Research Article

Cell Biology

Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila

Universite Cote d'Azur, CNRS, INRAE, France
Faculty of Sciences III and Azm Center for Research in Biotechnology and its Applications, LBA3B, EDST, Lebanese University, Lebanon
Laboratory for Food Safety, University Paris-Est, French Agency for Food, Environmental and Occupational Health & Safety, France

Feb 27, 2023

https://doi.org/10.7554/eLife.80179

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Version of Record updated: March 17, 2023 (This version)
Version of Record published: February 27, 2023 (Go to version)
Accepted: February 5, 2023
Received: May 11, 2022
Preprint posted: April 13, 2022 (Go to version)

1. Of interest
Defects in lipid homeostasis reflect the function of TANGO2 in phospholipid and neutral lipid metabolism

Agustin Leonardo Lujan, Ombretta Foresti ... Vivek Malhotra

Research Article Updated Mar 27, 2023
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Abstract

Bacillus thuringiensis subsp. kurstaki (Btk) is a strong pathogen toward lepidopteran larvae thanks to specific Cry toxins causing leaky gut phenotypes. Hence, Btk and its toxins are used worldwide as microbial insecticide and in genetically modified crops, respectively, to fight crop pests. However, Btk belongs to the B. cereus group, some strains of which are well known human opportunistic pathogens. Therefore, ingestion of Btk along with food may threaten organisms not susceptible to Btk infection. Here we show that Cry1A toxins induce enterocyte death and intestinal stem cell (ISC) proliferation in the midgut of Drosophila melanogaster, an organism non-susceptible to Btk. Surprisingly, a high proportion of the ISC daughter cells differentiate into enteroendocrine cells instead of their initial enterocyte destiny. We show that Cry1A toxins weaken the E-Cadherin-dependent adherens junction between the ISC and its immediate daughter progenitor, leading the latter to adopt an enteroendocrine fate. Hence, although not lethal to non-susceptible organisms, Cry toxins can interfere with conserved cell adhesion mechanisms, thereby disrupting intestinal homeostasis and endocrine functions.

Editor's evaluation

The microbial pathogen Bacillus thuringiensis subsp. kurstaki (Btk) and its Cry toxins are used extensively to kill lepidopteran crop pests. Although Btk is not lethal to Drosophila, Jneid et al. present convincing evidence that Btk's Cry1A toxins disrupt Drosophila intestinal homeostasis by inducing enterocyte death, activating stem cell divisions, and promoting excess enteroendocrine differentiation via weakening of adherens junctions between stem cells and terminal daughter cells. These important findings raise the possibility that Btk and Cry-based insecticides may alter the intestinal lining of non-targeted animal species.

https://doi.org/10.7554/eLife.80179.sa0

Introduction

The gut lining is undergoing constant damage caused by environmental aggressors (pesticides, drugs, viruses, bacteria and toxins) ingested along with food. The gut quickly responds to these aggressions by accelerating its epithelium renewal to replace damaged cells. Over the past decade, studies in Drosophila melanogaster have contributed substantially to the understanding of the cellular and molecular mechanisms controlling the maintenance of intestinal homeostasis and regeneration. These mechanisms have proven to be highly conserved in the animal kingdom. In Drosophila, resident intestinal stem cells (ISCs) are the guarantors of this cell renewal process. Under normal conditions, asymmetric division of an ISC gives rise to a new ISC (to maintain the pool of ISC) and to a daughter progenitor cell that can commit to two different paths of differentiation (de Navascués et al., 2012; Goulas et al., 2012; O’Brien et al., 2011; Perdigoto et al., 2011; Tian and Jiang, 2014). The enteroblasts (EBs) and enteroendocrine precursors (EEPs) are the precursors of enterocytes (ECs) and enteroendocrine cells (EEs), respectively (Guo et al., 2021; Joly and Rousset, 2020; Pasco et al., 2015). ECs are the main intestinal epithelial bricks constituting an efficient barrier against aggressors and are therefore their first victims. The damaged or dying ECs emit cytokines, which stimulate the proliferation of ISCs to augment the pool of EBs that will differentiate into ECs to replace the damaged ones (Bonfini et al., 2016; Osman et al., 2012). Two mechanisms underlying intestinal regeneration have been described. The first one is the ‘cell renewal’ model, which occurs under weak aggression conditions that does not induce EC apoptosis. In this case, ISC proliferation is low and the neo-EBs differentiate into ECs. However, this provokes a transient excess of ECs due to the absence of prior EC death. The gut cell homeostasis is subsequently reestablished by the removal of old ECs (Loudhaief et al., 2017). The second mechanism, called ‘cell replenishment’ or ‘regenerative cell death’, occurs after a strong aggression that induces massive EC apoptosis. In this case, a rapid ISC proliferation is followed by the differentiation of EBs into ECs to replace the dying ones (Loudhaief et al., 2017; Vriz et al., 2014) without producing supernumerary ECs.

Bacillus thuringiensis (Bt) bacteria are largely used as microbial insecticides to fight crop pests. Bt is a Gram-positive sporulating bacterium belonging to the Bacillus cereus (Bc) group (Ehling-Schulz et al., 2019). It was first identified and characterized for its specific entomopathogenic properties due to the presence of a crystal containing specific Cry protoxins, which are produced during the bacteria sporulation (Rabinovitch et al., 2017). Among all the subspecies of Bt inventoried (http://www.bgsc.org/), spores of Bt subsp. Kurstaki (Btk) are used to specifically kill lepidopteran larvae that threaten crops, through a cocktail of Cry toxins made of Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa and Cry2Ab (Caballero et al., 2020). Cry toxins sequentially bind to different receptors present in the midgut to exert their cytotoxicity. Among those receptors, the ones named Bt-R that belong to the Cadherin transmembrane cell adhesion molecules are primordial for the Cry1A holotype of toxins, allowing them to bind to enterocyte brush borders. The other receptors—such as Alkaline phosphatases, Aminopeptidases N, and ABC transporters—appear to account for the cytotoxicity that Cry exert toward susceptible organisms (Adang et al., 2014; Gao et al., 2019; Li et al., 2020; Liu et al., 2018b). In susceptible insects, upon ingestion of spores and crystals, the basic midgut pH dissolves the crystals, releasing the Cry protoxins. Then, digestive enzymes cleave Cry protoxins (130kD and 72kD for proCry1A and proCry2A, respectively) into activated Cry toxins (around 67kD) allowing them to bind their midgut receptors. Thereby, Cry toxins form pores in the plasma membrane of ECs, ultimately leading to their death. An alternative mode of action of Cry toxins suggested that Cry binding to Cadherin induces an intracellular flux of Mg²⁺ resulting in EC apoptosis (Castella et al., 2019; Mendoza-Almanza et al., 2020). In both models, toxin-induced breaches within the gut lining allow bacteria (spores and vegetative cells) to reach the internal body cavity, generating a septicemia and subsequent death of the lepidopteran larvae within 2 or 3 days after ingestion of Btk spores (Mendoza-Almanza et al., 2020). It is assumed that Btk do not harm the intestine of non-susceptible organisms because, first, the intestinal pH is not suitable for the solubilization of the crystal of protoxins and, second, the Cry toxin receptors are absent from their gut epithelium (Rubio-Infante and Moreno-Fierros, 2016).

However, recent studies provide evidence that Btk also exhibits some adverse effects on non-susceptible organisms including humans. Indeed, Bt belongs to the B. cereus group to which some strains are well-known worldwide food-poisoning pathogens causing diarrheal-type illnesses (Jovanovic et al., 2021). Recently, Bt has also been implicated in foodborne outbreak events and the strains identified were indistinguishable from the commercial ones (Biggel et al., 2021; Bonis et al., 2021; Johler et al., 2018). Furthermore, we have shown that Btk spores and toxins at concentrations close to those recovered on vegetables after spraying induce growth defects and developmental delay in Drosophila larvae (Nawrot-Esposito et al., 2020). Increasing spore and toxin doses ultimately lead to larval lethality (Babin et al., 2020). Cry toxins produced by Btk are also used in genetically modified crops (GMCs) (ISAAA, 2017), and it has been reported that GMC-produced Cry1Ab toxin is found in agricultural water stream networks at abnormally high doses that may affect the survival rate of non-susceptible insects (Rosi-Marshall et al., 2007). In a similar vein, laboratory studies have demonstrated genotoxic activity of Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A in zebrafish rearing water (Grisolia et al., 2009). Based on all these data, our aim in this study was to decipher the interaction of Btk and its toxins with the intestinal epithelium using Drosophila melanogaster, an organism non-susceptible to Btk Cry toxin and a well-established model for studying host-pathogen interaction mechanisms.

Using environmental doses of spores and crystals of protoxins recovered on vegetables after treatment, we first showed that crystals of Btk Cry protoxins induced moderate enterocyte death that triggers a quick cell replenishment. We then demonstrated that the crystals diverted a higher number of progenitor cells from their initial EC fate toward an EE fate, generating an excess of EEs. Importantly, this effect was due to a weakened cell-cell interaction between ISC mother cells and progenitor daughter cells. We were able to rescue the crystal-dependent excess of EEs by specifically overexpressing the DE-Cadherin in ISC and progenitor daughters, reinforcing the strength of the adherens junction between these cells. Moreover, we found that among the five Btk Cry toxins, only the Cry1A holotype was able to induce this EE excess. Unexpectedly, we observed that Btk crystals are processed in the midgut of adult Drosophila as they are in that of susceptible-organisms, releasing activated Cry1A toxins. Hence, since our data demonstrate that Cry1A toxins disrupt conserved cellular processes, many non-susceptible organisms may exhibit an excess of EEs and consequently a disruption of their enteroendocrine functions.

Results

Crystals of Btk Cry protoxins induce EC death and stimulate proliferation of intestinal stem cells

During sporulation, Btk produces a crystal of protoxins that is lethal to lepidopteran larvae once ingested by the insect. To study the Btk effects on the non-susceptible organism Drosophila melanogaster, we orally infected flies with the SA11 Btk strain (hereafter named Btk^SA11), which is widely utilized in commercial microbial insecticides. A suspension of spores/crystals in water was deposited on the fly medium corresponding to 10⁶ CFU (Colony Forming Unit) of spores per female for 4 cm². The impact on the gut of the spores alone, or the toxins alone, was also monitored using a Btk strain devoid of protoxin crystals (Btk^ΔCry) or purified crystals, respectively (see Materials and methods).

The Gal4/UAS binary expression control system (Brand and Perrimon, 1993) allowed us to monitor first the effect of the spores/crystals on EC apoptosis by expressing the Caspase 3 sensor Casp::GFP; (Schott et al., 2017) under the control of the myo1A-Gal4 EC driver (myo1A>Casp::GFP). With this transgenic combination, the GFP is detectable only when the Caspase 3 is activated in ECs. As a negative control, we fed flies with water alone. In all our experiments, we focused our observations on the posterior midgut (R4 region, https://flygut.epfl.ch/overview) (Figure 1—figure supplement 1A; Buchon et al., 2013) because this region is known to show a high stem cell renewal activity (Marianes and Spradling, 2013) and exhibits the strongest phenotypes (see below). One day post-ingestion, Btk^SA11 or purified crystals induced moderate apoptosis of the ECs compared to the control (Figure 1A and Figure 1—figure supplement 1B). However, the treatment with spores alone devoid of protoxin crystals (Btk^ΔCry) did not induce EC death. Noteworthy, the overall morphology of the posterior midgut was not altered in the different conditions (Figure 1—figure supplement 1A).

Figure 1 with 2 supplements see all

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Crystals of *Btk* Cry protoxins induce EC death and stimulate proliferation of intestinal stem cells.

(A) EC apoptosis was monitored by expressing the Caspase 3 sensor (Casp:: GFP) using the *myo1A-GAL4* EC driver (*myo1A>Casp::GFP*). With this transgenic combination, the GFP is detectable only when the Caspase 3 is activated in ECs. Left panel: ×40 magnification of a R4 subregion. Green stars mark GFP-positive dying ECs. Scale bar = 20 µm. Right panel: quantification of dead ECs 24 hr post ingestion (PI) in the posterior midgut (R4 region). (B) Quantification of mitoses using the anti-PH3 antibody in the whole midgut 24 hr PI. (C) ISC density in the R4 region of *esg >GFP* flies 24, 72, and 120 hr PI. (D) EC density in the R4 region of *myo1A>GFP* flies 24, 72, and 120 hr PI. Data is reported as mean ± SEM. ns = not significant; * (p≤0.05); ** (p≤0.01), *** (p≤0.001).

Figure 1—source data 1 Cell type counting.: https://cdn.elifesciences.org/articles/80179/elife-80179-fig1-data1-v2.zip
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Induction of cell death is known to strongly induce ISC proliferation in the whole midgut (Biteau et al., 2008; Chatterjee and Ip, 2009; Jiang et al., 2009; Loudhaief et al., 2017). This prompted us to assess the number of ISC mitoses in the different conditions, using an Anti-phospho-Histone H3 antibody marking mitotic cells. As expected, ISC mitotic indexes were stronger upon oral infection with Btk^SA11 spores or purified crystals than in the control (Figure 1B). In Btk^ΔCry spore infection, mitotic figures were only moderately increased (Figure 1B). This is consistent with previous observations showing that a low dose (10⁶ CFU per Drosophila) of Btk vegetative cells (that do not produce and contain crystals of Cry protoxins) only moderately activates ISC proliferation without inducing EC apoptosis (Loudhaief et al., 2017). We confirmed this increase in ISC proliferation by analyzing ISC density and proportion in the posterior midgut (R4 region). To specifically mark ISCs, we expressed the GFP under the control of the Dl-Gal4 driver that is expressed in ISCs and EEPs (Joly and Rousset, 2020), and we co-stained with an anti-Prospero (Pros), an EEP and EE marker (ISCs were therefore GFP+, Pros-). While we observed an increase in ISC number with Btk^SA11 spores or purified crystals (both density and ratio increased), Btk^ΔCry spores did not induce any increase (Figure 1C and Figure 1—figure supplement 2A–B). This could be explained by the fact that the moderate stimulation of ISC proliferation by Btk^ΔCry spores was not sufficient to promote a detectable increase in global ISC number. Nonetheless, to verify that ISC daughter cells committed to a process of differentiation upon Btk^ΔCry or Btk^SA11 spore ingestion, we used the ReDDM Drosophila genetic tool (Antonello et al., 2015) under the control of the Dl-Gal4 driver (Dl-ReDDM). This tool allows us to follow the progeny of the ISCs because they express a stable RFP (H2B::RFP) while the mother cells (the ISCs) express a labile GFP. After shifting the flies to 29 °C to activate the Dl-ReDDM tool, the GFP was only expressed in Dl + cells (i.e. the ISCs and EEPs) while the H2B::RFP was expressed in Dl + cells but also stably transmitted to the progeny. We also used an anti-Pros to label EEPs and EEs. Consequently, ISCs were recognized by their expression of both GFP and RFP; EEPs expressed GFP, RFP and Pros, EEs expressed only Pros, EBs and ECs expressed only the RFP. As expected, both Btk^ΔCry or Btk^SA11 spore ingestion promoted ISC daughter cell differentiation (Figure 1—figure supplement 1C) but surprisingly we observed an abnormal elevated number of EEP doublets upon ingestion of Btk^SA11 spore (see below).

We next monitored the density and ratio of ECs using the fly strain myo1A>GFP allowing the expression of GFP in all ECs. Btk^ΔCry spores induced an increase of EC density at days 1 and 3 post-ingestion though their ratio was not altered; the right density of ECs was recovered 5 days after ingestion (Figure 1D and Figure 1—figure supplements 1D and 2A, C). Along with the low number of dying EC (Figure 1A) and the moderate induction of ISC proliferation (Figure 1B), our data strongly suggest that Btk^ΔCry spores weakly damage the intestinal epithelium, reminiscent of the ‘cell renewal’ process previously described after infection with poorly virulent bacteria (Loudhaief et al., 2017). On the contrary, ingestion of the Btk^SA11 spores or purified crystals provoked a decrease in total EC number 3 days post-ingestion (both density and ratio dropped down) (Figure 1D and Figure 1—figure supplements 1D and 2A, C) that we attributed to EC apoptosis (Figure 1A; Loudhaief et al., 2017). A normal number of ECs was restored 5 days post-ingestion for Btk^SA11 spores and to a lesser extent for purified crystals (Figure 1D and Figure 1—figure supplements 1D and 2A, C). Hence, Btk^SA11 spores or purified crystals launch a process of regenerative cell death, inducing a strong proliferation of ISCs to quickly replenish the gut lining as previously described for strong pathogens (Vriz et al., 2014). Importantly, the ingestion of purified crystals containing the Btk Cry-protoxins recapitulates the midgut phenotypes caused by the ingestion of Btk^SA11 spores.

