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MUCOSAL BIOLOGY

Gata4 and Hnf1 are partially required for the expression of specific intestinal genes during development

¹School of Medicine, University of Amsterdam, Amsterdam, The Netherlands; ²Division of Gastroenterology and Nutrition, Department of Medicine, Children's Hospital Boston, Boston, Massachusetts; ³School of Medicine, Erasmus University Rotterdam, Rotterdam, The Netherlands; ⁴School of Medicine, University of Utrecht, Utrecht, The Netherlands; ⁵Department of Pediatrics, Harvard Medical School, Boston, Massachusetts; and ⁶Dorothy R. Friedman School of Nutrition Science and Policy, Tufts University, Boston, Massachusetts

Submitted 9 September 2006 ; accepted in final form 25 January 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The terminal differentiation phases of intestinal development in mice occur during cytodifferentiation and the weaning transition. Lactase-phlorizin hydrolase (LPH), liver fatty acid binding protein (Fabp1), and sucrase-isomaltase (SI) are well-characterized markers of these transitions. With the use of gene inactivation models in mature mouse jejunum, we have previously shown that a member of the zinc finger transcription factor family (Gata4) and hepatocyte nuclear factor-1 (Hnf1) are each indispensable for LPH and Fabp1 gene expression but are both dispensable for SI gene expression. In the present study, we used these models to test the hypothesis that Gata4 and Hnf1 regulate LPH, Fabp1, and SI gene expression during development, specifically focusing on cytodifferentiation and the weaning transition. Inactivation of Gata4 had no effect on LPH gene expression during either cytodifferentiation or suckling, whereas inactivation of Hnf1 resulted in a 50% reduction in LPH gene expression during these same time intervals. Inactivation of Gata4 or Hnf1 had a partial effect (50% reduction) on Fabp1 gene expression during cytodifferentiation and suckling but no effect on SI gene expression at any time during development. Throughout the suckling period, we found a surprising and dramatic reduction in Gata4 and Hnf1 protein in the nuclei of absorptive enterocytes of the jejunum despite high levels of their mRNAs. Finally, we show that neither Gata4 nor Hnf1 mediates the glucocorticoid-induced precocious maturation of the intestine but rather are downstream targets of this process. Together, these data demonstrate that specific intestinal genes have differential requirements for Gata4 and Hnf1 that are dependent on the developmental time frame in which they are expressed.

lactase-phlorizin hydrolase; liver fatty acid binding protein; sucrase-isomaltase; intestinal differentiation; cytodifferentiation; weaning

Lactase-phlorizin hydrolase (LPH), liver fatty acid binding protein (Fabp1), and sucrase-isomaltase (SI) are intestinal proteins important for nutrition during different stages of development and are also established markers for the transitions that occur in intestinal development (18, 20, 25, 35, 36, 38, 40, 44, 45, 47). LPH and SI are microvillus membrane disaccharidases that hydrolyze milk lactose and -disaccharides found in solid foods, respectively, whereas Fabp1 is a cytoplasmic protein important for intracellular lipid transport. In rodents, LPH and Fabp1 are first detected at the beginning of cytodifferentation in preparation for a critical function after birth, continue to be expressed at high levels during the suckling period, and decline during weaning. In contrast, SI is undetectable before weaning and increases to adult levels during weaning. Thus, LPH and Fabp1 are markers of cytodifferentiation during fetal development, whereas LPH, Fabp1, and SI are indicators of the well-orchestrated patterns of absorptive enterocyte gene expression that occur during postnatal development.

Although much is known about the patterns of LPH, Fabp1, and SI gene expression during development, the mechanisms underlying these patterns remain to be fully elucidated. Transgenic studies have shown that the 5'-flanking regions of LPH, Fabp1, and SI direct appropriate tissue, cell-type, and temporal patterns of expression (21, 24, 26, 27, 40, 48), and highly conserved transcription factor binding sites in the proximal promoters of LPH, Fabp1, and SI have been identified for the Gata family of zinc finger transcription factors that bind the consensus DNA sequence (A/T)GATA(A/G), as well as the hepatocyte nuclear factor-1 (Hnf1) and caudal (Cdx) families of homeodomain proteins (5, 6, 8–10, 12, 14, 15, 22, 28, 42, 46, 53). Gata4 and Hnf1 are the predominant members of their respective families in nuclear extracts from mouse intestinal epithelial cells that bind to the LPH and SI promoters (4, 5, 49), and both Gata4 and Hnf1 activate the LPH, Fabp1, and SI promoters in cell culture overexpression experiments (5, 6, 8, 9, 12, 22, 28, 42, 49). Gata4 and Hnf1 physically associate and synergistically activate the LPH, Fabp1, and SI genes (5, 9, 49) through an evolutionarily conserved pathway (49, 50), and we have postulated that this interaction is a means to achieve high levels of intestine-specific gene expression in vivo (20).

