DrosophilaEGFR pathway coordinates stem cell proliferation and gut remodeling following infection
- Nicolas Buchon†1Email author,
- Nichole A Broderick†1,
- Takayuki Kuraishi1 and
- Bruno Lemaitre1Email author
© Buchon et al; licensee BioMed Central Ltd. 2010
Received: 29 November 2010
Accepted: 22 December 2010
Published: 22 December 2010
Gut homeostasis is central to whole organism health, and its disruption is associated with a broad range of pathologies. Following damage, complex physiological events are required in the gut to maintain proper homeostasis. Previously, we demonstrated that ingestion of a nonlethal pathogen, Erwinia carotovora carotovora 15, induces a massive increase in stem cell proliferation in the gut of Drosophila. However, the precise cellular events that occur following infection have not been quantitatively described, nor do we understand the interaction between multiple pathways that have been implicated in epithelium renewal.
To understand the process of infection and epithelium renewal in more detail, we performed a quantitative analysis of several cellular and morphological characteristics of the gut. We observed that the gut of adult Drosophila undergoes a dynamic remodeling in response to bacterial infection. This remodeling coordinates the synthesis of new enterocytes, their proper morphogenesis and the elimination of damaged cells through delamination and anoikis. We demonstrate that one signaling pathway, the epidermal growth factor receptor (EGFR) pathway, is key to controlling each of these steps through distinct functions in intestinal stem cells and enterocytes. The EGFR pathway is activated by the EGF ligands, Spitz, Keren and Vein, the latter being induced in the surrounding visceral muscles in part under the control of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Additionally, the EGFR pathway synergizes with the JAK/STAT pathway in stem cells to promote their proliferation. Finally, we show that the EGFR pathway contributes to gut morphogenesis through its activity in enterocytes and is required to properly coordinate the delamination and anoikis of damaged cells. This function of the EGFR pathway in enterocytes is key to maintaining homeostasis, as flies lacking EGFR are highly susceptible to infection.
This study demonstrates that restoration of normal gut morphology following bacterial infection is a more complex phenomenon than previously described. Maintenance of gut homeostasis requires the coordination of stem cell proliferation and differentiation, with the incorporation and morphogenesis of new cells and the expulsion of damaged enterocytes. We show that one signaling pathway, the EGFR pathway, is central to all these stages, and its activation at multiple steps could synchronize the complex cellular events leading to gut repair and homeostasis.
An important function of epithelial surfaces is to maintain the barrier between an organism's internal and external environments. This is especially true for the gut epithelium because of the magnitude of its surface and exposure to both ingested material and the indigenous microbiota [1, 2]. Gut integrity is maintained in large part through epithelial renewal, which is sustained by the proper activation and differentiation of stem cells embedded in the gut . In mammals, the intestinal epithelium displays one of the most rapid turnover rates of any tissue. Stem cells, located in the basal crypts, proliferate continuously to completely turn over the gut every 3 to 4 days . Similar to mammals, the Drosophila adult midgut is sustained by intestinal stem cells (ISCs), which self-renew and produce a population of nondividing, undifferentiated ISC daughters, termed enteroblasts [4, 5]. Some of these progenitors remain in a transient state in the gut, while the majority differentiate into the two principal cell types of the intestinal epithelium: absorptive enterocytes and secretory enteroendocrine cells. The turnover of enterocytes is continuous, and it is thought that the entire Drosophila gut epithelium is renewed in 7 to 10 days . In addition to this function in basal maintenance, epithelial renewal is also critical in the host response to acute damage to the gut.
In this line, several reports have demonstrated that, in Drosophila, ingestion of cytotoxic compounds or damage by enteric pathogens increases epithelial renewal through ISC proliferation [6–11]. We and others further demonstrated that the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is required for bacteria-induced epithelium renewal [7, 9, 10]. These studies showed that stressed and/or damaged enterocytes produce a secreted ligand, Upd3, which activates the JAK/STAT pathway in ISCs to promote both their division and their differentiation, establishing a homeostatic regulatory loop. Interestingly, flies unable to renew their epithelium succumb to infection, demonstrating that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection [7, 9, 10]. These studies also identified natural stimuli that provoke ISC activation, providing a powerful model system to study epithelium renewal and its genetic control.
