Murine Intestine Research Articles

A Nitrogen Metabolic Enzyme Provides Salmonella Fitness Advantage by Promoting Utilization of Microbiota-Derived Carbon Source.

Microbes support their growth in vertebrate hosts by exploiting a large variety of dietary components as nutrients, which determines the composition of gut microbiota. A pathogen Salmonella expands by utilizing 1,2-propanediol, a microbiota-fermented product, during mucosal inflammation. However, it remains largely unknown how the pathogen decides which nutrient to consume from the complex mixture in the gut. Here, we show that Salmonella enterica serovar Typhimurium utilizes 1,2-propanediol by EIIANtr (a nitrogen-metabolic PTS component implicated in virulence)-mediated regulation of the pdu operon, thereby expanding in the murine intestine. Propionyl-CoA, a metabolic intermediate produced by 1,2-propanediol catabolism, elevates EIIANtr protein amounts, entailing positive feedback, thereby boosting the 1,2-propanediol-utilization process. EIIANtr promotes pdu expression by enhancing glutathione synthesis. CRP (cAMP receptor protein) induces pdu genes by increasing EIIANtr expression in response to glucose availability. Notably, EIIANtr-mediated 1,2-propanediol-utilization conferred a growth benefit even under high glucose conditions which reduces CRP activity. The EIIANtr-mediated activation is likely conserved in pathogenic enterobacteria including Escherichia coli. Collectively, our findings suggest that Salmonella promotes its fitness by precisely modulating the utilization system for microbiota-derived carbon source. They also suggest that Salmonella may integrate signals, processed via EIIANtr, into its metabolic program as well as virulence circuit.

A diffusible signal factor of the intestine dictates Salmonella invasion through its direct control of the virulence activator HilD.

Successful intestinal infection by Salmonella requires optimized invasion of the gut epithelium, a function that is energetically costly. Salmonella have therefore evolved to intricately regulate the expression of their virulence determinants by utilizing specific environmental cues. Here we show that a powerful repressor of Salmonella invasion, a cis-2 unsaturated long chain fatty acid, is present in the murine large intestine. Originally identified in Xylella fastidiosa as a diffusible signal factor for quorum sensing, this fatty acid directly interacts with HilD, the master transcriptional regulator of Salmonella, and prevents hilA activation, thus inhibiting Salmonella invasion. We further identify the fatty acid binding region of HilD and show it to be selective and biased in favour of signal factors with a cis-2 unsaturation over other intestinal fatty acids. Single mutation of specific HilD amino acids to alanine prevented fatty acid binding, thereby alleviating their repressive effect on invasion. Together, these results highlight an exceedingly sensitive mechanism used by Salmonella to colonize its host by detecting and exploiting specific molecules present within the complex intestinal environment.

Inhibition of Na+ /H+ exchanger isoform 3 improves gut fluidity and alkalinity in cystic fibrosis transmembrane conductance regulator-deficient and F508del mutant mice.

Constipation and intestinal obstructive episodes are major health problems in cystic fibrosis (CF) patients. Three FDA-approved drugs against constipation-prone irritable bowel syndrome were tested for their ability to increase luminal fluidity and alkalinity in cystic fibrosis transmembrane conductance regulator (CFTR) null (cftr-/- ) and F508del mutant (F508delmut/mut ) murine intestine. Guanylate cyclase C agonist linaclotide, PGE1 analogue lubiprostone and intestine-specific NHE3 inhibitor tenapanor were perfused through a ~3 cm jejunal, proximal or mid-distal colonic segment in anaesthetized cftr-/- , F508delmut/mut and WT mice. Net fluid balance was determined gravimetrically and alkaline output by pH-stat back titration. Basal jejunal fluid absorptive rates were significantly higher and basal HCO3- output was significantly lower in cftr-/- and F508delmut/mut compared to WT mice. In cftr-/- and F508delmut/mut mice, all three drugs significantly inhibited the fluid absorptive rate and increased alkaline output in the jejunum and tenapanor and lubiprostone, but not linaclotide, in the colon. After tenapanor pre-incubation, linaclotide elicited a robust fluid secretory response in WT jejunum, while no further change in absorptive rates was observed in cftr-/- and F508delmut/mut jejunum, suggesting that the increase in gut fluidity and alkalinity by linaclotide in CF gut is mediated via NHE3 inhibition. Lubiprostone also inhibited fluid absorption in cftr-/- and F508delmut/mut jejunum via NHE3 inhibition but had a residual NHE3-independent effect. Linaclotide, lubiprostone and tenapanor reduced fluid absorption and increased alkaline output in the CF gut. Their application may ameliorate constipation and reduce obstructive episodes in CF patients.

