Intestinal stem cell ablation reveals differential requirements for survival in response to chemical challenge

Abstract

The Drosophila intestine is maintained by multipotent intestinal stem cells (ISCs). Although increased intestinal stem cell (ISC) proliferation has been correlated with a decrease in longevity, there is some discrepancy regarding whether a decrease or block in proliferation also has negative consequences. Here we identify headcase (hdc) as a novel marker of ISCs and enteroblasts (EBs) and demonstrate that Hdc function is required to prevent ISC/EB loss through apoptosis. Hdc depletion was used as a strategy to ablate ISCs and EBs in order to test the ability of flies to survive without ISC function. While flies lacking ISCs showed no major decrease in survival under unchallenged conditions, flies depleted of ISCs and EBs exhibited decreased survival rates in response to damage to mature enterocytes (EC) that line the intestinal lumen. Our findings indicate that constant renewal of the intestinal epithelium is not absolutely necessary under normal laboratory conditions, but it is important in the context of widespread chemical-induced damage when significant repair is necessary.

1. Introduction

The long-term maintenance of adult tissues is facilitated by stem cells that are capable of integrating systemic and local signals to maintain homeostasis or respond to altered tissue demands, when necessary [1]. Many adult tissue stem cell populations are relatively quiescent under homeostatic conditions but can be activated to divide in response to damage or wounding in order to increase the number of stem and progenitor cells to facilitate tissue repair [2]. Therefore, tight regulation of stem cell behavior (proliferation, survival, and differentiation) is necessary to maintain tissue function under homeostatic conditions as well as in response to damage [3][4].

The Drosophila intestine shows remarkable similarity, on a cellular and molecular level, to the human intestine, including the regulation of stem cell activity via highly conserved pathways, such as the Wnt, Notch, Hippo, and Epidermal Growth Factor Receptor signaling pathways [5]. Drosophila intestinal stem cells (ISCs) are located basally, immediately adjacent to the basement membrane and in close proximity to the visceral muscle (Figure 1 A–C). The majority of multipotent ISCs divide asymmetrically to generate a new ISC and a transient enteroblast (EB) that differentiates into an EC through activation of the Notch pathway [6][7]. A smaller subset of ISCs, on the other hand, gives rise to enteroendocrine (EE) cells through asymmetric divisions (ISC+EE) or direct differentiation [8][9][10][11].

Figure 1. Headcase is a novel marker of ISCs/EBs in the Drosophila intestine.

A) Anatomical organization of the adult Drosophila intestine. B) Immunofluorescence image of the posterior midgut showing the four different cell types found in this epithelium: ISCs and EBs are generally found in nests, and express escargot (reflected by UAS-GFP expression under the control of a esgGal4 driver, white bracket, green cells), secretory enteroendocrine cells (EE, Prospero positive, red, nuclear signal, white arrow) and absorptive enterocytes (large polyploid cells, white arrowhead); Armadillo (red) marks the membrane of all cells; C) Schematic of the posterior midgut epithelium; ISCs and EBs are found in close association with the basement membrane (BM); ISCs (green) can either divide symmetrically generating more ISCs or asymmetrically giving rise to one ISCs and one daughter cells that will differentiate either as an EE (red) or an EC (Blue); D) Staining with a Hdc antibody shows co-localization with esg expression, which marks ISC and EBs within the posterior midgut; inset shows GFP signal (Hdc) in ISC/EB nests (white brackets); E) hdc reporter line (enhancer trap) shows the same expression pattern as detected with Hdc antibody; F) Within the ISC/EBs nests, generally one GFP positive cell is positive for Delta (white arrow, ISC marker) and one GFP positive cell is negative for this marker (red arrow, EBs); Scale bars 20μm