Despite these studies, it remains unclear how ISC activation and epithelium renewal are globally coordinated. Gut repair upon bacterial infection not only involves the proliferation of ISCs but also necessitates the elimination of damaged cells and integration of new cells into the epithelium, two processes that have not received attention. Moreover, genome-wide profiling of the gut response to damage caused by infection indicates that other pathways are induced , suggesting that additional pathways could contribute to the regulation of epithelium renewal.
In the present work, we have measured the morphological changes that occur in the gut of Drosophila in response to ingestion of a nonlethal pathogenic bacterium, Erwinia carotovora carotovora 15 (Ecc15). We show that infection induces a dramatic remodeling of the gut, which is required to repair the loss of nearly half its cells. This repair occurs initially through the immediate differentiation of enteroblasts, a pool of undifferentiated progenitors, and is completed through stem cell proliferation and differentiation. Our study demonstrates the complexity of epithelium renewal in response to infection, as it encompasses three different processes: (1) the proliferation and differentiation of ISCs, (2) the proper morphogenesis of new enterocytes, and (3) the delamination and anoikis of damaged enterocytes. We further demonstrate that one signaling pathway, the epidermal growth factor receptor (EGFR) pathway, is key to controlling these three cellular and morphogenetic events, therefore ensuring gut homeostasis following infection.
Infection with Ecc15induces a major remodeling of the gut
At 8 hours postinfection, the total number of gut cells began increasing as a consequence of stem cell proliferation, which peaked 12 hours postinfection (Figure 1a and Additional files 1C and 2A). Furthermore, we observed an expansion of the green fluorescent protein (GFP) signal arising from an ISC-specific marker (delta-Gal4 UAS-nlsGFP) (Figure 1c), which correlates with the increase in mitotic index in these guts. ISC proliferation and their subsequent differentiation into enterocytes were sufficient to restore the morphology of the gut to preinfection dimensions by 48 hours postinfection (Figures 1a and 1b). However, the proportions between different cell populations in the gut remained different from unchallenged conditions, but were restored by 120 hours postinfection (Additional files 1D and 2B). These results indicate that, following infection with Ecc15, epithelium repair occurs as a biphasic response, comprised of an immediate differentiation of preformed progenitor cells followed by epithelial replenishment through ISC proliferation and differentiation.
Altogether, our quantitative analysis reveals the profound plasticity of the Drosophila gut, which maintains its integrity and barrier function despite the loss of half of its cells. This integrity is maintained through the coordinated removal of dead cells by anoikis and their replacement through a biphasic repair response.
The EGFR/mitogen-activated protein kinase pathway is required cell-autonomously to promote ISC proliferation induced by infection
The activation of the EGFR pathway in progenitor cells led us to examine the role of this pathway in controlling ISC activity upon bacterial infection. To examine this, we expressed double-stranded RNA (dsRNA or RNAi) or dominant-negative constructs targeting EGFR pathway components in ISCs using an esgGal4 TS , which expresses Gal4 under thermosensitive conditions (see Materials and Methods). Inactivation of the EGFR/MAPK pathway with UAS-EGFR DN , UAS-Ras-IR and UAS-Raf-IR in ISCs did not significantly affect the number of ISCs in the gut (Figure 3c and Additional file 6A) or the mitotic index in unchallenged conditions (Figure 3d). However, in sharp contrast to wild-type flies, Ecc15 infection did not increase the number of mitotic cells (PH3-positive; Figure 3d) or change the pattern and distribution of escargot-GFP-positive cells (Figure 3c) in the gut of flies with reduced EGFR signaling in ISCs, indicating a lack of induction of epithelium renewal. Similarly, ectopic expression of Argos or the phosphatase MAPK phosphatase 3 (MKP3) (two negative regulators of the EGFR pathway), as well as RNAi against the transcription factor Pointed, also blocked ISC proliferation induced by Ecc15 (Additional file 6A), indicating that a canonical EGFR/MAPK pathway acts in ISCs to promote proliferation. In agreement with these observations, flies with reduced levels of EGFR pathway activity in ISCs exhibited a higher mortality to Ecc15 infection because of a failure in gut repair (Additional file 7). Conversely, overexpression of an activated form of the EGFR receptor (UAS-EGFR ACT ) in ISCs was sufficient to induce proliferation along the gut in the absence of infection (Figures 3c and 3d), although the resulting cells are abnormally shaped and do not appear as fully differentiated enterocytes (Additional file 6B). Altogether, these results indicate that the EGFR pathway is both required and sufficient in ISCs to promote their proliferation in response to infection.