Btk^SA11 spores induced an increase in EB, EEP and EE numbers

During the course of our experiments we observed that many more EEs were apparently present in the flies that had ingested Btk^SA11 spores compared with the control flies (Figure 2—figure supplement 1A–C). As ECs derive from EBs and EEs from EEPs, we assessed the amount and the identity of the precursors in the different conditions of infection (H₂O; Btk^ΔCry spores, Btk^SA11 spores and purified crystals). To count the EBs, we used a Gal4 strain of Drosophila driving GFP expression specifically in EBs (Su(H)>CD8::GFP). As expected, a significant increase in the number of EBs was observed between the first and the fifth day after ingestion of Btk^ΔCry, Btk^SA11 or purified crystals (Figure 2A and Figure 1—figure supplement 2A, D). The EEP density was assessed using two markers: the GFP expressed in ISCs and progenitors (EBs and EEPs) using the esg-Gal4 driver (esg >GFP) and a Pros staining which labels EEPs and EEs. Cells expressing both GFP and Pros corresponded to EEPs. While Btk^ΔCry spores did not modify the density of EEPs, ingestion of either Btk^SA11 spores or purified crystals resulted in an increase in EEPs from day one onwards (Figure 2B and D). Since EEPs must differentiate into EEs, we then counted the differentiated EEs that were GFP-/Pros+. No difference in EE density was obtained with Btk^ΔCry spores compared to the control, whereas there was a net increase with the Btk^SA11 spores and with the purified crystals (Figure 2C and D, Figure 1—figure supplement 2A and E, and Figure 2—figure supplement 1A–D). Interestingly, although this event was rare, we also observed that EEPs could undergo a cycle of mitosis that might contribute to the increase in EEPs and EEs (Figure 2E; Biteau and Jasper, 2014; Li et al., 2014; Zeng and Hou, 2015). Noticeably, we never observed such an increase in EEPs and EEs in the anterior part of the midgut (Figure 2—figure supplement 1E–I). Altogether, our results showed that the protoxin crystals of Btk^SA11 were responsible for the excess of EEs in the posterior midgut.

Figure 2 with 1 supplement see all

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*Btk^SA11* spores induce an increase in EB, EEP and EE numbers.

(**A–D**) Flies were fed with water, *Btk^ΔCry* spores, *Btk^SA11* spores or Crystals. (**E-E"**) Flies were fed with *Btk^SA11* spores. (**A–C**) Control (water-ctrl): blue; *Btk^ΔCry* spores: green; *Btk^SA11* spores: purple; Crystals: beige. (A) EB density in the R4 region of *Su(H)>CD8::GFP* flies 24, 48, and 72 h PI. (**B and C**) EEP (B) and EE (C) density in the R4 region of *esg >GFP* flies 24, 48, and 72 hr PI. (**D-E"**) R4 region of *esg >GFP* flies labeled with anti-Pros (Red). GFP was expressed in ISCs, EBs and EEPs, and Pros was expressed in EEPs (yellow arrows in D) and EEs (red arrows in D). (**E-E’’**) PH3 staining (blue) marks mitosis. Pink arrows point to dividing EEPs and blue arrows point to dividing ISCs. ×40 magnification. Scale bar = 20 µm. Data is reported as mean ± SEM. ns = not significant; * (p≤0.05); ** (p≤0.01), *** (p≤0.001).

Figure 2—source data 1 Cell type counting.: https://cdn.elifesciences.org/articles/80179/elife-80179-fig2-data1-v2.zip
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EE excess arises from newborn EEPs after ingestion of crystals of Btk^SA11 protoxins

To demonstrate that the excess of EEPs and EEs arose from proliferating ISCs caused by the ingestion of protoxin crystals, we used the ReDDM cell lineage tracing system using the esg-Gal4 driver (esg-ReDDM flies). We chose to analyze the progeny at day 3 post-ingestion (Figure 3A), when the increase in EEs reached its peak (Figure 2C and Figure 1—figure supplement 1E). According to the expression of specific cell markers and nucleus size, we could identify different cell types either that existed before the ingestion or that appeared after the ingestion of Btk^ΔCry spores, Btk^SA11 spores or purified crystals. Identities of the different cell types were defined as follows: ISCs and EBs were GFP+/RFP+/DAPI +with small nuclei (although EBs were bigger cell than ISCs); EEPs were GFP+/RFP+/Pros+/DAPI+; new EEs were RFP+/Pros+/DAPI+; old EEs were Pros+/DAPI+, new ECs were RFP+/DAPI + with big polyploid nuclei and old ECs were DAPI + with very big nuclei (Figure 3B–E"). In the control experiments, a few newborn ECs (red arrows in Figure 3B and B', and Figure 3G) and rare newborn EEs (Figure 3F) appeared 3 days post-ingestion, reflecting the relative steady state of the cellular homeostasis. As expected for poorly virulent bacteria, ingestion of Btk^ΔCry spores induced the appearance of newborn ECs (red arrows in Figure 3C and C' and Figure 3G) and only rare newborn EEs resulted (pink arrow in Figure 3C–C" and Figure 3F). Similarly, ingestion of Btk^SA11 spores or purified crystals promoted the appearance of newborn ECs (red arrows in Figure 3D, D', E and E' and Figure 3G) but, strikingly, a high number of newborn EEs appeared (pink arrows in Figure 3D–E" and Figure 3F). However, we could not rule out the possibility that the Btk^SA11 spores altered EB behavior, pushing them toward an EE fate. To verify this possibility, we carried out a ReDDM lineage tracing using the Su(H)-Gal 4 driver that is specifically expressed in EBs (Su(H)-ReDDM flies). In this case, no newborn EEs were detectable upon ingestion of the Btk^SA11 spores while many newborn ECs were present (Figure 3—figure supplement 1A–D), indicating that EBs are not the source of the increase in the number of EEs. These data confirm previous observations that EEs never develop from Su(H)+EBs (Biteau and Jasper, 2014; Zeng and Hou, 2015).

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EE excess arises from newborn EEPs after ingestion of *Btk^SA11* crystals.

(A) Schema of the experimental design for the *esg-ReDDM* cell lineage used in this entire figure. (**B-E"**) R4 region of *esg-ReDDM* flies. Midguts were stained for Pros (blue) and DAPI which marks nuclei (white in B, **C, D and E**). (**B-E’’**) show the different cell types which either existed before the ingestion (green and red) or arise after the ingestion (red only) of water (B-B", Ctrl), *Btk^∆Cry* spores (**C-C"**) or *Btk^SA11* spores (**D-D"**) and Crystals (**E-E’’**). ISCs were GFP + RFP + DAPI +with small nuclei; EBs were GFP + RFP + DAPI +with bigger nuclei; EEPs were GFP + RFP + Pros +DAPI + ; new EEs were RFP+, Pros + DAPI + ; old EEs were Pros +DAPI + ; new ECs were RFP + DAPI + with polyploid big nuclei and old ECs were DAPI +with very big nuclei. 40 X magnification. Scale bar = 20 µm. (F) Counting old EEs (Pros + RFP-) and new EEs (Pros + RFP + ) in the conditions described in (**B–E**). (G) Counting old ECs (DAPI+) and new ECs (DAPI + RFP + ) in the conditions described in (**B–E**) n=number of 40 x images analyzed Data is reported as mean ± SEM. ns = not significant; * (p≤0.05); *** (p≤0.001).

Figure 3—source data 1 Cell type counting.: https://cdn.elifesciences.org/articles/80179/elife-80179-fig3-data1-v2.zip
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Altogether, our data demonstrate that ingestion of Btk^SA11 spores damages the intestinal epithelium, stimulating ISC proliferation. However, some of the progenitors make the choice to commit to an EEP/EE fate instead of an EB/EC fate. Consequently, there is a lack of new ECs to replace the dying ones and there is an excess of EEs. Interestingly, the crystals containing the Cry protoxins can recapitulate all the Btk^SA11 spore effects. In contrast, the effect of Btk^ΔCry spores is less damaging for the gut epithelium. In this case, ISC proliferation is only weakly stimulated and the progenitors make the choice to commit to the EB/EC fate to replace the damaged ones.

Crystals of Btk^SA11 protoxins decrease ISC-progenitor cell-cell adhesion

It is well established that the Notch (N) signaling pathway governs progenitor differentiation and cell lineage choice in the adult Drosophila midgut (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006; Ohlstein and Spradling, 2007; Pasco et al., 2015). Indeed, the transmembrane ligand Delta (Dl) expressed in ISCs binds to its N receptor present on the surface of progenitors. This induces the cleavage of the intracellular domain of N and its relocation into the nucleus to activate its target genes (Perdigoto and Bardin, 2013). Upon N activation, progenitors differentiate into EBs and then into ECs while in the absence/weak activation of N signaling, progenitors commit to an EEP/EE fate (Beehler-Evans and Micchelli, 2015; Guo and Ohlstein, 2015; Ohlstein and Spradling, 2007; Sallé et al., 2017). A prolonged and/or strong interaction between the ISC and its progenitor is necessary to reach the threshold of the N signaling activation sufficient to commit the progenitor to the EB/EC fate. A shorter and/or weaker interaction between the ISC and its progenitor weakly induces the N pathway, pushing the progenitor towards the EE fate (Guisoni et al., 2017; Sallé et al., 2017). The adherens junctions between ISCs and progenitors formed by E-Cadherins and Connectins intervene to prolong the contact between Dl and N, favoring the EB/EC fate (Choi et al., 2011; Falo-Sanjuan and Bray, 2021; Maeda et al., 2008; Zhai et al., 2017). As the consensus receptors for Cry1A toxins in target organisms are members of the Cadherin family (Adang et al., 2014), we wondered whether the Btk^SA11 could interfere with the function of the adherens junctions. We hypothesized that ISC-progenitor interaction could be reduced via interference of Cry toxins with Cadherins, modifying the progenitor cell fate and thus explaining the excess in EE number seen after ingestion of Btk^SA11 spores or purified crystals. To test this hypothesis, we labelled the intestines of esg >GFP Drosophila fed with Btk^ΔCry spores, Btk^SA11 spores or purified crystals with the anti-Armadillo (Arm)/β-catenin antibody that strongly marks the adherens junctions. We observed an intense labeling at the level of the junctions between pairs of GFP + cells in the control (Figure 4—figure supplement 1A–A’’, E) or following intoxication by Btk^ΔCry spores (Figure 4—figure supplement 1B–B, E). Strikingly, this labeling became less intense following ingestion of Btk^SA11 spores or purified crystals (Figure 4—figure supplement 1C–E), and correlated with an increase in EEPs number. To verify that weakening of cell junctions corresponded to cell shift towards an EEP fate, we used a Drosophila line expressing the endogenous DE-Cadherin (DE-Cad) fused to a Tomato tag in which progenitor cells (ISCs, EBs and EEPs) were labelled with GFP, while EEPs and EEs were marked with Pros (Figure 4). Using this genetic background, we first confirmed the decrease in the proportion of cells showing strong junctions between pairs of GFP + cells upon feeding with Btk^SA11 spores or purified crystals (Figure 4E). Second, as expected, we observed that the weak Tomato::DE-Cad labelling between a GFP + cell (an ISC or an EB) and its neighboring cells was correlated with the expression of the EEP marker Pros (blue stars in Figure 4A–D' and Table 1). Together, our results suggested that crystals of Cry protoxins produced by Btk^SA11 are responsible for the increase in the number of EEPs/EEs, this effect being associated with a decrease in intercellular adhesion between ISCs and progenitors.

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Btk crystals decrease ISC-Progenitor cell-cell adhesion.

(**A–E**) *esg >UAS GFP, Tomato::shg Drosophila* midgut R4 region 24 hr PI of water (A-A” Ctrl), *Btk^∆Cry* spores (**B-B”**), *Btk^SA11* spores (**C-C”**) or crystals (**D-D”**). (**A-D"**) Midguts are labelled for Pros (blue), DE-Cadherin (red) and ISCs and progenitors (green). Red arrows point to the high intensity of adherens junctions staining between ISC and progenitors. Yellow arrows point to the weak intensity of adherens junction staining. Note that the high intensity of adherens junction staining is associated with ISC/EB interaction while the weak intensity of adherens junction staining is associated with ISC/EEP interaction (blue stars mark EEPs). ×40 magnification. Scale bar = 10 µm. (E) Graph representing the percentage of the different categories of cell contact intensity between ISCs and progenitors. n=number of cell pairs analyzed. Weak = Contact Intensity/Membrane Intensity <1.4; Mild = 1.4 < Contact Intensity/Membrane Intensity <1.6; Strong = Contact Intensity/Membrane Intensity >1.6.

Figure 4—source data 1 Ratio of contact intensity.: https://cdn.elifesciences.org/articles/80179/elife-80179-fig4-data1-v2.zip
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Table 1

Cell junction intensity ratio measurement between pairs of progenitors.

Cadherin::RFP labeling intensity were measured first at the cell junction between pairs of progenitors and second around the rest of the cell membrane (see Figure 4—figure supplement 1F–G). Ratio correspond to the Junction intensity/the rest of the membrane. Prospero positive progenitors were both GFP+/Pros+ (see Figure 4). Yellow highlight labels Pros + progenitors with a weak intensity ratio. Orange highlight labels Pros + progenitors with a medium intensity ratio. Red highlight labels Pros + progenitors with a strong intensity ratio.

H2OIntensity ratio	Pros +	Btk^ΔCry Intensity ratio	Pros +	Btk^SA11Intensity ratio	Pros +	Crystals Intensity ratio	Pros +
2,29		1,46		1,49	Yes	1,27	Yes
1,37		2,27		1,38	Yes	2,03
1,43		2,25		1,91		0,78	Yes
1,75		1,92		1,52		1,18	Yes
1,87		1,51		1,89		1,89
2,37		2,19		1,88		2,56
2,54		1,96		1,19	Yes	2,20
1,77		1,91		2,27		3,12
1,45		1,35	Yes	1,63		4,05
1,49		1,94		1,39		1,91
1,83		2,46		1,54	Yes	1,70
1,76		2,93		1,87		1,76
2,20		2,29		2,55		2,17
1,25	yes	3,60		1,63		1,55
2,48		3,03		1,84		2,06
1,12	yes	2,12		1,31	Yes	1,77
1,75		2,30		0,79	Yes	1,62
1,67		1,36	Yes	1,55
1,43		2,91		1,98
2,41		1,60		1,89
2,64		2,82		1,65
2,49		1,55		1,78
1,60	Yes	3,00		1,21	Yes
2,95		1,88		1,86	Yes
2,67		2,32		1,07	Yes
2,31		3,69		2,24
2,24		2,93		2,20
2,45		1,87		1,08	Yes
1,49		2,55		1,46
2,06		1,70		1,72
3,67		2,80		2,29
2,57		3,74		1,74
2,33		2,27		0,92	Yes
1,50		1,92
2,10		1,79
1,47		2,39
1,76

Increasing adherens junction strength rescues crystal-dependent cell fate diversion

To confirm that the crystals of Btk^SA11 interfered with progenitor fate by disturbing adherens junctions, we wondered whether increasing the strength of cell adhesion between ISCs and progenitors could rescue the right number of EEPs/EEs. Thus, we overexpressed the DE-Cad in these cells using the esg-ReDDM flies (Figure 5A–G and Figure 5—figure supplement 1A–E). We analyzed the identity of newborn cells 3 days after ingestion of water (control), Btk^ΔCry or Btk^SA11 spores, or purified crystals. First of all, we verified that DE-Cad overexpression in ISCs and progenitor cells (esg + cells) did not blocked ISC proliferation (Figure 5—figure supplement 1E compared to Figure 3—figure supplement 1E). Interestingly, in flies overexpressing the DE-Cad fed with Btk^SA11 spores or purified crystals, we observed a rescue in the number of EEs (blue arrows in Figure 5A–D', and Figure 5E compared to Figure 3F). In agreement, the number of ISC-progenitor pairs with a strong interaction was increased (Figure 5G compared to Figure 4E). Furthermore, as expected, more newborn ECs appeared (red arrows in Figure 5B–D' and Figure 5F compared to Figure 3G), strongly suggesting that increasing the cell adhesion between ISCs and progenitors rescued the progenitor fate disturbance generated by the Btk^SA11 crystals. Surprisingly, overexpressing the Connectin, another cell adhesion molecule, which mediates hemophilic cell-cell adhesion (Zhai et al., 2017), in both ISCs and progenitors did not rescue the number of EEs following feeding with Btk^SA11 spores or purified crystals (Figure 5—figure supplement 2). The emergence of new Pros + cells were still considerable upon ingestion of Btk^SA11 or crystals at the expense of new ECs (Figure 5—figure supplement 2I and J). Of note, the overexpression of the Connectin in esg+ cells did not impact ISC proliferation (Figure 5—figure supplement 2K). To confirm the disturbance of DE-Cad-dependent cell adhesion by purified crystals, we carried out an aggregation assay in Drosophila S2 cell culture. Indeed, S2 cells do not endogenously express the DE-Cad and display only a weak cell-cell adhesion phenotype (Toret et al., 2014). Transfection of S2 cells with a plasmid encoding a DE-Cad::GFP fusion resulted in large aggregate formation as early as 1 hr post-agitation (Ctrl in Figure 5H and Figure 5—figure supplement 1F). Adding purified crystals to the cell culture medium strongly reduced the size of aggregates. Interestingly, purified Cry1Ab and Cry1Ac protoxins have the same effect although Cry1Ab needed a longer time to reduce the size of S2 cell aggregates (Figure 5H–H" and Figure 5—figure supplement 1F). Because Cadherins serve as receptors for the Cry1A toxin family in target Lepidoptera (Adang et al., 2014), our data suggest that in non-target organisms such as Drosophila melanogaster, Cry1A toxins could interfere physically with the well-conserved E-Cadherin.

