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rgn gene is required for gut cell homeostasis after ingestion of sodium dodecyl sulfate in Drosophila
Gene 549 (2014) 141–148
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rgn gene is required for gut cell homeostasis after ingestion of sodium dodecyl sulfate in Drosophila Jichuan Pan, Li Hua Jin ⁎ College of Life Science, Northeast Forestry University, No. 26 Hexing Road, Xiangfang District, Harbin 150040, PR China
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Article history: Received 5 December 2013 Received in revised form 12 July 2014 Accepted 22 July 2014 Available online 23 July 2014 Keywords: Mutant Survival Melanization 7-Aminoactinomycin D Antimicrobial peptide
a b s t r a c t Resistance and resilience constitute the two complementary aspects of epithelial host defenses in Drosophila. Epithelial cell homeostasis is necessary for the recovery of damages caused by stress or infections. However, the genes responsible for gut epithelial homeostasis remain poorly understood. Here, we show that rgnG4035 mutant flies have higher mortality than wild-type flies after ingestion of sodium dodecyl sulfate (SDS). Excessive melanization and increased necrotic cells in the gut contribute to the reduced survival of rgnG4035 mutant flies following SDS ingestion. rgn mutant flies have a defect in the replenishment of intestinal stem cells (ISCs) following gut damage. The antimicrobial peptide (AMP) expression is affected in rgnG4035 mutant fly guts. Together, our study provides evidence that rgn gene is essential for gut cell homeostasis following damage in Drosophila. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Epithelial host defenses in Drosophila are composed of two complementary facets, resistance and resilience (Ferrandon, 2012). Resistance involves the activation of various immune responses induced by stress or infections. Resilience refers to the capacity to endure and repair damages caused by infections or the host's own immune response (Ferrandon, 2012). To maintain homeostasis, immune and developmental mechanisms must be tightly regulated in the gut (Buchon et al., 2009a; Royet, 2011). Dysregulation of inflammatory responses and tissue regeneration could lead to inflammatory bowel diseases and colorectal cancer in mammals (Garrett et al., 2010). Drosophila is an advantageous model species because of the genetic amenability and high degree of conservation between Drosophila and mammals (Apidianakis and Rahme, 2011). Advances in the research of Drosophila intestinal physiology and pathology suggest that this model can be used to elucidate many aspects of human intestinal diseases (Apidianakis and Rahme, 2011). In Drosophila, several physical and chemical tools have been identified to function in gut immunity (Royet, 2011). Peritrophic matrix (PM) which protects the host from ingested microbes and prevents the damaging action of pore-forming toxins secreted by pathogens is the first physical barrier of host defenses (Ferrandon, 2012; Royet, 2011). In addition, reactive oxygen species (ROS) and antimicrobial Abbreviations: SDS, sodium dodecyl sulfate; DSS, dextran sulfate sodium; AMPs, antimicrobial peptides; ISCs, intestinal stem cells; 7-AAD, 7-Aminoactinomycin D. ⁎ Corresponding author. E-mail address: [email protected] (L.H. Jin).
http://dx.doi.org/10.1016/j.gene.2014.07.057 0378-1119/© 2014 Elsevier B.V. All rights reserved.
peptides (AMPs) act synergistically to restrict growth and proliferation of infectious and commensal bacteria in Drosophila gut (Ferrandon, 2012; Royet, 2011). AMPs controlled by immune deficiency (IMD) pathway play a crucial role in response to ROS-resistant pathogens and limiting commensal bacteria population in Drosophila gut immunity (Ryu et al., 2006). Because gut epithelium is in constant exposure to stress and microbes from the environment, immune response and epithelial renewal require strict coordination to maintain gut homeostasis (Buchon et al., 2009a). A number of studies have shown that gut epithelium should be renewed by the division and differentiation of intestinal stem cells (ISCs) during aging, stress and intestinal infections (Apidianakis and Rahme, 2011; Buchon et al., 2009b). ISCs are located along the basement membrane of Drosophila midgut epithelium and one ISC can divide asymmetrically to generate a new ISC and a post-mitotic enteroblast, which further differentiates either into an enterocyte or an enteroendocrine cell (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). Several signaling pathways such as Notch, JAK/STAT, JNK, Wingless and EGFR have been reported to play important roles in ISC proliferation (Buchon et al., 2009a; Jiang and Edgar, 2009; Lin et al., 2008; Ohlstein and Spradling, 2006). However, genes responsible for epithelial renewal and the mechanisms for maintaining gut epithelial homeostasis remain elusive. Previous study has shown that Rgn protein sequence contains a C-type lectin domain at its N-terminus (McClure et al., 2008). Drosophila C-type lectins are reported to have carbohydrate binding activities and function in immune defense via the hemocyte-mediated pathogen recognition (Tanji et al., 2006). Two rgn hypomorphic alleles, whose homozygous animals are semi-lethal have been described by McClure et al. (2008), and rgn gene was proposed to participate in the imaginal disc
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regeneration by controlling the blastema formation time in Drosophila (McClure et al., 2008). Reg is the ortholog of rgn in mammals. In mouse, Reg acts as a growth factor which plays a crucial role in pancreatic regeneration (Takasawa et al., 2006). Moreover, it has been demonstrated that Reg is required for generation and maintenance of gastric mucosal structure (Ose et al., 2006). Knockout of Reg results in decreased proliferation of intestinal stem cells in mouse small intestine (Ose et al., 2006). In this paper, we use rgn mutant flies to investigate the function of rgn gene in epithelial host defenses. Our study demonstrates that rgn gene is essential for the maintenance of epithelial homeostasis in Drosophila gut. 2. Materials and methods 2.1. Drosophila stocks Drosophila melanogaster strains were cultured on standard cornmeal-yeast medium at 25 °C and 60% humidity unless indicated otherwise. W1118 was used as the wild-type (WT) strain. The rgnG4035 mutant (# 21628) with a P-element in the 5′ untranslated region of rgn gene was obtained from GenExel library (http://genexel.kaist.ac. kr). UAS–rgn–RNAi (# 31067) was purchased from Vienna Drosophila RNAi Center (http://stockcenter.vdrc.at). Act5C–Gal4 and Tub–Gal4 were obtained from Bloomington Stock Center. NP3084–Gal4 (Nehme et al., 2007) was obtained from the Drosophila Genetic Resource at the National Institute of Genetics (http://www.shigen.nig.ac.jp/fly/nigfly/). esg–Gal4 (Micchelli and Perrimon, 2006) was obtained from Drosophila Genomics Resource Center. 2.2. Survival experiments Groups of thirty 3–5 days old adult flies (15 females and 15 males) were dehydrated for 2 h in an empty vial and then transferred to a fly vial with food solution and maintained at 25 °C. The food solution containing 0.5% (w/v) sodium dodecyl sulfate (SDS) (Sigma) or 3% dextran sulfate sodium (DSS) (MP Biomedicals) with 5% (w/v) sucrose was added to filter disks that completely covered the vial bottom. The food solution and filter disks were changed every day and dead flies were recorded.
