Figure 2. Loss of Hnf4a does not compromise goblet cell maturation in healthy colon mucosa but in long term inflamed colon mucosa. (A) Alcian blue staining of control mice aged of 12 months. (B) Healthy colon region of 12 month-old mutant mice showing normal size and number of goblet cells (Scale bar, 100 mm). Electron microscopy shows a similar maturation level of goblet cells in control (C) and mutant mice (D) (Scale bar, 2 mm). (E) Small size goblet cells in hyperplasic epithelium of 12 month-old mutant mice. (F) Drastic reduction in goblet cell number in hyperplasic crypts (Scale bar, 100 mm). (G) qRT-PCR of Muc2 mRNA and (H) Muc3 mRNA in Hnf4a mutants (Open bars) and controls (Black bars). Expression levels are shown as mean values (6 SEM) relative to controls and were normalized with the Hprt gene. **P#0.01 and ***P#0.001. doi:10.1371/journal.pone.0007609.g002 with IBD, some of the Hnf4a mutant mice displayed a characteristic phenotype with relapsing episodes of hematochezia and rectal prolapse (data not illustrated). To further assess the expression of modulators of the immune response, a cytokine Hnf4a and Spontaneous IBD antibody array assay was performed on Hnf4a colon and control total protein extracts (Table S1). This analysis identified several cytokines and chemokines to be significantly up-regulated in the Hnf4a colon mutant mice (Figure 3A). An ELISA confirmed that the epithelial chemokine C-X-C ligand 1 (CXCL1) was significantly elevated in these samples (Figure 3B). This up- regulation was further correlated to the increased detection of the myeloperoxidase (MPO) signal associated with leukocytes that are recruited following the chemoattractant properties of CXCL1 released at inflammation site (Figure 3C, D). NFkB activity was significantly increased in Hnf4a colon mutant mice as determined by the level of phosphorylated IkBa (Figure 3E). Overall, these observations demonstrated that the loss of Hnf4a results in spontaneous chronic inflammation resembling inflammatory bowel disease. Expression of Cell Death, Cancer and Gastrointestinal Diseases Related Genes Is Altered in the Colon of Hnf4a Mutant Mice Gene expression profiling was performed to clarify the gene network associated with the loss of Hnf4a and the long term morphological changes observed in colonic crypts. A statistical analysis (p#.05) predicted 485 unique mouse transcripts to be modulated between control and mutant animals (differential ratio $2.0, Table S2). To gain insight into how these modifications relate to colonic function, IPA software was utilized to categorize these modified genes by biological function and to extrapolate interaction networks among them. This analysis identified cell death and cell cycle as being the most important cellular processes predicted to be affected in the Hnf4a colon (Figure S1). Of interest, the most significant diseases and disorders that were predicted to occur based on modifications in gene transcripts expression was cancer and gastrointestinal diseases. Identification of these specific molecular-physiological networks led us to show that specific cleavage of caspase-3, a critical executioner during cell apoptosis, was significantly increased in the colon of mutant Hnf4a mice (Figure 4A and B). However, the cleavage of caspase-3 was not significantly altered in young adult mice (data not shown). The expression of the E2f2 transcript, a marker for both cell proliferation and apoptosis [21], remained stable in early postnatal life but became significantly increased in the colon of 12 months old mice (Figure 4C). The expression of the aurora kinase A (AurkA) transcript, a regulator of mitosis that is up- regulated in patients with ulcerative colitis [22], was also significantly up-regulated in the long term in colon of Hnf4a mutant mice (Figure 4C). These observations were consistent with the long term significant increase in colon crypt length observed in 12 months old Hnf4a mutant mice (Figure 4D) and the drastic increase in Ki67 labeling in elongated crypts of Hnf4a colon mice as compared to controls (Figure 4E). Thus, apoptosis and cell proliferation was increased in the long term in colon disease of Hnf4a mutant mice probably as a consequence to the inflamma- tory response. Ion Channel/Transporters Are Modulated Early in Hnf4a Colon Mutant Mice Hnf4a induces epithelial polarity and barrier function in intestinal epithelial cells in vitro [5] and is a crucial regulator of multiple genes involved in tight and adherent junctions in hepatocytes [4]. We thus looked for transcript variation in components of cell-to-cell junctional complexes to monitor whether loss of Hnf4a could affect these transcriptional targets. Surprisingly, this analysis identified very few modifications in the PLoS ONE | www.plosone.org 4 October 2009 | Volume 4 | Issue 10 | e7609