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Increasing adherens junction strength rescues crystal-dependent cell fate diversion.

(**A–G**) *esg-ReDDM >DE Cad Drosophila* midgut R4 region. These flies specifically overexpress the DE-Cad in ISCs and progenitors. Flies were fed with water (A-A', and blue in E and F, Ctrl), *Btk^∆Cry* spores (B, B’, and green in E and F), *Btk^SA11* spores (C, C’ and purple in E and F) or Crystal (D-D' and beige in E and F) and observed 72 h PI (see Figure 3A for the experimental design). In (**A-D'**) blue arrows point to old EEs and red arrows newborn ECs. (E) Number of old EEs (Pros + RFP-) and new EEs (Pros + RFP + ) and (F) number of old ECs (DAPI +RFP-) and new ECs (DAPI + RFP + ) in the conditions described in (**A–D**). (**A-D'**) 40 X magnification. Scale bar = 20 µm. (G) Graph representing the percentage of the different categories of cell contact intensity between ISCs and progenitors in the experimental conditions shown in Figure 5—figure supplement 1A–D’. Weak = Contact Intensity/Membrane Intensity <1.4; Mild = 1.4 < Contact Intensity/Membrane Intensity <1.6; Strong = Contact Intensity/Membrane Intensity >1.6. n=number of cell pairs analysed. (**H-H"**) Cell aggregation assays on S2 cells expressing the DE-Cadherin::GFP. Cells placed under constant rotation were incubated with or without Bt crystals or purified Cry protoxins (Cry1Ab or Cry1Ac) for 1 hr (G), 2 hr (G') or 3 hr (G"). Each scatter plot represents the area (µm2) of all objects (aggregates or individual cells) obtained from three independent experiments. Representative images of cell aggregates formed in aggregation assays are shown in Figure 5—figure supplement 1F data. In (**E and F**), n=number of 40 X images analyzed. In (G), n=number of cell pairs analyzed. Data is reported as mean ± SEM. ns (non-significant), * (p≤0.05), ** (p≤0.01), *** (p≤0.001).

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Cry1A toxins mimic Btk^SA11 spore effects

Btk^SA11 produces five different Cry toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, and Cry2Ab) (Caballero et al., 2020). We investigated whether the increase in EEP/EE number was due to all toxins present in the crystals or to only one family of toxins (i.e. the Cry1A or Cry2A family). We made use of the Btk^Cry1Ac strain (referred to as 4D4 in https://bgsc.org/) which produces crystals composed only of the Cry1Ac protoxin. We fed esg >GFP flies either with spores of Btk^Cry1Ac or with purified Cry1Ac crystals. In both conditions, we observed a significant increase in the number of EEPs and EEs 3 days post-ingestion compared to controls (Figure 6A and Figure 6—figure supplement 1A, C, D). To verify whether other toxins of the Cry1A family induced a similar rise in EEP/EE number, we generated a Btk^Cry1Ab strain (see Material and Methods) producing only the Cry1Ab toxins (Figure 6—figure supplement 1I). Similar to the Btk^Cry1Ac spores, Btk^Cry1Ab spores induced an increase in EEP/EE numbers (Figure 6B and Figure 6—figure supplement 1B). Unfortunately, no Btk strain yielding only Cry2A-containing crystals was available and we were unsuccessful in generating one. To overcome this, we used heterologous expression of Cry toxins in E coli. We first checked whether Cry1Ac protoxin produced and purified from Escherichia coli (E. coli) was indeed able to induce an increase in EEP/EE numbers. We also forced the activation of the Cry1Ac protoxin into an activated form in vitro (see Materials and methods). Interestingly, both the protoxin and the activated form of Cry1Ac were able to induce the expected phenotype, although the activated Cry1Ac form was more efficient (Figure 6C and Figure 6—figure supplement 1E, F). Conversely, both Cry2Aa protoxin and its activated form purified from E coli were unable to increase the number of EEP/EEs (Figure 6C and Figure 6—figure supplement 1G and H). Therefore, our data demonstrate that ingestion of Cry1A toxins was sufficient to induce a rise in the numbers of both EEPs and EEs.

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Cry1A toxins mimic *Btk* crystal effects.

(**A–C**) *esg >GFP* flies fed with water (blue, Ctrl), *Btk^Cry1Ac* spores (fuchsia in A), Cry1Ac crystals (orange in A), *Btk^Cry1Ab* spores (rose in B), Cry1Ac protoxins (light khaki in C), Cry1Ac activated toxins (khaki in C), Cry2Aa protoxins (light grey in C) and Cry2Aa activated toxins (grey in C). ns (non-significant). Data is reported as mean ± SEM. *** (p≤0.001). (**A and B**) Density of EEPs or EEs in the R4 region 72 hr PI. (C) Density of Pros + cells in the R4 region 72 hr PI. (**D and E**) Western Blot from dissected intestines using a polyclonal Anti-Cry1A antibody detecting both the protoxins and the activated forms of Cry1A family of toxins.(D) (left lane) 0 h corresponds to *Btk^SA11* spores extemporaneously resuspended in water. (Right part of the blot) *Btk^SA11* spores incubated ex vivo (control) in water at 25 °C for the period of the experiment. We mainly detect the protoxin forms of Cry1A at 130 kDa (arrowhead). (Left part of the blot). Proteins extract from midguts of flies fed by the same *Btk^SA11* preparation (T 0 h) at 4 hr and 1, 3, and 6 days PI. The 130 kDa protoxins are still visible. The 67 kDa activated form appears as early as 4 hr (arrow). 6 days PI no more toxins are detected in the midgut. (E) Flies fed 2 days with water (Ctrl, left part) or with purified crystals (right part). Protoxins (130 kDa) are present in the insoluble fraction (Mb) in both the anterior (ant) and the posterior (post) midgut. The 67 kDa activated forms are present in the insoluble fraction of both the anterior and posterior midgut and in the soluble fraction (Cyto) of the posterior midgut. Actin was used as western blot loading control, especially for the insoluble fraction.

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Cry1A Protoxins from Btk crystals are activated in the Drosophila midgut

Our data above showed that purified activated Cry1Ac toxin was more efficient for inducing an EEP/EE excess than the purified Cry1Ac protoxin. Interestingly, the magnitude of EEP/EE excess was similar using either Cry1Ac crystals or purified activated Cry1Ac toxin (compare Figure 6A and C), suggesting that Cry1Ac protoxins contained in the crystals were activated in the Drosophila intestine. However, the admitted model proposes that protoxins can be activated in vivo only in the intestine of the susceptible lepidopteran owing to the presence of appropriate digestive proteases specifically functioning at the basic pH and reducing conditions encountered in the larval midgut of lepidopteran (Pardo-López et al., 2013; Soberón et al., 2009; Vachon et al., 2012). We therefore wondered whether the effects we observed in vivo were due to the crystal on its own (i.e. protoxins) or to the activated Cry toxins after processing in the Drosophila midgut. We first monitored by western Blot the processing of the Cry1A toxin family in the fly midgut fed with Btk^SA11 spores. We used an anti-Cry1A antibody raised against the activated form of the toxin, which therefore recognizes both forms (Babin et al., 2020). As a control, we incubated Btk^SA11 spores in vitro in water, at 25 °C for 4 hr, 1 days, 3 days and 6 days. Under these conditions, the protoxin form of Cry1A at 130kD was predominant and stable for at least 3 days before fading (control in Figure 6D, right part of the blot). This observation is in agreement with the fact that the half-life of Cry1A crystals has been estimated at about 1 week in soil or under laboratory conditions at 25 °C (Hung et al., 2016). We used the same initial Btk^SA11 spore preparation (0 hr) to feed the flies. We further dissected intestines and extracted total proteins at different times post-feeding. Interestingly, as early as 4 hr, we observed the 67kD activated form of the Cry1A toxins (Figure 6D left part). Noteworthy, the 130kD protoxin forms were still present, as the flies kept ingesting spores and crystals throughout the experiments. Six days after ingestion, almost no more protoxins or toxins were detectable due to the instability of the crystals (Figure 6D). Thus, our data show that the crystals can be processed in the midgut of adult Drosophila to give rise to active forms of the Cry1A toxins.

As mentioned previously, the increase in EEPs/EEs number was prominent in the posterior midgut (Figure 2—figure supplement 1). Indeed, it has been previously observed that posterior midgut is more prone to ISC proliferation and EE differentiation or tumor formation (Beebe et al., 2010; Marianes and Spradling, 2013; Tamamouna et al., 2020). Nonetheless, a differential processing of Cry1A protoxins along the midgut could also participate to this difference. Thereby, we fed flies with purified Btk^SA11 crystals and 24 hr later, we dissected and crushed the intestines by separating the anterior midgut from the posterior part. Furthermore, we performed subcellular fractioning by separating the soluble fraction (considered as the cytosol-enriched fraction) and the insoluble fraction containing membranes. As expected, actin is mainly found in insoluble fraction (lysis buffer detergent-free) and was consequently used as house-keeping protein for this cellular compartment. Interestingly, we observed that protoxins were already activated in the anterior part of the midgut (the 67kD form) but were only found in insoluble fraction, containing membranes (Figure 6E). In the posterior midgut, we detected a stronger quantity of the 67kD-activated form both in the soluble and insoluble fraction, suggesting an internalization by epithelial cells (Figure 6E). In this part of the midgut, smaller forms appeared mainly associated with the membrane, probably resulting from degradation processes. Notably, we did not detect the 130kD protoxins in the cytoplasmic fraction of both anterior and posterior midguts, suggesting that the protoxins remained associated with membranes. Moreover, total processing was never achieved since some protoxins still remained detectable. Altogether, our data show that crystals containing Cry1A protoxins are processed all along the adult Drosophila midgut to generate activated Cry1A toxins.

Cry1A toxins cross the intestinal barrier independently of cell adhesion strength and cell death

DE-Cad/adherens junctions are located basally in the posterior Drosophila midgut (Chen et al., 2018; Chen and St Johnston, 2022; Loudhaief et al., 2017; St Johnston and Ahringer, 2010) and are therefore protected from direct contact with the luminal content (i.e. from the Cry1A toxins ingested along with the food) by the septate junctions (i.e. the tight junctions) located apically. To reach the ISC-progenitor pairs that are also located basally within the intestinal epithelium, Cry1A toxins can follow different routes. Indeed, toxins could weaken the septate junctions, penetrating the intercellular space to directly interact with DE-Cadherins. An alternative might be that Cry-induced cell death would cause a leaky midgut favoring the toxin passage. To address this question, we first overexpressed either DE-Cad or Connectin in ECs using myo1A^ts-Gal4 UAS-GFP driver. We assumed that increasing the amount of cell-cell adhesion molecules would strengthen the sealing of the intestinal epithelium, thereby limiting intercellular passage of Cry toxins. As expected, DE-Cad overexpression in ECs partially rescued the increased number of EEs due to Btk^SA11 or crystal ingestion (Figure 7B–G and N and Figure 7—figure supplement 1A–F) while, surprisingly, Connectin overexpression did not (Figure 7H–J and N and Figure 7—figure supplement 1G–I). These data suggest that strengthening EC-EC interaction on its own is not sufficient to prevent Cry toxins to reach basal ISC-progenitor cell junctions. However, the partial rescue obtained with the DE-Cad overexpression suggest that DE-Cad in ECs could trap Cry1A toxins away from the ISC-progenitor cell junctions, limiting the amount of toxins able to interfere. We then blocked EC death by overexpressing the anti-apoptotic factor p35 in ECs, but we did not rescue EE increase induced by Btk^SA11 or crystal ingestion (Figure 7K–N and Figure 7—figure supplement 1J–L). Together our data suggest that Cry1A toxins can reach the basally located ISC through routes that do not depend on cell adhesion strength or cell death. Further investigations are therefore needed to clearly identify the cellular mechanisms involved.

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Cry1A toxins likely cross the intestinal barrier through EC transcytosis.

(A) Schema of the experimental design for the *myo1A-GAL4 UAS-GFP tub-GAL80^ts* (*myo1At^s >GFP*) overexpression in ECs used in this entire figure. (**B–N**) R4 region of midguts of flies fed with water (Ctrl, B, E, H, K and blue in N), *Btk^SA11* spores (C, F, I, L and green in N) and crystals (D, G, J,M and beige in N) and labelled for Pros. ×40 magnification. Scale bar = 20 µm. (**B–D**) *myo1At^s >GFP* midguts. (**E–G**) *myo1At^s >GFP* midguts overexpressing DE-Cad. (**H–J**) *myo1At^s >GFP* midguts overexpressing Connectin (Con). (**K–L**) *myo1At^s >GFP* midguts overexpressing the anti-apoptotic p35 factor. (N) Counting of EEPs/EEs (Pros + cells) in the different conditions described in (**B–M**). Data is reported as mean ± SEM. ** (p≤0.01), *** (p≤0.001).

Figure 7—source data 1 Counting of Prospero positive cells.: https://cdn.elifesciences.org/articles/80179/elife-80179-fig7-data1-v2.zip
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Discussion

Our results show that the Cry1A toxin family of Btk disrupts the gut cellular homeostasis of the non-susceptible organism Drosophila melanogaster. Cry1A induces EC death coupled to an increase in ISC proliferation to replace the damaged ECs. Importantly, Cry1A toxins also altered intestinal cell composition by weakening DE-Cadherin-dependant cell-cell adhesion which is normally highly enriched in adherens junctions linking ISCs to their immediate progenitors (Choi et al., 2011; Maeda et al., 2008; Ohlstein and Spradling, 2006; Zhai et al., 2017). As a consequence, progenitors are pushed toward the EE path of differentiation instead of the EC, owing to reduced activation of N signaling in progenitors (Figure 8; Guo and Ohlstein, 2015; Maeda et al., 2008; Ohlstein and Spradling, 2006; Ohlstein and Spradling, 2007; Pasco et al., 2015; Zhai et al., 2017). Our data confirmed that the duration and/or the strength of the cell-cell contact between the ISC and its progenitor daughter cell is important to drive the progeny toward an appropriate fate. Indeed, it has been shown both in Drosophila and in mammalian cell culture that adherens junctions are crucial to reinforce the contact between neighboring cells to allow the activation of N signaling (Falo-Sanjuan and Bray, 2021; Shaya et al., 2017; Zhai et al., 2017), N being necessary for the EB-EC cell fate choice.

Figure 8

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Cry1A toxins interfere with progenitor fate behavior.

**Notch ON:** in *Drosophila*, 90% of ISC daughter cells commit to the EB/EC fate owing to the strong activation of the Notch signaling pathway in the EBs. The adherens junction DE-Cadherin (DE-Cad)-dependent are required to permit the interaction between the Delta ligand in ISC and the Notch receptor in EB. **Notch OFF:** Ingestion of Cry1A toxins impedes the DE-Cad homophilic interaction between the ISCs and their progenitor daughter cells, reducing the activation of Notch signaling in progenitors. Consequently, progenitors adopt an EEP/EE fate.

Only the Cry1A family of toxins induces an increased number of EEs while Cry2A toxins do not display any phenotype. Previous studies showed that Cry1A and Cry2A bind to different receptors in the intestinal epithelium of susceptible lepidopteran larvae, and proteins of the Cadherin family appear essential for Cry1A, but not Cry2A, binding (Adang et al., 2014; Gao et al., 2019; Hernández-Rodríguez et al., 2013; Li et al., 2020; Liu et al., 2018a). Drosophila possesses 17 genes encoding for members of the Cadherin superfamily (Hill et al., 2001). Cad88C is the most similar to Bt-R Cadherin of susceptible Lepidoptera, but shares only 17% of identity, (Stevens et al., 2017) and is poorly expressed in the Drosophila midgut (see http://flygutseq.buchonlab.com/ and https://flygut.epfl.ch/). However, Cry1A effects on Drosophila progenitor cell fate appear to depend specifically on the DE-Cadherin present in the adherens junctions, since overexpression of DE-Cadherin in progenitor cells (ISCs, EBs and EEPs) can overcome Cry1A-induced impacts while another component of the adherens junctions (Connectin) cannot. Interestingly, the percentage of identity between the Cry1A binding regions (CBRs) shared by the orthologs of Cadherin-type receptors in different susceptible lepidopteran species ranges from 21% to 66% (Li et al., 2021; Shao et al., 2018). Hence, the presence of a well-conserved primary consensus sequence within the CBRs cannot explain the specificity of binding. In agreement, it has been recently shown in susceptible Lepidoptera that only two dipeptides within the CBRs are essential for high-affinity binding of Cry1A to its Cadherin receptor (Liu et al., 2018a). These two dipeptides are not conserved in the CBRs of the Bt-R orthologs in different lepidopteran species targeted by Cry1A toxins (Li et al., 2021). These data suggest that binding of Cry1A to the receptor relies more on a conserved conformation of the CBRs than on a conserved primary sequence. In addition, alignment of the Helicoverpa armigera (a Cry1A susceptible lepidopteran) CBR sequence (Gao et al., 2019) and the DE-Cadherin sequence between amino acids 164 and 298 shows 24% identity and 47% similarity (using BlastP, https://blast.ncbi.nlm.nih.gov/). Altogether, these results support a possible binding of Cry1A toxins to Drosophila intestinal DE-cadherins, although this could occur with a low affinity.