Multiple EGFR ligands are involved in ISC proliferation
A key mechanism of EGFR pathway activation is through binding of EGFR ligands (EGFs) . Drosophila has four EGFs: Spitz, Keren, Gurken and Vein. Vein is produced as a secreted protein that does not require further processing, while the other three ligands require maturation by the protease Rhomboid. Vein, Keren and rhomboid are transcriptionally induced in the gut upon infection with Ecc15  (Figure 3a), while spitz but not gurken is expressed in the gut (data not shown), indicating that three of the four ligands could potentially activate the EGFR pathway in the adult gut.
Expression of veinin visceral muscles is mediated by the JAK/STAT pathway
The JAK/STAT pathway is activated in ISCs through the release of Upd3 by damaged enterocytes and controls both proliferation and differentiation of ISCs upon infection [7, 9, 10]. Use of a STAT-GFP reporter transgene revealed that the JAK/STAT pathway is activated not only in ISCs, as previously described [7, 10, 17–19], but also in some visceral muscles in response to infection (Figure 4c and Additional file 9). Additionally, specific knockdown of the JAK/STAT pathway using a dominant-negative form of the receptor Domeless or a STAT-RNAi in the visceral muscles reduced ISC proliferation (Figure 4d), uncovering a new role of the JAK/STAT pathway in this tissue. We hypothesize that the production of Upd3 by enterocytes could activate the JAK/STAT pathway in visceral muscles, which would then indirectly regulate ISC proliferation through the production of the EGF Vein. In agreement with this hypothesis, depletion of JAK/STAT activity in visceral muscles or reduction of upd3 in enterocytes partially decreased the level of vein expression (Figure 4e). These results indicate that, in addition to its role in progenitor cells, the JAK/STAT pathway indirectly contributes to ISC proliferation through the transcriptional activation of the EGF vein in visceral muscles.
The JAK/STAT and EGFR pathways synergize in ISCs to promote cell proliferation
EGFR pathway is required in enterocytes for proper adult gut morphogenesis
The above experiments point to a role of EGFR in enterocyte morphogenesis. Nevertheless, they rely on the use of the Myo1AGal4 driver that continuously expresses Gal4 along the entire development. As a consequence, we could not conclude whether the effect of the EGFR pathway occurred in mature enterocytes, in enterocytes undergoing maturation during epithelium renewal or earlier during the development of the gut. Therefore, we examined the impact of EGFR using the Myo1AGal4 thermosensitive (Myo1AGal4 TS ) driver, which enabled us to activate or downregulate the EGFR pathway specifically in adult enterocytes to avoid developmental effects. Guts of Myo1AGal4 TS ; UAS-EGFR DN flies shifted to the restrictive temperature for 4 to 5 days are initially of normal size, indicating that the effect of EGFR on the gut was not due to an immediate alteration in the shape of mature enterocytes (Figure 6c). However, we observed a significant increase in gut length when flies were incubated for 3 weeks at the restrictive temperature (Additional file 11E), a time frame in which there is significant renewal of the gut during normal aging. This suggests that the requirement of the EGFR pathway for proper enterocyte shape occurs during their maturation. To confirm this observation, we analyzed the gut length of Myo1AGal4 TS ; UAS-EGFR DN flies orally infected with Ecc15, a challenge that considerably accelerates cell renewal in the gut. Figure 6c shows that 48 hours postinfection, "regenerated" guts from flies with reduced EGFR signaling in enterocytes were 20% longer than in wild-type flies. Conversely, guts of flies overexpressing an activated form of EGFR were shorter compared to guts from wild-type flies 48 hours postinfection (Figure 6c). Altogether, we conclude that the EGFR pathway regulates cell morphogenesis during enterocyte maturation and that this process is critical during normal development of the adult gut, compensatory renewal following infection and aging.
The EGFR pathway is required to coordinate delamination and anoikis of enterocytes during infection
Our results support a role of the EGFR pathway in both ISC proliferation and the shaping of enterocytes. Surprisingly, the guts of flies with reduced levels of EGFR or Ras activity in enterocytes did not decrease in length and increase in width to the same extent as the guts of wild-type flies (Figure 6c), suggesting a role for the EGFR pathway in cell sloughing.