A RAC-GEF network critical for early intestinal tumourigenesis

RAC1 activity is critical for intestinal homeostasis, and is required for hyperproliferation driven by loss of the tumour suppressor gene Apc in the murine intestine. To avoid the impact of direct targeting upon homeostasis, we reasoned that indirect targeting of RAC1 via RAC-GEFs might be effective. Transcriptional profiling of Apc deficient intestinal tissue identified Vav3 and Tiam1 as key targets. Deletion of these indicated that while TIAM1 deficiency could suppress Apc-driven hyperproliferation, it had no impact upon tumourigenesis, while VAV3 deficiency had no effect. Intriguingly, deletion of either gene resulted in upregulation of Vav2, with subsequent targeting of all three (Vav2−/−Vav3−/−Tiam1−/−), profoundly suppressing hyperproliferation, tumourigenesis and RAC1 activity, without impacting normal homeostasis. Critically, the observed RAC-GEF dependency was negated by oncogenic KRAS mutation. Together, these data demonstrate that while targeting RAC-GEF molecules may have therapeutic impact at early stages, this benefit may be lost in late stage disease.

Immune Cell Isolation from Murine Intestine for Antibody Array Analysis.

Gut mucosal immune cells play an essential role in health due to their ability to orchestrate host signaling events in response to exogenous antigens. These antigens may originate from microorganisms including viruses, commensal or pathogenic bacteria, or single-celled eukaryotes, as well as from dietary foodstuff-derived proteins or products. A critical technological capacity to understand host responses to antigens is the ability to efficiently isolate and functionally characterize immune cells from intestinal tissues. Additionally, after characterization, it is of paramount importance to understand the exact functions of these immune cells under different disease states or genetic variables. Here, we outline methods for immune cell isolation from murine small and large intestines with the goal of undertaking a functional analysis of isolated cell types using antibody array platforms.

Gut commensals suppress interleukin-2 production through microRNA-200/BCL11B and microRNA-200/ETS-1 axes in lamina propria leukocytes of murine large intestine

The role of microRNAs (miRNAs) in how microbiota influence the host intestinal immune system is not fully understood. We compared the expression profiles of miRNAs and mRNAs in lamina propria leukocytes (LPL) in the large intestines of germ-free (GF) and specific pathogen-free (SPF) mice. Microarray analysis revealed different expression profiles of miRNAs and mRNAs between GF and SPF mice. Quantitative real time-PCR (qRT-PCR) showed that the level of miR-200 family members was significantly higher in SPF mice than in GF mice. In silico prediction followed by qRT-PCR suggested that Bcl11b, Ets1, Gbp7, Stat5b, and Zeb1 genes were downregulated by the miR-200 family. Western blotting revealed that the expression of BCL11B and ETS-1, but not ZEB1, in large intestinal LPL was significantly lower in SPF mice than in GF mice. Interleukin (IL)-2 production in cultured LPL upon stimulation with phorbol 12-myristate 13-acetate and ionomycin for 24 h was significantly lower in SPF mice than in GF mice. Conventionalization of GF mice substantially recapitulated SPF mice in terms of the expression of miR-200 family members and their target genes and IL-2 production in large intestinal LPL. Considering that BCL11B and ETS-1 reportedly function as transcription factors to activate the Il2 gene, we propose that the presence of gut commensals suppresses IL-2 production in large intestinal LPL, at least in part through post-transcriptional downregulation of Bcl11b and Ets1 genes by miR-200 family members.

Expression of serotonin receptor HTR4 in glucagon-like peptide-1-positive enteroendocrine cells of the murine intestine.