We recently investigated the importance of Gata4 and Hnf1 in vivo for intestinal gene expression by using gene-inactivation approaches (3, 4). Inactivation of Gata4 in adult mouse jejunum produces a shift to an ileal-like phenotype but no obvious consequences in weight, behavior, skin, or general physiology (3). Germline Hnf1 knockout mice survive into adulthood and demonstrate sterility, diabetes, delayed growth rate, and liver dysfunction (23). In both models, LPH and Fabp1 mRNA abundances in adult jejunum were reduced 90%, whereas that of SI was surprisingly not affected by the inactivation of either Gata4 or Hnf1 (3, 4). These data thus indicate that, in adult mouse intestine, both Gata4 and Hnf1 are necessary for the expression of the LPH and Fabp1 genes, consistent with our model of coregulation, but are dispensable for SI gene expression. The goal of the present study is to use these models to define the requirement of Gata4 and Hnf1 for the regulation of LPH, Fabp1, and SI gene expression in the developing mouse small intestine, specifically focusing on cytodifferentiation and the weaning transition.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Mice. All animal studies were performed under protocols approved by the Institutional Animal Care and Use Committee of Children's Hospital Boston. Mice were housed in the Animal Research at Children's Hospital facility under standard conditions with 12-h light-dark cycles and were given food and water ad libitum.

Two gene-inactivation models were used, including an inducible, intestine-specific Gata4 inactivation model (3) and a germline Hnf1-null model (4, 23), all in a C56Bl/6 background. To inactivate Gata4 in the small intestinal epithelium, Gata4^flox/flox mice (33) were crossed with transgenic Villin-CreER^T2 mice (11) to generate Gata4^flox/flox, Villin-CreER^T2 positive (Cre+) study animals (mutant) and Gata4^flox/flox, Villin-CreER^T2 negative (Cre–) controls. To inactivate Gata4 in the intestine, tamoxifen (1 mg/20 g body wt; Sigma-Aldrich, St. Louis, MO) was administered to timed-pregnant females for 5 consecutive days beginning at E12.5 or for 4 consecutive days beginning at postnatal day (P) 7, as described (2). Gata4-mutant mice produce a truncated, transcriptionally inactive form of Gata4 that is capable of site-specific binding to DNA elements and thus has the potential for dominant negative activity in vivo (3). Hnf1–/– mice survive into adulthood but are sterile; therefore mating of Hnf1+/– parents is required to generate both null and wild-type study animals (23). All mice were genotyped by using PCR on tail DNA as previously described (4).

Mice were killed for study at various time points beginning at E13.5 and extending throughout postnatal development. Study mice or pregnant females were anesthetized with Avertin anesthesia (2,2,2-tribromoethanol, 240 mg/kg body wt; Sigma) prior to dissection. For fetal mice, embryos were removed from the mother and transferred to a Petri dish containing 1x PBS, and tissue was isolated by using a dissecting microscope. For postnatal mice, tissue was extracted through a midline incision and transferred to a glass plate on a bed of wet ice. All tissues were collected between 1300 and 1600 to avoid any fluctuations in gene expression due to circadian cycles (34).

RNA isolation. RNA was isolated from snap-frozen fetal and postnatal mouse tissues. From fetal animals, RNA was isolated from either the entire small intestine or, in the case of selected E17.5 pups, from intestinal segments separated into equal lengths, where segment 1 was the most proximal 20%, segment 2 the next 20%, segment 3 the middle 20%, segment 4 the next 20%, and segment 5 the most distal 20% of the small intestine. From postnatal mice, RNA was isolated from 30–50 mg of small intestine (0.5–1.0 cm) obtained from the geometric center (segment 3, jejunum). RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA). To ensure that all traces of DNA were removed, RNA samples were treated with DNase (DNA-free; Ambion, Austin, TX) for 1 h at 37°C, following the manufacturer's instructions. RNA samples were quantified by optical density at A260 nm and were checked for absence of degradation on an agarose gel.

Semiquantitative and real-time RT-PCR. Semiquantitative and real-time RT-PCR were conducted as previously described (4, 49). For both PCR reactions, complementary DNA (cDNA) was synthesized by using iScript (BioRad). Primer pairs were designed by using Beacon Designer software (Premier Biosoft International, Palo Alto, CA) and were optimized as described (3, 4). Primer sequences are available upon request. Semiquantitative RT-PCR experiments were terminated in the linear range of amplification. Real-time RT-PCR was conducted by using an iCycler and iQ SYBRgreen Supermix (Bio-Rad, Hercules, CA). All real-time RT-PCR data were corrected for Gapdh and were expressed relative to the calibrator, which was adult jejunal RNA from a single mouse, unless otherwise indicated.

Immunofluorescence. Immunofluorescence was conducted on mouse tissue as previously described (3). Following dissection, mouse tissues were immediately immersed in a freshly made solution of buffered 4% paraformaldehyde and incubated for 4 h at 4°C, then resuspended in 70% ethanol overnight. Tissue was embedded in paraffin, and 5-µm sections were prepared for immunohistochemistry in the Department of Pathology, Children's Hospital Boston. Following tissue deparaffinization and rehydration, antigen retrieval was conducted by boiling slides for 10 min in 10 mM sodium citrate (pH 6). The slides were then slow-cooled and washed in 1x PBS. After being blocked (10% donkey serum in 1x PBS) for 1 h in a humidified chamber, the primary antibody was pipetted onto slides and incubated overnight at 4°C. After being washed, the fluorescent secondary antibody was pipetted onto slides and incubated for 4 h at room temperature. Due to cross-reactivity, sequential addition of the two secondary antibodies with extensive washing in between was necessary for Gata4-Hnf1 coimmunofluorescence experiments.