Fig. 1. Confirmation of the P-element inserted rgnG4035 mutant fly. (A) Relative positions and orientations of PCR primers used to confirm the P-element insertion. The insertion point for P-element is 590 bp upstream of the ATG translation start site. Primers F and R are complementary to the rgn genomic DNA surrounding the P-element insertion site, while the pry4 primer is complementary to the P-element. PCR fragments amplified by primers F and R or by primers pry4 and R are indicated I and II respectively. (B) PCR fragments I and II amplified from wild-type (WT) and rgnG4035 flies. (C) The mRNA levels of rgn in WT and rgnG4035 using RT-qPCR. Data were from three independent experiments.
Transcriptase (Fermentas). The real-time PCR was performed using SYBR Premix Ex Taq II (TaKaRa) on 7500 Real Time PCR System (Applied Biosystems). The mRNA level for each sample was normalized to control rp49 values. The primer sequences used in this study are available on request.
2.3. Imaging and 7-Aminoactinomycin D staining Female flies were used for gut dissection, because of the bigger size. For live imaging, guts were dissected at room temperature in 1 × phosphate-buffer saline (PBS) and immediately observed under an Axioskop 2 plus microscope (Zeiss). For 7-Aminoactinomycin D (7AAD) staining, 10–15 guts were dissected and immediately fixed in 4% paraformaldehyde for 30 min; then stained with 5 μg/ml 7-AAD (Invitrogen) for 30 min and washed 5 min in 1× PBS for three times. DNA was stained with 0.1 μg/ml DAPI (Sigma). The guts were finally mounted in 50% glycerol and posterior midguts were imaged under an Axioskop 2 plus microscope (Zeiss). 2.4. Antibodies The primary antibodies included rabbit anti-cleaved caspase 3 (Cell Signaling; 1:50) and rabbit anti-phospho-histone-H3 (Millipore; 1:200). Standard immunohistochemical method was used (Jin et al., 2009). 2.5. RT-qPCR Total RNA was extracted from 30 to 35 dissected guts using Trizol reagent according to the manufacturer's protocols (Invitrogen). The first stranded cDNA was synthesized by using RevertAid Reverse
Fig. 2. The mRNA levels of rgn in different developmental stages and tissues. (A) The expression analysis of rgn in different developmental stages by RT-qPCR. (B) The expression analysis of rgn in different tissues of the third instar larva by RT-qPCR. Data were from three independent experiments.
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2.6. Statistical analysis Each experiment was repeated independently three times unless otherwise indicated. The log-rank test performed in SPSS 17.0 was used for statistical analysis of fly survival curves. Error bars represented the standard deviation. Statistical significance was calculated using the Student's t-test in Data Processing System (DPS version 7.05). *P b 0.05 was considered significant and **P b 0.01 was considered more significant. ns: no significant difference. 3. Results 3.1. rgnG4035 mutant disrupts the rgn transcript rgnG4035 mutant fly that carries a P-element in the 5′ untranslated region of rgn gene was generated by GenExel library using EP element established by P. Rørth (1996). To verify the P-element insertion, PCR with primers specific to the 3′ end of the P-element (pry4) and to the 5′ untranslated region sequence of rgn surrounding the P-element insertion site (F and R) was performed (Fig. 1A). The F and R primer pairs amplified the specific fragment I from WT, failed to amplify this fragment from rgnG4035. On the contrary, the pry4 and R primer pairs amplified the specific fragment II from rgnG4035, failed to amplify this fragment from WT (Fig. 1B). The mRNA level of rgn was significantly
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decreased in rgnG4035 compared with WT by RT-qPCR analysis (Fig. 1C and Supplementary Fig. S1). These results suggest that the P-element is correctly inserted in the 5′ untranslated region of rgn, and rgnG4035 fly can be used as a mutant to study rgn gene function. Because rgnG4035 contains Gal4 binding sites at the 5′ untranslated region, we crossed rgn G4035 with Act5C–Gal4 to re-express the rgn gene (Act–gal4NUAS–rgn). 3.2. The expression pattern of rgn gene in Drosophila We analyzed the rgn gene expression in different developmental stages and various tissues of the third instar larva using RT-qPCR. During early embryo stages, the rgn was almost undetectable (Fig. 2A). While high levels of rgn were detected in the late embryo, larva, pupa and adult stages, the expression in adult stage was the highest (Fig. 2A). Likewise, high levels of rgn were detected in all larval tissues, especially in wing disc, brain and malpighian tubule (Fig. 2B). The patterns of rgn expression suggest an association with rapid cell growth and dividing tissues. 3.3. rgn mutant flies have reduced resistance to SDS To assess the function of rgn in the maintenance of Drosophila gut homeostasis, adult flies were fed with 5% sucrose containing 0.5% SDS
Fig. 3. The survival rates of rgn mutant and control flies following ingestion of SDS or DSS. (A) The survival rates of rgnG4035 mutant and control flies feeding with 5% sucrose at 25 °C. (B) The survival rates of rgnG4035 mutant and control flies feeding with 0.5% SDS at 25 °C. (C) The survival rates of rgn RNAi and control flies feeding with 0.5% SDS at 29 °C. (D) The survival rates of rgnG4035 mutant and control flies feeding with 3% DSS at 25 °C. (E) The survival rates of rgn RNAi and control flies feeding with 3% DSS at 29 °C. Three replicates were used for the determination of the survival rates. Error bars represent the standard deviation. P-value was calculated by log-rank test.
