Gata4 is essential for the maintenance of jejunal-ileal identities in the adult mouse small intestine

Abstract

Gata4, a member of the zinc finger family of GATA transcription factors, is highly expressed in duodenum and jejunum but is nearly undetectable in distal ileum of adult mice. We show here that the caudal reduction of Gata4 is conserved in humans. To test the hypothesis that the regional expression of Gata4 is critical for the maintenance of jejunal-ileal homeostasis in the adult small intestine in vivo, we established an inducible, intestine-specific model that results in the synthesis of a transcriptionally inactive Gata4 mutant. Synthesis of mutant Gata4 in jejuna of 6- to 8-week-old mice resulted in an attenuation of absorptive enterocyte genes normally expressed in jejunum but not in ileum, including those for the anticipated targets liver fatty acid binding protein (Fabp1) and lactase-phlorizin hydrolase (LPH), and a surprising induction of genes normally silent in jejunum but highly expressed in ileum, specifically those involved in bile acid transport. Inactivation of Gata4 resulted in an increase in the goblet cell population and a redistribution of the enteroendocrine subpopulations, all toward an ileal phenotype. The gene encoding Math1, a known activator of the secretory cell fate, was induced approximately 75% (P < 0.05). Gata4 is thus an important positional signal required for the maintenance of jejunal-ileal identities in the adult mouse small intestine.

FIG. 1.

Gata4 is expressed in absorptive enterocytes on villi and throughout the crypt epithelium in the adult mouse jejunum. (A to D) Immunofluorescence showing that Gata4 (red) is not expressed in the nuclei (DAPI, blue) of chromogranin A (green) enteroendocrine cells. Arrowheads indicate the absence of Gata4 in the nucleus of a specific enteroendocrine cell. (E to H) Serial section of a PAS-stained goblet cell, showing absence of Gata4 immunofluorescence (red) in the nucleus (DAPI, blue) of this cell (arrowhead). (I to M) Coimmunofluorescence for Gata4 (red) and Ki67 (green), showing that Gata4 is expressed in proliferating epithelial cells of the upper crypt (yellow). (N to P) Coimmunofluorescence for Gata4 (red) and lysozyme (green), showing that Gata4 is expressed in the nuclei (DAPI, blue) of Paneth cells. (Q and R) Serial section using a blocking peptide, showing that the nuclear Gata4 expression in Paneth cells is not due to nonspecific fluorescence. (S to U) Coimmunofluorescence for Gata6 (green) and Gata4 (red), showing that these Gata factors are coexpressed in the absorptive enterocytes on villi (yellow).

FIG. 2.

The absence of Gata4 in adult ileum is conserved in humans. (A) Real-time RT-PCR analysis of Gata4 mRNA abundance along the length of the adult mouse small intestine, showing that the level of Gata4 mRNA is significantly lower in the distal ileum than in all other segments (, P < 0.05; mean ± SEM, n = 3). The calibrator was a pooled sample of jejunal RNA. (B and C) Immunofluorescence of adult human intestinal epithelium for Gata4 (red) and nucleic acid by DAPI (blue), showing the presence of Gata4 in jejunum (B) and the absence of Gata4 in ileum (C). (D and E). H&E staining of adult human jejunum and ileum, showing intact morphology.

FIG. 3.

Villin-CreER^T2-mediated recombination of the Gata4^flox allele in mouse small intestine. (A) Schematic representation of the Gata4 and Villin-CreER^T2 alleles. The Gata4^flox allele encodes wild-type Gata4, whereas the Gata4^Δex2 allele represents the Gata4 locus after Cre-mediated recombination. Arrows indicate locations of primers used for genotyping, and the inset shows the PCR products that distinguish the wild-type (wt) (350 bp) from the floxed (390 bp) Gata4 alleles. (B) Semiquantitative RT-PCR analyses of exon 2 with the F1 and F2 primers reveal normal Gata4 expression in stomach, pancreas, and heart but null expression in the small intestines of the mutant mice, with the exception of duodenum (segment 1), where Gata4 mRNA remains detectable.

FIG. 4.