The primary antibodies used were goat anti-Hnf1 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-Gata4 (1:100; Santa Cruz), goat anti-Gata4 (1:400; Santa Cruz), rabbit anti-Cdx2 (1:500; gift from D. Silberg, University of Pensylvania), rabbit anti-LPH (1:500; gift from K. Y. Yeh, Louisiana State University), rabbit anti-Fabp1 (1:1,000; gift from J. Gordon, Washington University), and rabbit anti-SI (1:500; gift from K. Y. Yeh, Louisiana State University). The secondary antibodies used were Alexa Fluor 594 donkey anti-goat IgG, Alexa Fluor 488 donkey anti-rabbit IgG, and Alexa Fluor 488 goat anti-mouse IgG (1:500; Molecular Probes). In most experiments, a solution containing 4',6-diamino-2-phenylindole dihydrochloride (DAPI) nucleic acid stain (2 µg/ml; Molecular Probes) in PBS was added to reveal the nuclei.

Isolation of nuclear and nonnuclear extracts. Nuclear extracts were isolated as previously described (49) from pooled mucosal scrapings of 4-cm segments at the geometric center of the small intestine (midjejunum) from P4, P7, P14, P21, P28, and adult (6–12 wk) mice. In selected experiments, nuclear and nonnuclear fractions were isolated similarly from P10 mice. The epithelial scrapings were resuspended in hypotonic buffer [in mM: 10 HEPES (pH 7.9), 10 KCl, 1.5 MgCl₂, 1.0 PMSF, and 1.0 DTT, plus protease inhibitor cocktail] and centrifuged at 5,000 rpm for 5 min at 4°C. The cell pellet was resuspended in hypotonic buffer, incubated on ice for 5 min, and homogenized in a precooled Dounce homogenizer with 20 strokes using the loose pestle. After centrifugation at 8,000 rpm for 5 min at 4°C, the supernatant in selected experiments was saved (–70°C) as the nonnuclear fraction. The nuclei were resuspended in low-salt buffer [20 mM HEPES (pH 7.9), 20 mM KCl, 1.5 mM MgCl₂, 25% glycerol, protease inhibitor cocktail, 1.0 mM PMSF, and 1.0 mM DTT] followed by the slow addition of high-salt buffer [20 mM HEPES (pH 7.9), 1.2 M KCl, 1.5 mM MgCl₂, 25% glycerol, protease inhibitor cocktail, 1.0 mM PMSF, and 1.0 mM DTT]. After extraction at 4°C for 30 min with vigorous mixing every 5 min, the sample was centrifuged at 14,000 rpm for 15 min at 4°C and the supernatant was saved as the nuclear fraction (–70°C).

Western blotting. Western blot analysis was performed as described previously (4) by using 20–80 µg of nuclear or nonnuclear extracts. The primary antibodies included affinity-purified goat polyclonal antibodies for Gata4 or Hnf1 (Santa Cruz), a mouse monoclonal antibody for Gata4 (Santa Cruz), or rabbit polyclonal antibodies for Fabp1 (gift of J. Gordon, Washington University). All blots were stripped and reprobed with anti-mouse -actin.

EMSA. EMSAs were performed by using labeled, double-stranded oligonucleotides containing well-characterized binding sites for Gata or Hnf1 families of transcription factors, as described previously (22). These included the Gata binding site present in the Xenopus Fabp1 promoter (X-GATA; 5'-GGAGATCCCTGTACAGATATGGGGAGAC-3') (17) and the Hnf1 binding site present in the rat -fibrinogen promoter (-Fib; 5'-CAAACTGTCAAATATTAACTAAAGGGAG-3') (7). Supershift EMSAs were conducted by using affinity-purified goat polyclonal antibodies for Gata4 or Hnf1 (Santa Cruz).

Dexamethasone treatment. To investigate the role of Gata4 and Hnf1 in hormonally induced precocious weaning, a model was used in which wild-type, and Gata4-mutant Hnf1–/– mice were treated with dexamethasone (Sigma-Aldrich) at P10 essentially as described (25). Dexamethasone was injected intraperitoneally at 1.0 µg/g body wt. Negative controls included littermates injected with vehicle (0.8% ethanol in 1x PBS). After 4 or 24 h, mice were killed and the jejuna (segment 3) were collected for the isolation of RNA and nuclear extracts as well as for sectioning.

