Hepatocyte Nuclear Factor 4α Is Essential for Embryonic Development of the Mouse Colon
2006; Elsevier BV; Volume: 130; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2006.01.003
ISSN1528-0012
AutoresWendy Garrison, Michele A. Battle, Chuhu Yang, Klaus H. Kaestner, Frances M. Sladek, Stephen A. Duncan,
Tópico(s)FOXO transcription factor regulation
ResumoBackground & Aims: Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor that has been shown to be required for hepatocyte differentiation and development of the liver. It has also been implicated in regulating expression of genes that act in the epithelium of the lower gastrointestinal tract. This implied that HNF4α might be required for development of the gut. Methods: Mouse embryos were generated in which Hnf4a was ablated in the epithelial cells of the fetal colon by using Cre-loxP technology. Embryos were examined by using a combination of histology, immunohistochemistry, DNA microarray, reverse-transcription polymerase chain reaction, electrophoretic mobility shift assays, and chromatin immunoprecipitation analyses to define the consequences of loss of HNF4α on colon development. Results: Embryos were recovered at E18.5 that lacked HNF4α in their colons. Although early stages of colonic development occurred, HNF4α-null colons failed to form normal crypts. In addition, goblet-cell maturation was perturbed and expression of an array of genes that encode proteins with diverse roles in colon function was disrupted. Several genes whose expression in the colon was dependent on HNF4α contained HNF4α-binding sites within putative transcriptional regulatory regions and a subset of these sites were occupied by HNF4α in vivo. Conclusions: HNF4α is a transcription factor that is essential for development of the mammalian colon, regulates goblet-cell maturation, and is required for expression of genes that control normal colon function and epithelial cell differentiation. Background & Aims: Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor that has been shown to be required for hepatocyte differentiation and development of the liver. It has also been implicated in regulating expression of genes that act in the epithelium of the lower gastrointestinal tract. This implied that HNF4α might be required for development of the gut. Methods: Mouse embryos were generated in which Hnf4a was ablated in the epithelial cells of the fetal colon by using Cre-loxP technology. Embryos were examined by using a combination of histology, immunohistochemistry, DNA microarray, reverse-transcription polymerase chain reaction, electrophoretic mobility shift assays, and chromatin immunoprecipitation analyses to define the consequences of loss of HNF4α on colon development. Results: Embryos were recovered at E18.5 that lacked HNF4α in their colons. Although early stages of colonic development occurred, HNF4α-null colons failed to form normal crypts. In addition, goblet-cell maturation was perturbed and expression of an array of genes that encode proteins with diverse roles in colon function was disrupted. Several genes whose expression in the colon was dependent on HNF4α contained HNF4α-binding sites within putative transcriptional regulatory regions and a subset of these sites were occupied by HNF4α in vivo. Conclusions: HNF4α is a transcription factor that is essential for development of the mammalian colon, regulates goblet-cell maturation, and is required for expression of genes that control normal colon function and epithelial cell differentiation. The primary functions of the gastrointestinal tract are to process ingested food, absorb nutrients and water, and regulate energy homeostasis.1Radtke F. Clevers H. Self-renewal and cancer of the gut two sides of a coin.Science. 2005; 307: 1904-1909Crossref PubMed Scopus (594) Google Scholar, 2Stainier D.Y. No organ left behind tales of gut development and evolution.Science. 2005; 307: 1902-1904Crossref PubMed Scopus (89) Google Scholar, 3Badman M.K. Flier J.S. The gut and energy balance visceral allies in the obesity wars.Science. 2005; 307: 1909-1914Crossref PubMed Scopus (408) Google Scholar, 4Pennisi E. 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Mammalian hepatocyte differentiation requires the transcription factor HNF-4alpha.Genes Dev. 2000; 14: 464-474PubMed Google Scholar, 26Parviz F. Matullo C. Garrison W.D. Savatski L. Adamson J.W. Ning G. Kaestner K.H. Rossi J.M. Zaret K.S. Duncan S.A. Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis.Nat Genet. 2003; 34: 292-296Crossref PubMed Scopus (488) Google Scholar, 27Hayhurst G.P. Lee Y.H. Lambert G. Ward J.M. Gonzalez F.J. Hepatocyte nuclear factor 4alpha (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis.Mol Cell Biol. 2001; 21: 1393-1403Crossref PubMed Scopus (894) Google Scholar To directly address the role of HNF4α in development of the colon, mice were derived in which the Hnf4a gene was ablated in the colonic epithelial cells by using the Cre-loxP system.28Parviz F. Li J. Kaestner K.H. Duncan S.A. Generation of a conditionally null allele of hnf4alpha.Genesis. 2002; 32: 130-133Crossref PubMed Scopus (35) Google Scholar Examination of these embryos revealed that loss of HNF4α disrupts formation of crypt topology in fetal colons by E18.5. Loss of crypts was accompanied by reduced goblet cell maturation and proliferation of epithelial cells. Gene expression and HNF4α DNA–binding analyses revealed that loss of HNF4α is required for expression of several target genes that are central to colon function. The derivation of Hnf4a+/− (Hnf4atm1Dnl), Hnf4aloxP/loxP (Hnf4atm1Sad), and Foxa3Cre (Tg[Foxa3-cre]1Khk) mice has been described previously.28Parviz F. Li J. Kaestner K.H. Duncan S.A. Generation of a conditionally null allele of hnf4alpha.Genesis. 2002; 32: 130-133Crossref PubMed Scopus (35) Google Scholar, 29Lee C.S. Sund N.J. Behr R. Herrera P.L. Kaestner K.H. Foxa2 is required for the differentiation of pancreatic alpha-cells.Dev Biol. 2005; 278: 484-495Crossref PubMed Scopus (158) Google Scholar, 30Chen W.S. Manova K. Weinstein D.C. Duncan S.A. Plump A.S. Prezioso V.R. Bachvarova R.F. Darnell Jr, J.E. Disruption of the HNF-4 gene, expressed in visceral endoderm, leads to cell death in embryonic ectoderm and impaired gastrulation of mouse embryos.Genes Dev. 1994; 8: 2466-2477Crossref PubMed Scopus (494) Google Scholar Mating of Hnf4a+/− and Hnf4aloxP/loxP with Foxa3Cre mice produced Hnf4a+/−Foxa3Cre and Hnf4aloxP/+ Foxa3Cre male mice that were used as studs. These males were mated with Hnf4aloxP/loxP female mice to generate control Hnf4aloxP/+Foxa3Cre or experimental Hnf4aloxP loxPFoxa3Cre and Hnf4aloxP/−Foxa3Cre embryos. Analysis of HNF4α loss of function was restricted to the colon because deletion of HNF4α was inefficient in the small intestine. Noon on the day of the appearance of a vaginal plug was considered as 0.5 days postcoitum, and the genotype of all embryos was determined by polymerase chain reaction (PCR) analysis of genomic DNA. The Medical College of Wisconsin's IACUC committee approved all animal experiments and procedures. Five millimeters of colon lying 5 mm distal to the cecum was dissected from E18.5 embryos in phosphate-buffered saline, fixed overnight with 4% paraformaldehyde (Sigma, St Louis, MO) in phosphate-buffered saline, and embedded in paraffin. Five-micrometer paraffin sections were used in all staining procedures. H&E, alcian blue, and β-galactosidase staining were performed by using standard histological protocols.31Bancroft J.D. Gamble M. Theory and practice of histological techniques. Elsevier Science, London, UK2002Google Scholar Immunohistochemistry was performed by using antigen retrieval in citrate buffer as described previously,26Parviz F. Matullo C. Garrison W.D. Savatski L. Adamson J.W. Ning G. Kaestner K.H. Rossi J.M. Zaret K.S. Duncan S.A. Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis.Nat Genet. 2003; 34: 292-296Crossref PubMed Scopus (488) Google Scholar except for PECAM1 (platelet/endothelial cell adhesion molecule 1) staining in which sections were treated with 0.25% trypsin for 20 minutes at room temperature. Primary antibodies recognized HNF4α (sc-6556, 1:500; Santa Cruz, Santa Cruz, CA), E-cadherin (#610181, 1:16,000; BD Transduction Laboratories, San Jose, CA), Ki-67 (sc-7846, 1:500; Santa Cruz), phospho-Histone H3 (#06-570, 1:1500; Upstate, Charlottesville, VA), acetylated tubulin (T6793, 1:1000; Sigma, St Louis, MO), α smooth-muscle actin (A2547, 1:1600; Sigma), laminin (L9393, 1:500; Sigma), and PECAM1 (#553370, 1:50; Pharmingen, San Jose, CA). Micrographs were collected by using