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(w/v) to induce intestinal damage. The survival rate of rgnG4035 mutant was decreased compared with WT and Act–gal4NUAS–rgn after ingestion of SDS for six days (Fig. 3B). Meanwhile, no significant difference in survival was observed between rgnG4035 mutant and control flies which were fed with 5% sucrose only (Fig. 3A). We further compared the survival of flies in which rgn was knocked down by crossing rgn– RNAi transgenic line with Gal4 lines. After ingesting 0.5% SDS for four days at 29 °C, the survival rates were decreased in RNAi flies compared with control flies (Fig. 3C). We also fed flies with 3% DSS (w/v) which had been reported as an effective cell-damage compound for Drosophila gut (Amcheslavsky et al., 2009). The similar phenotype was observed between control and rgn mutant flies after ingestion of DSS (except NP3084–gal4NUAS–rgnRNAi) (Figs. 3D and E). These results indicate that rgnG4035 mutant and rgn RNAi flies have reduced resistance to SDS and DSS. 3.4. Excessive gut melanization is a major cause of accelerating death of rgnG4035 mutant flies following SDS ingestion Previous studies have shown that SDS is a potent chemical reagent to trigger melanization in the fly gut (Chen et al., 2010; Li et al., 2013). To assess the morphological changes, we observed the dissected guts from adult flies which had been fed with SDS for 96 h. Most guts appeared to be shriveled and the length was reduced after ingestion of SDS in both rgn mutant and control flies (Fig. 4A and Supplementary Fig. S2). Melanotic masses were found at the boundary between the
mid and hindgut in most rgnG4035 mutant flies (Fig. 4B). The melanization rates were 17.3%, 76.2% and 32.2% in WT, rgnG4035 and Act– gal4NUAS–rgn fly guts respectively (Fig. 4C). These observations suggest that SDS causes more severe damage to rgn G4035 mutant guts and the reduced survival rate of rgnG4035 mutant may be related with the excessive melanization.
3.5. Increased necrotic cells contribute to the excessive gut melanization and high mortality of rgnG4035 mutant flies following SDS ingestion To further investigate whether the gut melanization and increased mortality resulted from cell death after ingestion of SDS, we stained dissected guts which had been exposed to SDS for 72 h with 7Aminoactinomycin D (7-AAD). 7-AAD can label necrotic cells which have compromised permeable membranes (Franc et al., 1999; Silva et al., 2007). rgnG4035 mutant guts showed an increased number of necrotic cells compared with WT and Act–gal4NUAS–rgn (Fig. 5A). The average number of necrotic cells in WT, rgnG4035 and Act–gal4NUAS–rgn posterior midguts was 120, 201 and 141 respectively (Fig. 5B). We also used antibodies against caspase 3 which marks apoptosis, and observed increased staining in both rgn mutant and control fly guts (Fig. 5C). But no significant difference was observed between rgnG4035 mutant and controls (Fig. 5D) (see Discussion). These results indicate that the excessive melanization and high mortality of rgnG4035 mutant after ingestion of SDS are due to increased necrotic cells in the gut.
Fig. 4. The excessive melanization in the gut of rgnG4035 contributes to the increased mortality following ingestion of SDS. (A) The length of guts is reduced after SDS ingestion. Scale bar: 500 μm. (B) Nomarski images of control and melanization guts. The arrow points to the melanization site. Scale bar: 100 μm. (C) The melanization rates in the guts of rgnG4035 mutant and control flies which had been fed with SDS for 96 h (n: number of analyzed flies).
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Fig. 5. The necrotic or apoptotic cells in rgnG4035 mutant guts compared with control flies following SDS ingestion. (A) Guts which had been fed with SDS for 72 h were stained with 7-AAD. Nuclei were stained with DAPI. Scale bar: 50 μm. (B) The comparison of gut necrotic cell quantity between rgnG4035 mutant and control flies which had been fed with SDS for 72 h. The number of necrotic cells was quantified using ImageJ software (10–15 guts were examined to quantify necrotic cells for each genotype). Error bars represent the standard deviation. P-value was calculated by Student's t-test. **P b 0.01. ns: no significant difference. (C) Guts which had been fed with SDS for 16 h were stained with caspase 3. Nuclei were stained with DAPI. 10–12 guts were examined for each genotype. Scale bar: 50 μm. (D) Quantification of fluorescence intensity of caspase 3 in the fly guts. ns: no significant difference.
3.6. rgn mutant flies have a defect in compensatory division of ISCs following SDS ingestion
gut (Fig. 6C) (see Discussion). These findings suggest that rgn mutant flies have a defect in the replenishment of ISCs following gut damage.
To study the ISC differentiation in fly gut following ingestion of SDS, we used anti-phospho-histone-H3 (PH3) staining, which can be used to assess the number of mitotic ISCs. rgnG4035 mutant guts showed a decreased number of PH3 positive cells compared with WT and Act– gal4NUAS–rgn (Fig. 6A). The average number of PH3 positive cells per midgut was 36, 10 and 26 in WT, rgnG4035 and Act–gal4NUAS–rgn respectively (Fig. 6B). We also targeted rgn in intestinal progenitor cells (ISCs and enterobasts) or in the whole midgut by using NP3084–gal4 or esg– gal4 drivers. Decreased number of PH3 positive cells was observed in NP3084–gal4NUAS–rgnRNAi fly gut compared with NP3084–gal4N+ (Fig. 6C). However, no significant difference of PH3 positive cell numbers was observed between esg–gal4NUAS–rgnRNAi and esg–gal4N+fly
3.7. The expression of AMPs is affected in rgnG4035 mutant fly gut following SDS ingestion To determine the AMP expression in fly guts following ingestion of SDS, four AMP transcripts (Drs: Drosomycin, Dpt: Diptericin, CecA2: Cecropin A2, CecA1: Cecropin A1) were analyzed using RT-qPCR. The result revealed that both Drs and Dpt were strongly induced in WT and rgnG4035 fly guts following SDS ingestion (Figs. 7A and B). But the induction seemed restricted to 4 h in WT fly gut, while the induction in rgnG4035 fly gut was much higher and occurred at 16 h (Figs. 7A and B). Moreover, CeCA1 and CeCA2 were not highly induced following SDS ingestion at 4 and 16 h in WT and rgnG4035 mutant guts (the fold
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Fig. 6. The number of phosphor-H3-positive cells in rgn mutant guts comparing with control flies following ingestion of SDS. (A) Guts which had been orally exposed to 0.5% SDS for 16 h were stained with antibody against phosphor-H3. Nuclei were stained with DAPI. Scale bar: 50 μm. (B and C) Quantification of PH3-positive cells per midgut of flies which had been fed with 0.5% SDS for 16 h. 8–10 guts were examined for each genotype. **P b 0.01. ns: no significant difference.