In Drosophila, maintenance of a functional intestinal epithelium has been shown to be an important determinant of health and viability at the organismal level [12][13][14][15]. The ISCs are the only proliferative cells in the intestine, and in the absence of challenges, the female posterior midgut epithelium is renewed roughly every 12 days [16]. However, ISC division increases significantly in response to a variety of stimuli and/or damage, including loss of ECs, infection with pathogenic bacteria, and oxidative stress [17][18][19]. Importantly, it has been demonstrated that significantly increased or decreased ISC proliferation, achieved through genetic manipulation of the insulin/IGF signaling (IIS) pathway, resulted in shortened lifespan [12]. In addition, data indicate that impairment of intestinal regeneration results in lower survival rates when flies are infected with enteropathogenic bacteria [19][20][21]. In contrast, Petkau et al. demonstrated that disrupting the proliferative program of ISCs and ECs causes damage to the intestinal epithelium without significantly affecting lifespan, even after microbial infection [22]. Damage caused by chemical agents, such as the DNA damaging agent bleomycin or dextran sulphate sodium (DSS), also induces a regenerative response in the intestine [17]; however, it has not been determined whether ISC activity is important for organismal survival in these models. A recent study described an approach to ablate ISCs/EBs, but the impact on lifespan was not addressed [23].

Here, we identify headcase (hdc) as a novel marker of ISCs and EBs and demonstrate that loss of hdc function results in progressive loss of these progenitor cells via apoptosis. As targeted depletion of hdc in ISCs and EBs resulted in elimination of these cell types, this strategy was used to successfully ablate ISCs/EBs from young flies to determine the effects of ISC loss on intestinal homeostasis and survival. Loss of ISCs did not compromise survival of the flies under homeostatic conditions; however, flies depleted of ISCs had lower survival rates than flies capable of intestinal regeneration after bleomycin-induced EC damage. Our results confirm that renewal of the intestinal epithelium is not absolutely required if flies are maintained under unchallenged conditions. In contrast, ISC activity and intestinal regeneration promotes survival in response to damage.

2. Material and Methods

2.1 Fly husbandry and stocks

Flies were raised on standard cornmeal-molasses-agar medium. Female progeny from experimental crosses were collected and maintained with less than 30 flies per vial. Flies were turned onto fresh food vials every two days. The following fly stocks used were from the Bloomington Drosophila Stock Center (BDSC), Vienna Drosophila Stock Center (VDRC), or generous gifts from the fly community as indicated: Gal80^ts; UAS-lacZ^NLS; UAS-p35 (Bloomington stock center #7018, #3956 and #5073); esgGal4, UASGFP (gift from Norbert Perrimon); esgGal4,2xYFP; Su(H)Gal80, tub-Gal80ts (gift from Steven Hou) hdcRNAi lines used were from Vienna Drosophila stock center and labeled as UAS-HdcRNAi¹ (VDRC#45069) and UAS-HdcRNAi² (VDRC#104322). Wild-type flies were Oregon R. More detailed information about these stocks can be found at Flybase (http://flybase.bio.indiana.edu).

2.2 Antibodies

Intestines were stained with: mouse anti-Hdc (1:3) (gift from R. White); mouse anti-Armadillo (1:20) and mouse anti-Prospero (1:100) (Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences); rabbit anti-phospo-histone H3 (1:200) (Milipore); rabbit anti-GFP (1:5000) (Molecular Probes). Secondary antibodies were diluted 1:500 (Molecular Probes).

2.3 Immunostaining and microscopy

Immunofluorescence (IF) microscopy was performed on whole-mount intestines dissected directly in 4% PFA, left in fixative solution for 1h, followed by 3×10 minutes washes with PBST (0.5% Triton-X100) and blocked for 1 hour in PBST/BSA (3% BSA in 1X PBS). After blocking primary antibodies were incubated at 4°C overnight followed by 3×10 minutes washes with PBST and addition of secondary antibodies at a concentration of 1:500. After 2 hours incubation with secondary antibodies, 3×10 minutes washes with PBST were performed and finally samples were mounted in Vectashield mounting medium with DAPI (Vector Laboratories). Images were obtained using either a Zeiss LSM 710 Laser Scanning confocal microscope or Zeiss Axiovert 200 microscope equipped with an Apotome.

Four images were taken from each midgut for each ISC/EB, quantification: one image from the top plus one image from the bottom layer of the intestines for the first two fields of view of the posterior midgut (after the pyloric ring) on a 40× objective. In Figures 2 E and F, Figure 4 E and F, Supplemental Figure 1 C, and Supplemental Figure 2 D, H and L, graph points represent average number of the four images taken for each midgut. Based on a recent characterization of anatomical and functional compartments of the posterior midgut this corresponds to the P3-P4 regions [24]. Images were analyzed in AxioVision (version 4.8; Carl Zeiss) and Adobe Photoshop software (Mountain View, CA). Images were originally obtained as Z-stacks, and ImageJ was used to generate maximum or average projections from each channel, which were saved as individual TIFF or JPEG files. Images processed using CellProfiler pipelines. For ISC/EB counts included in Figures 2, 4 and Supplemental Figure 1, a mask was designed to score GFP⁺ nuclei, which were quantified relative to total cell number (cell nuclei determined using DAPI).