a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI) and images from control and experimental samples were processed identically using Adobe Photoshop 7.0 (Adobe, San Jose, CA). Cell counts were taken from digital images of 3 sections from each embryo analyzed. Thickness of the circular muscle was measured using Metamorph software (Molecular Devices, Sunnyvale, CA). Data were subjected to statistical analyses by using analysis of variance (ANOVA) (StatView 5.0.1; Adept Scientific Inc, Acton, MA) and were considered significant at P < .001. Affymetrix GeneChip Mouse Expression 430A Arrays were used for the analyses. Total RNA was extracted from fetal colons using Qiagen-RNeasy kit (Qiagen, Valencia, CA). Biotin-labeled complementary RNA probes were generated from 5 μg of total RNA by using a bioarray transcript labeling kit (Affymetrix). After hybridization, array data were analyzed by using DNA-Chip Analyzer (dChip) software (http://www.dchip.org). The microarray data (Supplementary Table 1; see supplemental material online at www.gastrojournal.org) presented in this manuscript have been deposited in National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) and are accessible through Gene Expression Omnibus series accession number GSE3116. Reverse-transcriptase PCR (RT-PCR) was performed as described previously21Duncan S.A. Nagy A. Chan W. Murine gastrulation requires HNF-4 regulated gene expression in the visceral endoderm tetraploid rescue of HNF-4-/- embryos.Development. 1997; 124: 279-287Crossref PubMed Google Scholar by using oligonucleotide sequences shown in Supplementary Table 2 (see supplemental material online at www.gastrojournal.org). Amplification of Hprt was used to standardize loading and reactions lacking reverse transcriptase were included to confirm the absence of contaminating DNA. Radiolabeled probes were incubated at room temperature for 20 minutes with liver or Cos-7 cell nuclear extracts in the presence or absence of HNF4α antiserum or a negative control antibody (anti-Pes132Lerch-Gaggl A. Haque J. Li J. Ning G. Traktman P. Duncan S.A. Pescadillo is essential for nucleolar assembly, ribosome biogenesis, and mammalian cell proliferation.J Biol Chem. 2002; 277: 45347-45355Crossref PubMed Scopus (101) Google Scholar). For competition analyses, double-strand oligonucleotide sequence representing the HNF4α-binding site (H4.2133Yang C, Liao H, Bolotin E, Evans J, Ellrott K, Jiang T, Sladek FM. Differences in HNF4a cis-regulatory elements in the human and mouse genomes. (in press)Google Scholar) from the ApoCIII promoter34Sladek F.M. Zhong W. Lai E. Darnell Jr, J.E. Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily.Genes Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (872) Google Scholar was labeled by using [P32]-deoxyadenosine triphosphate and incubated with 1 μg of nuclear extracts from Cos-7 cells or Cos-7 cells expressing exogenous HNF4α in the presence or absence of 150-fold molar excess of each test binding site as described previously.35Jiang G. Nepomuceno L. Hopkins K. Sladek F.M. Exclusive homodimerization of the orphan receptor hepatocyte nuclear factor 4 defines a new subclass of nuclear receptors.Mol Cell Biol. 1995; 15: 5131-5143Crossref PubMed Scopus (176) Google Scholar Alternatively, radiolabeled binding sites were incubated with mouse liver nuclear extract prepared as described previously.34Sladek F.M. Zhong W. Lai E. Darnell Jr, J.E. Liver-enriched transcription factor HNF-4 is a novel member of the steroid hormone receptor superfamily.Genes Dev. 1990; 4: 2353-2365Crossref PubMed Scopus (872) Google Scholar Complexes were resolved by electrophoresis in 4% polyacrylamide gels in 0.25X Tris-borate-EDTA buffer at 400 V for 1 hour. The oligonucleotide sequences representing putative HNF4α-binding sites used in electrophoretic mobility shift assay (EMSA) reactions are provided in Supplementary Table 2. Brains and colons were isolated from wild-type CD1 mice, fixed with 1% formaldehyde, and cells dissociated by using a BD Medimachine (BD Biosciences, #340587). Chromatin immunoprecipitation (ChIP) assays were performed using the Upstate ChIP Assay Kit (Upstate #17-295) following the manufacturer's instructions with anti-HNF4α (Santa Cruz sc-8987) or anti-Pes132Lerch-Gaggl A. Haque J. Li J. Ning G. Traktman P. Duncan S.A. Pescadillo