Serotonin (5-hydroxytryptamine [5-HT]) synthesized and released in enterochromaffin (EC) cells participates in various functions in the gastrointestinal tract by acting on a diverse range of 5-HT receptors (HTRs) expressed on smooth muscle, enteric neurons, and epithelial cells. We previously observed that genes encoding HTR2A, HTR2B, and HTR4 are expressed in murine intestinal organoids, suggesting the expression of these HTRs in intestinal epithelial cells. The present study investigated the localization of these HTRs in the murine intestine by immunofluorescence staining. HTR2A, HTR2B, and HTR4 localized in individual solitary cells in the epithelium, while HTR2C was observed in the lamina propria. In the epithelium, HTR2A, HTR2B, and HTR4 colocalized with 5-HT, and HTR4 colocalized with glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). Murine intestinal organoids show a colocalization pattern that is similar to in vivo HTR2A and HTR4 with 5-HT, GLP-1, and PYY. Intraperitoneal and intragastric administration of tegaserod, an HTR4 agonist, failed to alter plasma GLP-1 levels in fasted mice. However, intragastric but not intraperitoneal administration of tegaserod reduced dietary lipid-induced increases of plasma GLP-1 levels. This action of tegaserod was inhibited by co-administration of RS39604, an HTR4 antagonist. These results suggest that murine ileal GLP-1/PYY-producing enteroendocrine (EE) cells express HTR4, while 5-HT-producing EC cells express HTR2A, HTR2B, and HTR4. In addition, the observations regarding in vivo GLP-1 secretion suggest that HTR4 signaling in ileal EE cells suppresses dietary lipid-induced GLP-1 secretion. We thus propose that EC and EE cells may interact with each other through paracrine signaling mechanisms.

The mycobiome in murine intestine is more perturbed by food arsenic exposure than in excreted feces

Arsenic is a global pollutant that can accumulate in rice and has been confirmed to disturb the gut microbiome. By contrast, the influence on the gut mycobiome is seldom concerned because fungi comprise a numerically small proportion of the whole gut microcommunity. To expand the detection of the mycobiome in different gut sections of mammals and investigate the influence of food arsenic on the gut mycobiome in the digestive tract, we treated mice with feeds containing different compositions of arsenic species (7.3% sodium arsenate, 72.7% sodium arsenite, 1.0% sodium monomethylarsonate, and 19.0% sodium dimethylarsinate) in rice at a total arsenic dose of 30 mg/kg. After 60 days of exposure, the feces of four different sites, the ileum, cecum, colon, and excreted feces, were collected and analyzed by internal transcribed spacer gene sequencing. Among the samples, the major fungal phyla were Ascomycota, Basidiomycota, and Zygomycota; the top 10 fungal genera were Aspergillus, Verticillium, Penicillium, Cladosporium, Alternaria, Fusarium, Ophiocordyceps, Trametes, Mucor, and Nigrospora. In control mice, along the murine digestive tract, the mycobial richness and composition were significantly changed; Aspergillus and Penicillium possessed the higher ability to be stabilized in the murine gut, and larger proportions of positive correlations were observed among the major fungi. After arsenic exposure, the fungal composition was more disturbed in the intestinal tract than in feces. Along the digestive tract, arsenic can trigger larger mycobial variations, and the sensitivities of major fungi to arsenic were changed. Thus, the murine intestinal spatial mycobiota are more perturbed than excreted fecal mycobiota after food arsenic exposure. Feces are insufficient to be selected as a representative of the gut mycobiota in arsenic exposure studies.

The Lactobacillus brevis 47 f Strain Protects the Murine Intestine from Enteropathy Induced by 5-Fluorouracil.

We report that the results of our study indicate that Lactobacillus brevis 47 f strain isolated from the faeces of a healthy individual prevents the manifestations of experimental mucositis induced by treatment of Balb/c mice with the anticancer drug 5-fluorouracil (5 FU; 100 mg/kg i.p. × 3 days). The presence of damage to the intestine and the colon was determined by a morphometric analysis of specimens including the height of villi, the amount of goblet cells and infiltrating mononuclear cells, and the expression of the proliferative Ki-67 antigen. Changes in the lipid peroxidation in the blood and the intestine were determined by severalfold increase of the concentration of malonic dialdehyde. Oral administration of L. brevis 47 f strain prior to 5 FU decreased the drug-induced morphological and biochemical changes to their respective physiological levels; the ability of intestinal epitheliocytes to express Ki-67 was partially restored. These effects of L. brevis 47 f strain were more pronounced or similar to those of the reference compound Rebamipid, a quinoline derivative known to protect the gut from drug-induced toxicity. Thus, the new lactobacilli strain attenuates the severity of 5 FU-induced enteropathy.