Statistical analyses. Statistically significant differences were determined by Student's t-test or analysis of variance followed by the Tukey-Kramer multiple-comparison test.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Gata4 and Hnf1 differentially regulate LPH and Fabp1 gene expression during cytodifferentiation. The mouse intestinal epithelium undergoes rapid cytodifferentiation beginning at E14.5, resulting in the formation of nascent villi and synthesis of intestinal differentiation markers by E17.5. The mRNAs for the markers of cytodifferentiation, LPH and Fabp1 (18, 35, 44), were not detectable by real-time RT-PCR in whole intestine at E13.5 or E14.5, were just detectable at E15.5, and reached their highest levels by E17.5 (Fig. 1A). LPH and Fabp1 mRNAs were highest throughout the proximal half of the small intestine (segments 1–3) and declined to nearly undetectable levels in the distal gut (segment 5), as determined by semiquantitative RT-PCR (Fig. 1B). These data confirm that LPH and Fabp1 are markers for cytodifferentiation of the mouse midgut.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Lactase-phlorizin hydrolase (LPH) and liver fatty acid binding protein 1 (Fabp1) mRNAs are induced in the mouse small intestine during cytodifferentiation. A: LPH and Fabp1 mRNAs are first detected in mouse small intestine at embryonic day (E) 15.5. LPH and Fabp1 mRNAs were quantified by real-time RT-PCR in whole intestine of mouse embryos obtained from timed-pregnant mothers. Data were obtained from a single mouse at each time point. B: LPH and Fabp1 mRNAs at E17.5 are highly expressed in the proximal half of mouse small intestine and decline distally. RNA was isolated from five equidistant segments of a mouse embryo at E17.5 as described in MATERIALS AND METHODS, and the abundances of LPH and Fabp1 mRNAs were determined by semiquantitative RT-PCR. Gapdh is shown as a positive control.

Hnf1

Hnf1

View larger version (48K): [in this window] [in a new window]   Fig. 2. Gata4 and hepatocyte nuclear factor-1 (Hnf1) are coexpressed in the intestinal epithelial cells of the presumptive small intestine during cytodifferentiation. A: Gata4 and Hnf1 mRNAs demonstrate an increasing pattern during cytodifferentiation. Gata4 and Hnf1 mRNAs were quantified by real-time RT-PCR in whole intestine of mouse embryos obtained from timed-pregnant mothers. Data were obtained from a single mouse in each age group. B: Gata4 mRNA at E17.5 is highly expressed in the proximal half of mouse small intestine and declines distally, whereas Hnf1 mRNA is low in proximal intestine and increases distally. RNA was isolated from five equidistant segments of a mouse embryo at E17.5 as described in MATERIALS AND METHODS, and the abundance of Gata4 and Hnf1 mRNA was determined by semiquantitative RT-PCR. C–H: Gata4 and Hnf1 are coexpressed in epithelial cells of presumptive small intestine at E13.5 (C–E) and E16.5 (segment 3; F–H). Immunofluorescence was conducted using mouse anti-Gata4 and goat anti-Hnf1. Colocalization of Gata4 and Hnf1 is indicated by merged photomicrographs as indicated (E and H).

Hnf1

Hnf1

Hnf1

View larger version (12K): [in this window] [in a new window]   Fig. 3. Gata4 and Hnf1 are differentially required for LPH and Fabp1 gene expression during cytodifferentiation. A: timed-pregnant mothers were treated with five daily injections of tamoxifen () beginning at E12.5. Mice were killed for analysis at E17.5 (arrow). B: Gata4 is specifically inactivated in the midgut of Gata4-mutant mice at E17.5. Semiquantitative RT-PCR for Gata4 was conducted on RNA isolated from the midgut (segment 3) and heart of a representative control and Gata4-mutant mouse as indicated. C: Gata4 is dispensable for LPH gene expression but partially required for Fabp1 gene expression, whereas Hnf1 is partially required for both LPH and Fabp1 gene expression. Real-time RT-PCR for LPH (left) and Fabp1 (right) mRNA was conducted on RNA isolated from E17.5 midgut (segment 3) of control and Gata4-mutant mice (top) as well as wild-type (+/+) and Hnf1-null (–/–) mice (bottom). The calibrator was midgut RNA from one of the control mice. Data are means ± SD of n = 3–5 mice. *P < 0.05 compared with control or +/+ mice.

Nuclear Gata4 and Hnf1 are paradoxically reduced before weaning despite high levels of their respective mRNAs.