To investigate further the role of EGFR in the delamination process, we examined histological sections of guts upon infection. In contrast to wild-type flies, we did not observe cell blebbing, the multilayering of enterocytes or loss of cells into the lumens of flies depleted for EGFR in enterocytes (Figure 7b and Additional files 3and 4). The lack of cell sloughing was further confirmed by a strong reduction of aspecific yellow fluorescence in the guts of these flies (Additional file 4C). We previously reported that in wild-type flies enterocytes undergo apoptosis only in the lumen following detachment from the epithelium layer (Figure 2e). In sharp contrast, apoptotic enterocytes with fragmented nuclei were observed within the epithelium of flies depleted of EGFR in enterocytes, as revealed by caspase 3 activity and 4',6-diamidino-2-phenylindole (DAPI) staining (Figure 7c and quantification in Figure 7d). Conversely, the overexpression of the EGFR pathway in enterocytes induced high levels of cell delamination in the absence of infection (Figure 7d and Additional files 3 and 4A). Accordingly, clones of enterocytes expressing activated forms of EGFR or Ras (UAS-EGFR ACT and UAS-Ras v12 , respectively) rapidly disappeared from the epithelium compared to wild-type clones, suggesting an increased rate of delamination (Additional file 4). Similarly, overexpression of the EGFR ligand Keren resulted in a thick, multilayering of the gut due to the accumulation of cells in the lumen (Additional file 3). The detached cells in these guts differed from those in wild-type flies in that they did not undergo cell death, had normal nuclei and did not contain enlarged vacuoles (Figure 7c and quantification in Figure 7d).
These results indicate multiple roles of the EGFR pathway in enterocytes in response to infection, where it is required for early cell blebbing, modulation of cell junction dynamics and the normal process of enterocyte death. We conclude that a wild-type level of EGFR pathway activity is required in enterocytes for the proper coordination of their delamination and anoikis upon infection.
To date, studies on epithelium renewal have mainly focused on stem cell activity and its role in basal epithelium maintenance [17–19]. Recently, several studies have shown that ISC activity is strongly stimulated in response to damage-inducing agents and infectious bacteria [6, 8, 9, 11, 21]. Importantly, these stimuli induce ISC activity to an extent that facilitates the identification of underlying regulatory networks. Using this approach, we have performed quantitative analysis of gut remodeling at different time points following infection with Ecc15. We demonstrate that restoration of normal gut morphology involves more than the activation of ISCs, but rather requires the coordination of ISC proliferation and differentiation, the incorporation and morphogenesis of new cells and the expulsion of damaged enterocytes. We further demonstrate that the EGFR pathway is central to these three steps following bacterial infection.
Epithelium renewal: A more complex picture
Our quantitative analysis of distinct cell populations in the gut identified two phases of compensatory repair that occur following bacterial infection. Initially, the pool of preexisting precursor cells (enteroblasts) rapidly differentiate to buffer the loss of cells. This first phase does not require ISC proliferation and likely serves the same function as the process of epithelium restitution described in mammals, where neighboring cells migrate into damaged regions to maintain gut integrity . This early step is followed by a longer, regenerative phase involving ISC proliferation and differentiation, which is capable of repairing the entire gut in approximately 2 days. The precision with which the gut is regenerated is striking, returning to its original dimensions within a small percentage of variation.
Our quantitative analysis also revealed the striking regenerative capacity of the gut, which maintains its integrity despite the loss of nearly half of the enterocytes and a 40% reduction of its size. This massive cell loss is first observed as a multilayering of enterocytes that contributes to the characteristic increase in gut width observed upon infection. Subsequently, cells are expelled into the lumen. These detached enterocytes appear highly vacuolized and undergo cell death only when they have left the epithelium. This process is similar to anoikis, a mechanism of cell death induced by the loss of cell adhesion that occurs in the guts of mammals . While the delamination of cells correlates temporally with the drastic shortening of the gut, we cannot exclude the involvement of additional processes. One hallmark of the response of the mammalian gut to infection is the contraction of visceral muscles . The contribution of visceral muscles to gut shrinking in Drosophila remains to be determined. Globally, the combination of the gut shortening with cells dying after leaving the epithelium through anoikis provides an efficient mechanism to maintain gut integrity and may explain its ability to resist infection and the action of damaging agents.