change b 1.5) (Figs. 7C and D). These results demonstrate that the induction time and expression level of AMPs in rgnG4035 mutant fly gut are not as the same as in WT. 4. Discussion Our data demonstrate the function of rgn gene in Drosophila epithelial host defenses following gut cell damage. Previous studies have reported that some chemical reagents such as sodium dodecyl sulfate
(SDS), dextran sulfate sodium (DSS) and bleomycin could lead to enterocyte damage and destroy the homeostasis of the intestinal epithelium (Buchon et al., 2009a, 2009b; Li et al., 2013). Since rgnG4035 mutant flies do not have any visible morphological or developmental abnormalities, and no difference in longevity is observed between WT and rgnG4035 maintained under customary conditions (data not shown), excessive gut damage and defect in damage repair might be the reason why rgnG4035 mutant flies have reduced resistance to SDS. This is supported by evidence that high melanization is observed in
Fig. 7. The mRNA levels of AMPs in WT and rgnG4035 mutant guts following ingestion of SDS. Expression levels of AMP genes in WT and rgnG4035 mutant guts which had been orally exposed to 0.5% SDS for 0, 4 and 16 h using RT-qPCR analysis. Drs: Drosomycin, Dpt: Diptericin, CecA2: Cecropin A2, CecA1: Cecropin A1. Data were from three independent experiments.
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rgnG4035 mutant fly guts following ingestion of SDS (Figs. 4B and C). The melanization reaction regulated by serine proteases and serpins is a prominent immune response in Drosophila (De Gregorio et al., 2002; Scherfer et al., 2008). In addition, the deposition of melanin at wound sites could help the host sequester the intruding microorganisms, and melanization reaction also cooperates with other immune responses such as blood clotting, wound healing, cellular defense and synthesis of antimicrobial peptides (Cerenius et al., 2008; Tang et al., 2008). However, excessive melanization generates high oxidative and toxic intermediates that could kill the fly (Chen et al., 2010; Scherfer et al., 2008). In our study, the excessive melanization reaction induced by SDS should be detrimental and led to the disruption of gut integrities, which correlates with the death of flies. Epithelial renewal is necessary for the recovery of fly guts from damage. To maintain gut homeostasis, dead cells should be removed and replenished by the division and differentiation of intestinal stem cells (Amcheslavsky et al., 2009; Apidianakis and Rahme, 2011). In this study, rgnG4035 mutant shows an increased number of necrotic cells and decreased number of compensatory division ISCs in the gut compared with control flies in response to SDS (Figs. 5A and B and 6A–C). This suggests that rgnG4035 mutant flies have defective abilities in enduring and repairing damage caused by SDS. Because the dead cells could not be replaced appropriately, severe damage and impaired epithelial renewal process lead to gut epithelial homeostasis disruption. We also found that the expression of rgn was elevated after SDS challenge in fly gut (Supplementary Fig. S3), which implies rgn functions in maintaining gut homeostasis following damage. Notably, no difference in compensatory division of ISCs following SDS ingestion was found between esg–gal4NUAS–rgnRNAi and esg– gal4N+fly gut (Fig. 6C), which hints that rgn function does not take place in enterocyte precursor cells (ISCs and enteroblasts), but in enterocytes, enteroendocrine cells, gut muscle cells or other tissues. It has been reported that the damage might be sensed by the Hippo and JNK pathways in enterocytes, then led to the production of Upd cytokines and relayed to ISCs to activate ISC proliferation (Karpowicz et al., 2010; Ren et al., 2010; Shaw et al., 2010). If rgn is implicated in these pathways should be investigated in further study. Antimicrobial peptides (AMPs) are required for protecting against pathogens and controlling commensal bacteria population in Drosophila gut (Ryu et al., 2006, 2008). Because the detergent SDS could lyse the microbiota, it is unlikely to trigger a strong gut immune response following SDS ingestion. We speculate that the induction of AMPs is an immune response to control gut indigenous microbiota population after ingestion of SDS. Surprisingly, rgnG4035 mutant flies reveal no sensitivity to Erwinia carotovora carotovora 15 (Ecc 15) and Pseudomonas aeruginosa (a pathogenic bacterium could cross the intestinal barrier via oral ingestion and cause systemic bacteremia) (Limmer et al., 2011) (Supplementary Fig. S4A and B). Likewise, rgnG4035 mutant flies have no increased sensitivity to septic injury by other pathogens (such as Enterobacter cloacae and Listeria monocytogenes) (data not shown). These results suggest that rgnG4035 mutant may have no defect in response to invasive pathogens even though the AMP expression is affected in the gut. It has been reported that ingested Ecc 15 could trigger the delamination and anoikis (a specific process of apoptosis induced by the loss of cell attachment) of damaged enterocytes (Buchon et al., 2010). In addition, we observed increased caspase 3 staining in fly guts which had been exposed to SDS for 16 h, but the staining was weak (Fig. 5C), it appeared that most gut cells were prone to necrosis rather than apoptosis following ingestion of SDS. Therefore, the absence of phenotype with ingested Ecc 15 may be due to the fact that Ecc 15 triggers anoikis, whereas SDS may essentially provoke necrosis. The precise mechanism by which SDS causes gut epithelial cell damages remains unknown. It has been reported that SDS could enhance drug and peptide absorption by increasing cell membrane and tight junction permeability in human intestinal epithelium (Anderberg and Artursson, 1993).