Hnf1

Hnf1

View larger version (28K): [in this window] [in a new window]   Fig. 4. Nuclear Gata4 and Hnf1 are paradoxically reduced before weaning despite high levels of their respective mRNAs. A: Gata4 and Hnf1 mRNA abundances decrease during the weaning transition. Gata4 and Hnf1 mRNAs were quantified in wild-type jejunum (segment 3) at postnatal day (P) 7, P14, P21, and P28 by real-time RT-PCR. Data are means ± SD of n = 3–5 mice. *P < 0.05 compared with P7. B: Gata4 and Hnf1 protein levels increase during the weaning transition. Gata4 and Hnf1 were quantified in nuclear extracts isolated from wild-type jejunum at P7, P14, P21, and P28 by Western blot analysis using polyclonal goat antibodies for Gata4 and Hnf1. Blots were reprobed with a -actin antibody to demonstrate similar protein loading in each lane. C: Gata4 and Hnf1 are reduced before weaning compared with adults. Gata4 and Hnf1 were quantified in nuclear extracts from wild-type jejunum at P4, P7, and P10 and in adults by Western blot analysis using a mouse monoclonal Gata4 antibody and a goat polyclonal Hnf1 antibody. Forty and 80 µg of protein were used for the Gata4 and Hnf1 blots, respectively. D: Gata4 and Hnf1 were not detected in nonnuclear fractions of P10 mouse jejunum. Gata4 and Hnf1 were quantified in nuclear and nonnuclear extracts isolated from wild-type jejunum at P10 and in nuclear extracts from wild-type (WT) knockout adult jejunum by Western blot analysis using polyclonal goat antibodies for Gata4 and Hnf1. Fabp1 immunoblot was conducted to verify the enrichment of cytoplasmic proteins in the nonnuclear fractions. E: Gata and Hnf1 binding activity were not detected in nuclear extracts from P7 mouse jejunum. With the use of well-characterized sites for Gata (X-GATA) and Hnf1 (-Fib) binding, EMSAs were conducted using nuclear extracts obtained from P7 and P28 mouse jejunum. Specific complexes (closed arrowhead) that supershifted (open arrowhead) with specific Gata4 (G4) or Hnf1 (H1a) antibodies were found only for extracts from P28 mice.

Gata4 and Hnf1 are readily detected in the nuclei of villi and intervillus regions of embryonic intestine by immunofluorescence (see Fig. 2). However, just after birth (P1), Gata4 was expressed only in the intervillus regions, not on villi (Fig. 5, A and B), and Hnf1 could not be detected in either compartment (Fig. 5C). From P4 to P10, neither Gata4 nor Hnf1 were detected, as exemplified by immunofluorescence of P7 intestine (Fig. 5, D–G). As a positive control, all sections during this time interval positively stained for Cdx2 (Fig. 5, H and I), an intestinal nuclear transcription factor that is expressed in the intestinal epithelium throughout development (39). At P14, Gata4 was detected only in the nuclei of cells in the crypts and lower villi (Fig. 5, J and K), whereas Hnf1 was not detected (data not shown). By P21, Gata4 and Hnf1 were coexpressed throughout the villus epithelium (Fig. 5, L–M), as in adults. Together, these studies demonstrate that both the Gata4 and Hnf1 genes are expressed at high levels throughout postnatal development, as indicated by their high levels of mRNA (see Fig. 4A), but their protein products are expressed at low levels during suckling.

View larger version (56K): [in this window] [in a new window]   Fig. 5. Gata4 and Hnf1 expression is attenuated in jejunum before weaning. A–C: immunofluorescence in P1 jejunum showing Gata4 in the nuclei of the intervillus regions, but not on villi (A and B). Hnf1 was not detected in either compartment at this age (C). D–I: immunofluorescence in P7 jejunum reveals an inability to detect either Gata4 (D and E) or Hnf1 (F and G). Cdx2 is readily detected in the nuclei throughout the crypt and villus epithelium (H and I). J and K: immunofluorescence in P14 jejunum showing Gata4 in crypt nuclei but not on villi. L–N: coimmunofluorescence showing coexpression of Gata4 and Hnf1 in the P21 jejunum. White arrowheads show specific nuclear immunofluorescence, and yellow arrowheads indicate the absence of specific nuclear immunofluorescence.

Gata4 and Hnf1 differentially regulate target gene expression during postnatal development.

View larger version (20K): [in this window] [in a new window]   Fig. 6. Gata4 is inducibly inactivated in the mouse jejunum during postnatal development. A: postnatal mice were treated with four daily injections of tamoxifen () beginning at P7. Mice were killed for analysis at P10, P20, and P30 (arrows). The weaning transition is indicated by the dotted line. B: Gata4 is inactivated in the jejunum of Gata4-mutant mice during postnatal development. Semiquantitative RT-PCR for Gata4 mRNA was conducted on RNA obtained from the jejunum of representative control and Gata4-mutant mice at P10, P20, and P30. Gapdh is shown as a positive control.

View larger version (67K): [in this window] [in a new window]   Fig. 7. Inactivation of Gata4 in jejunum reveals gene-specific, developmental regulation. A: LPH, Fabp1, and SI mRNA abundances are differentially affected by the inactivation of Gata4 during development. Real-time RT-PCR for LPH (top), Fabp1 (middle), and SI (bottom) mRNAs were conducted on RNA isolated from jejunum (segment 3) of control and Gata4-mutant mice at P10, P20, and P30. Data are means ± SD of n = 3–5 mice. *P < 0.05 compared with controls. B–M: immunofluorescence for LPH (B–E), Fabp1 (F–I), and SI (J–M) in P10 control (B, F, J), P10 Gata4-mutant (C, G, K), P30 control (D, H, L), and P30 Gata4-mutant (E, I, M) mice.