Susceptibility of lepidopteran larvae to CryA1 toxins relies on the presence of a secondary receptor such as Alkaline phosphatases, Aminopeptidases N, or ABC transporters (Adang et al., 2014; Gao et al., 2019; Li et al., 2020; Liu et al., 2018b). None of the orthologs of these receptors has been shown to be strongly expressed in the Drosophila midgut (Li et al., 2021; Stevens et al., 2017), which could explain why binding of Cry1A to DE-cadherins does not lead to the death of adult Drosophila. Interestingly, in Drosophila larvae, heterologous expression of ABCC2 from Bombix mori or Plutella xylostella, or Aminopeptidases N from Manduca sexta was sufficient to induce Cry1A-dependent death (Gill and Ellar, 2002; Obata et al., 2015). Conversely, heterologous expression of the Bombix mori Cadherin Bt-R receptor in Drosophila is not sufficient to induce death upon exposure to Cry1A but strongly enhances death when co-expressed with BmABCC2 (Obata et al., 2015). Together, these data suggest that, in Drosophila, in the absence of a Cadherin receptor displaying a high affinity to Cry1A toxin, endogenous Cadherins with reduced affinity toward Cry1A could step in to enhance the death potential of Cry1A toxins in the context of heterologous expression of a secondary receptor from susceptible Lepidoptera. In agreement, it has been previously demonstrated in Drosophila larvae that increasing the dose of ingested Btk spores and crystals could ultimately lead to death (Babin et al., 2020; Cossentine et al., 2016), arguing that the receptors (i.e. the Cadherins and the secondary receptors) present in the Drosophila midgut have lower affinities for Cry1A toxins.

Cry toxin activities in susceptible organisms rely not only on the presence of specific host receptors in the midgut, but also on the extreme midgut pH and reducing environment allowing crystal solubilization, as well as the enzymatic capacity of digestive proteins, both of which are involved in the conversion of protoxins into active toxins (Fiuza et al., 2017; Mendoza-Almanza et al., 2020; Pardo-López et al., 2013; Shao et al., 2013). Nevertheless, our data along with another recent study (Stevens et al., 2017) suggest that crystals can be solubilized and then protoxins activated in the Drosophila midgut. Therefore, more investigations will be necessary to understand how crystals are processed and protoxins activated in the intestine of non-susceptible organisms. What makes the difference between Cry-susceptible and non-susceptible organisms is likely the affinity of Cry toxins for midgut host receptors. The higher the affinity of the toxins, the greater the cellular damage/death and the greater the risk of death. In addition, the capacity of regeneration of the midgut epithelium also plays an important role to overcome Bt pathogenicity (Castagnola and Jurat-Fuentes, 2016). If the regeneration of the intestinal epithelium is more efficient than the destructive capacity of Cry toxins, the host will survive. However, whatever the host is (susceptible or non-susceptible), Bt uses a three-pronged strategy to improve its degree of virulence. First, it tries to disrupt the epithelial barrier function by damaging or killing ECs. Second, it diverts the behavior of the progenitor cells toward the wrong fate, thus limiting the amount of ECs produced that are necessary to replace the damaged ones and to maintain the midgut integrity. Third, killing ECs also reduces the capacity of these cells to produce reactive oxygen species and antimicrobial peptides known to be involved in antimicrobial defenses (Allaire et al., 2018; Capo et al., 2016; Kim and Lee, 2014).

The Bt subspecies kurstaki and aizawai are widely used as microbial pesticide to fight lepidopteran pests worldwide (Casida and Bryant, 2017), both subspecies partially producing the same Cry1A toxins (Caballero et al., 2020). Likewise, 180 millions of hectares of genetically modified crops express Cry1A toxins. Consequently, Cry1A toxins are present in the food, the feed and in the environment, implying that many organisms might be affected. As the mechanisms of intestinal progenitor fate choice are conserved in the animal kingdom (Guo et al., 2021; Joly and Rousset, 2020; Zwick et al., 2019), it would be interesting to investigate whether Cry1A toxins can also promote an increased number of EEs in other organisms (vertebrates and invertebrates). EEs, through the production of neuropeptides and hormones, are involved in the regulation of many physiological functions such as feeding behavior, metabolism and immune response (Guo et al., 2021; Nässel and Zandawala, 2019; Watnick and Jugder, 2019). Consequences of this increase in EE number could be, for example, metabolic dysfunctions or inflammatory pathologies. More studies are needed to understand the physiological impacts of this change in intestinal cellular composition on organismal health.

Materials and methods

Bacterial strains

Btk^∆Cry (identified under the code 4D22, González et al., 1982), Btk^Cry1Ac (identified under the code 4D4), and the E. coli strains producing Cry1Ac (identified under the code ECE53), Cry1Ab (identified under the code ECE54) and Cry2Aa (identified under the code ECE126) were obtained from the Bacillus Genetic Stock Center (https://bgsc.org/). The strain Btk SA-11 (Btk^SA11) was isolated from the commercial product Delfin.

Generation of Btk^Cry1Ab

The mutant Btk^Cry1Ab producing only Cry1Ab as crystal toxin was obtained from the WT strain Btk^SA11, by a procedure of plasmid curing, as follows. After isolation on TSA-YE agar (Biomérieux, 18 hr culture at 30 °C), the strain Btk^SA11 was sub-cultured successively 3 times in 10 mL of brain heart Infusion (BHI, Oxoid) broth at 42 °C with agitation, for 64, 48, and 36 hr, respectively. The first BHI culture was inoculated from an isolated colony, and the subsequent cultures were inoculated with 100 µl of the previous ones. Clones from the last culture were isolated on TSA-YE agar after serial dilution, then subcultured on the sporulating medium hydrolysate of casein tryptone (HCT) + 0.3% Glc, in order to verify the absence of crystal production, using phase-contrast microscopy (NF EN ISO 7932/Amd1). A panel of crystal-negative isolates were then subjected to whole genome sequencing as described hereafter. The genomic DNA of Btk^Cry1Ab and Btk^SA11 was extracted using the KingFisher cell and Tissue DNA kit (ThermoFisher) and sequenced using Illumina technology at the Institut du Cerveau et de la Moelle Epinière (ICM) platform, as previously described (Bonis et al., 2021), (NCBI accession numbers SAMN23436140 and SAMN23455549, respectively). The mutant Btk^Cry1Ab was selected for the single presence of cry1Ab as cry toxin plasmid gene. The absence of cry genes in Btk^Cry1Ab, with the exception of cry1Ab, was confirmed from raw reads using KMA (Clausen et al., 2018).

Spore production

From isolated colonies on LB agar Petri dish, 4x5 mL of Bt pre-culture was carried out. The pre-culture was used for sowing 4x500 mL of PGSM medium (0.75% casamino acids, 0.34% KH₂PO₄, 0.435% K₂HPO₄, 0.75% glucose, 1.25 mM CaCl₂, 0.123% MgSO₄, 0.002% MnSO₄, 0.014% ZnSO₄, 0.02% FeSO₄) and allowed to grow and sporulate in an incubator shaker at 30 °C, 180 rpm for 2 weeks. In order to eliminate vegetative cells, culture was heated 1 hr at 70 °C and then centrifuged 15 min at 7500 g. The pellet was resuspended with a Dounce homogenizer in 1 L of 150 mM NaCl and placed for 30 min on roller agitation at room temperature. After centrifugation (15 min, 7500 g), the pellet was washed twice with sterile water. The final pellet was resuspended in 30 mL of sterile water, dispatched in 1 mL weighed tubes and lyophilized for 24–48 hr. The spore mass was determined by the difference between the full and the empty tubes weights.

Spore titer

The lyophilized spores were resuspended in sterile water to obtain a concentration of 50 mg/mL. This solution was diluted serially (100 µL in 900 µL of sterile water) to obtain 10^–1 to 10^–9 dilutions. 100 µL of dilutions 10^–5 to 10^–9 were plated on LB agar and incubated at 30 °C overnight. The number of colonies was counted for each dilution and reported to the mass of spores plated. The experience was renewed three times. The mean of these ratios allows us to determine the titer of spores in CFU (colony forming unit)/g.

Crystal purification

A total of 2x1 g of Btk^SA11 lyophilized spores were resuspended in 2x30 mL of sterile water and placed on roller agitation at 4 °C for 5 hr. Then, the solution was sonicated for 4 cycles of 15 s/15 s with a frequency of 50% (Fisherbrand Model 505 Sonic Dismembrator). 6x10 mL were deposited on a discontinuous sucrose gradient (67%/72%/79%) and centrifuged overnight in SW28 swinging buckets at 100,000 g at 4 °C (ultracentrifuge Thermo-Sorval WX Ultra 80). The crystals were collected at the 72%/79% and 72% /67% interfaces with a micropipette and dispatched by 10 mL in centrifuge tubes (Beckman Avanti JE, rotor JA 25.50). A total of 25 mL of sterile water were added in each tube, vortexed and centrifuged at 4 °C at 16,000 g for 15 min. Each pellet was resuspended with 20 mL of sterile water and centrifuged again in the same conditions. Each final pellet was resuspended in 2 mL of sterile water, aliquoted by 1 mL in weighed microtubes and lyophilized 24 hr-48hr. The crystal mass was determined by the difference between the full and the empty tubes’ weights.

Cry protoxin production

2x15 mL of LB ampicillin (50 mg/mL) were inoculated with two colonies of E. coli expressing the desired Cry toxin, and allowed to grow at 37 °C, 220 rpm overnight. 4x5 mL of the overnight preculture were added to 4x500 mL of LB ampicillin (50 mg/mL) and allowed to grow at 37 °C and 200 rpm until DO₆₀₀=0.6–0.7. Cry protein expression was induced by adding 500 µL of 1 M IPTG (isopropyl β-D-1-thiogalactopyranoside) in each culture. The cultures were left at 37 °C and 200 rpm overnight and then centrifuged at 6500 g, 15 min at 22 °C. Pellets were pooled in two batches and resuspended 100 mL of cold WASH buffer (20 mM Tris, 10 mM EDTA, 1% Triton X-100, pH 7.5) and incubated 5 min at 4 °C. 500 µL of lysozyme (20 mg/mL) were added in each solution and incubated 15 min at 30 °C and then 5 min at 4 °C. The solutions were sonicated for 6 cycles of 15 s/15 s at 40% (Fisherbrand Model 505 Sonic Dismembrator) and centrifuged 10 min at 10,000 g, at 4 °C. The pellets were washed twice with 100 ml of WASH buffer and centrifuged in the same conditions. The last pellets were weighed, resuspended in CAPS buffer (50 mM CAPS, 0.3% lauroyl-sarcosine, 1 mM DTT pH11) to obtain a final concentration of 100 mg/mL, placed for 30 min under roller agitation at room temperature and centrifuged 10 min at 10,000 g, 4 °C. The supernatant was dialyzed twice against 50 volume of PBS1x, 0.1 mM DTT and twice against 50 volume of PBS1X at 4 °C and centrifuged 10 min at 20,000 g at 4 °C. The supernatant was conserved at –20 °C until purification or digestion (activation) by trypsin.

Cry toxin activation

Half of the produced supernatant (see above) was dosed by the Bradford method (Protein Assay Dye Reagent Concentrate, Biorad #500–0006) and digested with 1% trypsin (weight/weight) (trypsin from bovine pancreas, Sigma #T1005) at 37 °C for 72 hr. The Cry toxins were then purified by FPLC (after 72 hr, the trypsin is fully degraded).

Cry toxin purification

The Cry toxins produced from E. coli (activated or not) were purified by FPLC (Äkta, UPC900/P920/INV907/FRAC950) on a 1 mL benzamidine column (HiTrap Benzamidine FF (high sub), GE Healthcare #17-5143-01) with PBS1X as charge buffer, PBS1X, 1 M NaCl as buffer for non-specific link and 100 mM glycin pH 3 as elution buffer.

Fly stocks and genetics

The following stocks are listed at the Bloomington Drosophila Stock Center (https://bdsc.indiana.edu/): WT canton S (#64349). w; Sco/CyO; tub-GAL80^ts/TM6b (#7018). w; tub-GAL80^ts; TM2/TM6b (#7019). w; esg-GAL4^NP5130 (#67054). w; UAS-GFP/TM3 Sb (#5430). w; UAS-shg-R (DE-Cadherin)(#58494); y w, shg::Tomato (#58789).

Other stocks

w;; Dl-GAL4/TM6b (Zeng et al., 2010). w; tub-GAL80^ts; Dl-GAL4 UAS-GFP/TM6b (this study). w; esg-GAL4^NP5130 UAS-GFP (Shaw et al., 2010). w; esg-GAL4^NP5130 UAS-GFP; tubGAL80^ts (Apidianakis et al., 2009). w; Su(H)GBE-GAL4, UAS-CD8::GFP (Zeng et al., 2010). w; Su(H)GBE-GAL4/SM6β; tub-GAL80^ts UAS-GFP/TM6b (this study). w; myo1A-GAL4 and w; myo1A-Gal4; tubGal80ts UAS-GFP/TM6b (Shaw et al., 2010). w; myo1A-GAL4 UAS-GFP/CyO (Apidianakis et al., 2009). w; UAS-GFP::CD8; UAS-H2B::RFP/TM2 (Antonello et al., 2015). w; UAS-CD8::GFP; UAS-H2B::RFP, tub-GAL80^ts/TM2 (Antonello et al., 2015). w; esg-GAL4, UAS-CD8::GFP; UAS-H2B::RFP, tub-GAL80^ts/TM6b (Antonello et al., 2015). w; UAS-CD8::GFP; Dl-GAL4, UAS-H2B::RFP/TM6b (this study). w;; UAS-GC3Ai^G7S (UAS-Casp::GFP) (Schott et al., 2017). w; UAS-connectin (Zhai et al., 2017). UAS-p35 (Amcheslavsky et al., 2009).

Cell lineage

Dl-ReDDM experiments

w; tub-GAL80^ts; Dl-GAL4 UAS-GFP/TM6b females were crossed to w; UAS-GFP::CD8; UAS-H2B::RFP/TM6 males at 18 °C. Progeny were kept at 18 °C until emergence. Flies were transferred at 25 °C for 5 days before infection, and then transferred at 29 °C for 2 days (Figure 1—figure supplement 1C).

esg-ReDDM experiments

w; esg-GAL4 UAS-GFP; UAS-H2B::RFP, tub-GAL80^ts/TM6b females were crossed to WT males at 18 °C. Progeny were kept at 18 °C until emergence. Flies were transferred at 25 °C for 5 days before infection, and then transferred at 29 °C for 3 days (Figure 3).

Su(H)-ReDDM experiments

w; Su(H)-GAL4/SM6β; tub-GAL80^ts UAS-GFP/TM6b females were crossed to w; UAS-GFP::CD8; UAS-H2B::RFP/TM6 males at 18 °C. Progeny were kept at 18 °C until emergence. Flies were transferred at 25 °C for 5 days before infection, and then transferred at 29 °C for 3 days (Figure S3).

DE-Cadherin and Connectin overexpression

w; esg-Gal4 UAS-GFP; UAS-H2B::RFP, tub-GAL80^ts/TM6b females were crossed to UAS-DE-Cadherin males. Progeny were kept at 18 °C until emergence. Flies were transferred at 25 °C for 5 days before infection, and then transferred at 29 °C for 3 days (Figure 5 Figure 5—figure supplement 1 and Figure 5—figure supplement 2).

Drosophila rearing and oral infection

Drosophila were reared on standard medium (0.8% Agar, 2.5% sugar, 8% corn flour, 2% yeast) at 25 °C with a 12 hr light/12 hr dark cycle. For oral infection, after 2 hr of starvation to synchronize the food intake, 5- to 6-day-old non-virgin females were transferred onto a fly medium vial covered with a filter disk soaked with water (control) or a suspension of spores (corresponding to 10⁶ CFU of spores per 4 cm² and per individual female; Loudhaief et al., 2017; Nawrot-Esposito et al., 2020), crystals, protoxins, or activated toxins. The quantity of crystals, protoxin, or activated toxins deposited on the filter disc corresponded to 30% of the spore weight, Btk crystals representing between 25% and 30% of the total weight of the 1:1 spore/crystal mix (Agaisse and Lereclus, 1995 Monro, 1961 ; Murty et al., 1994). Flies were kept feeding on the contaminated media until dissection in all the experiments.

