Functions of HNF1 Family Members in Differentiation of the Visceral Endoderm Cell Lineage (2003)

The two members of the hepatocyte nuclear factor 1 (HNF1) transcription factor family, HNF1 and variant HNF1 (vHNF1), show a strong homology in their atypical POU-homeodomain and dimerization domain but differ in their transactivation domains. Moreover, two vHNF1 isoforms generated by alternative splicing are present in all tissues expressing this gene. vHnf1-deficient mouse embryos die soon after implantation due to defective visceral endoderm formation, an extraembryonic tissue essential for development and survival of the embryo proper. In contrast, invalidation of Hnf1, which is expressed at later developmental stages than vHnf1, does not lead to embryonic lethality or developmental defects. To examine the specific or potential equivalent functions of vHNF1 isoforms and HNF1 during the process of visceral endoderm differentiation, we stably reexpressed these factors in vHnf1-deficient embryonic stem cells. Analysis of these embryonic stem cells upon differentiation into embryoid bodies shows that vHNF1 isoforms exhibit specific behaviors depending on particular target genes and cooperate in the establishment of a functional visceral endoderm. Furthermore, forced expression of HNF1 in vHnf1-deficient embryonic stem cells fully restores the formation of a mature visceral endoderm with the correct expression profile of early and late markers of this lineage. Thus, in this context, HNF1 functionally replaces both vHNF1 isoforms, suggesting that the different developmental functions of these transcription factors are mainly due to the acquisition of novel expression patterns. The two members of the hepatocyte nuclear factor 1 (HNF1) transcription factor family, HNF1 and variant HNF1 (vHNF1), show a strong homology in their atypical POU-homeodomain and dimerization domain but differ in their transactivation domains. Moreover, two vHNF1 isoforms generated by alternative splicing are present in all tissues expressing this gene. vHnf1-deficient mouse embryos die soon after implantation due to defective visceral endoderm formation, an extraembryonic tissue essential for development and survival of the embryo proper. In contrast, invalidation of Hnf1, which is expressed at later developmental stages than vHnf1, does not lead to embryonic lethality or developmental defects. To examine the specific or potential equivalent functions of vHNF1 isoforms and HNF1 during the process of visceral endoderm differentiation, we stably reexpressed these factors in vHnf1-deficient embryonic stem cells. Analysis of these embryonic stem cells upon differentiation into embryoid bodies shows that vHNF1 isoforms exhibit specific behaviors depending on particular target genes and cooperate in the establishment of a functional visceral endoderm. Furthermore, forced expression of HNF1 in vHnf1-deficient embryonic stem cells fully restores the formation of a mature visceral endoderm with the correct expression profile of early and late markers of this lineage. Thus, in this context, HNF1 functionally replaces both vHNF1 isoforms, suggesting that the different developmental functions of these transcription factors are mainly due to the acquisition of novel expression patterns. Duplication of developmental control genes is thought to be an evolutionary mechanism for the generation of novel functions allowing for increased diversity in complex organisms. Acquisition of new functions principally involves the evolution of cis-regulatory sequences, although coding sequences have also evolved, albeit at significantly lower levels (1Force A. Lynch M. Pickett F.B. Amores A. Yan Y.L. Postlethwait J. Genetics. 