Figure 2. Headcase depletion leads to ISC/EB loss via apoptosis.

A) Quantification of percentage of ISCs/EBs per total cell number in conditions described in B–F; B) Intestines from 10-day old control flies showing ISC/EBs (green, B′), enteroendocrine cells (Prospero⁺, red, B″) and ECs (polyploid cells in B; DAPI, blue); C) Intestine from 10-day old HdcRNA¹ fly in which hdc expression was depleted by expressing UAS-HdcRNAi¹ specifically in ISCs/EBs in the adult. Note loss of ISC/EBs (compare B′ to C′); D) Complete loss of ISC/EBs was observed in intestines from 10-day old HdcRNA² flies (compare B′ with D′); E and F) Expression of apoptosis inhibitor P35 rescues ISC/EB loss observed with UAS-HdcRNAi¹ or UAS-HdcRNAi² (compare C′/D′ with E′/F′); Scale bars, 20μm.

Figure 4. Regeneration is required for survival after intestinal damage.

A) Intestines from control flies fed a 5% sucrose solution for 48h. ISCs/EBs (green), mitotic cells [phosphohistone H3⁺ (pH3) red], nuclei (DAPI, blue). (B) Intestines from flies fed bleomycin for 48h. (B′) Note that bleomycin feeding induces an increase in esg+ (GFP⁺) cells; C) Intestines from control flies aged for 10 days and then fed bleomycin for 7 days. Note increase in esg+ (GFP⁺) cells; D) Intestines from HdcRNAi² flies aged for 10 days and then fed bleomycin for 7 days. E) Quantification of ISCs/EBs before and after 7 days of bleomycin treatment in control and HdcRNAi² flies; n>20 for all conditions; F) Quantification of the number of mitotic cells (pH3⁺) before and after 48h of bleomycin treatment in control and HdcRNAi² flies and HdcRNAi²+P35; n>15 for all conditions with the exception of HdcRNAi²+P35 after bleomycin where N=12; G) Survival curves for control and HdcRNAi² flies after bleomycin feeding (initiated at day 10), p-value < 0.01 log-rank (Mantel-Cox) test; n>180 for both genotypes. Scale bars 20μm

2.4. Bleomycin feeding

Bleomycin was dissolved in MQ H20 at a final concentration of 2.5μg/mL and 5% sucrose solution. For control a 5% sucrose solution was used. 750uL of control or bleomycin solutions were used to dampen half of a Kimwipe that was placed at a bottom of an empty food vial. Solutions/Kimwipes were changed every day.

2.5 Survival curves

Flies were transferred to fresh food vials every two days, with 20–30 flies in each vial. The number of dead flies was scored each day and survival rates were measured as the total number of flies dead until that specific day divided by the total number of flies analyzed. More than 200 flies were used for each genotype/experiment (n=2).

2.6 Assay for intestinal barrier function (Smurf assay)

Food was prepared using standard fly food medium with blue dye (FD&C blue dye #1) added at a concentration of 2.5 % w/v. Flies were fed blue food for 24 hours at indicated time points and then scored as Smurf+ if dye could be seen outside of the digestive tract as described in [13]. More than 150 flies were used for each genotype/time point (n=3).

3. Data analysis

Statistical analysis and graphical display were performed using the Prism5 (GraphPad) software. Graphs where cell counts are plotted show mean +/− SEM and one-way ANOVA Tukey’s multiple comparisons test was used for significance test (*P<0,05; **P<0,01; ***P<0,001; ****P<0,0001). On Figure 4 panel F, where the number of pH3 positive cells per DAPI is shown, a Mann Whitney U test was used to compare situations before and after bleomycin feeding. For lifespan analysis described in Figures 3A and 4G, a log rank test was performed. For analysis of loss of intestinal barrier function (“Smurf” assay) in Figure 3D, a Fisher’s exact test was used.

Figure 3. Loss of intestinal regeneration capacity does not alter maximal lifespan under homeostatic conditions.