Dissection, immunostaining and microscopy

Dissection, fixation and immunostaining were performed as described by Micchelli, 2014. Dilutions of the various antibodies were: mouse anti-Armadillo N27A1 at 1:50 (DSHB), mouse anti-Connectin-C1-427 at 1/200 (DSHB), mouse anti-Prospero MR1A at 1:200 (DSHB), rabbit anti-PH3 at 1:1000 (Millipore, 06–570), Rabbit anti-Cleaved Caspase-3 at 1/600 (Cell Signalling Asp175 #9661), Goat anti-mouse AlexaFluor-647 at 1/500 (Molecular Probes Cat# A-21235), Goat anti-mouse AlexaFluor-546 at 1/500 (Molecular Probes Cat# A-11003), Goat anti-rabbit AlexaFluor-647 at 1/500 (Thermo Fisher Scientific Cat# A32733), Goat anti-rabbit AlexaFluor-546 at 1/500 (Thermo Fisher Scientific Cat# A-11010). For microscopy, guts were mounted in Fluoroshield DAPI medium (Sigma, # F6057) and immediately observed on a Zeiss Axioplan Z1 with Apotome 2 microscope. Images were analyzed using ZEN (Zeiss), ImageJ and Photoshop software. Image acquisition was performed at the microscopy platform of the Sophia Agrobiotech Institute (INRAE1355-UCA-CNRS7254 – Sophia Antipolis).

DNA constructs

The full-length expression construct of DE-cadherin Full Length fused to GFP (DEFL) was introduced into pUAST as previously described (Oda and Tsukita, 1999).

Cell aggregation assay

Drosophila Schneider S2 cells (S2-DRSC, DGRC Stock 181; https://dgrc.bio.indiana.edu//stock/181) were obtained from DGRC (Drosophila Genomics Resource). We tested this cell line for contamination of mycoplasma (MycoFluor Mycoplasma Detection Kit, Invitrogen). We have not tested cell line identity because (1) S2 is the only cell line used in our lab and in a dedicated culture cell room, (2) this is the only Drosophila cell line present at the institute, (3) all experiments were conducted using the same batch of initial frozen ampoule received from the DGRC, and (4) transfection results were compared with control data experiment conducted in the same culture passage at the same time. S2 cells were cultured in Schneider’s medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 25 °C in a non-humidified ambient air-regulated incubator (Gallet et al., 2006). For S2 aggregation assay, 2.2 10⁶ cells were plated in 25 cm² flask for each condition. After 6 hr, transient transfection was performed by mixing transfection reagent (TransIT–2020; Mirus Bio) with a reagent-to-DNA ratio of 3:1. A total of 3 µg plasmid DNA per T25 was used, corresponding to a 5:1 mixture of pUAST-DEFL and pWA-GAL4. Approximately 46 hr after transfection, the cells were collected into 15 mL tubes and centrifuged for 5 min at 400 g. The pellet was resuspended in 2 mL of fresh medium supplemented in CaCl₂ to obtain a final concentration of 7.4 mM and separated into single cells by repeated pipetting. Of this cell suspension, 500 µL were added to a well of a 24-well microplate. To allow cell-cell adhesion (aggregation), the microplate was placed under constant agitation on a rotary platform at 150 rpm at 25 °C for the indicated time (1 hr, 2 hr, and 3 h) with or without protoxins (at a final concentration 35 µg/mL). Cell aggregates formed in the wells were observed using an inverted Fluorescent microscope (Nikon, Eclipse TE2000-U). Images for the florescence of GFP was acquired using a CCD camera (ORCA ER, Hamamatsu Photonics). The same parameter settings were used to acquire images (objectives, gain, exposure time …). The area of fluorescent aggregates and individual cells were measured using Fiji software (Schindelin et al., 2012). The average area of a S2 cell in our condition is about 15.5 µm². Hence, quantification of the aggregates area was performed excluding all objects smaller than 15 µm². The mean area of aggregates were calculated after background subtraction. Three independent experiments were performed for each condition. Values in µm² were represented in GraphPad Prism 7 software as scatter-plot view. Statistical analysis was conducted using GraphPad Prism 7 software. The significance of the difference between CTR and exposed conditions was assessed using one-way ANOVA and Tukey’s post hoc tests. Statistical parameters for each experiment can be found within the corresponding figure legends.

Western blot

Figure 6D Twenty Drosophila (5–6 day-old non-virgin females) were orally infected with spores and reared at 25 °C with a 12 hr/12 hr day/night cycle for the indicated time. At the same time, 10⁷ CFU of spores in 50 µL of sterile water were incubated in the same conditions. After 4, 24, 72, or 144 hr, Drosophila midguts were dissected in PBS1x with anti-proteases (cOmplete Tablets EDTA free EASY Pack, Roche #04693132001). Then midguts were transferred on ice into a 2 ml microtube containing 200 µL of PBS1X with anti-proteases and crushed one minute at 30 Hz with a Tissue Lyser (Qiagen, Tissue Lyser LT).

Figure 6E Twenty Drosophila (5–6 day-old non-virgin females) were orally infected with water or purified crystals and reared at 25 °C with a 12 hr/12 hr day/night cycle for the indicated time. Flies were dissected and anterior and posterior regions of midguts were separated and lysed in a hypotonic buffer (25 mM HEPES, pH7.5, 5 mM MgCl₂, 5 mM EDTA, 5 mM DTT, 2 mM PMSF, 10 µg/mL leupeptin, 10 µg/mL pepstatin A) on ice. Midguts were crushed one minute at 30 Hz with the Tissue Lyser. Homogenates were centrifuged at 20,000 g for 45 min at 4 °C. The supernatant was the soluble fraction (considered as cytosol-enriched fraction) and the pellet was the insoluble fraction (containing membranes).

Proteins were dosed by Bradford method (Protein Assay Dye Reagent Concentrate, Biorad #500–0006). Twenty µg of midgut proteins and 2.10⁴ CFU of spores were deposited on a 8.5% SDS-polyacrylamide gel. After migration at 100 V for 1h30, proteins were transferred onto a PVDF membrane (Immobilon-P Membrane, Millipore #IPVH00010) (120mA/gel, 1 hr) in a semi-dried transfer system with a transfer buffer (200 mM glycine, 25 mM Tris base, 0.1% SDS, pH7.4, 20% methanol). Membranes were saturated with 5% milk in TBS-T (140 mM NaCl, 10 mM Tris base, 0.1% Tween 20, pH 7.4) for 1 hr and incubated overnight at 4 °C with a homemade anti-Cry1A antibody (Babin et al., 2020) or anti-actin monoclonal antibody (ACTN05, C4, Thermo Fisher Scientific) diluted at 1/7500 in TBS-T 3% BSA. After three washes of 10 min each with TBS-T, membranes were incubated with an anti-rabbit antibody coupled with HRP (Goat Anti-Rabbit (IgG), Invitrogen #G21234) diluted 1/7500 in TBS-T 2% milk for 1 hr at room temperature. Membranes were rinsed three times with TBS-T and once with TBS. Western blots were revealed with enhanced chemiluminescence (luminol and hydrogen peroxide, homemade) on an autoradiographic film (Amersham Hyperfilm ECL, GE Healthcare #28906837).

Measurement, counting, and statistical analysis

In all the data presented, the pictures and counting were always performed in the posterior part of the R4 region (http://flygut.epfl.ch/) named R4bc in the flygut site (see also Buchon et al., 2013; Marianes and Spradling, 2013; Figure 1—figure supplement 1A). Experiments were independently repeated at least three times.

Cell type counting

midgut images were taken at ×40 magnification within the R4bc region with same microscope settings. A region of interest (ROI) of about 20,000 μm² were applied in which all cells were DAPI labelled. We utilized the Zeiss ZEN 2 (blue edition) to quantify cell number positive for a given marker. Data were represented as cell density (e.g. number of cells/20,000 μm²) except in Figure 1—figure supplement 2 where ratio of each cell type was represented over total cell number. For a given cell type, when we noticed changes (increase or decrease) in both cell density and ratio, we assumed that this corresponded to global changes in the so-called cell type number.

Cell junction intensity measurement

For both Arm or Tomato::DE-Cad labelling, we compared the average staining intensity at the junction between neighboring progenitors with the average intensity around the rest of cell membrane and then calculate the ratio between both values (see Figure 4—figure supplement 1F, G). Only junctions between individual paired cells were analyzed (we excluded clusters containing more than two cells). To assess junction strengths, we fixed the following scale: a weak junction when the ratio of staining intensity was <1.4, a mild junction when the ratio was between 1.4 and 1.6 and a strong junction when the ratio was >1.6.

Statistics

Effects of treatments were analyzed using Kyplot or GraphPad Prism 7. When ‘n’ was equal or superior to 30, statistical analysis was performed using a parametric t-test. An F-test was systematically done before applying the t-test to verify the homogeneity of variances. When ‘n’ was inferior to 30, we used the non-parametric pairwise comparisons of the Mann-Whitney test. Differences were considered significant when *p≤0.05, **p≤0.01, ***p≤0.001. Error bars in all the graphics correspond to standard error of the mean (SEM).

Appendix 1

Appendix 1—key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
strain, strain background (B. thuringiensis)	Btk SA-11	Isolated from commercial product	Delfin
strain, strain background (B. thuringiensis)	Btk^∆Cry	BGSC	#4D22
strain, strain background (B. thuringiensis)	Btk producing Cry1Ac	BGSC	#4D4
strain, strain background (B. thuringiensis)	Btk producing Cry1Ab	This study		Materials and Methods: Generation of Btk^Cry1Ab
strain, strain background (Escherichia coli)	producing Cry1Ab	BGSC	#ECE54
strain, strain background (Escherichia coli)	producing Cry1Ac	BGSC	#ECE53
strain, strain background (Escherichia coli)	producing Cry2Aa	BGSC	#ECE126
genetic reagent (D. melanogaster)	WT canton S	https://bdsc.indiana.edu/	#64349
genetic reagent (D. melanogaster)	w; Sco/CyO; tub-GAL80^ts/TM6b	https://bdsc.indiana.edu/ M. Vidal	#7018
genetic reagent (D. melanogaster)	w; tub-GAL80^ts; TM2/TM6b	https://bdsc.indiana.edu/	#7019
genetic reagent (D. melanogaster)	w;; Dl-GAL4/TM6b	S. Hou and X. Zeng; Zeng et al., 2010
genetic reagent (D. melanogaster)	w; tub-GAL80^ts; Dl-GAL4 UAS-GFP/TM6b	This study		Can be obtained from Gallet's lab
genetic reagent (D. melanogaster)	w; esg-GAL4^NP5130	https://bdsc.indiana.edu/ N. Tapon	#67054
genetic reagent (D. melanogaster)	w; esg-GAL4^NP5130 UAS-GFP	N. Tapon; Shaw et al., 2010
genetic reagent (D. melanogaster)	w; esg-GAL4^NP5130 UAS-GFP; tubGAL80^ts	Y. Apidianakis; Apidianakis et al., 2009
genetic reagent (D. melanogaster)	w; Su(H)GBE-GAL4, UAS-CD8::GFP	M. Vidal; Zeng et al., 2010
genetic reagent (D. melanogaster)	w; Su(H)GBE-GAL4/SM6; tub-GAL80^ts UAS-GFP/TM6b	This study		Can be obtained from Gallet's lab
genetic reagent (D. melanogaster)	w; myo1A-GAL4	N. Tapon; Shaw et al., 2010
genetic reagent (D. melanogaster)	w; myo1A-GAL4 UAS-GFP/CyO	Y. Apidianakis; Apidianakis et al., 2009
genetic reagent (D. melanogaster)	w; UAS-GFP/TM3 Sb	https://bdsc.indiana.edu/	#5430
genetic reagent (D. melanogaster)	w; UAS-GFP::CD8; UAS-H2B::RFP/TM2	T. Reiff and M. Dominguez; Antonello et al., 2015
genetic reagent (D. melanogaster)	w; UAS-CD8::GFP; UAS-H2B::RFP, tub-GAL80^ts/TM2	T. Reiff and M. Dominguez; Antonello et al., 2015
genetic reagent (D. melanogaster)	w; esg-GAL4, UAS-CD8::GFP/CyO; UAS-H2B::RFP, tub-GAL80ts/TM6b	T. Reiff and M. Dominguez; Antonello et al., 2015
genetic reagent (D. melanogaster)	w; UAS-CD8::GFP; Dl-GAL4, UAS-H2B::RFP/TM6b	This study		Can be obtained from Gallet's lab
genetic reagent (D. melanogaster)	w;; UAS-GC3Ai^G7S (UAS-Casp::GFP)	M. Suzanne; Schott et al., 2017
genetic reagent (D. melanogaster)	w; UAS-shg-R (DE-Cadherin)	https://bdsc.indiana.edu/	#58494
genetic reagent (D. melanogaster)	w; UAS-connectin	JP Boquete and B. Lemaitre; Zhai et al., 2017
genetic reagent (D. melanogaster)	y w, shg::Tomato	https://bdsc.indiana.edu/	#58789.
genetic reagent (D. melanogaster)	w;UAS-p35	Tony Ip; Amcheslavsky et al., 2009
genetic reagent (D. melanogaster)	Dl-ReDDM (w/w; UAS-CD8::GFP/UAS-CD8::GFP; Dl-GAL4, UAS-H2B::RFP/UAS-H2B::RFP, tub-GAL80^ts)	This study		Can be obtained from Gallet's lab
genetic reagent (D. melanogaster)	esg-ReDDM (w/w⁺; esg-GAL4, UAS-CD8::GFP/+; UAS-H2B::RFP, tub-GAL80^ts/+)	This study		Can be obtained from Gallet's lab
genetic reagent (D. melanogaster)	Su(H)-ReDDM (w/w; Su(H)-GAL4/UAS-GFP::CD8; tub-GAL80^ts UAS-GFP/UAS-H2B:RFP)	This study		Can be obtained from Gallet's lab
cell line (D. melanogaster)	Drosophila melanogaster Schneider 2 (S2) cells	S2-DGRC Stock 181	RRID:CVCL_Z992
antibody	Mouse monoclonal anti-Armadillo (ß-catenin) antibody	DSHB	Cat# N27A1 RRID:AB_528089	1/50
antibody	Mouse monoclonal anti-Connectin antibody	DSHB	Cat# Connectin C1.427, RRID:AB_1066083	1/200
antibody	Mouse monoclonal anti-Prospero antibody	DSHB	Cat# MR1A RRID:AB_528440	1/200
antibody	Rabbit polyclonal anti-Cleaved Caspase-3 (Asp175) antibody	Cell Signalling	Cat# 9661 RRID:AB_2341188	1/600
antibody	Rabbit polyclonal anti-phospho-Histone H3 (Ser10) antibody	Millipore	Cat# 06–570 RRID:AB_31017	1/1000
antibody	Rabbit polyclonal anti-Cry1A antibody	Babin et al., 2020		WB: 1/7500 i
antibody	Mouse monoclonal anti-actin antibody (ACTN05, C4) antibody	Invitrogen	Thermo Fisher Scientific Cat# MA5-11866, RRID:AB_10985365	WB: 1/2000
antibody	Goat anti mouse IgG (H+L) secondary antibody, AlexaFluor-647	Invitrogen	Molecular Probes Cat# A-21235, RRID:AB_2535804	1/500
antibody	Goat polyclonal anti mouse IgG (H+L) secondary antibody, AlexaFluor-546	Invitrogen	Molecular Probes Cat# A-11003, RRID:AB_141370	1/500
antibody	Goat polyclonal anti-rabbit IgG (H+L) secondary antibody, AlexaFluor-647	Invitrogen	Thermo Fisher Scientific Cat# A32733, RRID:AB_2633282	1/500
antibody	Goat polyclonal anti-rabbit IgG (H+L) secondary antibody, AlexaFluor-546	Invitrogen	Thermo Fisher Scientific Cat# A-11010, RRID:AB_253407	1/500
recombinant DNA reagent	pUAST-DECadherintagged with GFP (DEFL)	Oda and Tsukita, 1999		Materials and Methods: Cell aggregation assay
recombinant DNA reagent	pWA-Gal4	Gift from L. Ruel
Software, algorithm	Image J	http://imagej.nih.gov	RRID:SCR_003070
Software, algorithm	Fiji	http://fiji.sc	RRID:SCR_002285
Software, algorithm	ZEN 2 (blue edition)	Zeiss
Software, algorithm	Photoshop CS2	Adobe
Software, algorithm	GraphPad Software	GraphPad Prism	RRID:SCR_002798	GraphPad Prism 7.0
Chemical compound, drug	PBS 10 x	Euromedex	ET330
Chemical compound, drug	Formaldehyde 16%	Thermo Fisher Scientific	Cat# 28908
Chemical compound, drug	Fluoroshield-DAPI	Sigma	Cat# F6057
Chemical compound, drug	Tween 20	VWR	Cat# 28829.296
Chemical compound, drug	Acrylamide/Bis-acrylamide	Sigma	Cat# A3699
Commercial assay, kit	Invitrogen MycoFluor Mycoplasma Detection Kit	Thermo Fisher Scientific	Cat# 10063202
Other	Amersham Hyperfilm	GE Healthcare	Cat# 28906837	Commercial product
Other	Bovin Serum Albumin	Sigma	Cat# A9647	Commercial product
Other	Schneider’s insect medium	Sigma-Aldrich	Cat# S0146	Commercial product
Other	TransIT–2020	Mirus Bio	Cat# MIR5400	Commercial product
other	Zeiss Axioplan Z1 with Apotome 2 microscope	Zeiss		Microscope
other	Zeiss Confocal LSM 810	Zeiss		Microscope

Data availability

Genomic DNA of BtkCry1Ab and BtkSA11 was sequenced and is available at NCBI accession SRR17044437 and SRR17036893, respectively. All data generated or analysed during this study are included in the manuscript and supporting files.