1999; 151: 1531-1545Crossref PubMed Google Scholar). The genes encoding the transcription factors hepatocyte nuclear factor 1 (HNF1α and Tcf-1) and variant HNF1 (vHNF1, HNF-1β, and Tcf-2) have arisen by duplication of an ancestral gene at the onset of vertebrate evolution. They belong to a distinct subclass of the homeobox transcription factor family characterized by a highly conserved N-terminal DNA binding domain and a more divergent C-terminal transactivation domain. The DNA binding domain is composed of three conserved regions: the N-terminal dimerization domain, an atypical POU-specific (POUs) 1The abbreviations used are: POUs, POU-specific domain; POUH, POU homeodomain; VE, visceral endoderm; ES, embryonic stem; EB, embryoid bodies; CAT, chloramphenicol acetyltransferase; HEK, human embryonic kidney; RSV, Rous sarcoma virus; GAPDH, glyceralde-hyde phosphate dehydrogenase; PBS, phosphate-buffered saline; RT, reverse transcriptase. domain and a divergent POU homeodomain (POUH), containing a loop of 21 amino acids between helices 2 and 3. The POUs and POUH domains are required for sequence specificity and DNA recognition (2Tomei L. Cortese R. De Francesco R. EMBO J. 1992; 11: 4119-4129Crossref PubMed Scopus (32) Google Scholar). Recent three-dimensional structure analysis indicates that the POUs domain interacts with the atypical insertion of the homeodomain to create a stable interface, further distinguishing HNF1/vHNF1 from other flexible POU-homeodomain factors (3Chi Y. Frantz J. Hansen L. Dhe-Paganon S. Shoelson S. Mol. Cell Biol. 2002; 10: 1129-1137Scopus (80) Google Scholar). Because of these particular structural features, HNF1 and vHNF1 bind DNA as dimers or heterodimers and display indistinguishable DNA-binding sequence specificity (4Rey-Campos J. Chouard T. Yaniv M. Cereghini S. EMBO J. 1991; 10: 1445-1457Crossref PubMed Scopus (239) Google Scholar, 5De Simone V. De Magistris L. Lazzaro D. Gerstner J. Monaci P. Nicosia A. Cortese R. EMBO J. 1991; 10: 1435-1443Crossref PubMed Scopus (151) Google Scholar, 6Bach I. Mattei M.G. Cereghini S. Yaniv M. Nucleic Acids Res. 1991; 19: 3553-3559Crossref PubMed Scopus (90) Google Scholar, 7Cereghini S. FASEB J. 1996; 10: 267-282Crossref PubMed Scopus (473) Google Scholar). HNF1 is however a more powerful activator than vHNF1, implying that these two factors display distinct regulatory properties in vivo (4Rey-Campos J. Chouard T. Yaniv M. Cereghini S. EMBO J. 1991; 10: 1445-1457Crossref PubMed Scopus (239) Google Scholar, 8Ringeisen F. Rey-Campos J. Yaniv M. J. Biol. Chem. 1993; 268: 25706-25711Abstract Full Text PDF PubMed Google Scholar). vHNF1 and HNF1 are co-expressed in the yolk sac endoderm and in developing liver, kidney, and pancreas, although with different spatio-temporal patterns (9Lazzaro D. De Simone V. De Magistris L. Lehtonen E. Cortese R. Development. 1992; 114: 469-479Crossref PubMed Google Scholar, 10Ott M.O. Rey-Campos J. Cereghini S. Yaniv M. Mech. Dev. 1991; 36: 47-58Crossref PubMed Scopus (84) Google Scholar, 11Cereghini S. Ott M.O. Power S. Maury M. Development. 1992; 116: 783-797PubMed Google Scholar, 12Barbacci E. Reber M. Ott M. Breillat C. Huetz F. Cereghini S. Development. 1999; 126: 4795-4805Crossref PubMed Google Scholar, 13Coffinier C. Barra J. Babinet C. Yaniv M. Mech. Dev. 1999; 89: 211-213Crossref PubMed Scopus (180) Google Scholar). Transcription of the vHnf1 gene precedes that of HNF1 starting from the earliest stages of differentiation of these organs. HNF1 appears later, during the maturation stages. Notably, vHnf1 is uniquely expressed in the primitive and visceral endoderm from embryonic day 4.5 to 7.5 and in the liver, pancreatic, and ureteric buds at embryonic days 9.5–11 (12Barbacci E. Reber M. Ott M. Breillat C. Huetz F. Cereghini S. Development. 1999; 126: 4795-4805Crossref PubMed Google Scholar). HNF1 appears in the visceral endoderm of the yolk sac around embryonic day 8.5 and in the liver and pancreatic buds at embryonic day 10.5, after specification of these organs from the gut endoderm (11Cereghini S. Ott M.O. Power S. Maury M. Development. 1992; 116: 783-797PubMed Google Scholar, 14Nammo T. Yamagata K. Hamaoka R. Zhu Q. Akiyama T. Gonzalez F. Miyagawa J. Matsuzawa Y. Diabetologia. 2002; 45: 1142-1153Crossref PubMed Scopus (31) Google Scholar). In addition, vHnf1 is expressed in the forming neural tube, the lungs, and the genital tract, where HNF1 is absent (12Barbacci E. Reber M. Ott M. Breillat C. Huetz F. Cereghini S. Development. 1999; 126: 4795-4805Crossref PubMed Google Scholar, 13Coffinier C. Barra J. Babinet C. Yaniv M. Mech. Dev. 1999; 89: 211-213Crossref PubMed Scopus (180) Google Scholar, 15Reber M. Cereghini S. Mech. Dev. 2001; 100: 75-78Crossref PubMed Scopus (35) Google Scholar). Consistent with these embryonic expression patterns, vHnf1-deficient mouse embryos die shortly after implantation due to defective visceral endoderm (VE) formation (12Barbacci E. Reber M. Ott M. Breillat C. Huetz F. Cereghini S. Development. 