The EGFR and JAK/STAT pathways are both required for ISC proliferation
Our epistatic analysis indicates that both the EGFR and JAK/STAT pathways are required to promote ISC proliferation. However, inhibition of the EGFR pathway completely blocks proliferation induced by overexpression of either Upd3 or Domeless in ISCs, whereas JAK/STAT depletion in ISCs only partially reduces the proliferation induced by activation of the EGFR pathway. This suggests a more central role for the EGFR pathway in ISC proliferation. Since our epistatic analysis involved partial loss of function of genes as a result of the use of RNAi and dominant negative proteins, it is difficult to establish a hierarchy between these two pathways. Future studies should address the respective roles of these pathways in ISCs, as well as the mechanisms by which they induce ISC proliferation. Of note, synergy between these two pathways has already been described in Drosophila, where both STAT and Ras are required in germ cells and tumor cells to promote cell proliferation and migration [25, 26]. Moreover, mild expression of the proto-oncogene Ras V12 in the gut promotes a preoncogenic state that, in combination with infection, induces dysplasia . An implication of the EGFR pathway in ISC proliferation is supported by a very recent study  that shows that the EGFR and JAK/STAT pathways are required in ISCs for the proliferation induced by the lack of Hippo signaling in enterocytes. Although both are required for ISC proliferation, the JAK/STAT and EGFR pathways also have distinct functions: the JAK/STAT pathway is required for enteroblast differentiation, while the EGFR pathway is required for proper gut morphogenesis and cell sloughing.
A dual role of EGFR in enterocytes
Our study points to a dual role of the EGFR pathway in the morphogenesis and sloughing of enterocytes. Flies with reduced EGFR activity in enterocytes have a characteristic long and thin gut that results from the flattening of enterocytes (scheme Additional file 11D), indicating that aberrant cellular morphogenesis has repercussions on the morphology of the tissue as a whole. The requirement of the EGFR pathway for enterocyte shape appears to occur during the maturation of newly synthesized enterocytes and affects at least three different morphogenetic events in the gut: the initial development of the adult gut, the basal maintenance upon aging and the accelerated renewal that occurs in response to damage induced by infection. In agreement, the guts of flies with reduced EGFR signaling in progenitors (esgGAL4 TS ; UAS-EGFR DN or Su(H)GBEGal4; UAS-EGFR DN ) are longer, indicating that EGFR modulates enterocyte shape at the late progenitor to young enterocyte transition (Additional file 15). The effect of the EGFR pathway on cell morphogenesis is supported by previous work reporting that EGFR affects tracheae and wing vein remodeling through its impact on E-cadherin-based cell adhesion [28–30]. A role of EGFR in enterocyte-to-enterocyte adhesion is also suggested by our observation that E-cadherin and Armadillo change their subcellular localization during epithelium renewal. Along with these studies, our results point to a general role of EGFR in epithelium morphogenesis. It would be interesting to investigate whether differences in the level of EGFR signaling determine the type of epithelia, squamous or columnar, encountered along the gut. In this sense, we observed that the largest increase in the gut length of flies defective in EGFR is mostly due to the intense flattening of cells in a region of the gut that is normally highly folded and composed of columnar enterocytes. Interestingly, our survival analysis points to a key role of EGFR-mediated enterocyte morphogenesis in the maintenance of the integrity of the gut, as shown by the increased susceptibility of EGFR-deficient flies to infection.