The following data sets were generated

1. Fichant A
2. Bonis M
(2023) NCBI Sequence Read Archive
ID SRR17044437. SA11.

https://www.ncbi.nlm.nih.gov/sra/?term=%09SRR17044437
1. Fichant A
2. Bonis M
(2023) NCBI Sequence Read Archive
ID SRR17036893. SA11cry1Ab.

https://www.ncbi.nlm.nih.gov/sra/?term=SRR17036893

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Decision letter

Lucy Erin O'Brien

Reviewing Editor; Stanford University School of Medicine, United States
Arturo Casadevall

Senior Editor; Johns Hopkins Bloomberg School of Public Health, United States
Karen Bellec

Reviewer; University of Glasgow, United Kingdom
Young Kwon

Reviewer; University of Washington, United States
Yiorgos Apidianakis

Reviewer; University of Cyprus, Cyprus

Our editorial process produces two outputs: i) public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Arturo Casadevall as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Karen Bellec (Reviewer #1); Young Kwon (Reviewer #2); Yiorgos Apidianakis (Reviewer #3).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Essential revisions:

1. In several instances (e.g. Figure 1D, Figure 2 C and D), conclusions about changes in numbers of cells are based upon plots that count number of cells per unit area. However, cell number per unit area is a measure of cell density, not overall cell number. Please either provide total cell counts for a defined midgut region (e.g. R4), or else modify these conclusions. Alternatively, the ratio of particular cell types to total cells could be helpful as a measure of the relative abundance of different cell populations. In addition, could images that illustrate overall organ morphology be included? Such information would provide a sense of the relative sizes of the organs.

2. Please provide evidence to support the use of the Δ-ReDDM system as a readout of symmetric versus asymmetric fate outcomes. (a) Does degradation of the ReDDM GFP signal occur in the same time frame as activation of the enteroblast-specific SuH marker? (For instance, is ReDDM GFP signal absent from SuH-lacZ cells in both control and experimental conditions?) (b) Cell contact was used to classify two progenitor cells as siblings from the same mother stem cell division. Please either justify this claim or else revise the interpretation of these data. (c) Can you respond to Reviewer 3's question about co-expression of two GFP transgenes in the Δ-ReDDM genotypes?

3. Please address the Reviewers' requests for improved data presentation. (individual values in graphs, scale bars in images, loading controls for Westerns, etc). In addition, please quantify the correlation between Tomato::DE-Cad and EEP markers (described in lines 283-284).

4. The Reviewers raised questions about visibility and interpretation of the Arm, E-cad, and caspase signals in multiple figure panels. In cases where improved visibility would be helpful, please present split channel images. In cases where alternative interpretations are raised (e.g., could Arm levels be affected by proliferation rate?), please evaluate these alternatives either through new data or textual discussion.

5. Please clarify the methodological details requested by the Reviewers. (a) Better descriptions are needed of how quantitative analyses were performed. (b) In some cases, the criteria used to identify particular cell types or populations were ambiguous (e.g. new cells versus old cells, sibling cells versus neighbor cells, diploid enteroblasts identified via nuclear size), (c) To avert reader confusion, might you consider referring to enteroendocrine cells by the standard abbreviation EE, rather than EEC?

Reviewer #1 (Recommendations for the authors):

1) Data representation

Different figures should be modified to improve the representation of the data all along the manuscript:

– Authors should plot individual values in the graphs shown all along the manuscript instead of using histograms.

– Scale bars should be included both in the pictures and in the legends.

– Figure 5G-G': errors bars should put in another color. The scale of the graph could be changes to have a better overview of the distribution of the different points.

For each figure, the signification of the error bars (sd or sem, etc..) should be included in the legends and/or in the Material and Methods.

2) Experiments

– Loading controls of the Western blots are missing: authors should provide a control to indicate that the same amount of sample has been loaded in each condition by staining another protein. This is particularly true for the Western blots shown in Figure 6D and Figure 6E

– Figure S1A-S1B: How were the authors able to confirm that two cells came from the same mother cell? This question is particularly true for BtkSA11 where there is a lot of neighboring labelled cells. Details about the way to quantify should be added in the Material and Methods (quantification in a small area only? n=number of divisions analyzed?)

Same figure: The picture of Btk∆Cry seems to show lot of symmetric cell divisions giving rise to cells that are GFP+RFP+Pros- (=ISCs) but surprisingly, the Figure 1C does not show this increase.

– L181: Authors conclude: "Therefore, consistent with previous studies, the mode of ISC division appeared to reflect the degree of intestinal damage, the symmetric mode being preferentiality adopted when the damage was greater (i.e., when cell death occurred)." This mode of division seems to be also preferentially adopted when there is intestinal damage but no cell death, as in the case of Btk∆Cry.

Reviewer #2 (Recommendations for the authors):

Overall, the manuscript is in a good shape.

Reviewer #3 (Recommendations for the authors):

The described work is about assessing Drosophila midgut histopathology upon consumption of an entomopathogenic strain of B. thuringiensis and its Cry1A toxins, which are lethal to lepidoptera, but non-lethal to Drosophila. Thus, Drosophila is characterized a non-susceptible organism. The authors tested if this "non-susceptible host" is nevertheless histopathologically susceptible. They convincingly show that it is, because the mechanism of action of the Cry1A toxins on progenitor cell E-Cadherin is functionally (but not biochemically) revealed in flies and in Drosophila S2 cells.

To improve the manuscript, I suggest the following:

1. Add "Drosophila midgut" in the title, because generalizations need to be omitted.

2. Lines 89-92: reference to B. cereus should not add to the proof about Btk. So need to phrase this accordingly. Different species and different strains have different effects.

3. Figure 1A: how do Caspase 3 sensor and Caspase 3 antibody compare? While both are of a value, the authors measure the first, but show the latter. Why?

4. Dl-ReDDM experiments: The M and Ms read "w; tub-GAL80ts; Dl-GAL4 UAS-GFP/TM6b females were crossed to w; UAS-GFP::CD8; UAS-H2B::RFP/TM6 males". This means that the progeny express two different UAS-GFP transgenes, right? This issue applies also to ReDDM experiments with the other GAL4 lines. What is the perdurance of UAS-GFP in each case and in combination with UAS-GFP::CD8?

5. Line 224: Why the authors chose esg-ReDDM flies instead of Dl-ReDDM? The latter makes clear what ISC is, while esg based ReDDM confuses the distinction between ISC and EB.

6. Line 229: "EBs were GFP+ RFP+ DAPI+ with bigger nuclei". Why bigger? EBs are presumably diploid.

7. Figure S3: The terms "old cells" and "new cells" seems misleading, because by "old cells" the authors mean EEs of Su(H)-independent origin and by "new cells" EEs of Su(H)-dependent origin.

8. Lines 293-4: "the decrease in Tomato::DE-Cad 283 labelling between an ISC and its progenitor was correlated with the expression of EEP markers". The authors should measure the extend of this correlation. Also, neighboring esg+ cells do not equal "ISC and its progenitor".

9. The following statement is imprecise "we no longer observed an increase in the number of EECs (blue arrows in Figure 5A-D, and Figure 5E)". Comparing Figure 3F and Figure 5E, there is an increase in EEs in both cases, but the increase is more prominent in 3F.

10. I suggest replacing the term EEC(s) with EE(s) to abide to previous nomenclature and make it easier to distinguish from the term EEP(s).

11. Figure panels 5G and S5I are missing from the PDF available for this review.

12. Figure S2I ("old" and "new" pros+ cells are plotted together) refers to the anterior midgut only and to see what is going on in the posterior is unclear (information is not plotted). One needs to extrapolate maybe by comparing with Figure 3F ("old" and "new" pros+ cells are plotted separately).

13. Lines 363-4. To explain the discrepancy between the anterior and the posterior midgut in terms of EEs and their progenitors, the authors should resort to Tamamouna et al. 2020 (PMID: 32513656) and Marianes and Spradling 2013 (PMID: 23991285), where such gut compartmentalization and anterior vs. posterior mitosis and propensity for EE and ISC accumulation has been thoroughly discussed.

14. Line 492. What does "fortuitously mean? How was the selection process arranged? Was there a random mutagenesis involved?

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila midgut" for further consideration by eLife. Your revised article has been evaluated by Arturo Casadevall (Senior Editor) and by Lucy O'Brien (Reviewing Editor), in consultation with the three original reviewers.

We all agreed that the revised manuscript addresses many, but not all, of the Essential Revision Items raised in the first round of review. In particular, concerns about cell density (Essential Revision Item #1) have been addressed, and the manuscript is much improved in terms of the data presentation and methodological detail (Essential Revision Items #3-5).

However, the overall consensus was that fundamental concerns still remain about the classification of division fate outcomes in the manuscript (Essential Revision Item #2). The authors were requested to perform two important controls to justify their classification: (1) examine whether degradation of the Δ-driven ReDDM GFP signal occurs in the same time frame as activation of an enteroblast-specific SuH marker, such as Su(H)-lacZ, and (2) justify their claim that progenitor cells that are in contact are sibling cells. As we explain in detail below ("Essential Revision Item #2 – Rationale"), the Response does not adequately address either of these points. Hence, we are not able to recommend the publication of the manuscript in its current form.

In light of the fact that fate outcomes are only one aspect of the study's overall conclusions, and that experiments to demonstrate division fate outcomes would represent substantial additional effort, we may consider publication if the authors were willing to revise their manuscript so as to refrain from drawing conclusions regarding symmetric and asymmetric fates. (It would be acceptable to include a speculative statement about fate outcomes, clearly indicated as such.) Without textual revisions that address these issues, or without new, direct evidence as originally requested, we are sorry that we are unable to move forward with this manuscript.

In addition, a few other items arose during the consultation; these can all be addressed by textual revision:

– Findings from prior studies described in lines 167-171 are described inaccurately (see explanation below), and two papers that contradict the authors' assertion were not cited.

– Other portions of the manuscript, such as the Figure 7 results, draw conclusions that are not supported by data. Careful revision of the study's results to avoid over-interpretation and to make clear distinctions between data-based conclusions and interesting, but speculative, remarks will also be necessary for a revised manuscript to be considered further.

– The title appears to be missing a "the". Two grammatically correct alternatives would be:

"Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in the Drosophila midgut" or "Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila"

Essential Revision Item #2 – Rationale

(1) In terms of Point (1) above, the authors did not perform the requested experiment to examine whether degradation of the Δ-driven ReDDM GFP signal occurs in the same timeframe as activation of an enteroblast-specific SuH marker. Instead, they offer two alternative justifications for the validity of their current approach. However, as detailed below, neither of these justifications is valid.

The first justification the authors give for drawing conclusions about cell identity from ReDDM hinges on the half-life of GFP in the ReDDM system being less than 24 hours. The authors cite Antonello 2015 as evidence for the < 24-hour half-life, but a quantitative measurement of GFP half-life in the Antonello study could not be identified in either the main figures or the supplemental figures. (If this is mistaken, please point out where these data are found in the paper.) In addition, the authors also claim that since they examined tissues two days after ReDDM induction, the GFP transmitted to most daughter cells has begun to be degraded. This statement appears to assume that all cell divisions happened at the time that ReDDM was induced. However, this assumption is erroneous. Stem cells continue to cycle throughout the two-day post-induction period, and with fixed tissues, it cannot be ascertained when during the two-day period a division actually occurred. It is likely that some stem cell divisions occurred a short time prior to fixation.

The second justification that the authors provide is two images of DeltaGAL4-driven ReDDM showing that, following feeding with the Btk mutant, there is an increased number of GFP-/RFP+ cells compared to control guts. In both images, the authors designate the GFP-/RFP+ cells as enteroblasts and the GFP+/RFP+ cells as stem cells, and they conclude there are more enteroblasts following Btk treatment because there are more red cells. Without a positive marker of enteroblast fate, however, the authors' reasoning is neither scientifically nor logically valid. Crucially, enteroblasts are not defined by the absence of Δ (for instance, loss of Δ does not result in loss of stem cells, but rather results in excess stem cells and enteroendocrine cells -- Ohlstein 2007 Science). Instead, enteroblasts are defined by the activation of Notch targets such as the Su(H)GBE Notch reporter. Hence Notch activation and Δ downregulation are not necessarily synchronous. Rather, Δ downregulation may lag behind Notch activation, such that new enteroblasts transiently exist in a Δ-positive, Notch-activated state. Indeed, Zhai 2017 PLoS Geneticsand Tian 2022 EMBO J have found that under injury conditions there is a marked increase in Su(H)+ enteroblasts that are also Δ+.

(2) In terms of Point (2) above, the authors do not present data to support the claim that a GFP-/RFP+ cell adjacent to a GFP+/RFP+ cell is the division progeny of the GFP+/RFP+ cell, and they state that two days after induction is too short a time frame for enteroblasts to come into contact with a stem cell that is not its mother stem cell. By contrast, Martin 2018 (eLife), which analyzed stem cell divisions in live movies, found that approximately one-third of sibling cells separate from each other after birth. The duration of these movies was ~16 hours, which is much shorter than the 2-day period the authors used. In addition, Martin et al. observed that some of these sibling cells subsequently associated with other progenitor cells. These findings undermine the authors' assertion and underscore the importance of performing lineage tracing to deduce lineage relationships. While we appreciate that some other journals have previously published midgut studies that assume progenitor cell pairs are siblings, our view is that publication in eLife requires more rigorous evidence such as lineage tracing or direct monitoring of live divisions.

Finally, the statement that "upon environmental challenges, ISCs divide symmetrically" (Author's response and manuscript lines 167-171) is not supported by the literature. The deNavascues 2012, Goulas 2012, Perdigoto 2011, and Tian 2014 studies examine homeostasis only; hence, these results do not speak to what happens when there are environmental challenges. In addition, Goulas and Perdigoto do not explicitly examine asymmetric versus symmetric division fate outcomes. O'Brien 2011 examined feeding-induced growth, which is not an environmental challenge but rather a normal physiological mechanism; hence, these results also do not speak to what happens upon environmental challenges. Although Zhai 2017 speculates on symmetric fate outcomes in the discussion, no conclusions are drawn about the fate outcomes of cell pairs in the data presented in the Results section.

By contrast, there are two studies that did examine division fate outcomes in injured guts, and that were not cited in either the Response or the manuscript: Jin 2017 (Stem Cell Reports) and Tian 2017 (PNAS). Both studies used Twin-spot MARCM – the gold standard for symmetric/asymmetric division fate outcomes – and found that the majority of divisions still exhibit asymmetric fate outcomes following injury. Their injury conditions of Pseudomonas infection and bleomycin ingestion are arguably in a similar realm to the current study's Btk treatment. Their results contradict the authors' assertion that stem cells divide symmetrically following environmental challenges and raise the possibility the authors' method is inaccurate.

Additional remarks from individual reviewers follow below:

Reviewer #1 (Recommendations for the authors):

Thanks to the authors for addressing the questions raised in the previous reviews and for their detailed answers.

In the previous reviews, we highlighted the lack of some quantifications; controls, and information about the methods of analysis for some key experiments (Arm staining; rescue experiments..). The authors addressed all the questions when experimentally possible and modified the text accordingly. They also modified the graphs as suggested. Altogether, these modifications greatly improved the manuscript and reinforced the conclusions proposed by the authors.

Reviewer #2 (Recommendations for the authors):

The authors have appropriately addressed most of the comments. However, I have a concern regarding the results presented in Figure 7. The results raise interesting ideas about how toxins might reach ISCs. The presented experiments do not directly test the ideas. Therefore, the conclusions are not appropriately supported. I suggest the authors revise the text to clearly separate the conclusions drawn from the experimental data and their speculations. Alternatively, the whole data can be removed as those are not essential for the story.