A) Survival curves of control flies (esgGAL4/UAS-LacZ;Gal80^ts) compared to flies lacking ISCs/EBs (esgGAL4/UAS-HdcRNAi²);Gal80^ts; n>200 flies for each genotype; p-value < 0.01 log-rank (Mantel-Cox) test; B) Intestine from a 30 day-old control fly and C) intestine from a HdcRNAi² fly; note that long-lived EEs (nuclear red, Prospero, white arrow) and ECs (large nuclei, DAPI, white arrowhead) are detected 20 days post-ablation of ISCs/EBs; D) percentage of control and HdcRNAi² flies of that displayed loss of barrier function, as determined by Smurf assay, at 10, 20 and 30 days. p-value > 0.05 (Fisher’s exact test) for 30d time point. Scale bar 20μm.

4. Results

4.1. Headcase is required for maintenance of ISCs/EBs in the Drosophila midgut

Given conflicting data regarding the relationship between changes in ISC proliferation and lifespan, we wanted to investigate the consequence of ISC ablation on fly survival. Previous results from our lab demonstrated that depletion of the hdc gene in the Drosophila testis led to loss of somatic support cells due to apoptosis [25]. In addition to its role in preventing cell death in the testis stem cell niche, Hdc appears to play a similar role in the eye [26]. In addition to a role in preventing cell death, Hdc has been shown to have roles in other biological processes, including tracheal branching and neural development [27][28][29]. Furthermore, the human homolog of hdc, HECA, has been shown to be mis-expressed in certain types of cancer [30][31]. However, a clear molecular function for hdc remains to be elucidated in the fly or in mammalian systems.

The transcription factor Escargot is expressed in ISCs/EBs throughout the digestive tract (Figure 1 B) [7] and is often used as a marker of ISC/EBs. Immunofluorescence using a Hdc monoclonal antibody revealed Hdc expression in ISCs and EBs within the posterior midgut (Figure 1 D) as evident by expression within esg positive cells. A reporter line for hdc [32] mimicked the expression pattern revealed by Hdc antibodies, and expression of hdc in ISCs was confirmed by co-staining with antibodies to Delta, an ISC- specific marker (Figure 1 E–G). Therefore, Hdc represents a novel marker for ISCs and EBs in the intestine. Although hdc and esg share the same expression pattern in the posterior midgut, hdc does not appear to be a direct target of Esg in the intestine [33][34]. Notably, expression of hdc was also detected in stem/early progenitor cells in other tissues, including the ovary and testis ([25] and data not shown).

Given its expression pattern and previous implication in preventing cell death, we tested whether hdc is required for maintenance of ISC/EBs in the posterior midgut. An esgGAL4 ‘driver’ was used to direct expression of two independent RNAi lines targeting the hdc transcript in ISCs and EBs in the adult fly (UAS-hdc^RNAi1 and UAS-hdc^RNAi2 [29]; see Materials and Methods for details). For simplicity, these genotypes (esgGal4, UAS-GFP/UAS-LacZ;Gal80^ts, esgGal4, UAS-GFP/UAS-hdc^RNAi1;Gal80^ts and esgGal4, UAS-GFP/UAS-hdc^RNAi2;Gal80^ts) will be referred from now on as control, HdcRNAi¹ and HdcRNAi², respectively. In order to avoid possible developmental lethality or defects, expression of the GAL4-UAS system was restricted to adults by using the temperature sensitive repressor of GAL4, Gal80^ts, and raising the flies at the permissive temperature (18°C) to suppress GAL4 activity and transgene expression. Upon eclosion, flies were shifted to 29°C to induce expression of the RNAi hairpins targeting hdc.

Two days after knock-down of hdc expression in ISC/EBs, similar numbers of GFP+ cells were observed in intestines from control, HdcRNAi¹ and HdcRNAi² flies (Figure 2A; Supplemental Figure 1A–C). In contrast, after 10 days of hdc depletion, a significant loss of ISCs and EBs was observed in guts dissected from both HdcRNAi¹ and HdcRNAi² flies (Figure 2 A–D), when compared to controls. Indeed, very few, if any, ISCs/EBs were observed in most intestines upon expression of HdcRNAi² after 10 days (Figure 2 A, D). Notably, esg+ gastric stem cells (GSSCs) were observed in the stomach-like copper cell region (CCR) of HdcRNAi² flies, although the total number of these cells were not quantified, nor compared to controls [44]. Depletion of hdc specifically in either ISCs or EBs also resulted in loss of the corresponding cell type after 10 days of depletion, indicating that both ISCs and EBs require hdc function for maintenance (Supplemental Figure 1 D–K). The anti-apoptotic baculovirus protein P35 has been shown to efficiently suppress cell death in Drosophila [35]. Consequently, co-expression of P35 with HdcRNAi¹ or HdcRNAi² efficiently suppressed ISC/EB loss (Figure 2 A, E–F). Therefore, hdc appears to prevent apoptosis in intestinal progenitor cells, similar to its role in hub cells in the testis [25] and ommatidia of the eye [26].