Surprisingly, we observed that altering the EGFR pathway activity in enterocytes strongly affected the delamination process. We observed that, upon infection, reduction of EGFR pathway activity in enterocytes decreased the sloughing of cells from the epithelium and led to apoptosis of enterocytes still within the layer. Conversely, expression of an activated form of this receptor, stimulated cell sloughing, but subsequent enterocyte death was not observed. We conclude that the EGFR pathway is essential for the proper execution of anoikis in response to infection. The presence of dead cells within the epithelium and reduced delamination could explain the disruption of gut barrier integrity observed in flies with reduced EGFR activity in enterocytes. Our data are compatible with the idea that EGFR pathway provides a transient survival signal to damaged cells, enabling them to delaminate and leave the epithelium before dying. This prosurvival effect of EGFR would explain why the overexpression of the EGF ligand Keren is sufficient to induce cell delamination, but there is no consecutive apoptosis. It is tempting to speculate that the loss of accessible growth factors such as EGFs by delaminated cells ends this transient survival period and switches the cell to enter anoikis. However, it remains unclear whether the function of EGFR in enterocyte delamination is dependent or not of EGFR ligands. We also observed a clear role of EGFR in the remodeling of the adherens junction during epithelium renewal. These findings are consistent with a number of studies highlighting the role of this pathway in anoikis in mammals. Early loss of E-cadherin from cell-to-cell junctions is involved in the onset of anoikis in human enterocytes . Moreover, using an in vitro model of culture of villus epithelium, Lugo-Martínez et al.  observed that inhibition of EGFR maintains E-cadherin at the membrane and decreases anoikis in mammals. We similarly observed a modulation of E-cadherin and Armadillo localization in the Drosophila gut during epithelium renewal, suggesting that a disassembly of E-cadherin-mediated junctions occurs during cell detachment. In agreement with this observation, overexpression of cadherin or dlg in enterocytes reduces the gut shortening normally observed with infection (Additional file 13E). Future studies should investigate whether EGFR mediates its effect through the disassembly of E-cadherin adherens junctions and what downstream components of the pathway are involved. In addition, it is not yet clear whether loss of E-cadherin is a cause or a consequence of cell detachment. In our study, we cannot exclude the possibility that the effects of EGFR on cell sloughing could also be a consequence of the abnormal morphogenesis of newly synthesized enterocytes, whose growth may aid in physically pushing damaged enterocytes out of the layer. Alternatively, new enterocytes may require the space created by delaminating enterocytes to shape properly. These scenarios are not mutually exclusive, and it is possible that the EGFR pathway separately modulates the integration of new enterocytes into the epithelia and their elimination by sloughing, thereby controlling the flux of intestinal cells.
Importantly, the implication of EGFR in three crucial stages of epithelium renewal, ISC proliferation, the synthesis and morphogenesis of new enterocytes and the elimination of damaged cells, could explain the synchronization of the complex cellular events that maintain gut homeostasis. The release of signals from damaged enterocytes that promote ISC proliferation and differentiation, in addition to enterocyte morphogenesis and elimination, provides a homeostatic loop coordinating gut repair. In this line, the cytokine Upd3 is a good candidate because it is produced by damaged enterocytes and capable of activating both the JAK/STAT and EGFR pathways, directly or through the production of Vein in muscles (Figure 8).
Our data provide new insights into the complex events regulating gut remodeling upon infection. Our cellular analysis identified striking similarities between the Drosophila and mammalian gut epithelium response to damage and suggests the conservation of some regulatory networks. Interestingly, stimulation of stem cell activity by invasive, bacteria-like Salmonella has been demonstrated to induce proliferation in mammalian guts , and gut infection is proposed to favor the development of cancer [34, 35]. Our study provides potential mechanisms to explain these links. Moreover, the EGFR pathway is involved in the maintenance of gut barrier integrity in mammals and is important in preventing the development of colitis . Thus, the use of infection to study Drosophila ISCs provides not only a powerful model to dissect stem cell regulation but also to elucidate the complex mechanisms that maintain tissue homeostasis and gut morphogenesis.
OregonR, CantonS, flies or flies carrying one copy of the Myo1A-Gal4, how-Gal4 or esg-Gal4 transgene were used as wild-type controls. A complete list of stocks is provided in supplementary materials (Additional file 16 and associated references [17, 20, 37–47]). For experiments, we used adult flies carrying one copy of the UAS construct (RNAi or dominant negative (DN)) combined with one copy of the Gal4 driver. The F1 progeny carrying both the UAS construct and the Gal4 driver were raised at 18°C until 3 days of adult development and then transferred to 29°C for optimal efficiency of the UAS/GAL4 system. Drosophila stocks were maintained at 23°C using standard fly medium (maize flour, dead yeast, agar and fruit juice) devoid of living yeast. Conditional esgGal4 TS , howgal4 TS or Myo1AGal4 TS animals were obtained by crossing virgin females (esg/how/Myo1A-Gal4, UAS-GFP; tub-Gal80 TS ) with males expressing a UAS construct. F1 progeny were raised at 18°C, and the activity of the Gal4 system was controlled by placing 3-day-old F1 adults at either restrictive (29°C, Gal80TS off, Gal4 system on) or permissive (18°C, Gal80TS on, Gal4 system off) temperatures. The clonal labeling of mature enterocytes was performed by shifting flies of the genotype yw,hsFLP;act FRT yellow FRT Gal4, UAS-GFP/CyO to 29°C for 1 day. At this temperature, the basal FLP expression is enough to enable flipout events in enterocytes (during their polyploidization) and GFP induction. To induce somatic recombination in flies expressing the mosaic analysis with a repressible cell marker system  or yw,hsFLP;act FRT yellow FRT Gal4, UAS-GFP/CyO, 3-day-old adult flies were heat-shocked for 60 minutes at 37°C for 3 consecutive days. Three days after or 1 week after, guts were dissected for immunostaining. Adherens junctions were marked by a DE-cadherin-GFP fusion protein expressed ubiquitously under the control of the ubi promoter . Septate junctions were marked by a Dlg-GFP construct expressed under the control of its own promoter . Autophagy markers were expressed under the control of the Myo1A-Gal4 driver and are described elsewhere .