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In summary, our results indicate that rgnG4035 mutant flies have reduced resistance to SDS. Excessive melanization and increased necrotic cells in the gut contribute to the high mortality of rgnG4035 mutant flies following ingestion of SDS. rgn mutant flies have a defect in the replenishment of ISCs following gut damage. The AMP expression is affected in rgnG4035 mutant gut. Taken together, our study provides evidence that rgn gene is required for the maintenance of epithelial homeostasis in Drosophila gut. Further work will be required to investigate which conserved signaling pathways act synergistically with rgn gene in maintaining epithelial homeostasis. Our findings provide helpful information in understanding the process of epithelial renewal as well as therapies for inflammatory bowel diseases in human. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.07.057. 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rgn gene is required for gut cell homeostasis after ingestion of sodium dodecyl sulfate in Drosophila Jichuan Pan, Li Hua Jin ⁎ College of Life Science, Northeast Forestry University, No. 26 Hexing Road, Xiangfang District, Harbin 150040, PR China
a r t i c l e
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Article history: Received 5 December 2013 Received in revised form 12 July 2014 Accepted 22 July 2014 Available online 23 July 2014 Keywords: Mutant Survival Melanization 7-Aminoactinomycin D Antimicrobial peptide
a b s t r a c t Resistance and resilience constitute the two complementary aspects of epithelial host defenses in Drosophila. Epithelial cell homeostasis is necessary for the recovery of damages caused by stress or infections. However, the genes responsible for gut epithelial homeostasis remain poorly understood. Here, we show that rgnG4035 mutant flies have higher mortality than wild-type flies after ingestion of sodium dodecyl sulfate (SDS). Excessive melanization and increased necrotic cells in the gut contribute to the reduced survival of rgnG4035 mutant flies following SDS ingestion. rgn mutant flies have a defect in the replenishment of intestinal stem cells (ISCs) following gut damage. The antimicrobial peptide (AMP) expression is affected in rgnG4035 mutant fly guts. Together, our study provides evidence that rgn gene is essential for gut cell homeostasis following damage in Drosophila. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Epithelial host defenses in Drosophila are composed of two complementary facets, resistance and resilience (Ferrandon, 2012). Resistance involves the activation of various immune responses induced by stress or infections. Resilience refers to the capacity to endure and repair damages caused by infections or the host's own immune response (Ferrandon, 2012). To maintain homeostasis, immune and developmental mechanisms must be tightly regulated in the gut (Buchon et al., 2009a; Royet, 2011). Dysregulation of inflammatory responses and tissue regeneration could lead to inflammatory bowel diseases and colorectal cancer in mammals (Garrett et al., 2010). Drosophila is an advantageous model species because of the genetic amenability and high degree of conservation between Drosophila and mammals (Apidianakis and Rahme, 2011). Advances in the research of Drosophila intestinal physiology and pathology suggest that this model can be used to elucidate many aspects of human intestinal diseases (Apidianakis and Rahme, 2011). In Drosophila, several physical and chemical tools have been identified to function in gut immunity (Royet, 2011). Peritrophic matrix (PM) which protects the host from ingested microbes and prevents the damaging action of pore-forming toxins secreted by pathogens is the first physical barrier of host defenses (Ferrandon, 2012; Royet, 2011). In addition, reactive oxygen species (ROS) and antimicrobial Abbreviations: SDS, sodium dodecyl sulfate; DSS, dextran sulfate sodium; AMPs, antimicrobial peptides; ISCs, intestinal stem cells; 7-AAD, 7-Aminoactinomycin D. ⁎ Corresponding author. E-mail address: [email protected] (L.H. Jin).
http://dx.doi.org/10.1016/j.gene.2014.07.057 0378-1119/© 2014 Elsevier B.V. All rights reserved.
peptides (AMPs) act synergistically to restrict growth and proliferation of infectious and commensal bacteria in Drosophila gut (Ferrandon, 2012; Royet, 2011). AMPs controlled by immune deficiency (IMD) pathway play a crucial role in response to ROS-resistant pathogens and limiting commensal bacteria population in Drosophila gut immunity (Ryu et al., 2006). Because gut epithelium is in constant exposure to stress and microbes from the environment, immune response and epithelial renewal require strict coordination to maintain gut homeostasis (Buchon et al., 2009a). A number of studies have shown that gut epithelium should be renewed by the division and differentiation of intestinal stem cells (ISCs) during aging, stress and intestinal infections (Apidianakis and Rahme, 2011; Buchon et al., 2009b). ISCs are located along the basement membrane of Drosophila midgut epithelium and one ISC can divide asymmetrically to generate a new ISC and a post-mitotic enteroblast, which further differentiates either into an enterocyte or an enteroendocrine cell (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006). Several signaling pathways such as Notch, JAK/STAT, JNK, Wingless and EGFR have been reported to play important roles in ISC proliferation (Buchon et al., 2009a; Jiang and Edgar, 2009; Lin et al., 2008; Ohlstein and Spradling, 2006). However, genes responsible for epithelial renewal and the mechanisms for maintaining gut epithelial homeostasis remain elusive. Previous study has shown that Rgn protein sequence contains a C-type lectin domain at its N-terminus (McClure et al., 2008). Drosophila C-type lectins are reported to have carbohydrate binding activities and function in immune defense via the hemocyte-mediated pathogen recognition (Tanji et al., 2006). Two rgn hypomorphic alleles, whose homozygous animals are semi-lethal have been described by McClure et al. (2008), and rgn gene was proposed to participate in the imaginal disc
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regeneration by controlling the blastema formation time in Drosophila (McClure et al., 2008). Reg is the ortholog of rgn in mammals. In mouse, Reg acts as a growth factor which plays a crucial role in pancreatic regeneration (Takasawa et al., 2006). Moreover, it has been demonstrated that Reg is required for generation and maintenance of gastric mucosal structure (Ose et al., 2006). Knockout of Reg results in decreased proliferation of intestinal stem cells in mouse small intestine (Ose et al., 2006). In this paper, we use rgn mutant flies to investigate the function of rgn gene in epithelial host defenses. Our study demonstrates that rgn gene is essential for the maintenance of epithelial homeostasis in Drosophila gut. 2. Materials and methods 2.1. Drosophila stocks Drosophila melanogaster strains were cultured on standard cornmeal-yeast medium at 25 °C and 60% humidity unless indicated otherwise. W1118 was used as the wild-type (WT) strain. The rgnG4035 mutant (# 21628) with a P-element in the 5′ untranslated region of rgn gene was obtained from GenExel library (http://genexel.kaist.ac. kr). UAS–rgn–RNAi (# 31067) was purchased from Vienna Drosophila RNAi Center (http://stockcenter.vdrc.at). Act5C–Gal4 and Tub–Gal4 were obtained from Bloomington Stock Center. NP3084–Gal4 (Nehme et al., 2007) was obtained from the Drosophila Genetic Resource at the National Institute of Genetics (http://www.shigen.nig.ac.jp/fly/nigfly/). esg–Gal4 (Micchelli and Perrimon, 2006) was obtained from Drosophila Genomics Resource Center. 2.2. Survival experiments Groups of thirty 3–5 days old adult flies (15 females and 15 males) were dehydrated for 2 h in an empty vial and then transferred to a fly vial with food solution and maintained at 25 °C. The food solution containing 0.5% (w/v) sodium dodecyl sulfate (SDS) (Sigma) or 3% dextran sulfate sodium (DSS) (MP Biomedicals) with 5% (w/v) sucrose was added to filter disks that completely covered the vial bottom. The food solution and filter disks were changed every day and dead flies were recorded.