Reviewer #3 (Recommendations for the authors):

Taking the authors' words (in their response to reviewers): "ISCs mostly divide asymmetrically while upon environmental challenges ISCs divide symmetrically (de Navascues et al., 2012; Goulas et al., 2012; O'Brien et al., 2011; Perdigoto et al., 2011; Tian and Jiang, 2014; Zhai et al., 2017)". Thus, being responsive to Bacillus thuringiensis infection and its Cry1A toxins does not equal B. thuringiensis being a hazard to Drosophila or other flies. Such treatments merely elicit a defense response against damaging agents, which are mild enough for flies to cope with (survive). I suggest the authors refrain from conclusions (should there be any) about B. thuringiensis being very hazardous to flies.

https://doi.org/10.7554/eLife.80179.sa1

Author response

Essential revisions:

1. In several instances (e.g. Figure 1D, Figure 2 C and D), conclusions about changes in numbers of cells are based upon plots that count number of cells per unit area. However, cell number per unit area is a measure of cell density, not overall cell number. Please either provide total cell counts for a defined midgut region (e.g. R4), or else modify these conclusions.

In our analysis, we generally took images in the middle of the R4 region (in general 2 pictures/gut) to avoid variations due to differences in R4 length or to mis determination of the R4 region boundaries. Then we performed our cell counting in a region of interest (ROI) of about 20.000µm² because this corresponds to the mean analyzable surface on a picture taken at the 40X magnification.

We provided more explanation in Material and Methods – section measurement, counting and statistical analysis lines 729-748. For more clarity, we also added images in Figure 1—figure supplement 1A delimiting the R4 region in the different feeding conditions.

Alternatively, the ratio of particular cell types to total cells could be helpful as a measure of the relative abundance of different cell populations.

We thank the reviewer for this measurement suggestion. We have now added all ratio in Figure 1—figure supplement 2.

We have also modified the text all along the manuscript to integrate this method of analysis. Noteworthy when both cell density and cell ratio changes (increased or decreased) for a given cell type, we assumed that this corresponded to a global changes in the so-called cell type number.

In the figure legends, we also replaced "cell number" by "cell density".

In addition, could images that illustrate overall organ morphology be included? Such information would provide a sense of the relative sizes of the organs.

We have added whole posterior midgut views at 5X magnification in Figure 1—figure supplement 1A, and we have modified the text accordingly (lines 144-145).

2. Please provide evidence to support the use of the Δ-ReDDM system as a readout of symmetric versus asymmetric fate outcomes. (a) Does degradation of the ReDDM GFP signal occur in the same time frame as activation of the enteroblast-specific SuH marker? (For instance, is ReDDM GFP signal absent from SuH-lacZ cells in both control and experimental conditions?)

It is widely admitted that Dl-Gal4 driver is only expressed in ISCs and enteroendocrine precursors (EEPs), and never reported to be expressed in Enteroblasts (Su(H)+ cells) (Biteau and Jasper, 2014; Guo and Ohlstein, 2015; Joly and Rousset, 2020; Zeng and Hou, 2015).

In the ReDDM system, the half-life of the GFP is less than 24h while the H2B::RFP we used is at least 2 weeks (Antonello et al., 2015). So even if GFP expressed in ISCs might be transmitted to EB daughter cells, the GFP intensity in EBs would be very week due to the short half-life of GFP. Since we took pictures 2 days after ReDDM induction (see Figure 1 —figure supplement 1D), we assume that the GFP transmitted to most of EB daughter cells has begun to fade. Indeed, increasing the intensity using Zeiss or Photoshop software allows to distinguish a weak GFP staining in the putative EBs. However, we consider this faint staining as background.

Author response image 1 shows a Dl-ReDMM midgut fed with water (Control), 2 days after temperature shift at 29°C (i.e. the induction of GFP and RFP). We have intentionally saturated the fluorescence intensity.

1st: GFP+/RFP+ cells (corresponding to ISCs) are mainly isolated cells.

2nd: RFP+/GFP- small nucleated cells (that are likely EBs) are only rarely labeled by a weak green florescence even when GFP intensity is intentionally increased.

Author response image 1

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Author response image 2 is an example of a Dl-ReDMM experiment using flies fed with Btk^ΔCry that stimulate the production of EBs. Many RFP+/GFP- are produced and the GFP is barely detectable in a few EBs even when we forced the intensity of the fluorescence.Therefore, we believe in our conclusions based on the use of the Dl-ReDDM tool.

Author response image 2

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(b) Cell contact was used to classify two progenitor cells as siblings from the same mother stem cell division. Please either justify this claim or else revise the interpretation of these data.

In control conditions, EEs are rarely found in close contact with an ISC (see all our control condition images in our figures and figure supplements). Therefore, an EEP GFP+/RFP+/Pros+ in direct contact with an ISC GFP+/RFP+ has most likely inherited the GFP from this neighboring ISC. A GFP-/RFP+ cell with a small nucleus (an EB) contacting a GFP+/RFP+ ISC is likely to be its daughter cell. Although we can imagine that a GFP-/RFP+ EB contacting an GFP+/RFP+ ISC could arise from a distant GFP+/RFP+ ISC, we took images 2 days after induction of the ReDDM system limiting the time frame allowing a distant EB to come in close contact to an ISC which is not its mother cells. This is explained in the text lines 172-187.

Moreover using these criteria, we found ratio of symmetric/asymmetric divisions close to what it has been previously described: ISCs mostly divide asymmetrically while upon environmental challenges ISC divide symmetrically (de Navascues et al., 2012; Goulas et al., 2012; O'Brien et al., 2011; Perdigoto et al., 2011) (Tian and Jiang, 2014; Zhai et al., 2017). These references have been added line 184-186.

We have also added a sentence in the Material and Methods section lines 627-630.

(c) Can you respond to Reviewer 3's question about co-expression of two GFP transgenes in the Δ-ReDDM genotypes?

Indeed, we used two copies of GFP in F1 flies because Dl-Gal4 driver was not strongly expressed. When Dl-Gal4 drives the expression of one GFP allele, the GFP is weakly expressed (see the expression of the CD8::GFP in Author response image 3, red labelling is Prospero). Therefore, this led us to use two GFP copies in order to label most mother cells in the Dl-ReDDM experiments. In addition, if the GFP was weakly detectable while the RFP was strongly expressed, these cells would have been considered as progenies and not as mother cells. Of note, we also combined the membrane tethered (GFP::CD8) and a nuclear GFP to increase the probability to detect the cells.

Author response image 3

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Similarly, we expressed two copies of the GFP in the Su(H)-ReDDM experiments to track most EBs (mother cells). We also confirmed that Su(H)-Gal4 expression was restricted to EBs with no leak in other cell type since the progenies in the Su(H)-ReDDM experiments were only ECs (RFP+/GFP).

3. Please address the Reviewers' requests for improved data presentation. (individual values in graphs, scale bars in images, loading controls for Westerns, etc). In addition, please quantify the correlation between Tomato::DE-Cad and EEP markers (described in lines 283-284).

i) We have now added the individual values in the graphs of figure 1 (and supplement), figure 2 (and supplement), figure 3—figure supplement 1, figure 5 (and supplement) and figure 6.

ii) We have added the scale bars on figures and the magnification in the legends.

iii) We have added the loading control for Western Blots.

For figure 6D, we cannot use classical loading controls such as actin since Bt does not have actins. So we used Coomassie blue staining of the acrylamide gel. The gel is presented in Figure 6 —figure supplement 1J.

For figure 6E: Loading controls have been realized after blotting membranes with the monoclonal Actin antibody from Invitrogen (ACTN05, C4). As we used detergent-free cell lysis buffer, actin was mainly found in the insoluble fraction (Orlando et al., 2001). This is now explained in the text lines 389-392, in the Material and Methods lines 717-718 and in Figure 6E legend.

Moreover, we always standardized the amount of proteins we have deposited in the each well of the gels: 20µg of midgut proteins and 2.10⁴ CFU of spores (right part of the gel in figure 6D) were deposited. This is explained lines 710-7119.

iv) To include this quantification, we have rebuilt the related figures. Tomato::DE-Cad images and quantification are now presented in figure 4. Arm staining and quantification have been moved in figure 4—figure supplement 1A-E. We have also illustrated our method of quantification in figure 4—figure supplement 1F-G. Finally, we have included the measurement value in Table 1. We have edited the text and accordingly lines 285-297. In the Material and Methods, we added a section to explain better the way we proceeded to perform this analysis (lines 736-742).

4. The Reviewers raised questions about visibility and interpretation of the Arm, E-cad, and caspase signals in multiple figure panels. In cases where improved visibility would be helpful, please present split channel images. In cases where alternative interpretations are raised (e.g., could Arm levels be affected by proliferation rate?), please evaluate these alternatives either through new data or textual discussion.

i) We have now added a new figure 4 (the old figure 4 has been combined with the Figure 4—figure supplement 1), and we drew table 1 to facilitate the interpretation of our quantification and improve the visibility. Details of the quantification methods have been added lines 736-742. See also our response to reviewer 1 point 2.

ii) We have now put the myo1A>Casp::GFP labelling in figure 1A. The anti-cleaved Caspase staining has been moved in Figure 1—figure supplement 1B with split channels. We have also adapted the text to these modifications.

iii) See our response to Reviewer 1 point 2. We have also improved the Arm visibility in Figure 5—figure supplement 1 A-D' (previously Figure S4A-D).

5. Please clarify the methodological details requested by the Reviewers. (a) Better descriptions are needed of how quantitative analyses were performed.

We provided the reviewers a detailed explanation of our quantitative analyses and added new texts and sections in the Material and Methods (lines 627-630 and 727-742).

(b) In some cases, the criteria used to identify particular cell types or populations were ambiguous e.g. new cells versus old cells, sibling cells versus neighbor cells, diploid enteroblasts identified via nuclear size,

We addressed these issues accordingly: see our responses to the Editor point 2(b), to the Reviewer 1 point 2 and to the Reviewer 3 points 6 and 7.

(c) To avert reader confusion, might you consider referring to enteroendocrine cells by the standard abbreviation EE, rather than EEC?

This have been corrected.

Reviewer #1 (Recommendations for the authors):

1) Data representation

Different figures should be modified to improve the representation of the data all along the manuscript:

– Authors should plot individual values in the graphs shown all along the manuscript instead of using histograms.

Done for graphs excepted for those that analyzed the old versus new cells. Putting all individuals value on the graph makes the graph harder to read. So we kept the initial presentation.

– Scale bars should be included both in the pictures and in the legends.

Scale bar added.

– Figure 5G-G': errors bars should put in another color. The scale of the graph could be changes to have a better overview of the distribution of the different points.

These were not the error bars but the averages. We have put the bars in black. We tried to change the scale but we were losing the highest points. We also tried different kinds of graphs and we found that the ones presented in the figures 5 were the most representative and easy to read.

For each figure, the signification of the error bars (sd or sem, etc..) should be included in the legends and/or in the Material and Methods.

This has been added to Methods lines 747-748.

2) Experiments

– Loading controls of the Western blots are missing: authors should provide a control to indicate that the same amount of sample has been loaded in each condition by staining another protein. This is particularly true for the Western blots shown in Figure 6D and Figure 6E

See our response to the "Essential Revisions (for the authors) point 3. To summarize, we have added the loading control. Moreover, we always standardized the amount of proteins we deposited in each well of the gels: 20µg for midgut proteins and 2.10⁴ CFU of spores (right part of the gel) were deposited. This is explained lines 710-712. Noteworthy, anti-Actin cannot be used for controlling the loading charge of Bt spores that do not have actins.

– Figure S1A-S1B: How were the authors able to confirm that two cells came from the same mother cell? This question is particularly true for BtkSA11 where there is a lot of neighboring labelled cells. Details about the way to quantify should be added in the Material and Methods (quantification in a small area only? n=number of divisions analyzed?)

We kindly refer the reviewer to our response to the "Essential Revisions, point 2b". We have added the explanations in the Material and Methods lines 627-630 and 725-748.

Indeed, n correspond to the number of samples analyzed. We have now specify to what n corresponds in the figure legends (intestines/40X images, cells or cell pairs).

Same figure: The picture of Btk∆Cry seems to show lot of symmetric cell divisions giving rise to cells that are GFP+RFP+Pros- (=ISCs) but surprisingly, the Figure 1C does not show this increase.

Both genotypes are different. In Figure 1C, Dl-Gal4 UAS-GFP/+ is used at 25°C. In Figure S1A-S1B (now Figure 1—figure supplement 1C-D), the genotype is Dl-ReDDM with the shift from 18°C to 29°C. Moreover, we noticed that the induction of the ReDDM system induces a stress (shifting flies from 18°C to 29°C) increasing the number of Esg+ cells and probably enhancing the symmetric division, giving rise to two ISCs.

Importantly, there are also EB-EB symmetric divisions (visible on the Figure 1—figure supplement 2D) that buffers the increase in ISCs in the Dl-Gal4 UAS/+ genotype (Figure 1C). Whether the divisions give rise to 2 EBs or 2 ISCs, this does not change the rate of symmetric vs asymmetric divisions presented in Figure 1 —figure supplement 1C.

– L181: Authors conclude: "Therefore, consistent with previous studies, the mode of ISC division appeared to reflect the degree of intestinal damage, the symmetric mode being preferentiality adopted when the damage was greater (i.e., when cell death occurred)." This mode of division seems to be also preferentially adopted when there is intestinal damage but no cell death, as in the case of Btk∆Cry.

We apologizes that our sentence was not well formulated. "Damage" does not means systematically "cell death". Cells can be damaged without resulting in cell death we already demonstrated this in (Loudhaief et al., 2017). So we have removed "(i.e., when cell death occurred)" line 187.

Reviewer #3 (Recommendations for the authors):

The described work is about assessing Drosophila midgut histopathology upon consumption of an entomopathogenic strain of B. thuringiensis and its Cry1A toxins, which are lethal to lepidoptera, but non-lethal to Drosophila. Thus, Drosophila is characterized a non-susceptible organism. The authors tested if this "non-susceptible host" is nevertheless histopathologically susceptible. They convincingly show that it is, because the mechanism of action of the Cry1A toxins on progenitor cell E-Cadherin is functionally (but not biochemically) revealed in flies and in Drosophila S2 cells.

We thank the reviewer for appreciating our findings and for all his constructive comments.

To improve the manuscript, I suggest the following:

1. Add "Drosophila midgut" in the title, because generalizations need to be omitted.

This has been fixed.

2. Lines 89-92: reference to B. cereus should not add to the proof about Btk. So need to phrase this accordingly. Different species and different strains have different effects.

We have rephrased the sentence as requested.

3. Figure 1A: how do Caspase 3 sensor and Caspase 3 antibody compare? While both are of a value, the authors measure the first, but show the latter. Why?

We have now modified and put in figure 1A the images corresponding to Caspase 3 sensor. The anti-cleaved-Casp3 staining has been moved in figure 1—figure supplement 1B.

4. Dl-ReDDM experiments: The M and Ms read "w; tub-GAL80ts; Dl-GAL4 UAS-GFP/TM6b females were crossed to w; UAS-GFP::CD8; UAS-H2B::RFP/TM6 males". This means that the progeny express two different UAS-GFP transgenes, right? This issue applies also to ReDDM experiments with the other GAL4 lines. What is the perdurance of UAS-GFP in each case and in combination with UAS-GFP::CD8?

We kindly refer the reviewer to our response to Editors' Essential Revisions point 2c.

5. Line 224: Why the authors chose esg-ReDDM flies instead of Dl-ReDDM? The latter makes clear what ISC is, while esg based ReDDM confuses the distinction between ISC and EB.

This choice was made based on technical reasons. Dl-ReDDM fly strain is very difficult to amplify and maintain. To perform several repetitions with high number of individuals, we decided to use esg-ReDDM to be sure to get enough F1 flies of the good genotypes. However, we carried out the Su(H)-ReDDM control to check that EBs did not transdifferentiate into EEs.

6. Line 229: "EBs were GFP+ RFP+ DAPI+ with bigger nuclei". Why bigger? EBs are presumably diploid.

We apologize for this mistake. Not "bigger nuclei" but "small". This has been corrected.

7. Figure S3: The terms "old cells" and "new cells" seems misleading, because by "old cells" the authors mean EEs of Su(H)-independent origin and by "new cells" EEs of Su(H)-dependent origin.

Now this is the Figure 3—figure supplement 1A-D. This is a great remark.

By new cells, we mean cells that have now appeared after the induction of the Su(H)-ReDDM system and that express the RFP. If cells express the RFP, this means that these RFP+/GFP- cells arise from Su(H) mother cells. This also mean that these RFP+/GFP- cells did not exist before the induction of the ReDDM system. Therefore, we consider these RFP+/GFP- cells as "newborn cells" compared to cells already present before the ReDDM induction. We have now added the counting of ECs in this Su(H)-ReDDM experiments (Figure 3—figure supplement 1D). In this latter experiment we do observe new enterocytes (RFP+/GFP-). Therefore, we clearly demonstrate that EBs can only give birth to "new" ECs and not to "new" EEs. We confirm the previous observation by Zeng and Hou (Zeng and Hou, 2015) showing that EEs never develop from Su(H)+ EBs. We modified the text lines 252-253.

8. Lines 293-4: "the decrease in Tomato::DE-Cad 283 labelling between an ISC and its progenitor was correlated with the expression of EEP markers". The authors should measure the extend of this correlation. Also, neighboring esg+ cells do not equal "ISC and its progenitor".