Bacterial strains and infection experiments
E. carotovora carotovora 15 is a Gram-negative bacterium that induces a strong local immune response  and is described elsewhere . For oral infection, 3-day-old to 5-day-old flies were incubated for 2 hours at 29°C in an empty vial before being transferred to a fly vial with infection solution and maintained at 29°C. The infection solution was obtained by mixing an equal volume of 100× concentrated pellet from an overnight culture of Ecc15 (optical density OD600 = 200) with a solution of 5% sucrose (1:1) and deposited on a filter disk that completely covered the surface of standard fly medium. Flies were incubated for 1 day at 29°C on the contaminated filter, after which they were transferred to fresh vials. In the case of survival analysis, flies were continuously exposed to filters contaminated with Ecc15 and flipped every 2 days into new vials containing a filter contaminated with a fresh pellet of Ecc15. Survival was monitored every day.
Quantification of Ecc15 was determined from three individual replicates of five flies at 0.5, 1, 2, 4, 8, 12, 24 and 48 hours following infection with Ecc15. Dissected midguts were placed in 1 mL of phosphate-buffered saline (PBS) in a 1.5-mL screw top microcentrifuge tube containing glass beads. The samples were homogenized using a Precellys 24 (Bertin Technologies, France), and then dilutions were plated on Luria Broth Agar (MP biomedicals, Illkirch, France) and incubated at 29°C. Colonies were counted after 24 hours. For hemolymph colony-forming unit counts, flies were surface sterilized in 70% ethanol and pricked in the thorax (once per side) with a thin sterile needle. The exuding drops of hemolymph were collected with a pipette and transferred to a 0.5-mL microcentrifuge tube for dilution plating as described above.
For live imaging, guts were dissected in PBS and immediately mounted in the antifading agent Citifluor AF1 (Citifluor Ltd, London, UK). For immunofluorescence, guts were dissected in PBS, fixed for 20 minutes in 0.1% Tween 20-PBS (PBT) with 4% paraformaldehyde, rinsed in PBT and then incubated with primary antibodies (dilution 1:50 anti-Armadillo (Developmental Studies Hybridoma Bank, Iowa city, Iowa, USA), 1:500 anti-PH3 (Millipore, Billerica, Massachusetts, USA), 1:500 anti-β-galactosidase (Promega, Madison Wisconsin, USA), 1:300 anti-phospho-ERK (in tributyltin; Cell Signaling, Boston, Massachusetts, USA), 1:500 anti-phospho-JNK (Cell Signaling), 1:500 anticleaved caspase3 (Cell signaling) and 1:1,000 anti-GFP (Roche, Rotkreuz, Switzerland)) or Rhodamine-Phalloidin (dilution 1:50 (Invitrogen, Basel, Switzerland)) in PBT + 1% bovine serum albumin. Primary antibodies were revealed with Alexa488- or Alexa594-coupled antimouse antibodies (Invitrogen), and nuclei were stained with DAPI (Sigma, Saint Louis, Missouri, USA). Guts were then scanned with an Axioplot imager (Zeiss, Feldbach, Switzerland) and recomposed using the program MosaiX (Zeiss).