Fig. 1. Confirmation of the P-element inserted rgnG4035 mutant fly. (A) Relative positions and orientations of PCR primers used to confirm the P-element insertion. The insertion point for P-element is 590 bp upstream of the ATG translation start site. Primers F and R are complementary to the rgn genomic DNA surrounding the P-element insertion site, while the pry4 primer is complementary to the P-element. PCR fragments amplified by primers F and R or by primers pry4 and R are indicated I and II respectively. (B) PCR fragments I and II amplified from wild-type (WT) and rgnG4035 flies. (C) The mRNA levels of rgn in WT and rgnG4035 using RT-qPCR. Data were from three independent experiments.
Transcriptase (Fermentas). The real-time PCR was performed using SYBR Premix Ex Taq II (TaKaRa) on 7500 Real Time PCR System (Applied Biosystems). The mRNA level for each sample was normalized to control rp49 values. The primer sequences used in this study are available on request.
2.3. Imaging and 7-Aminoactinomycin D staining Female flies were used for gut dissection, because of the bigger size. For live imaging, guts were dissected at room temperature in 1 × phosphate-buffer saline (PBS) and immediately observed under an Axioskop 2 plus microscope (Zeiss). For 7-Aminoactinomycin D (7AAD) staining, 10–15 guts were dissected and immediately fixed in 4% paraformaldehyde for 30 min; then stained with 5 μg/ml 7-AAD (Invitrogen) for 30 min and washed 5 min in 1× PBS for three times. DNA was stained with 0.1 μg/ml DAPI (Sigma). The guts were finally mounted in 50% glycerol and posterior midguts were imaged under an Axioskop 2 plus microscope (Zeiss). 2.4. Antibodies The primary antibodies included rabbit anti-cleaved caspase 3 (Cell Signaling; 1:50) and rabbit anti-phospho-histone-H3 (Millipore; 1:200). Standard immunohistochemical method was used (Jin et al., 2009). 2.5. RT-qPCR Total RNA was extracted from 30 to 35 dissected guts using Trizol reagent according to the manufacturer's protocols (Invitrogen). The first stranded cDNA was synthesized by using RevertAid Reverse
Fig. 2. The mRNA levels of rgn in different developmental stages and tissues. (A) The expression analysis of rgn in different developmental stages by RT-qPCR. (B) The expression analysis of rgn in different tissues of the third instar larva by RT-qPCR. Data were from three independent experiments.
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2.6. Statistical analysis Each experiment was repeated independently three times unless otherwise indicated. The log-rank test performed in SPSS 17.0 was used for statistical analysis of fly survival curves. Error bars represented the standard deviation. Statistical significance was calculated using the Student's t-test in Data Processing System (DPS version 7.05). *P b 0.05 was considered significant and **P b 0.01 was considered more significant. ns: no significant difference. 3. Results 3.1. rgnG4035 mutant disrupts the rgn transcript rgnG4035 mutant fly that carries a P-element in the 5′ untranslated region of rgn gene was generated by GenExel library using EP element established by P. Rørth (1996). To verify the P-element insertion, PCR with primers specific to the 3′ end of the P-element (pry4) and to the 5′ untranslated region sequence of rgn surrounding the P-element insertion site (F and R) was performed (Fig. 1A). The F and R primer pairs amplified the specific fragment I from WT, failed to amplify this fragment from rgnG4035. On the contrary, the pry4 and R primer pairs amplified the specific fragment II from rgnG4035, failed to amplify this fragment from WT (Fig. 1B). The mRNA level of rgn was significantly
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decreased in rgnG4035 compared with WT by RT-qPCR analysis (Fig. 1C and Supplementary Fig. S1). These results suggest that the P-element is correctly inserted in the 5′ untranslated region of rgn, and rgnG4035 fly can be used as a mutant to study rgn gene function. Because rgnG4035 contains Gal4 binding sites at the 5′ untranslated region, we crossed rgn G4035 with Act5C–Gal4 to re-express the rgn gene (Act–gal4NUAS–rgn). 3.2. The expression pattern of rgn gene in Drosophila We analyzed the rgn gene expression in different developmental stages and various tissues of the third instar larva using RT-qPCR. During early embryo stages, the rgn was almost undetectable (Fig. 2A). While high levels of rgn were detected in the late embryo, larva, pupa and adult stages, the expression in adult stage was the highest (Fig. 2A). Likewise, high levels of rgn were detected in all larval tissues, especially in wing disc, brain and malpighian tubule (Fig. 2B). The patterns of rgn expression suggest an association with rapid cell growth and dividing tissues. 3.3. rgn mutant flies have reduced resistance to SDS To assess the function of rgn in the maintenance of Drosophila gut homeostasis, adult flies were fed with 5% sucrose containing 0.5% SDS
Fig. 3. The survival rates of rgn mutant and control flies following ingestion of SDS or DSS. (A) The survival rates of rgnG4035 mutant and control flies feeding with 5% sucrose at 25 °C. (B) The survival rates of rgnG4035 mutant and control flies feeding with 0.5% SDS at 25 °C. (C) The survival rates of rgn RNAi and control flies feeding with 0.5% SDS at 29 °C. (D) The survival rates of rgnG4035 mutant and control flies feeding with 3% DSS at 25 °C. (E) The survival rates of rgn RNAi and control flies feeding with 3% DSS at 29 °C. Three replicates were used for the determination of the survival rates. Error bars represent the standard deviation. P-value was calculated by log-rank test.
