Hnf4a ^IEC-KO mice, RNA-seq and environmental regulation of HNF4A-dependent genes. Related to Figs. 1 and 2. (A) Body weight, spleen weight, colon length, and cecum weight of 10-wk-old mice. Mean with SD. N = 4 for each group. (B) Representative H&E staining of ileum, cecum, and colon of 11–12-wk-old mice. Scale bars were shown for WT ileum and the same for all other groups: 2×, 0.5 mm; 20×, 50 μm. (C) Pathology was evaluated based on H&E staining of the ileum, cecum, and colon from 11–12-wk-old mice. Samples with a score >0 had focal epithelial loss without inflammation (likely due to artifacts during sample processing). N = 3 for each group. (D) Colonic microbiome of 3-wk-old mice was determined by shotgun sequencing. Taxa composition at the species level (mean with SEM) and α diversity indicated by Shannon entropy (min to max) were shown. N = 9 for each group. (E) Gating strategy for flow sorting IECs. (F) GO enrichment analysis of DE genes in cecal IECs from RNA-seq. (G) 8-wk-old mice were given water (NT) or antibiotic cocktails (ABX) ad lib for 4 wk. RNA levels of indicated genes in the colonic IECs were determined by qPCR and shown as 2^−dCT. Median. N = 6 for each group. (H) Mice were treated with 2% DSS for 7 d. Expression of indicated genes in the colonic IECs was determined by qPCR and shown as 2^−dCT. Median. N = 5 for each group. (I) Expression of Ang4 in the ileal and colonic IECs from 3-wk-old mice was determined by qPCR and shown as 2^−dCT. Median. N = 10–12 for each group. (G) ChIP-seq dataset (GSM1266727) was visualized on the UCSC genome browser (Kent et al., 2002; Rosenbloom et al., 2013). HNF4A enrichment at the Btnl1 and Ang4 loci was shown. Each dot represented individual mouse. Data was combined from two independent experiments or one representative experiment of two independent experiments and analyzed by Mann-Whitney tests (A and G–I). *, P < 0.0332; **, P < 0.0021.