is essential for nucleolar assembly, ribosome biogenesis, and mammalian cell proliferation.J Biol Chem. 2002; 277: 45347-45355Crossref PubMed Scopus (101) Google Scholar antibodies. Oligonucleotide primers used for PCR amplification of precipitated genomic DNA are given in Supplementary Table 2. Hnf4a mRNA has been previously localized to the fetal gastrointestinal tract.36Duncan S.A. Manova K. Chen W.S. Hoodless P. Weinstein D.C. Bachvarova R.F. Darnell Jr, J.E. Expression of transcription factor HNF-4 in the extraembryonic endoderm, gut, and nephrogenic tissue of the developing mouse embryo HNF-4 is a marker for primary endoderm in the implanting blastocyst.Proc Natl Acad Sci U S A. 1994; 91: 7598-7602Crossref PubMed Scopus (319) Google Scholar, 37Taraviras S. Monaghan A.P. Schutz G. Kelsey G. Characterization of the mouse HNF-4 gene and its expression during mouse embryogenesis.Mech Dev. 1994; 48: 67-79Crossref PubMed Scopus (149) Google Scholar However, to facilitate cell-specific disruption of the Hnf4a gene using the Cre-loxP system required clarification of which specific cell types in the developing gut expressed HNF4α protein. Figure 1 shows the result of immunohistochemistry staining analyses of mouse embryos using an antibody (Santa Cruz, anti-HNF4α C19) that specifically recognizes the carboxyl end of all major isoforms of HNF4α.26Parviz F. Matullo C. Garrison W.D. Savatski L. Adamson J.W. Ning G. Kaestner K.H. Rossi J.M. Zaret K.S. Duncan S.A. Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis.Nat Genet. 2003; 34: 292-296Crossref PubMed Scopus (488) Google Scholar At E9.5, HNF4α protein was undetectable in the foregut but could be identified in the developing hepatic rudiment as well as in the epithelium of the midgut and hindgut (Figure 1A and B). By E11.5, staining became more pronounced in these tissues presumably reflecting an increase in the abundance of HNF4α (Figure 1C and D). At this developmental time, HNF4α protein was detected throughout the epithelium of the lower gastrointestinal tract with a rostral boundary of expression occurring within the developing stomach (Figure 1C). HNF4α expression was maintained in all epithelial cells during the conversion of the gut epithelium from psuedostratified to simple columnar (Figure 1E–H). The restriction of HNF4α protein to the epithelium of both small and large intestine continued throughout development into adulthood (not shown) with expression found in both absorptive and secretory cell lineages of the intestinal epithelium. Development of Hnf4a knockout embryos arrests during gastrulation because of defects in visceral endoderm function.30Chen W.S. Manova K. Weinstein D.C. Duncan S.A. Plump A.S. Prezioso V.R. Bachvarova R.F. Darnell Jr, J.E. Disruption of the HNF-4 gene, expressed in visceral endoderm, leads to cell death in embryonic ectoderm and impaired gastrulation of mouse embryos.Genes Dev. 1994; 8: 2466-2477Crossref PubMed Scopus (494) Google Scholar, 36Duncan S.A. Manova K. Chen W.S. Hoodless P. Weinstein D.C. Bachvarova R.F. Darnell Jr, J.E. Expression of transcription factor HNF-4 in the extraembryonic endoderm, gut, and nephrogenic tissue of the developing mouse embryo HNF-4 is a marker for primary endoderm in the implanting blastocyst.Proc Natl Acad Sci U S A. 1994; 91: 7598-7602Crossref PubMed Scopus (319) Google Scholar This has prevented the use of Hnf4a−/− embryos to study the role of HNF4α in gut development. The Cre-loxP system was therefore used to circumvent this early embryonic lethality and to generate embryos lacking HNF4α in the fetal colonic epithelium. The production of mice (Hnf4aloxP/loxP) that contain an allele of Hnf4a in which exon2 is flanked by loxP elements has been described previously.26Parviz F. Matullo C. Garrison W.D. Savatski L. Adamson J.W. Ning G. Kaestner K.H. Rossi J.M. Zaret K.S. Duncan S.A. Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis.Nat Genet. 2003; 34: 292-296Crossref PubMed Scopus (488) Google Scholar, 28Parviz F. Li J. Kaestner K.H. Duncan S.A. Generation of a conditionally null allele of hnf4alpha.Genesis. 2002; 32: 130-133Crossref PubMed Scopus (35) Google Scholar Mice that are homozygous for this allele are healthy and fertile. However, in the presence of Cre, recombination between loxP sites generates a null allele.26Parviz F. Matullo C. Garrison W.D. Savatski L. Adamson J.W. Ning G. Kaestner K.H. Rossi J.M. Zaret K.S. Duncan S.A. Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis.Nat Genet. 2003; 34: 292-296Crossref PubMed Scopus (488) Google Scholar, 28Parviz F. Li J. Kaestner K.H. Duncan S.A. Generation of a conditionally null allele of hnf4alpha.Genesis. 2002; 32: 130-133Crossref PubMed Scopus (35) Google Scholar A line of transgenic mice (Foxa3Cre) in which Cre recombinase is expressed from a yeast artificial chromosome encompassing all Foxa3 transcriptional regulatory elements has also been previously described.29Lee C.S. Sund N.J. Behr R. Herrera P.L. Kaestner K.H. Foxa2 is required for the differentiation of pancreatic alpha-cells.Dev Biol. 2005; 278: 484-495Crossref PubMed Scopus (158) Google Scholar The tissue distribution of Cre activity in transgenic mice can be determined by breeding Cre mice to a LacZ reporter mouse (Gt[ROSA]26Sortm1Sho).38Mao X. Fujiwara Y. Orkin S.H. Improved reporter strain for monitoring Cre recombinase-mediated DNA excisions in mice.Proc Natl Acad Sci U S A. 1999; 96: 5037-5042Crossref PubMed Scopus (278) Google Scholar Using this approach confirmed that Cre recombinase activity was present from E8.5 onwards in the definitive endoderm and its derivatives including the epithelial cells of the colon (Figure 2A and B).29Lee C.S. Sund N.J. Behr R. Herrera P.L. Kaestner K.H. Foxa2 is required for the differentiation of pancreatic alpha-cells.Dev Biol. 2005; 278: 484-495Crossref PubMed Scopus (158) Google Scholar Foxa3Cre mice were therefore chosen to delete Hnf4a from the colon because Cre was active at the very onset of colon development in these mice. To disrupt HNF4α in the gastrointestinal tract, Hnf4aloxP/loxP mice or Hnf4α+/− mice were mated to Foxa3Cre mice. Double-heterozygous offspring were then bred to Hnf4aloxP/loxP mice to generate either Hnf4aloxP/loxPFoxa3Cre and Hnf4aloxP/−Foxa3Cre experimental embryos or Hnf4aloxP/+Foxa3Cre control embryos. In contrast to control embryos, colons could be recovered from Hnf4aloxP/loxPFoxa3Cre embryos at E18.5 in which HNF4α protein was undetectable by immunohistochemistry (Figures 2C and D). E18.5 was the latest stage at which HNF4α-null embryos could be recovered because Hnf4aloxP/loxPFoxa3Cre newborn mice also lacked HNF4α in the liver, which resulted in neonatal lethality as previously reported.26Parviz F. Matullo C. Garrison W.D. Savatski L. Adamson J.W. Ning G. Kaestner K.H. Rossi J.M. Zaret K.S. Duncan S.A. Hepatocyte nuclear factor 4alpha controls the development of a hepatic epithelium and liver morphogenesis.Nat Genet. 2003; 34: 292-296Crossref PubMed Scopus (488) Google Scholar The efficiency of ablating HNF4α in the colon of Hnf4aloxP/loxPFoxa3Cre embryos was variable, with <10% of the mice showing complete absence of HNF4α and the remaining colons exhibiting varying levels of chimerism (Figure 2E). The observed variation in loss of HNF4α in different Hnf4aloxP/loxPFoxa3Cre embryos most likely reflects variation in recombination efficiency. In an attempt to increase the efficacy of recovering HNF4α-null colons, Hnf4aloxP/−Foxa3Cre embryos were derived in which 40% had undetectable levels of HNF4α in the colon (Figure 2F). The continued presence of HNF4α in a subset of fetal colons could potentially confound any phenotypic analyses and so only embryos in which either HNF4α protein was judged absent by immunohistochemistry or Hnf4a mRNA was undetected by using RT-PCR were studied further. In addition, Hnf4aloxP/loxPFoxa3Cre and Hnf4aloxP/−Foxa3Cre embryos were used interchangeably because they displayed indistinguishable phenotypes. Examination of E18.5 HNF4α-null colons by histochemistry uncovered abnormalities in lower gut topology and tissue organization. H&E-stained sections of control colons revealed that the epithelium contained characteristic crypts extending from a relatively compact lumen (Figure 2G). In contrast, HNF4α-null colons showed no obvious sign of crypt formation with the mucosa arranged as a simple band surrounding a greatly expanded lumen (Figure 2H). To more accurately define the consequence of the absence of HNF4α on the development of the colon, the structure of various tunics was examined by using immunoh
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