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(w/v) to induce intestinal damage. The survival rate of rgnG4035 mutant was decreased compared with WT and Act–gal4NUAS–rgn after ingestion of SDS for six days (Fig. 3B). Meanwhile, no significant difference in survival was observed between rgnG4035 mutant and control flies which were fed with 5% sucrose only (Fig. 3A). We further compared the survival of flies in which rgn was knocked down by crossing rgn– RNAi transgenic line with Gal4 lines. After ingesting 0.5% SDS for four days at 29 °C, the survival rates were decreased in RNAi flies compared with control flies (Fig. 3C). We also fed flies with 3% DSS (w/v) which had been reported as an effective cell-damage compound for Drosophila gut (Amcheslavsky et al., 2009). The similar phenotype was observed between control and rgn mutant flies after ingestion of DSS (except NP3084–gal4NUAS–rgnRNAi) (Figs. 3D and E). These results indicate that rgnG4035 mutant and rgn RNAi flies have reduced resistance to SDS and DSS. 3.4. Excessive gut melanization is a major cause of accelerating death of rgnG4035 mutant flies following SDS ingestion Previous studies have shown that SDS is a potent chemical reagent to trigger melanization in the fly gut (Chen et al., 2010; Li et al., 2013). To assess the morphological changes, we observed the dissected guts from adult flies which had been fed with SDS for 96 h. Most guts appeared to be shriveled and the length was reduced after ingestion of SDS in both rgn mutant and control flies (Fig. 4A and Supplementary Fig. S2). Melanotic masses were found at the boundary between the
mid and hindgut in most rgnG4035 mutant flies (Fig. 4B). The melanization rates were 17.3%, 76.2% and 32.2% in WT, rgnG4035 and Act– gal4NUAS–rgn fly guts respectively (Fig. 4C). These observations suggest that SDS causes more severe damage to rgn G4035 mutant guts and the reduced survival rate of rgnG4035 mutant may be related with the excessive melanization.
3.5. Increased necrotic cells contribute to the excessive gut melanization and high mortality of rgnG4035 mutant flies following SDS ingestion To further investigate whether the gut melanization and increased mortality resulted from cell death after ingestion of SDS, we stained dissected guts which had been exposed to SDS for 72 h with 7Aminoactinomycin D (7-AAD). 7-AAD can label necrotic cells which have compromised permeable membranes (Franc et al., 1999; Silva et al., 2007). rgnG4035 mutant guts showed an increased number of necrotic cells compared with WT and Act–gal4NUAS–rgn (Fig. 5A). The average number of necrotic cells in WT, rgnG4035 and Act–gal4NUAS–rgn posterior midguts was 120, 201 and 141 respectively (Fig. 5B). We also used antibodies against caspase 3 which marks apoptosis, and observed increased staining in both rgn mutant and control fly guts (Fig. 5C). But no significant difference was observed between rgnG4035 mutant and controls (Fig. 5D) (see Discussion). These results indicate that the excessive melanization and high mortality of rgnG4035 mutant after ingestion of SDS are due to increased necrotic cells in the gut.
Fig. 4. The excessive melanization in the gut of rgnG4035 contributes to the increased mortality following ingestion of SDS. (A) The length of guts is reduced after SDS ingestion. Scale bar: 500 μm. (B) Nomarski images of control and melanization guts. The arrow points to the melanization site. Scale bar: 100 μm. (C) The melanization rates in the guts of rgnG4035 mutant and control flies which had been fed with SDS for 96 h (n: number of analyzed flies).
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Fig. 5. The necrotic or apoptotic cells in rgnG4035 mutant guts compared with control flies following SDS ingestion. (A) Guts which had been fed with SDS for 72 h were stained with 7-AAD. Nuclei were stained with DAPI. Scale bar: 50 μm. (B) The comparison of gut necrotic cell quantity between rgnG4035 mutant and control flies which had been fed with SDS for 72 h. The number of necrotic cells was quantified using ImageJ software (10–15 guts were examined to quantify necrotic cells for each genotype). Error bars represent the standard deviation. P-value was calculated by Student's t-test. **P b 0.01. ns: no significant difference. (C) Guts which had been fed with SDS for 16 h were stained with caspase 3. Nuclei were stained with DAPI. 10–12 guts were examined for each genotype. Scale bar: 50 μm. (D) Quantification of fluorescence intensity of caspase 3 in the fly guts. ns: no significant difference.
3.6. rgn mutant flies have a defect in compensatory division of ISCs following SDS ingestion
gut (Fig. 6C) (see Discussion). These findings suggest that rgn mutant flies have a defect in the replenishment of ISCs following gut damage.
To study the ISC differentiation in fly gut following ingestion of SDS, we used anti-phospho-histone-H3 (PH3) staining, which can be used to assess the number of mitotic ISCs. rgnG4035 mutant guts showed a decreased number of PH3 positive cells compared with WT and Act– gal4NUAS–rgn (Fig. 6A). The average number of PH3 positive cells per midgut was 36, 10 and 26 in WT, rgnG4035 and Act–gal4NUAS–rgn respectively (Fig. 6B). We also targeted rgn in intestinal progenitor cells (ISCs and enterobasts) or in the whole midgut by using NP3084–gal4 or esg– gal4 drivers. Decreased number of PH3 positive cells was observed in NP3084–gal4NUAS–rgnRNAi fly gut compared with NP3084–gal4N+ (Fig. 6C). However, no significant difference of PH3 positive cell numbers was observed between esg–gal4NUAS–rgnRNAi and esg–gal4N+fly
3.7. The expression of AMPs is affected in rgnG4035 mutant fly gut following SDS ingestion To determine the AMP expression in fly guts following ingestion of SDS, four AMP transcripts (Drs: Drosomycin, Dpt: Diptericin, CecA2: Cecropin A2, CecA1: Cecropin A1) were analyzed using RT-qPCR. The result revealed that both Drs and Dpt were strongly induced in WT and rgnG4035 fly guts following SDS ingestion (Figs. 7A and B). But the induction seemed restricted to 4 h in WT fly gut, while the induction in rgnG4035 fly gut was much higher and occurred at 16 h (Figs. 7A and B). Moreover, CeCA1 and CeCA2 were not highly induced following SDS ingestion at 4 and 16 h in WT and rgnG4035 mutant guts (the fold
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Fig. 6. The number of phosphor-H3-positive cells in rgn mutant guts comparing with control flies following ingestion of SDS. (A) Guts which had been orally exposed to 0.5% SDS for 16 h were stained with antibody against phosphor-H3. Nuclei were stained with DAPI. Scale bar: 50 μm. (B and C) Quantification of PH3-positive cells per midgut of flies which had been fed with 0.5% SDS for 16 h. 8–10 guts were examined for each genotype. **P b 0.01. ns: no significant difference.
