Cell Culture Full length claudin-15 (NM_021719.3, 1..1846) was amplified from mouse cDNA with iproof polymerase (Bio-Rad) and cloned in plenti6/V5 vector. Lentiviruses were produced and infection of T84 and IEC6 epithelial cell lines (obtained from the American Type Culture Collection, catalog no. CCL-248 and CRL-1592) was performed as described previously [13]. Stable T84 cell populations were seeded on Corning Transwell permeable supports and electrical resistance was measured each day following the reach of confluency with EVOM instrument (World Precision Instrument). Ion Transport and Permeability Measurement Two sections of colon from each mouse were used for epithelial ion transport and permeability measurements [19]. Each colonic segment was opened along the mesenteric border, rinsed, and mounted in an Ussing chamber. Tissues were bathed in oxygenated Krebs buffer containing 10 mM glucose (serosal side) or 10 nM mannitol (luminal side) at 37uC, and net active transport across the epithelium was measured via a short circuit current (Isc; mA) injected through the tissue under voltage-clamp conditions. 2 After a 15-min equilibration period, baseline Isc (mA/cm ) was recorded. Colon paracellular permeability was measured using recovery of 51-chromium-ethylenediaminetetraacetic acid (51Cr- EDTA). Briefly, 6 mCi of 51Cr-EDTA/ml was added to the luminal side of tissues. 100 ml samples were taken from the serosal side at 0, 30, 60, 90 and 120 min and compared to 100 ml of the radioactive sample (100%) obtained from the luminal side at time 0 [20]. Statistical Analyses All data were expressed as mean 6 SEM. Groups were compared with student t test unless otherwise noted (GraphPad Prism 5, GraphPad Software, SD). Statistical significance was defined as P,.05. Results Intestinal Epithelial Specific Deletion of Hnf4a Alters Adult Colonic Crypt Integrity and Result in Spontaneous Colonic Inflammation A mouse Hnf4a intestinal epithelial conditional knockout colony was generated exactly as described recently [11]. Total RNA isolated from the colon and subjected to qRT-PCR showed that mutant mice did not produce significant levels of wild-type Hnf4a mRNA (Figure 1A). Indirect immunofluorescence confirmed that production of the Hnf4a protein was abolished in the colon of mutant mice as compared to control mice (Figure 1B). Intestinal morphology was then compared in tissue sections prepared from control and mutant mice. No significant alteration of the intestinal mucosa was observed in young adult mice. Mild colonic crypt distortion was detected in 3 month old mutant mice. This phenotype worsened over time, becoming severe and fully penetrant for each 6 to 12 months old mutant mouse analysed (Figure 1). Histological analysis of colon sections from these mice revealed healthy crypt regions (Figure 1C) surrounded with abundant focal regions of colonic mucosa with signs of acute inflammation and epithelial destruction (Figure 1D), crypt hyperplasia associated with increased submucosal immune cell infiltrates (Figure 1E) and in rare occasion, early signs of neoplasia that was never observed in age-matched control mice (Figure 1F). Goblet cells in the colonic mucosa were stained with Alcian blue and no significant differences were observed between control Hnf4a and Spontaneous IBD Figure 1. Epithelial specific loss of Hnf4a leads to various signs of chronic colon inflammation. (A) qRT-PCR of Hnf4a mRNA at 7 days, 1 month and 12 months in mutants (open bars) relative to controls (black bars). Expression levels are shown as mean values (6SEM) relative to controls for each time point (3–8 mice per group) and were normalized with the Tbp housekeeping gene. **P#.01 and ***P#.001. (B) Indirect immunofluorescence of Hnf4a performed on controls and Hnf4a colon mice. A non-specific fluorescent signal was observed in the stroma of both control and mice. Scale bars, 50 mm. (C) H&E staining of a healthy mucosa in mutants. (D) Mucosa with signs of crypt distortion and crypt loss. (E) Increased crypt length and cell infiltrates. (F) Polyp formation. These photographs are representative of eight 12 month-old individuals all showing these morphological features. The formation of polyps was only observed in 2 independent Hnf4a colon mice. All scale bars are 100 mm except for panel F that is 200 mm. doi:10.1371/journal.pone.0007609.g001 (Figure 2A) and non-inflamed regions of the adult mutant mice (Figure 2B). Electron microscopy confirmed the similarities in goblet cell maturity between control (Figure 2C) and mutant mice (Figure 2D). However, the size of goblet cells was reduced in enlarged crypts (Figure 2E) and decreased in number in the more severely distorted crypts of the Hnf4a mutant mice (Figure 2F). Relative mRNA levels of Muc2, a specific marker of goblet cells, remained stable during the first 3 months but significantly declined in older Hnf4a mutant animals (Figure 2G). The expression of the Muc3 gene decreased early during post-natal life (Figure 2H). Consistent with the similarity to the histopathology seen in patients PLoS ONE | www.plosone.org 3 October 2009 | Volume 4 | Issue 10 | e7609