In Hnf1–/– mice, LPH mRNA abundance was 50% of that in wild-type jejunum at P7, P14, and P21, similar to that at E17.5, but was <10% of that in wild-type jejunum at P28 (Fig. 8A, top), similar to that in adult Hnf1-null mice (4). Fabp1 mRNA was reduced 50% in the Hnf1-null mice at P7 and P14, similar to that at E17.5 (see Fig. 3C), and was barely detectable at P21 and P28 (Fig. 8A, middle), similar to that in adults (4). SI mRNA increased during weaning in Hnf1+/+ and Hnf1–/– mice, with no significant difference between the two groups (Fig. 8A, bottom).

View larger version (74K): [in this window] [in a new window]   Fig. 8. Null expression of Hnf1 demonstrates gene-specific, developmental regulation. A: LPH, Fabp1, and SI mRNA abundances are differentially affected by null expression of Hnf1 during development. Real-time RT-PCR for LPH (top), Fabp1 (middle), and SI (bottom) mRNAs were conducted on RNA isolated from jejunum (segment 3) of Hnf1+/+ and Hnf1–/– mice at P7, P14, P21, and P28. Data are means ± SD of n = 3–5 mice. *P < 0.05 compared with controls. B–M: immunofluorescence for LPH (B–E), Fabp1 (F–I), and SI (J–M) in P7 Hnf1+/+ (B, F, J), P7 Hnf1–/– (C, G, K), P28 Hnf1+/+ (D, H, L), and P28 Hnf1–/– (E, I, M).

Hnf1

Hnf1

Hnf1

Gata4 and Hnf1 do not mediate the precocious weaning induced by glucocorticoids. Glucocorticoids are known to induce maturation of the small intestine, resulting in the precocious induction of intestinal enzymes such as SI (25). However, because this induction is characterized by an 8-h lag, it is thought to be a secondary effect. The primary response is likely mediated by intestinal transcription factors, and Gata factors have been implicated (32). To define the possible role of Gata4 as well as Hnf1 in mediating the glucocorticoid response in preweaning mice, we characterized the dexamethasone-induced response in the context of null intestinal expression of Gata4 or Hnf1. As shown in Fig. 9A (top), SI mRNA abundance was similarly induced 80-fold 24 h after dexamethasone administration in wild-type, Gata4-mutant, and Hnf1–/– mice, indicating that neither Gata4 nor Hnf1 is necessary to mediate the dexamethasone response on SI in vivo. LPH mRNA abundance was not affected by dexamethasone in wild-type mice but was significantly reduced by dexamethasone in both Gata4-mutant and Hnf1–/– mice (Fig. 9A, bottom). These data indicate that dexamethasone induces a process in which Gata4 and Hnf1 become regulatory for LPH gene expression, as in the postweaning situation. In wild-type mice, the mRNAs for both Gata4 and Hnf1 were significantly reduced by dexamethasone, both at 4 h (data not shown) and 24 h (Fig. 9B), similar to that which occurs after weaning. Together, these data indicate that Gata4 and Hnf1 are not required for the precocious maturation process induced by glucocorticoids but are likely downstream targets of this process.

View larger version (13K): [in this window] [in a new window]   Fig. 9. Dexamethasone induces a precocious weaning response in jejunum in the presence or absence of Gata4 or Hnf1. A: dexamethasone induces SI mRNA levels in preweaning mice independently of Gata4 or Hnf1 but attenuates LPH mRNA levels specifically in the absence of Gata4 or Hnf1. Mice were treated with dexamethasone (1 µg/g body wt) on P10, and RNA was collected 24 h later from the jejunum of wild-type, Gata4-mutant, and Hnf1–/– mice. SI (top) and LPH (bottom) mRNA levels were quantified by real-time RT-PCR in n = 3–5 mice. *P < 0.05 and **P < 0.01 compared with untreated mice. B: dexamethasone attenuates Gata4 and Hnf1 mRNA levels in jejunum of preweaning mice. Wild-type mice were treated with dexamethasone on P10, and RNA was collected 24 h later from the jejunum. Gata4 and Hnf1 mRNA levels were quantified by real-time RT-PCR in n = 3–5 mice. *P < 0.05 compared with untreated mice.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

In mature jejunum of mice, we have previously shown that Gata4 and Hnf1 are necessary for LPH and Fabp1 gene expression, but not for SI gene expression (3, 4). In the present study, we found that the regulation of these target genes by Gata4 and Hnf1 during development differs from that in adults (Fig. 10), in that before weaning, including during cytodifferentiation, Gata4 and Hnf1 are either not required or only partially required for LPH and Fabp1 gene expression, contrasting with their indispensability after weaning. The partial Gata4 requirement for Fabp1 gene expression during cytodifferentiation is consistent with data from E18.5 mosaic Gata4-knockout mice, in which Fabp1 gene expression is attenuated in intestinal epithelial cells that do not express Gata4, as indicated by in situ hybridization (9). We also found that Gata4 and Hnf1 are not required for SI gene expression at any time during development. During the suckling period, we found a surprising and dramatic reduction in Gata4 and Hnf1 protein in the nuclei of absorptive enterocytes of the jejunum despite high levels of mRNA. Finally, we show that neither Gata4 nor Hnf1 mediate the precocious maturation of the intestine induced by glucocorticoids. Together, these data demonstrate that specific intestinal genes, including LPH and Fabp1, have differential requirements for Gata4 and Hnf1 that are dependent on the developmental time frame in which they are expressed.