Galectin-9 Is a Novel Regulator of Epithelial Restitution

Increasingly, the ß-galactoside binding lectins, termed galectins, are being recognized as critical regulators of cell function and organismal homeostasis. Within the context of the mucosal surface, galectins are established regulators of innate and adaptive immune responses, microbial populations, and several critical epithelial functions, including cell migration, proliferation, and response to injury. However, given their complex tissue distribution and expression patterns, their role within specific processes remains poorly understood. We took a genetic approach to understand the role of endogenous galectin-9 (Gal-9), a mucosal galectin that has been linked to inflammatory bowel disease, within the context of the murine intestine. Gal-9-deficient (Gal9-/-, also known as Lgals9-/-) animals show increased sensitivity to chemically induced colitis and impaired proliferation in the setting of acute injury. Moreover, Gal9-/--derived enteroids showed impaired growth exvivo. Consistent with a model in which endogenous Gal-9 controls epithelial growth and repair, Gal9-/- animals showed increased sensitivity to intestinal challenge in multiple models of epithelial injury, including acute irradiation injury and ectopic wound biopsies. Finally, regenerating crypts from patient biopsies showed increased expression of Gal-9, indicating these processes may be conserved in humans. Taken together, these studies implicate Gal-9 in the regulation of cellular proliferation and epithelial restitution after intestinal epithelial injury.

In vivo labeling of epithelial cell-associated antigen passages in the murine intestine.

The intestinal immune system samples luminal contents to induce adaptive immune responses that include tolerance in the steady state and protective immunity during infection. How luminal substances are delivered to the immune system has not been fully investigated. Goblet cells have an important role in this process by delivering luminal substances to the immune system through the formation of goblet cell-associated antigen passages (GAPs). Soluble antigens in the intestinal lumen are transported across the epithelium transcellularly through GAPs and delivered to dendritic cells for presentation to T cells and induction of immune responses. GAPs can be identified and quantified by using the ability of GAP-forming goblet cells to take up fluorescently labeled dextran. Here, we describe a method to visualize GAPs and other cells that have the capacity to take up luminal substances by intraluminal injection of fluorescent dextran in mice under anesthesia, tissue sectioning for slide preparation and imaging with fluorescence microscopy. In contrast to in vivo two-photon imaging previously used to identify GAPs, this technique is not limited by anatomical constraints and can be used to visualize GAP formation throughout the length of the intestine. In addition, this method can be combined with common immunohistochemistry protocols to visualize other cell types. This approach can be used to compare GAP formation following different treatments or changes to the luminal environment and to uncover how sampling of luminal substances is altered in pathophysiological conditions. This protocol requires 8 working hours over 2-3 d to be completed.

S3-4 The interaction between Candida species and bacteria in murine intestine

S3-4 The interaction between Candida species and bacteria in murine intestine

DNMT3B Oncogenic Activity in Human Intestinal Cancer Is Not Linked to CIMP or BRAFV600E Mutation.

SummaryApproximately 10% of human colorectal cancer (CRC) are associated with activated BRAFV600E mutation, typically in absence of APC mutation and often associated with a CpG island methylator (CIMP) phenotype. To protect from cancer, normal intestinal epithelial cells respond to oncogenic BRAFV600E by activation of intrinsic p53 and p16-dependent tumor suppressor mechanisms, such as cellular senescence. Conversely, CIMP is thought to contribute to bypass of these tumor suppressor mechanisms, e.g. via epigenetic silencing of tumor suppressor genes, such as p16. It has been repeatedly proposed that DNMT3B is responsible for BRAFV600E-induced CIMP in human CRC. Here we set out to test this by in silico, in vitro, and in vivo approaches. We conclude that although both BRAFV600E and DNMT3B harbor oncogenic potential in vitro and in vivo and show some evidence of cooperation in tumor promotion, they do not frequently cooperate to promote CIMP and human intestinal cancer.

