GATA4 is essential for jejunal function in mice

Abstract

Background & aims: Although the zinc-finger transcription factor GATA4 has been implicated in regulating jejunal gene expression, the contribution of GATA4 in controlling jejunal physiology has not been addressed.

Methods: We generated mice in which the Gata4 gene was specifically deleted in the small intestinal epithelium. Measurements of plasma cholesterol and phospholipids, intestinal absorption of dietary fat and cholesterol, and gene expression were performed on these animals.

Results: Mice lacking GATA4 in the intestine displayed a dramatic block in their ability to absorb cholesterol and dietary fat. Comparison of the global gene expression profiles of control jejunum, control ileum, and GATA4 null jejunum by gene array analysis revealed that GATA4 null jejunum lost expression of 53% of the jejunal-specific gene set and gained expression of 47% of the set of genes unique to the ileum. These alterations in gene expression included a decrease in messenger RNAs (mRNAs) encoding lipid and cholesterol transporters as well as an increase in mRNAs encoding proteins involved in bile acid absorption.

Conclusions: Our data demonstrate that GATA4 is essential for jejunal function including fat and cholesterol absorption and confirm that GATA4 plays a pivotal role in determining jejunal vs ileal identity.

Figure 1

GATA4 is efficiently eliminated from the intestinal epithelium. (A) Nuclear GATA4 protein (brown) was detected by IHC in adult mouse duodenum and jejunum but not in ileum. Scale bar = 50 _m. (B) IHC showed elimination of GATA4 protein in jejunum from adult Gata4 conditional knockout (cKO) mice. Boxed regions are shown at higher magnification below original images. Arrows indicate positive nuclear staining (brown) in control tissue and negative nuclear staining (blue) in cKO tissue. Scale bar = 50 _m (C) RT-PCR showed the absence Gata4 mRNA in jejunum of Gata4 cKO mice. Gata5 and Gata6 mRNA levels were unchanged in cKO jejunum compared with controls. Polr2a was used to normalize cDNA concentrations between samples.

Figure 2

Villous morphology is abnormal in GATA4 null jejunum. (A) H&E stained jejunum harvested from control and Gata4 conditional knockout (cKO) adult mice showed that GATA4 null villi are shorter and wider than those of controls. Alcian blue staining (deep blue) identified comparable numbers of goblet cells in the jejunum of control and mutant mice. IHC staining (brown) for acetylated tubulin (AT), α-smooth muscle actin (SMA), PECAM-1, and laminin (LAM) revealed no differences between control and GATA4 null jejunum. Scale bar = 50 _m. (B) The length and width (_m) of villi from control (n=7 mice, 151 villi) and Gata4 cKO mice (n= 12 mice, 250 villi) were determined using H&E stained micrographs of jejunum and NIH ImageJ software. Mean villus length and width were compared using a two-sample Student t test. Error bars represent standard error of the mean (SEM). * p≤0.01; **p≤0.0001

Figure 3

Mice lacking GATA4 in the intestinal epithelium are smaller than control mice. (A) Image of two 7 week old male littermates showing that the Gata4^loxP/−VilCre mouse (right) is smaller than the control mouse (left). (B) The size difference between control and mutant mice was quantified by weighing mice over a 10 week period spanning from weaning (3 weeks) to 12 weeks of age. Black lines (diamonds) in each graph show the mean weight (g) of control mice (n= 6 males, 7 females). Gray lines (squares) show the mean weight of Gata4^loxP/−VilCre mice (n=6 males, 12 females). Error bars represent SEM. A two-sample Student t test was used to determine p-values. *p<0.05; **p<0.01

Figure 4

Lipid and cholesterol metabolism are disrupted in mice lacking GATA4 in the intestinal epithelium. (A) Food consumption was measured over a 24-hour period for control (n=6) and Gata4 conditional knockout (cKO, n=10) adult male mice using the DietMax system. White bars, black bars, and gray bars show mean consumption during the light cycle, during the dark cycle, and in total, respectively. (B–D) Plasma isolated from control (n=7) and Gata4^loxP/−VilCre (n=7) adult male mice was assayed for glucose (mg/dL), cholesterol (mg/dL), and phospholipids (mg/dL). The average concentration of each is represented by black bars for controls and by gray bars for mutants. The mean level of plasma glucose (B) was not changed between control and GATA4 mutant mice. The mean levels of both plasma cholesterol (C) and plasma phospholipids (D) were significantly lower in Gata4^loxP/−VilCre mice compared with controls. (E) Dietary fat absorption was measured in control (n=7) and Gata4^loxP/−VilCre (n=8) male mice using a non-invasive, non-radioactive method. Mutant mice (gray bar) absorbed less dietary fat compared with controls (black bar). (F) Cholesterol absorption was measured in control (n=6) and Gata4^loxP/−VilCre (n=10) male mice using a fecal dual-isotope ratio method. Mutant mice (gray bar) absorbed less cholesterol compared with controls (black bar). Error bars show SEM. A two-sample Student t test was used to determine p-values (*p=0.013; **p<0.01; ***p<0.0001).

Figure 5

Ingenuity Pathway Analysis (IPA) of microarray data revealed lipid metabolism, small molecule biochemistry, and molecular transport as the functions most affected by loss of GATA4 in the jejunum. Analysis of genes differentially expressed (± ≥ 2.0, p ≤0.05) in GATA4 null intestines compared with controls by IPA identified the network shown, which involved the most genes from our array data, as functioning in lipid metabolism, small molecule biochemistry, and molecular transport. Because IPA limits each network to 35 nodes, we selected the 30 nodes representing up- and down-regulated genes and expanded the network to be maximally inclusive of interactions between differentially expressed genes. This uncovered eight additional interacting, differentially expressed genes. Blue circles/ovals represent down-regulated genes; yellow circles/ovals represent up-regulated genes. Solid lines/arrows represent direct interactions; dashed lines/arrows represent indirect interactions.

Figure 6

Loss of GATA4 in the jejunum resulted in a wide-scale shift in the jejunal gene expression profile from that characteristic of jejunum toward that of ileum. (A) Expression of genes encoding proteins involved in lipid metabolism was altered in GATA4 null jejunum. RT-PCR analysis of jejunum from three independent Gata4^loxP/+VilCre and three independent Gata4^loxP/−VilCre adult male mice confirmed that the mRNA levels of 20 genes classified as lipid metabolism genes by IPA were changed in mutant jejunum compared with control jejunum. Analysis of the level of Gata4 mRNA verified that mutant mice expressed no detectable Gata4. Polr2a was used to normalize cDNA concentrations between samples. An asterisk (*) denotes those genes that exceeded our dChip p-value threshold of p ≤0.05 (Apoc3, p = 0.05681; Fabp1, p =0.05287; Scarb1, p=0.05497). (B) Enterohepatic signaling was disrupted in mice lacking GATA4 in the intestine. RT-PCR analysis of livers from three independent control Gata4^loxP/+VilCre and three independent mutant Gata4^loxP/−VilCre adult male mice confirmed that the level of Cyp7a1 mRNA was decreased in Gata4 intestine-specific conditional knockout mice compared with control mice. Polr2a was used to normalize cDNA concentrations between samples. (C) We determined the gene sets expressed by control jejunum, control ileum, and conditional knockout (cKO) jejunum by microarray analysis. Comparison of control jejunum with control ileum revealed that although the majority of expressed genes were common to both tissues, there was a subset of jejunal-specific genes and a subset of ileal-specific genes. Comparison of the cKO expression profile revealed a large decrease in expression of jejunal genes and the induction of expression of ileal genes. J, jejunum; I, ileum.