View larger version (9K): [in this window] [in a new window]   Fig. 10. Summary of Gata4 and Hnf1 regulation of LPH, Fabp1, and SI gene expression during intestinal development. Schematic representation of LPH (top), Fabp1 (middle), and SI (bottom) gene expression in wild-type (solid line), Gata4-mutant (dashed line), and Hnf1–/– (dotted line) mice during development.

These data also reveal differential mechanisms underlying the regulation of genes whose expression patterns during development are strikingly similar. Both LPH and Fabp1 are highly expressed in more proximal regions of small intestine than distal regions and at higher levels before weaning than after weaning (Figs. 1, 7, and 8 and Refs. 18, 20, 35, 36, 40), and their promoters contain binding sites for similar sets of transcription factors (8, 22). However, although both are similarly regulated by Gata4 and Hnf1 in mature intestine (3, 4), they are differentially regulated by these two transcription factors before weaning. Both LPH and Fabp1 mRNAs are reduced 50% in Hnf1-null mice before weaning, but only Fabp1 mRNA is reduced in the Gata4-mutant mice before weaning (Figs. 3, 7, and 8). Furthermore, Fabp1 mRNA abundance is attenuated 90% by the inactivation of Gata4 at P20 or Hnf1 at P21, similar to that in adult mice, whereas LPH mRNA abundance is reduced <50% at these time points, similar to preweaning mice, indicating a differential in the timing of regulation by Gata4 or Hnf1. The differential regulation of LPH and Fabp1 by Gata4 before weaning highlights a target-specific gene regulation during development.

We have previously hypothesized that the induction of SI gene expression during postnatal development is regulated by the combinatory effect of a complex of transcription factors, including Gata4 and Hnf1 (5, 22). In addition, the abundance of nuclear Gata4 and Hnf1 proteins in the jejunum increases during the weaning transition, paralleling SI gene expression (Fig. 4B and Refs. 5, 22). Despite these compelling data for a combinatory role by Gata4 and Hnf1 in the activation of SI gene expression, we recently reported that the inactivation of Gata4 or Hnf1 had no effect on SI gene expression in adult mice (3, 4). Here, we show that the inactivation of Gata4 or Hnf1 had no effect on the initiation of SI gene expression during weaning. Together, these data demonstrate that Gata4 and Hnf1 are dispensable for SI gene expression throughout development. The future challenge, therefore, is to identify transcription factors essential for SI gene expression.

In our studies, we identified a paradoxical loss of Gata4 and Hnf1 protein in the nuclei of absorptive enterocytes beginning shortly after birth, continuing through the suckling period, and ending during the third week of life, when weaning occurs. At P4–P10, Gata4 and Hnf1 were greatly reduced in the nuclear fraction of jejunal extracts as determined by Western blot analysis (Fig. 4, B–D) and EMSA (Fig. 4E), and neither could be detected in the nuclei of villus enterocytes in the jejunum by immunofluorescence (Fig. 5, D–I). Interestingly, Gata4 and Hnf1 mRNA abundance remain high during this time interval (Fig. 4A), suggesting that the reduction in Gata4 and Hnf1 protein is not due to a decrease in transcription rate. We thus believe that either the mRNAs for these proteins are not transcribed and/or that their translation products are actively catabolized.

Although Gata4 and Hnf1 are greatly reduced or absent in the intestinal epithelial nuclei during suckling, they are nevertheless partially required for LPH and Fabp1 gene expression during this time frame (Fig. 7 and 8). One explanation is that at least some Gata4 and Hnf1 is normally present in the nucleus, as suggested by Western blot analysis for Gata4 using 40 µg of protein (Fig. 4C). However, Hnf1 could not be detected at P4 and P7 on Western blots using 80 µg of protein (Fig. 4C), suggesting that Hnf1 is not present during this time interval. Thus a second explanation is that Gata4 and/or Hnf1 is required earlier in intestinal development for later expression of putative target genes. Although this is a plausible explanation for Hnf1, in which a germline-null model was used, it is a less likely explanation for Gata4, which is inducibly inactivated after birth. Precedence for an early developmental requirement for later expression is shown in the liver, where embryonic, but not postnatal, reexpression of Hnf1 is capable of reactivating the silent phenylalanine hydroxylase gene in Hnf1-deficient hepatocytes (52).

Interestingly, regulation in the knockout models at P7–P14 (Figs. 7 and 8), when Gata4 and Hnf1 nuclear protein is decreased, is virtually identical to that which occurs at E17.5 (Fig. 3), when Gata4 and Hnf1 are normally present in epithelial nuclei, suggesting that the loss of nuclear Gata4 and Hnf1 after birth is not a critical regulatory mechanism for LPH and Fabp1 gene expression. Thus it is possible that the process of nuclear Gata4 and Hnf1 loss during suckling in mice is a regulatory process, but for other, as yet unknown, targets of Gata4 and Hnf1.