4.2. Ablation of ISCs/EBs does not lead to a significant decrease in survival under non-challenged conditions

It has recently been suggested that impairment of ISC function does not impact fly longevity under homeostatic conditions [22], challenging a previous model proposing that substantial decreases in ISC division result in shortened lifespan [12]. Given that depletion of hdc resulted in significant ablation of ISC/EBs, we wanted to address the impact of loss of intestinal renewal using our model. Depletion of ISCs/EBs resulted in a modest decrease in median lifespan but did not impact maximal lifespan (Figure 3 A). At day 30, midguts of control flies showed a dysplastic phenotype typically observed in aged guts characterized by an expansion of cells expressing the esg reporter [36](Figure 3 B–B″). In contrast, only rare esg⁺ cells were observed in HdcRNAi² flies, despite EEs and ECs (Figure 3 C–C″) being easily detectable, indicating that differentiated cells within the intestine are maintained for at least 20 days post-ISC ablation. Despite no significant increase in mortality after ISC ablation, we hypothesized that the lack of intestinal turnover mediated by ISCs would result in disruption of the intestinal barrier. Contrary to our prediction, an assay to determine intestinal barrier integrity (the “Smurf” assay [13][14]) revealed no significant increase in intestinal barrier dysfunction for HdcRNAi² flies, when compared to control flies (Figure 3 D).

Our data are consistent with findings that suggest that ISCs are largely dispensable for survival under unchallenged conditions, typically found in the laboratory. Notably, Petkau et al. reported that life spans were also not significantly altered after bacterial challenge, but this is in contrast to other studies that have shown that intestinal regeneration in flies is required for viability after infection with pathogenic bacteria [19][20]. Given these conflicting data, we wanted to investigate the consequence of ISC ablation on fly survival after chemical damage to ECs.

4.3. Intestinal regeneration is important for fly survival in the context of chemical-induced damage

The drug bleomycin has been used to induce DNA damage specifically in ECs, resulting in EC death [17]. Consequently, bleomycin feeding stimulates ISC proliferation to facilitate replacement of damaged cells. Posterior midguts from flies fed bleomycin for 48 hours showed an increase in ISC division, as revealed by staining with the mitotic marker phospho-histone H3 (pH3) and an increase in the number of cells expressing the ISC/EB marker esg in comparison to control flies fed with sucrose alone (Figure 4 A,B and E,F). In order to test whether a block in regeneration leads to a decrease in survival after bleomycin-induced EC damage, HdcRNAi² flies were kept on normal food for 10 days at 29°C to ablate ISCs/EBs, prior to exposure to bleomycin or growth on sucrose. After ISC/EB ablation (10days at 29°C), HdcRNAi² flies were exposed to bleomycin for 7 days, and intestines were compared to intestines from control flies subjected to the same feeding regime (10 days on normal food at 29°C, followed by 7 days of bleomycin feeding). In response to bleomycin, intestines from control flies exhibited an increase in the number of proliferative cells, as well as an increase in esg⁺ (GFP+) cells, characteristic of an ongoing regeneration response. In contrast, intestines from HdcRNAi² flies showed only rare GFP+ cells and no dividing cells, as expected for intestines in which progenitor cells had been ablated (Figure 4 C–F). In addition, these midguts were much thinner than controls, due to EC death without replacement (Figure 4D). In flies co-expressing HdcRNAi² and P35, proliferating ISCs were easily observed under homeostatic conditions, and an increase in pH3+ ISCs was observed in intestines of flies fed bleomycin, similar to controls (Figure 4F). We predicted that failure to replace ECs in HdcRNAi² flies would lead to increased mortality. Indeed, after 7 days of bleomycin exposure, flies expressing HdcRNAi² exhibited a significant decrease in survival (Figure 4G), highlighting the importance of regeneration in the context of damage.

