of midguts analysed (n) for each condition is indicated in the corresponding figure and figure legend.
Quantification of posterior midgut area
Midgut tissue was visualized by DAPI staining and the posterior midgut area (length x width) was measured with ImageJ (Extended Data Fig. 1c, d). Number of midguts analysed (n) for each condition is indicated in the corresponding figure and figure legend.
Quantification of lacZ reporters
Antibodies against β-galactosidase were used to detect lactate dehydrogenase-, Delta-, breathless-, bs/dSRF-, branchless- and trachealess-lacZ reporters. Pictures were taken with confocal microscopy and staining was quantified using ImageJ. For each gut quantified, the background staining signal was subtracted from the total signal of β-galactosidase detected in TTCs, ISCs/EBs or ECs. This value was then divided by the background signal to normalize the data. Number of cells (n) and midguts analysed for each condition are indicated in the corresponding figures and figure legends.
Quantification of cell death
Antibodies against Dcp1 were used to assess cell death in posterior midguts. Pictures were taken with confocal microscopy and Dcp1 staining intensity was measured relative to the surface of the gut area analysed. Number of midguts analysed (n) for each experiment are indicated corresponding in the figure and figure legend.
Quantification of pERK, Imp::GFP, Myc and Trachealess staining
Midguts stained with antibodies to detect these proteins included a methanol fixation step between the PFA fixation and PBST washing steps of the standard protocol, as described previously40. Images were acquired with confocal microscopy and staining was quantified using ImageJ. For each gut quantified, the background staining signal was subtracted from the total antibody signal within DAPI positive cells. This value was then divided by the background signal in order to normalize the data. The number of cells (n) and midguts analysed for each condition are indicated in the corresponding figures and figure legends.
Statistical analysis
Unless otherwise noted, in all figures: NS, not significant (p>0.05); *p < 0.05, **p < 0.01, ***p < 0.001 ****p< 0.0001. Prism 6 software (GraphPad) was used for statistical analyses. A two-tailed Student’s t-test was conducted to compare two sample groups. To test for significance in larger groups, two-way ANOVAs, corrected for multiple comparisons using Sidak’s statistical hypothesis testing, respectively, were used. P values less than 0.05 were considered significant. Information on sample size and statistical tests used for each experiment are indicated in figure legends. Unless otherwise noted, experiments represent a minimum of 2 independent biological replicates. Please see reporting summary for further details.
qRT-PCR
Trizol (Invitrogen) was used to extract total RNA from 30 midguts per biological replicate. cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). QuantiNova SYBR Green (Qiagen) was used for qPCR. Data were extracted and analysed using QuantStudio and Prism 6.0. Data from 5 biological replicates is presented as the mean fold change with standard error. Expression of target genes was measured and normalized to gapdh1 or act5c using standard curves. Primer sequences can be found in Supplementary Table 4.
Targeted DamID (TaDa), library preparation, sequencing and data analysis
dSRF-GAL4; tub-gal80ts (dSRFts>) animals were crossed to UAS-LT3-Dam or UAS-LT3-Dam-Pol II animals at 18°C. F1 progeny were collected every 48 hrs and aged for a further 7 days at 18°C before transferring to 29°C to induce adult restricted Dam protein expression for 24 hrs. During the last 16 hrs at 29°C, flies were fed a Sucrose or Sucrose + Pe solution. 60 midguts per condition per biological replicate were dissected in cold PBS and stored at −80°C. Methylated DNA fragments were isolated and next generation sequencing libraries were prepared as described previously45. Sequencing data from TaDa experiments were processed using a previously described pipeline62 and mapped to release 6.03 of the Drosophila genome. Transcribed genes were annotated for Pol II binding data using a custom Perl script46 (available at https://github.com/tonysouthall/Dam-RNA_POLII_analysis) and release 6.11 of the annotated Drosophila genome. Genes with significant RNA pol II binding were identified based on meeting a threshold of 1% FDR and > 0.2 log2 ratios. Briefly, a log2 ratio of the Dam-RNA Pol II read counts over control Dam-only read counts is calculated after quantile normalisation62 and if this ratio is higher than 0.2, then we would determine that this gene has significant RNA Pol II binding (Supplementary Table 1). Significance was assigned based on the signal from multiple GATC fragments and using a very stringent pipeline as a transcript had to have a false discovery ratio (FDR) of less than 1% in both replicates to be called significant.
Gene Ontology (GO) term analysis was performed using the R package ClusterProfiler63 to search for enriched GO terms (Supplementary Table 2). Three independent biological replicates were originally processed for each condition (Sucrose and Pe). However, after visual inspection of the sequencing tracks, one replicate from each condition was excluded from the analysis, due to poor DNA sample quality and unreliable sequencing data. Scattered plots showing the correlation between samples for each condition are provided (Extended data Fig. 7a).
Data availability
TaDa sequencing data is available through the University of Glasgow institutional repositories DOI: 10.5525/gla.researchdata.994 (2020). The entire set of raw data supporting this study will be made accessible through the same repository prior to publication.
Author contributions
J.P designed and carried out most experiments and analysed and interpreted the data. Y.Y. provided technical support throughout the study and perform RT-qPCRs. J.P. G.A, T.S and J.B.C. analysed the TaDa data. J.B.C conceived the project, designed experiments, analysed the data and supervised the study. J.P and J.B.C wrote the paper with contributions from the rest of the authors.
Acknowledgements
J.P. and J.B.C. are funded by a Wellcome Trust and Royal Society Sir Henry Dale Fellowship (Grant Number 104103/Z/14/Z; J.B.C.) and a Wellcome Trust Institutional Strategic Support Fund (ISSF) — Excellence and Innovation Catalyst Award to J.B.C. J.P. was partly funded by a BBSRC - Flexible Talent Mobility Account (FTMA) Award (BB/R506576/1). Y.Y. is supported by CRUK core funding to the CRUK Beatson Institute (A17196). T.D.S and G.N.A. were funded by a Wellcome Trust Investigator grant (104567; T.D.S.) and a BBSRC grant (BB/P017924/1; T.D.S. and G.N.A.)
We are thankful to Marta Llimargas, Markus Affolter, Irene Miguel-Aliaga, Andrea Brand, Cedric Polesello, Alessandro Scopelliti, Pablo Wappner, Ilan Davis, Florence Besse, Hugo Stoker, Gines Morata and Fisun Hamaratoglu Dion for generously sharing reagents and fly lines. We thank the Kyoto, Vienna and Bloomington Drosophila Stock Centres and the Drosophila Studies Hybridoma Bank for fly stocks and antibodies. We thank Colin Nixon (Beatson CRUK histology service) for IHC of mouse intestinal samples, Elaine McKenzie for help and training on the use of the hypoxia chamber, Margaret Mullin for assistance with TEM and David Strachan, John Halpin and Robert Insall (Beatson CRUK) for support with image quantification. We thank David McGuinness and Julie Galbraith (Glasgow Polyomics) for sequencing samples for TaDa and Rhoda Stefanatos for advice with RT-qPCRs. We thank Maté Naszai for help with the creation of custom ImageJ macros and Lynsey Carroll for providing mouse intestinal samples. We thank Jean-Philippe Parvy and multiple members of the Cordero lab for scientific discussion and advice on the project.