change b 1.5) (Figs. 7C and D). These results demonstrate that the induction time and expression level of AMPs in rgnG4035 mutant fly gut are not as the same as in WT. 4. Discussion Our data demonstrate the function of rgn gene in Drosophila epithelial host defenses following gut cell damage. Previous studies have reported that some chemical reagents such as sodium dodecyl sulfate
(SDS), dextran sulfate sodium (DSS) and bleomycin could lead to enterocyte damage and destroy the homeostasis of the intestinal epithelium (Buchon et al., 2009a, 2009b; Li et al., 2013). Since rgnG4035 mutant flies do not have any visible morphological or developmental abnormalities, and no difference in longevity is observed between WT and rgnG4035 maintained under customary conditions (data not shown), excessive gut damage and defect in damage repair might be the reason why rgnG4035 mutant flies have reduced resistance to SDS. This is supported by evidence that high melanization is observed in
Fig. 7. The mRNA levels of AMPs in WT and rgnG4035 mutant guts following ingestion of SDS. Expression levels of AMP genes in WT and rgnG4035 mutant guts which had been orally exposed to 0.5% SDS for 0, 4 and 16 h using RT-qPCR analysis. Drs: Drosomycin, Dpt: Diptericin, CecA2: Cecropin A2, CecA1: Cecropin A1. Data were from three independent experiments.
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rgnG4035 mutant fly guts following ingestion of SDS (Figs. 4B and C). The melanization reaction regulated by serine proteases and serpins is a prominent immune response in Drosophila (De Gregorio et al., 2002; Scherfer et al., 2008). In addition, the deposition of melanin at wound sites could help the host sequester the intruding microorganisms, and melanization reaction also cooperates with other immune responses such as blood clotting, wound healing, cellular defense and synthesis of antimicrobial peptides (Cerenius et al., 2008; Tang et al., 2008). However, excessive melanization generates high oxidative and toxic intermediates that could kill the fly (Chen et al., 2010; Scherfer et al., 2008). In our study, the excessive melanization reaction induced by SDS should be detrimental and led to the disruption of gut integrities, which correlates with the death of flies. Epithelial renewal is necessary for the recovery of fly guts from damage. To maintain gut homeostasis, dead cells should be removed and replenished by the division and differentiation of intestinal stem cells (Amcheslavsky et al., 2009; Apidianakis and Rahme, 2011). In this study, rgnG4035 mutant shows an increased number of necrotic cells and decreased number of compensatory division ISCs in the gut compared with control flies in response to SDS (Figs. 5A and B and 6A–C). This suggests that rgnG4035 mutant flies have defective abilities in enduring and repairing damage caused by SDS. Because the dead cells could not be replaced appropriately, severe damage and impaired epithelial renewal process lead to gut epithelial homeostasis disruption. We also found that the expression of rgn was elevated after SDS challenge in fly gut (Supplementary Fig. S3), which implies rgn functions in maintaining gut homeostasis following damage. Notably, no difference in compensatory division of ISCs following SDS ingestion was found between esg–gal4NUAS–rgnRNAi and esg– gal4N+fly gut (Fig. 6C), which hints that rgn function does not take place in enterocyte precursor cells (ISCs and enteroblasts), but in enterocytes, enteroendocrine cells, gut muscle cells or other tissues. It has been reported that the damage might be sensed by the Hippo and JNK pathways in enterocytes, then led to the production of Upd cytokines and relayed to ISCs to activate ISC proliferation (Karpowicz et al., 2010; Ren et al., 2010; Shaw et al., 2010). If rgn is implicated in these pathways should be investigated in further study. Antimicrobial peptides (AMPs) are required for protecting against pathogens and controlling commensal bacteria population in Drosophila gut (Ryu et al., 2006, 2008). Because the detergent SDS could lyse the microbiota, it is unlikely to trigger a strong gut immune response following SDS ingestion. We speculate that the induction of AMPs is an immune response to control gut indigenous microbiota population after ingestion of SDS. Surprisingly, rgnG4035 mutant flies reveal no sensitivity to Erwinia carotovora carotovora 15 (Ecc 15) and Pseudomonas aeruginosa (a pathogenic bacterium could cross the intestinal barrier via oral ingestion and cause systemic bacteremia) (Limmer et al., 2011) (Supplementary Fig. S4A and B). Likewise, rgnG4035 mutant flies have no increased sensitivity to septic injury by other pathogens (such as Enterobacter cloacae and Listeria monocytogenes) (data not shown). These results suggest that rgnG4035 mutant may have no defect in response to invasive pathogens even though the AMP expression is affected in the gut. It has been reported that ingested Ecc 15 could trigger the delamination and anoikis (a specific process of apoptosis induced by the loss of cell attachment) of damaged enterocytes (Buchon et al., 2010). In addition, we observed increased caspase 3 staining in fly guts which had been exposed to SDS for 16 h, but the staining was weak (Fig. 5C), it appeared that most gut cells were prone to necrosis rather than apoptosis following ingestion of SDS. Therefore, the absence of phenotype with ingested Ecc 15 may be due to the fact that Ecc 15 triggers anoikis, whereas SDS may essentially provoke necrosis. The precise mechanism by which SDS causes gut epithelial cell damages remains unknown. It has been reported that SDS could enhance drug and peptide absorption by increasing cell membrane and tight junction permeability in human intestinal epithelium (Anderberg and Artursson, 1993).
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In summary, our results indicate that rgnG4035 mutant flies have reduced resistance to SDS. Excessive melanization and increased necrotic cells in the gut contribute to the high mortality of rgnG4035 mutant flies following ingestion of SDS. rgn mutant flies have a defect in the replenishment of ISCs following gut damage. The AMP expression is affected in rgnG4035 mutant gut. Taken together, our study provides evidence that rgn gene is required for the maintenance of epithelial homeostasis in Drosophila gut. Further work will be required to investigate which conserved signaling pathways act synergistically with rgn gene in maintaining epithelial homeostasis. Our findings provide helpful information in understanding the process of epithelial renewal as well as therapies for inflammatory bowel diseases in human. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2014.07.057. 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