5. Discussion

Optimal intestinal function is increasingly being recognized for its importance for overall health, particularly with respect to infections and chronic inflammatory and neoplastic diseases [38][39]. Here we have used the Drosophila posterior midgut to address whether intestinal regeneration, supported by functional ISCs, is absolutely required under homeostatic conditions and in response to chemical-induced damage. Previous work noted that impairment of intestinal regeneration led to reduced lifespan [12]. However, this conclusion was recently challenged in another study in which blocking ISC proliferation, by expression of a cell cycle inhibitor, did not lead to a decrease in survival, even after challenge with pathogenic bacteria [22]. Both of these studies were based on genetic interventions that aimed to impair ISC proliferation.

Our goal was to design an alternative experimental paradigm based on ISC/EB ablation by inducing programmed cell death. Previous studies have attempted complete ablation of ISCs by expressing pro-apoptotic genes, which has proved challenging due to an apparent resistance of some stem cells to apoptosis [5][40][41][42] or embryonic or larval death due to leaky expression of pro-apoptotic factors (data not shown). Our strategy to ablate ISCs/EBs by depleting Hdc supports a model wherein ISCs are largely dispensable for intestinal function and fly survival in non-challenged conditions. In contrast, ISC-driven intestinal regeneration appears to be absolutely necessary once damage to ECs is induced with bleomycin. Previous studies have also indicated that an impairment of epithelial regeneration severely compromises organismal fitness; however, these studies were based on bacterial infection models. Additional chemical agents, such as H₂0₂ or dextran sulfate sodium (DSS), could also be tested to address the impact of reduced intestinal regeneration on fly survival [43][17]. As noted above, the GSSCs were not ablated in response to Hdc depletion, indicating stark differences between the various stem cell populations in the intestine. Future studies will focus on exploring these differences and the role of Hdc in ISCs and GSSCs in more depth. As ISCs in flies and mammals share many regulatory features, studies characterizing the effects of prolonged periods of impaired intestinal regeneration on intestinal function and organismal health could shed light on the impact of pathological conditions in which ISC behavior is altered and regeneration is compromised.

Supplementary Material

supplement. Supplemental Figure 1. Loss of ISCs/EBs is observed after hdc knock down in ISC and/or EBs.

A) Intestines from 2 day-old control flies showing ISC/EBs (green, A′), enteroendocrine cells (Prospero⁺, nuclear red, A″) and ECs (polyploid cells in A; DAPI, blue); B) and C) Intestines from 2-day old HdcRNAi¹ and HdcRNAi² flies showing normal complement of ISCs/EBs; D) Intestine from 10 day-old control fly. ISCs (green, YFP, D′), enteroendocrine cells (Prospero⁺, nuclear red, D″) and ECs (polyploid cells, DAPI, blue); Intestines from 10 day-old flies in which E) UAS-HdcRNAi¹ or F) UAS-HdcRNAi² was expressed in ISCs specifically. Note decrease in ISCs in E′ and F′; G) Quantification of ISCs in D–F; n=12 for Control and n>15 for both HdcRNAi¹ and HdcRNAi²; H) Intestine from 10 day-old control fly showing EBs (GFP⁺, green, H′), enteroendocrine cells (Prospero⁺, nuclear red, H″) and ECs (polyploid cells in H; DAPI, blue); Intestines from 10 day-old flies in which I) UAS-HdcRNAi¹ or J) UAS-HdcRNAi² was expressed in EBs specifically. Note decrease in EBs in I′ and J′; K) Quantification of percentage of EBs per total cell number in conditions described in H) to J); n>15 for all conditions; Scale bars, 20μm

Highlights.

• Headcase (Hdc) is a marker of intestinal stem cells (ISCs) and enteroblasts (EBs)

• Depletion of Hdc leads to loss of ISCs and EBs

• Ablation of ISCs does not compromise lifespan under homeostatic conditions

• ISCs are necessary for intestinal regeneration and survival in response to damage

Abbreviations list

arm

armadillo

EB

enteroblast

EC

enterocyte

EE

enteroendocrine

Esg

escargot

GFP

green fluorescent protein

Hdc

headcase

ISC

intestinal stem cell

pH3

phospo histone 3

pros

prospero

Su(H)

supressor of hairless