An inactive, truncated form of Gata4 is synthesized in the jejuna of Gata4 mutant mice. (A) Western blot analysis using an antibody specific for the C-terminal domain of Gata4, demonstrating a specific band of ∼54 kDa in the jejuna of control mice (Gata4) and the absence of this band in Gata4 mutant animals. In the Gata4 mutant mice, another specific band of ∼33 kDa reveals the presence of a truncated Gata4 protein (Gata4^Δex2). Identical results were obtained from three other mutant animals. (B) EMSA using a standardized GATA binding site as a probe (44) and nuclear extracts from control and Gata4 mutant mice, showing that both Gata4 and Gata4^Δex2 bind DNA. Supershift complexes (SC) are formed using an antibody directed against the C-terminal domain of Gata4. (C) Transient-cotransfection assay in HeLa cells, demonstrating that Gata4^Δex2 is transcriptionally inactive and does not demonstrate dominant-negative activity with Gata6. A human LPH promoter/human growth hormone reporter plasmid was cotransfected into HeLa cells with the empty expression vector pRC-CMV or individually or in combinations of expression vectors for Gata6, Gata4, and Gata4^Δex2. Transcriptional activity is expressed as the ratio of the amount of human growth hormone synthesized from the human LPH promoter/human growth hormone reporter relative to that of the metallothionein promoter fused to human growth hormone. Data are expressed as means ± SEMs (n = 5 assays). (D) Schematic representation of the predicted Gata4 protein synthesized in the jejuna of the Gata4 mutant mice, showing the deletion of the N-terminal activation domains (ActI and ActII) but intact zinc fingers (ZnI and ZnII), basic region (BR), and C-terminal domain (CTD).

FIG. 5.

The absorptive enterocyte gene expression program in jejunum is partially transformed into an ileal-like pattern in Gata4 mutant mice. (A to C) Cytoplasmic Fabp1 immunofluorescence (green) and DAPI nuclear staining (blue) of the absorptive enterocytes of control jejunum (A), mutant jejunum (B), and control ileum (C), showing a reduced expression of Fabp1 in mutant jejunum. (D to F) Microvillus membrane Asbt immunofluorescence (green) and DAPI nuclear staining (blue) of control jejunum (D), mutant jejunum (E), and control ileum (F), showing an induction of Asbt in mutant jejunum. (G) Real-time and semiquantitative RT-PCRs conducted on jejunal RNA from control and Gata4 mutant mice, showing differential effects on absorptive enterocyte gene expression. Real-time RT-PCR (left) is shown as a ratio of mRNA abundance of Gata4 mutant jejunum compared to controls (, P < 0.05; , P < 0.01; , P < 0.001) of genes normally expressed at higher levels in jejunum than in ileum (J>I), equally in jejunum and ileum (J=I), and at lower levels in jejunum than in ileum (J<I). Semiquantitative RT-PCR (right) is shown for two representative samples each from control and Gata4 mutant mice. A reaction without reverse transcriptase (No RT) served as a control for DNA contamination. (H) Comparison of LPH and Asbt mRNAs in control jejunum, mutant jejunum, and control ileum by real-time RT-PCR, showing that the transformation to an ileal-like phenotype is not complete. Data are means ± SEMs (n = 5). Bars with the same letter are significantly different from each other (P < 0.05). The calibrators were adult jejunal RNA for LPH and adult ileal RNA for Asbt. (I) Semiquantitative RT-PCR analysis for Gata5, Gata6, Cdx2, Hnf1α, c-Jun, c-Fos, Lrh1, and Gapdh on RNA from jejunum, showing that the mRNA abundances in control and Gata4 mutant mice are not different. A reaction without reverse transcriptase (No RT) served as a control for DNA contamination.

FIG. 6.

Secretory lineages are redistributed towards an ileal-like composition. (A to C) PAS staining of control jejunum (A), mutant jejunum (B), and control ileum (C), showing expansion of the goblet cell population in Gata4 mutant jejunum. (D to F) Real-time RT-PCR of Muc2 (D), CCK (E), and PYY (F) mRNA abundances in control jejunum, mutant jejunum, and control ileum, showing a redistribution toward an ideal pattern in Gata4 mutant jejunum. , P < 0.05 compared to control jejunum; data are means ± SEMs.

FIG. 7.

Proliferation, apoptosis, and signaling pathways in Gata4 mutant mice. (A and B) Immunofluorescence for the proliferation marker Ki67 (green) and DAPI (blue), showing that the proliferative compartment in control (A) and mutant (B) jejuna were not different. (C and D) Immunofluorescence for cleaved caspase-3 (green) and DAPI (blue), showing that cell death in control (C) and mutant (D) jejuna were not different. (E) Semiquantitative RT-PCR analysis of Tcf4, cyclin D1, Bmp4, Shh, Ihh, and Gapdh, showing that the mRNA abundances in control and Gata4 mutant mice are not different. (F) Semiquantitative RT-PCR analysis of Notch1, Hes1, Math1, neurogenin3 (Ngn3), and Gapdh, showing that jejunal mRNA abundances in control and Gata4 mutant mice are not different, with the exception of Math1, which is significantly greater by real-time RT-PCR (G) in both mutant jejunum (P < 0.05) and control ileum (P < 0.01) compared to control jejunum (data are means ± SEMs). The calibrator was adult jejunal RNA.

FIG. 8.

Model showing that Gata4 maintains a jejunal phenotype through (i) activation of a jejunal gene expression program in absorptive enterocytes, possibly through interactions with Hnf1α; (ii) repression of an ileal gene expression program in absorptive enterocytes through an as-yet-unknown mechanism; and (iii) alteration of cell fate specification, possibly through a Math1-dependent pathway.