We have now added the quantification in figure 4E and Table 1 to highlight this correlation. We have also improved the whole Figure 4 and added details for the quantification in Figure 4—figure supplement 1F-G.

We also adapted the text (lines 285-299) to fine-tune our interpretation in light of this new analysis. Indeed, what we have now defined as "mild" adherens junction intensity is between the ratio 1.4 and 1.6 instead of the previous ratio (1.3 to 1.6), because we observed most of the EEP progenitors arising from cell displaying a junction intensity with their mother cells below the 1.4 ratio (see Table 1). The material and Methods have also been implemented (lines 736-742).

9. The following statement is imprecise "we no longer observed an increase in the number of EECs (blue arrows in Figure 5A-D, and Figure 5E)". Comparing Figure 3F and Figure 5E, there is an increase in EEs in both cases, but the increase is more prominent in 3F.

The reviewer comment is right. There is a partial rescue in the increase of EEC when we overexpressed the DE-Cadherin. So, we have modified the text lines 308-310.

10. I suggest replacing the term EEC(s) with EE(s) to abide to previous nomenclature and make it easier to distinguish from the term EEP(s).

This has been fixed.

11. Figure panels 5G and S5I are missing from the PDF available for this review.

Sorry. This was a bug during the PDF building.

12. Figure S2I ("old" and "new" pros+ cells are plotted together) refers to the anterior midgut only and to see what is going on in the posterior is unclear (information is not plotted). One needs to extrapolate maybe by comparing with Figure 3F ("old" and "new" pros+ cells are plotted separately).

It is now added in the new Figure 2—figure supplement 2I to easily compare what is happening.

13. Lines 363-4. To explain the discrepancy between the anterior and the posterior midgut in terms of EEs and their progenitors, the authors should resort to Tamamouna et al. 2020 (PMID: 32513656) and Marianes and Spradling 2013 (PMID: 23991285), where such gut compartmentalization and anterior vs. posterior mitosis and propensity for EE and ISC accumulation has been thoroughly discussed.

We have now integrated these observations and these references lines 382-385.

14. Line 492. What does "fortuitously mean? How was the selection process arranged? Was there a random mutagenesis involved?

Fortuitously means by chance, since this experiment was originally carried out with the aim of generating a mutant depleted for the all cry genes, carried by distinct plasmids. After analysis of the sequencing data, we identified this partial mutant (Btk^Cry1Ab) that we used for further analysis. The selection process was therefore arranged at 2 levels: (i) by selection of the crystal-negative phenotype, and (ii) after analysis of the genomic sequences.

The part (lines 540 to 550) were modified for more clarity.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

In addition, a few other items arose during the consultation; these can all be addressed by textual revision:

– Findings from prior studies described in lines 167-171 are described inaccurately (see explanation below), and two papers that contradict the authors' assertion were not cited.

– Other portions of the manuscript, such as the Figure 7 results, draw conclusions that are not supported by data. Careful revision of the study's results to avoid over-interpretation and to make clear distinctions between data-based conclusions and interesting, but speculative, remarks will also be necessary for a revised manuscript to be considered further.

– The title appears to be missing a "the". Two grammatically correct alternatives would be:

"Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in the Drosophila midgut" or "Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila"

This has been corrected.

Essential Revision Item #2 – Rationale

(1) In terms of Point (1) above, the authors did not perform the requested experiment to examine whether degradation of the Δ-driven ReDDM GFP signal occurs in the same timeframe as activation of an enteroblast-specific SuH marker. Instead, they offer two alternative justifications for the validity of their current approach. However, as detailed below, neither of these justifications is valid.

The first justification the authors give for drawing conclusions about cell identity from ReDDM hinges on the half-life of GFP in the ReDDM system being less than 24 hours. The authors cite Antonello 2015 as evidence for the < 24-hour half-life, but a quantitative measurement of GFP half-life in the Antonello study could not be identified in either the main figures or the supplemental figures. (If this is mistaken, please point out where these data are found in the paper.) In addition, the authors also claim that since they examined tissues two days after ReDDM induction, the GFP transmitted to most daughter cells has begun to be degraded. This statement appears to assume that all cell divisions happened at the time that ReDDM was induced. However, this assumption is erroneous. Stem cells continue to cycle throughout the two-day post-induction period, and with fixed tissues, it cannot be ascertained when during the two-day period a division actually occurred. It is likely that some stem cell divisions occurred a short time prior to fixation.

The second justification that the authors provide is two images of DeltaGAL4-driven ReDDM showing that, following feeding with the Btk mutant, there is an increased number of GFP-/RFP+ cells compared to control guts. In both images, the authors designate the GFP-/RFP+ cells as enteroblasts and the GFP+/RFP+ cells as stem cells, and they conclude there are more enteroblasts following Btk treatment because there are more red cells. Without a positive marker of enteroblast fate, however, the authors' reasoning is neither scientifically nor logically valid. Crucially, enteroblasts are not defined by the absence of Δ (for instance, loss of Δ does not result in loss of stem cells, but rather results in excess stem cells and enteroendocrine cells -- Ohlstein 2007 Science). Instead, enteroblasts are defined by the activation of Notch targets such as the Su(H)GBE Notch reporter. Hence Notch activation and Δ downregulation are not necessarily synchronous. Rather, Δ downregulation may lag behind Notch activation, such that new enteroblasts transiently exist in a Δ-positive, Notch-activated state. Indeed, Zhai 2017 PLoS Geneticsand Tian 2022 EMBO J have found that under injury conditions there is a marked increase in Su(H)+ enteroblasts that are also Δ+.

(2) In terms of Point (2) above, the authors do not present data to support the claim that a GFP-/RFP+ cell adjacent to a GFP+/RFP+ cell is the division progeny of the GFP+/RFP+ cell, and they state that two days after induction is too short a time frame for enteroblasts to come into contact with a stem cell that is not its mother stem cell. By contrast, Martin 2018 (eLife), which analyzed stem cell divisions in live movies, found that approximately one-third of sibling cells separate from each other after birth. The duration of these movies was ~16 hours, which is much shorter than the 2-day period the authors used. In addition, Martin et al. observed that some of these sibling cells subsequently associated with other progenitor cells. These findings undermine the authors' assertion and underscore the importance of performing lineage tracing to deduce lineage relationships. While we appreciate that some other journals have previously published midgut studies that assume progenitor cell pairs are siblings, our view is that publication in eLife requires more rigorous evidence such as lineage tracing or direct monitoring of live divisions.

Finally, the statement that "upon environmental challenges, ISCs divide symmetrically" (Author's response and manuscript lines 167-171) is not supported by the literature. The deNavascues 2012, Goulas 2012, Perdigoto 2011, and Tian 2014 studies examine homeostasis only; hence, these results do not speak to what happens when there are environmental challenges. In addition, Goulas and Perdigoto do not explicitly examine asymmetric versus symmetric division fate outcomes. O'Brien 2011 examined feeding-induced growth, which is not an environmental challenge but rather a normal physiological mechanism; hence, these results also do not speak to what happens upon environmental challenges. Although Zhai 2017 speculates on symmetric fate outcomes in the discussion, no conclusions are drawn about the fate outcomes of cell pairs in the data presented in the Results section.

By contrast, there are two studies that did examine division fate outcomes in injured guts, and that were not cited in either the Response or the manuscript: Jin 2017 (Stem Cell Reports) and Tian 2017 (PNAS). Both studies used Twin-spot MARCM – the gold standard for symmetric/asymmetric division fate outcomes – and found that the majority of divisions still exhibit asymmetric fate outcomes following injury. Their injury conditions of Pseudomonas infection and bleomycin ingestion are arguably in a similar realm to the current study's Btk treatment. Their results contradict the authors' assertion that stem cells divide symmetrically following environmental challenges and raise the possibility the authors' method is inaccurate.

We are grateful to the editors for the exhaustive bibliographic analysis they provided. We are agreeing that the methods we used cannot provide solid conclusions on the mode of ISC division upon Bt/toxin ingestion and more experiments are required. Since the mode of ISC division is not the focus of our work and is not linked to the main message of our publication, we followed the editor’ suggestion and therefore modified the text by removing all the sentences referring to the mode of ISC division. We hope the changes we have made are satisfactory now and have taken into consideration all the points raised by the reviewers and editors regarding the division fate outcomes upon Bt/toxin ingestion.

We have modified the text lines 48-49, 110, 161-174, 242 and 610. We removed the graph related to the mode of ISC division in the Figure 1 —figure supplement 1. The figure legend has been corrected accordingly lines 1201-1206.

Additional remarks from individual reviewers follow below:

Reviewer #1 (Recommendations for the authors):

Thanks to the authors for addressing the questions raised in the previous reviews and for their detailed answers.

In the previous reviews, we highlighted the lack of some quantifications; controls, and information about the methods of analysis for some key experiments (Arm staining; rescue experiments..). The authors addressed all the questions when experimentally possible and modified the text accordingly. They also modified the graphs as suggested. Altogether, these modifications greatly improved the manuscript and reinforced the conclusions proposed by the authors.

Thank you very much for your suggestions that have increased the quality of our results.

Reviewer #2 (Recommendations for the authors):

The authors have appropriately addressed most of the comments. However, I have a concern regarding the results presented in Figure 7. The results raise interesting ideas about how toxins might reach ISCs. The presented experiments do not directly test the ideas. Therefore, the conclusions are not appropriately supported. I suggest the authors revise the text to clearly separate the conclusions drawn from the experimental data and their speculations. Alternatively, the whole data can be removed as those are not essential for the story.

We have included these experiments and data in the first revised version to address the question raised by reviewer #2 during the first round of revision. According to the reviewer's new issue, we have rewritten this part (Lines 387-411) to better describe our results and moderate our conclusions.

Reviewer #3 (Recommendations for the authors):

Taking the authors' words (in their response to reviewers): "ISCs mostly divide asymmetrically while upon environmental challenges ISCs divide symmetrically (de Navascues et al., 2012; Goulas et al., 2012; O'Brien et al., 2011; Perdigoto et al., 2011; Tian and Jiang, 2014; Zhai et al., 2017)". Thus, being responsive to Bacillus thuringiensis infection and its Cry1A toxins does not equal B. thuringiensis being a hazard to Drosophila or other flies. Such treatments merely elicit a defense response against damaging agents, which are mild enough for flies to cope with (survive). I suggest the authors refrain from conclusions (should there be any) about B. thuringiensis being very hazardous to flies.

We agree that Bt is not strongly hazardous to flies. Indeed, we have already demonstrated that Bt is a weak opportunistic bacteria causing moderate damage in the Drosophila intestine (Loudhaief et al., 2017). This is also what we mentioned clearly in this manuscript, lines 178-182. Of note, we have verified the text and we didn’t find the word hazardous in any section.

In our response to reviewers, we used the term "environmental challenge" (not used in the manuscript) to point to an environmental stimulus (not necessarily a strong aggression) that is perceived by the intestine as potentially harmful, requiring a local cellular response. But for sure, Bt is an opportunistic bacterium weakly virulent for Drosophila as well as for human since the main symptom is diarrhea.

https://doi.org/10.7554/eLife.80179.sa2

Article and author information

Author details

Rouba Jneid
1. Universite Cote d'Azur, CNRS, INRAE, Sophia Antipolis, France
2. Faculty of Sciences III and Azm Center for Research in Biotechnology and its Applications, LBA3B, EDST, Lebanese University, Tripoli, Lebanon
Present address
Department of Biology, Faculty of Science, University of Copenhagen, Copenhagen, Denmark

Contribution
Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing – original draft

Competing interests
No competing interests declared
Rihab Loudhaief

Universite Cote d'Azur, CNRS, INRAE, Sophia Antipolis, France

Present address
K8520 IWK Health Centre 8th Floor East Research 5850/5980 University Avenue Halifax, Nova Scotia, Canada

Contribution
Conceptualization, Investigation, Visualization

Competing interests
No competing interests declared
Nathalie Zucchini-Pascal

Universite Cote d'Azur, CNRS, INRAE, Sophia Antipolis, France

Contribution
Formal analysis, Investigation, Visualization

Competing interests
No competing interests declared
Marie-Paule Nawrot-Esposito

Universite Cote d'Azur, CNRS, INRAE, Sophia Antipolis, France

Contribution
Methodology

Competing interests
No competing interests declared
Arnaud Fichant
1. Universite Cote d'Azur, CNRS, INRAE, Sophia Antipolis, France
2. Laboratory for Food Safety, University Paris-Est, French Agency for Food, Environmental and Occupational Health & Safety, Maisons-Alfort, France
Contribution
Data curation, Formal analysis

Competing interests
No competing interests declared
Raphael Rousset

Universite Cote d'Azur, CNRS, INRAE, Sophia Antipolis, France

Contribution
Funding acquisition, Validation, Writing – review and editing

Competing interests
No competing interests declared
Mathilde Bonis

Laboratory for Food Safety, University Paris-Est, French Agency for Food, Environmental and Occupational Health & Safety, Maisons-Alfort, France

Contribution
Resources, Data curation, Methodology

Competing interests
No competing interests declared
Dani Osman

Faculty of Sciences III and Azm Center for Research in Biotechnology and its Applications, LBA3B, EDST, Lebanese University, Tripoli, Lebanon

Present address
UMR PIMIT (Processus Infectieux en Milieu Insulaire Tropical) CNRS 9192- INSERM 1187-IRD 249-Université de La Réunion, île de La Réunion, France

Contribution
Conceptualization, Funding acquisition, Validation, Writing – review and editing

Competing interests
No competing interests declared
Armel Gallet

Universite Cote d'Azur, CNRS, INRAE, Sophia Antipolis, France

Contribution
Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Project administration, Writing – review and editing

For correspondence
gallet@unice.fr

Competing interests
No competing interests declared

"This ORCID iD identifies the author of this article:" 0000-0002-2054-4780

Funding

Agence Nationale de la Recherche (ANR-15-IDEX-01)

Armel Gallet

Agence Nationale de la Recherche (ANR-13-CESA-0003-01)

Armel Gallet

Conseil Régional Provence-Alpes-Côte d'Azur

Armel Gallet

Conseil Départemental des Alpes Maritimes

Armel Gallet

Institut Olga Triballat

Armel Gallet

Office Français de la Biodiversité

Armel Gallet

Azm and Saade Association

Dani Osman

Université Côte d'Azur

Rouba Jneid

Ministère de l'Education Nationale, de l'Enseignement Superieur et de la Recherche

Rihab Loudhaief

Fondation pour la Recherche Médicale

Rihab Loudhaief

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We are grateful to all members of the BES team for fruitful discussions. We want to thank Arnaud Felten (GVB Unit, Anses Ploufragan) and Pierre-Emmanuel Douarre (SEL unit, Anses Maisons-Alfort) for their help regarding the WGS analysis of the mutant Btk^Cry1Ab. We also thank Laurent Ruel for providing pWA-Gal4. We are grateful to Hiroki Oda for providing the pUAST-DEFL. We thank Olivier Pierre from the microscopy platform of the Sophia Agrobiotech Institute for his help. Our thanks to the Université Côte d’Azur Office of International Scientific Visibility for English language editing of the manuscript. We also thank the Space, Environment, Risk and Resilience Academy of Université Côte d'Azur for their financial support. This work has been supported by the French government, through the UCAJEDI Investments in the Future project managed by the National Research Agency (ANR) with the reference number ANR-15-IDEX-01 and through the ANR-13-CESA-0003–01, by the Région Provence Alpes Côte d'Azur, by the Département des Alpes-Maritimes, by the Institut Olga Triballat (PR2016-19) and by the ANSES PNR-EST & ECOPHYTO II (EST-2017–2021). RJ was funded by the association AZM & SAADE (Lebanon) and Université Côte d'Azur (ATER). RL was funded by the Ministère de l’Education Nationale, de l’Enseignement Superieur et de la Recherche (MESR) and a grant from the Fondation pour la Recherche Médicale (FRM).

Senior Editor

Arturo Casadevall, Johns Hopkins Bloomberg School of Public Health, United States

Reviewing Editor

Lucy Erin O'Brien, Stanford University School of Medicine, United States

Reviewers

Karen Bellec, University of Glasgow, United Kingdom
Young Kwon, University of Washington, United States
Yiorgos Apidianakis, University of Cyprus, Cyprus

Publication history

Preprint posted: April 13, 2022 (view preprint)
Received: May 11, 2022
Accepted: February 5, 2023
Version of Record published: February 27, 2023 (version 1)
Version of Record updated: March 17, 2023 (version 2)

Copyright

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Rouba Jneid
Rihab Loudhaief
Nathalie Zucchini-Pascal
Marie-Paule Nawrot-Esposito
Arnaud Fichant
Raphael Rousset
Mathilde Bonis
Dani Osman
Armel Gallet

(2023)

Bacillus thuringiensis toxins divert progenitor cells toward enteroendocrine fate by decreasing cell adhesion with intestinal stem cells in Drosophila

eLife 12:e80179.

https://doi.org/10.7554/eLife.80179