Measurements in Figure 1a and Additional files 1 and 2 were determined from dissected guts fixed as described above and stained with DAPI. Individual guts (N = 20 to 40) were visualized and captured using an Axioplot imager (Zeiss). Full guts were scanned at ×10 magnification and then recomposed with MosaiX (Zeiss). Images from representative fields of the same guts were captured at ×20 magnification in Z-stacks, and full projections were counted. Measurements to the nearest micron were obtained using the measure functions within AxioVision software (Zeiss, Feldbach, Switzerland). Length was measured by tracing from the middle of the proventriculus along the midgut to the midgut-hindgut junction (indicated by the branching of the Malphigian tubules). Gut width measurements are based on the average of five measurements taken along the gut. Counts of midgut cells were estimated on the basis of values obtained from counting the different cell population in the projected Z-stack images and then multiplying the sum to the area of the whole gut. The distinction between cell types was based on GFP staining (progenitors expressing GFP under the control of the esgGal4 driver) and the level of polyploidy (nuclear size). New enterocytes were defined as having low polyploidy (intermediate-sized nuclei) and/or persisting GFP signal (due to residual escargot-GFP signal), while old enterocytes were defined as having high polyploidy (large-sized nuclei). To verify the estimated cell counts, full counts were conducted of each cell type from five wild-type guts. Cell density was determined by measuring the distance from the nucleus of a given cell to the nucleus of its nearest neighboring cell, or distance internuclei (DIN). The values for 20 cells in a single field per gut of N = 20 guts were measured. The same position in the gut was recorded each time, and the average distance for each genotype was plotted. Delamination was quantified by counting the number of attached cells (within the epithelium layer) or unattached cells (outside the epithelium layer) in histological sections (see below). The values of three sections of N = 16 guts were measured for each genotype with and without infection. For all measurements, similar regions were recorded and all samples were exposed to identical conditions.
Drosophila adults were dissected into PBS, and the guts were immediately fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in PBS for 4 hours at 4°C. The samples were rinsed three times in 0.1 M cacodylate buffer, then postfixed with 1% osmium tetroxide and 1.5% potassium ferrocyanide solution in 0.1 M cacodylate buffer for 40 minutes at room temperature, followed by 1% osmium tetroxide solution in 0.1 M cacodylate buffer for 40 minutes at room temperature. The samples were then treated with 1% uranyl acetate in water for 40 minutes at room temperature. Dehydration of the guts was performed in an ascending series of ethanol concentrations, and then the samples were embedded in Durcupan (Sigma). The guts were cut at 0.2 mm (semithin sections) for light microscopy with a Leica ultramicrotome (Leica, Wetzlar, Germany). Semithin sections were stained with 2% toluidine blue and observed under a Zeiss microscope. For ultrathin sections, guts were cut at 50 nm for transmission electron microscopy with a Leica ultramicrotome. Ultrathin sections were contrasted with lead citrate and observed with an electron microscope. Immunostaining on cryostat sections was performed using the protocol described by Baumann .
Total gut RNA was extracted from 30 dissected midguts with TRIzol reagent (Invitrogen). Template RNA (1 μg) was used to generate cDNA by reverse transcription and then analyzed by quantitative polymerase chain reaction (qPCR) with a LightCycler 2.0 and the SYBR Green I kit (Roche). Expression values were normalized to RpL32. Primers used to monitor mRNA quantification can be obtained upon request.
Mean fly mortality, mean mitosis per gut (PH3 counts) and mean relative gene expression and their corresponding standard errors were determined using PROC MEANS (SAS Institute, Cary, NC, USA). Means were separated for significance using Fisher's protected least significant difference test at P = 0.05.
- Ecc15 :
Erwinia carotovora carotovora 15
epidermal growth factor
extracellular signal-regulated kinase
intestinal stem cell
Janus kinase-signal transducers and activators of transcription
mitogen-activated protein kinase
We are grateful to our colleagues JP Boquete for technical assistance and D Osman for helpful comments on the manuscript. We thank M Crozatier, WM Deng, B Edgar, M Freeman, M Meister, C Micchelli, D Montell, J Pastor-Pareja, the Bloomington Stock Center, the Vienna Drosophila RNAi Center and the National Institute of Genetics for fly stocks and S Rosset and G Knott (École Polytechnique Fédérale de Lausanne BioEM Facility) for assistance with histological analysis. N Broderick is supported by a Human Frontier Long-Term Postdoctoral Fellowship. The Lemaitre laboratory is supported by an European Research Council Advanced Investigators Grant and the Swiss National Science Foundation (3100A0-12079/1).
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