The Adult Murine Intestine is Dependent on Constitutive Laminin-\u03b31 Synthesis

Laminin-γ1 is required for early embryonic development; however, the need for laminin-γ1 synthesis in adulthood is unknown. A global and inducible mouse model of laminin-γ1 deficiency was generated to address this question. Genetic ablation of the Lamc1 gene in adult mice was rapidly lethal. Despite global Lamc1 gene deletion in tamoxifen-induced mutant mice, there was minimal change in total cardiac, pulmonary, hepatic or renal laminin protein. In contrast, laminin-γ1 was significantly depleted in the small intestines, which showed crypt hyperplasia and dissociation of villous epithelium from adjacent mesenchyme. We conclude that the physiologic requirement for laminin-γ1 synthesis in adult mice is dependent on a tissue-specific basal rate of laminin-γ1 turnover that results in rapid depletion of laminin-γ1 in the intestine.

Bacterial immunogenic α-galactosylceramide identified in the murine large intestine: dependency on diet and inflammation

The glycosphingolipid, α-galactosylceramide (αGalCer), when presented by CD1d on antigen-presenting cells, efficiently activates invariant natural killer T (iNKT) cells. Thereby, it modulates immune responses against tumors, microbial and viral infections, and autoimmune diseases. Recently, the production of αGalCer by Bacteroidetes from the human gut microbiome was elucidated. Using hydrophilic interaction chromatography coupled to MS2, we screened murine intestinal tracts to identify and quantify αGalCers, and we investigated the αGalCer response to different dietary and physiologic conditions. In both the cecum and the colon of mice, we found 1-15 pmol of αGalCer per milligram of protein; in contrast, mice lacking microbiota (germ-free mice) and fed identical diet did not harbor αGalCer. The identified αGalCer contained a β(R)-hydroxylated hexadecanoyl chain N-linked to C18-sphinganine, which differed from what has been reported with Bacteroides fragilis Unlike β-anomeric structures, but similar to αGalCers from B. fragilis, the synthetic form of the murine αGalCer induced iNKT cell activation in vitro. Last, we observed a decrease in αGalCer production in mice exposed to conditions that alter the composition of the gut microbiota, including Western type diet, colitis, and influenza A virus infection. Collectively, this study suggests that αGalCer is produced by commensals in the mouse intestine and reveals that stressful conditions causing dysbiosis alter its synthesis. The consequences of this altered production on iNKT cell-mediated local and systemic immune responses are worthy of future studies.

Epithelial CD47 is critical for mucosal repair in the murine intestine in vivo

CD47 is a ubiquitously expressed transmembrane glycoprotein that regulates inflammatory responses and tissue repair. Here, we show that normal mice treated with anti-CD47 antibodies, and Cd47-null mice have impaired intestinal mucosal wound healing. Furthermore, intestinal epithelial cell (IEC)-specific loss of CD47 does not induce spontaneous immune-mediated intestinal barrier disruption but results in defective mucosal repair after biopsy-induced colonic wounding or Dextran Sulfate Sodium (DSS)-induced mucosal damage. In vitro analyses using primary cultures of CD47-deficient murine colonic IEC or human colonoid-derived IEC treated with CD47-blocking antibodies demonstrate impaired epithelial cell migration in wound healing assays. Defective wound repair after CD47 loss is linked to decreased epithelial β1 integrin and focal adhesion signaling, as well as reduced thrombospondin-1 and TGF-β1. These results demonstrate a critical role for IEC-expressed CD47 in regulating mucosal repair and raise important considerations for possible alterations in wound healing secondary to therapeutic targeting of CD47.

Control of Ion Transport by Tmem16a Expressed in Murine Intestine.

Cl– secretion by the human and murine intestinal epithelium occurs through the cystic fibrosis transmembrane conductance regulator (cftr). However, the Ca2+ activated Cl– channel Tmem16a was shown to contribute to Cl– secretion, mainly, but not exclusively, as a basolaterally located Cl– channel that controls basolateral Ca2+ signaling, and thus activation of basolateral Ca2+ dependent Sk4 K+ channels. In intestinal goblet cells, Tmem16a was shown to regulated Ca2+ signals required for exocytosis of mucus. Because a recent report denied the existence and functional role of Tmem16a in murine intestine, we reexamined in detail expression of mRNA and protein for Tmem16a in mouse colon. In experiments using short-circuited Ussing chamber and whole cell patch-clamp techniques, we further compared ion transport in wild type (WT) colon with that in mice with intestinal epithelial specific knockout of Tmem16a. As reported earlier we fully confirm expression of Tmem16a in colonic epithelial cells and the role of Tmem16a for both Ca2+-dependent and cAMP-regulated ion secretion.