Glucocorticoids like dexamethasone can induce the precocious maturation of the intestine, but the underlying mechanism has not been fully elucidated (1, 54). Characteristic of this process is a dramatic increase in SI mRNA abundance 24 h after the administration of glucocorticoids (25). Since the induction in SI mRNA is not apparent within the first 8 h (25), it is thought that the SI induction is not a direct response to glucocorticoids, but rather a secondary effect of early response genes on SI gene transcription (1). Recently, Oesterreicher and Henning (32) showed that in 8-day-old mice, Gata4 and Gata6 were both induced 4 h after dexamethasone treatment, as shown by supershift EMSAs and Western blot analysis, suggesting a role for Gata factors in the glucocorticoid-induced maturation of the intestine. To test the hypothesis that Gata4 or Hnf1 mediates this process, we conducted dexamethasone-induced precocious maturation experiments in our knockout models. SI mRNA was strongly induced in the presence or absence of Gata4 or Hnf1 (Fig. 9A), indicating that these proteins are not required for mediating the dexamethasone response on SI. We next defined the effect of dexamethasone on LPH mRNA abundance in our knockout models and found that LPH mRNA was reduced in the Gata4-mutant and Hnf1–/– mice (Fig. 9A) but not in wild-type controls. Our interpretation of these data is that dexamethasone induces precocious maturation to the point where Gata4 and Hnf1 become more regulatory for LPH gene expression. We also found a decrease in both Gata4 and Hnf1 mRNA abundance with dexamethasone treatment (Fig. 9B), which is also consistent with a maturation of the intestine, because Gata4 and Hnf1 mRNAs decline normally during weaning (Fig. 4A). Therefore, we believe that neither Gata4 nor Hnf1 mediates the glucocorticoid response but rather are downstream targets of this response.

Cre-mediated inactivation of Gata4 in our current model results in the synthesis of a truncated, transcriptionally inactive form of Gata4 (mutant Gata4) that is missing its NH₂-terminal activation domains but contains its functional zinc finger region (3). Since mutant Gata4 continues to bind DNA, it has the potential to act as a dominant negative Gata factor (3), masking the activity of other enterocyte Gata factors, such as Gata6, which is coexpressed with Gata4 in villus enterocytes (3, 9, 41). Furthermore, mutant Gata4 contains the functional zinc finger region, which has been shown to interact not only with DNA, but also with other proteins, such as Hnf1 (49) and friend of Gata (Fog) cofactors (16). Indeed, we have shown that, although the activation domains of Hnf1 are absolutely required for synergy, the Gata activation domains are dispensable for this activity (49, 50), suggesting that mutant Gata4 maintains the ability to mediate Gata4-Hnf1 synergy. Thus the phenotype attributed to the inactivation of Gata4 in our current model could represent a specific Gata4 function, a masked Gata6 function, and/or a function dependent on Gata4 activation domains. Furthermore, because Gata4 is inducibly inactivated rather than null for Gata4 from the earliest phases of intestinal development, it is possible that gene expression that is dependent on the presence of Gata4 early in development (before administration of tamoxifen) may not be revealed in the current model. Nevertheless, since the same Gata4 model (induction of mutant Gata4) is used throughout our studies, the differential regulation of target genes at diverse developmental time points continues to support different underlying mechanisms of regulation during development.

Our data show that the induction and maintenance of terminal differentiation by Gata4 and Hnf1 is highly dependent on the developmental time frame under study. Although Gata4 and Hnf1 are indispensable for the maintenance of expression of specific genes in the mature intestine (3, 4), consistent with a mechanism of coregulation, these transcription factors are only partially required for the same genes prior to the final maturation that occurs at weaning, arguing against a coregulation mechanism and implicating other mechanisms in the development of gut function. Understanding the individual and combined effects of intestinal transcription factors during development will continue to reveal important regulatory pathways essential for intestinal differentiation.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01-DK-61382 (S. D. Krasinski) and R37-DK-32658 (R. J. Grand), the Harvard Digestive Disease Center (5P30-DK-34854), and the Nutricia Research Foundation (T. Bosse and E. Beuling).

	ACKNOWLEDGMENTS

 
We thank Dr. W. T. Pu (Children's Hospital Boston, Boston, MA) for the floxed Gata4 mouse line, Dr. S. Robine (Institut Curie, Paris, France) for the Villin-CreER^T2 transgenic mouse line, and Dr. F. J. Gonzalez (National Institutes of Health, Bethesda, MD) for the Hnf1-null mouse line. We also thank Dr. J. Gordon (Washington University, St. Louis, MO) for the Fabp1 antibody, Dr. K. Y. Yeh (Louisiana State University, Shreveport, LA) for the LPH and SI antibodies, and Dr. D. Silberg (University of Pennsylvania, Philadelphia, PA) for the Cdx2 antibody. We also thank Lena Liu (Department of Pathology, Children's Hospital Boston, Boston, MA) for technical support.

	FOOTNOTES

 

Address for reprint requests and other correspondence: S. D. Krasinski, GI/Cell Biology, EN 720, Children's Hospital Boston, 300 Longwood Ave., Boston, MA 02115 (e-mail: stephen.krasinski{at}childrens.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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