Foodborne infection of mice with Salmonella Typhimurium.

The bacterial pathogen Salmonella enterica serovar Typhimurium is one of the most common causes of foodborne disease in humans and is also an important model system for bacterial pathogenesis. Oral inoculation of C57Bl/6 mice, which are genetically susceptible to Salmonella, results in systemic infection but the murine intestine is not efficiently colonized unless the intestinal microbiota is disrupted. Pretreatment of C57Bl/6 mice with streptomycin, followed by oral inoculation with Salmonella Typhimurium results in colitis resembling human intestinal Salmonellosis. The predominant method of delivery of bacteria is oral gavage, during which organisms are deposited directly into the stomach via a feeding needle. Although convenient, this method can be stressful for mice, and may lead to unwanted tracheal or systemic introduction of bacteria. Here, we developed a method for oral infection of mice by voluntary consumption of regular mouse chow inoculated with bacteria. Mice readily ate chow fragments containing up to 108 CFU Salmonella, allowing for a wide range of infectious doses. In mice pretreated with streptomycin, infection with inoculated chow resulted in reproducible infections with doses as low as 103 CFU. Mice not treated with streptomycin, as well as resistant Nramp1 reconstituted C57Bl/6J mice, were also readily infected using this method. In summary, voluntary consumption of chow inoculated with Salmonella represents a natural route of infection for foodborne salmonellosis and a viable alternative to oral gavage.

A direct comparison of mouse and human intestinal development using epithelial gene expression patterns

PURPOSE:Preterm infants are susceptible to unique pathology due to their immaturity. Mouse models are commonly used to study immature intestinal disease including necrotizing enterocolitis (NEC). Current NEC models are performed at a variety of ages, but data directly comparing intestinal developmental stage equivalency between mice and humans are lacking.METHODS:Small intestines were harvested from C57B1/6 mice at 3–4-day intervals from birth to P28 (n=8 at each age). Preterm human small intestine samples representing 17–23 weeks of completed gestation were obtained from the University of Pittsburgh Health Sciences Tissue Bank, and at term gestation during reanastamoses after resection for NEC (n=4–7 at each age). Quantification of intestinal epithelial cell types and mRNA for marker genes were evaluated on both species.RESULTS:Overall, murine and human developmental trends over time are markedly similar. Murine intestine prior to P10 is most similar to human fetal intestine prior to viability. Murine intestine at P14 is most similar to human intestine at 22–23 weeks completed gestation, and P28 murine intestine is most similar to human term intestine.CONCLUSION:Use of C57BL/6J mice to model the human immature intestine is reasonable, but the age of mouse chosen is a critical factor in model development.

Normal Calcium-Activated Anion Secretion in a Mouse Selectively Lacking TMEM16A in Intestinal Epithelium.

Calcium-activated anion secretion is expected to ameliorate cystic fibrosis, a genetic disease that carries an anion secretory defect in exocrine tissues. Human patients and animal models of the disease that present a mild intestinal phenotype have been postulated to bear a compensatory calcium-activated anion secretion in the intestine. TMEM16A is calcium-activated anion channel whose presence in the intestinal epithelium is contradictory. We aim to test the functional expression of TMEM16A using animal models with Cftr and/or Tmem16a intestinal silencing. Expression of TMEM16A was studied in a wild type and intestinal Tmem16a knockout mice by mRNA-seq, mass-spectrometry, q-PCR, Western blotting and immunolocalization. Calcium-activated anion secretion was recorded in the ileum and proximal colon of these animals including intestinal Cftr knockout and double mutants with dual Tmem16a and Cftr intestinal ablation. Mucus homeostasis was studied by immune-analysis of Mucin-2 (Muc2) and survival curves were recorded. Tmem16a transcript was found in intestine. Nevertheless, protein was barely detected in colon samples. Electrophysiological measurements demonstrated that the intestinal deletion of Tmem16a did not change calcium-activated anion secretion induced by carbachol or ATP in ileum and proximal colon. Muc2 architecture was not altered by Tmem16a silencing as was observed when Cftr was deleted from mouse intestine. Tmem16a silencing neither affected animal survival nor modified the lethality observed in the intestinal Cftr-null mouse. Our results demonstrate that TMEM16A function in the murine intestine is not related to electrogenic calcium-activated anion transport and does not affect mucus homeostasis and survival of animals.