1999; 126: 4795-4805Crossref PubMed Google Scholar, 16Coffinier C. Thepot D. Babinet C. Yaniv M. Barra J. Development. 1999; 126: 4785-4794PubMed Google Scholar). The VE is an extraembryonic tissue, source of nutrients and multiple signals essential for normal development of the pregastrulating mouse embryo. In addition to its nutritional and histotrophic role, the VE participates in other embryonic developmental processes including early anterior neural patterning (17Beddington R.S. Robertson E.J. Trends Genet. 1998; 14: 277-284Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar), cavitation of the ectoderm (18Coucouvanis E. Martin G.R. Development. 1999; 126: 535-546Crossref PubMed Google Scholar), and specification of hematopoietic and endothelial cell fates (19Belaoussoff M. Farrington S.M. Baron M.H. Development. 1998; 125: 5009-5018PubMed Google Scholar). vHnf1 homozygous mutant embryos lack a distinct extraembryonic VE and as a consequence the ectodermal cells are severely disorganized. In contrast, HNF1 is not required for normal development, although Hnf1-deficient mice die during postnatal life because of hepatic, pancreatic and renal dysfunction (20Pontoglio M. Barra J. Hadchouel M. Doyen A. Kress C. Bach J.P. Babinet C. Yaniv M. Cell. 1996; 84: 575-585Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 21Lee Y.H. Sauer B. Gonzalez F.J. Mol. Cell Biol. 1998; 18: 3059-3068Crossref PubMed Scopus (228) Google Scholar, 22Pontoglio M. Sreenan S. Roe M. Pugh W. Ostrega D. Doyen A. Pick A.J. Baldwin A. Velho G. Froguel P. Levisetti M. Bonner-Weir S. Bell G.I. Yaniv M. Polonsky K.S. J. Clin. Invest. 1998; 101: 2215-2222Crossref PubMed Scopus (279) Google Scholar). The sequential expression of vHNF1 and HNF1 in diverse tissues, along with their structural similarities, raise the question of whether the distinct function of these factors is defined by their different spatiotemporal expression patterns or by their intrinsic biochemical activities. An additional level of complexity in elucidating the precise function of these factors comes from the existence of different isoforms generated by either constitutive or regulated alternative splicing. Whereas in mice, transcription of the Hnf1 gene generates only one major transcript, two isoforms of vHNF1 designated vHNF1-A and -B, resulting from alternative exon usage, are always present, albeit at different levels, in all tissues where vHnf1 is expressed (8Ringeisen F. Rey-Campos J. Yaniv M. J. Biol. Chem. 1993; 268: 25706-25711Abstract Full Text PDF PubMed Google Scholar, 11Cereghini S. Ott M.O. Power S. Maury M. Development. 1992; 116: 783-797PubMed Google Scholar, 23Bach I. Yaniv M. EMBO J. 1993; 12: 4229-4242Crossref PubMed Scopus (112) Google Scholar). These two isoforms, which differ in the insertion of 26 amino acids between the POUs domain and the homeodomain, retain compatible dimerization domains and essentially identical DNA binding and transactivation domains. This pattern of alternative splicing is conserved through evolution from rodents to humans. It remains unclear why these two structurally highly similar proteins are co-expressed and whether they display distinct or complementary functions in vivo. In the present study, we have examined whether vHNF1-A, vHNF1-B, and HNF1 have similar or distinct functions during the process of VE specification upon differentiation of embryonic stem (ES) cells into embryoid bodies (EB). The EBs derived from ES cells homozygous deleted for vHnf1 display a specific block in the formation of a surrounding layer of VE and lack the expression of early and late VE markers, whereas other differentiation programs do not appear to be affected (12Barbacci E. Reber M. Ott M. Breillat C. Huetz F. Cereghini S. Development. 1999; 126: 4795-4805Crossref PubMed Google Scholar). We therefore took advantage of our homozygous vHnf1-deficient ES cells, which are deleted of the two vHNF1 spliced isoforms and in which activation of HNF1 is impaired upon differentiation, to stably reexpress vHNF1-A, vHNF1-B, or HNF1. Using this model system, we show here that vHNF1-A and vHNF1-B isoforms exhibit similar and complementary functions in the VE specification, whereas HNF1 alone can substitute for the function of both vHNF1 isoforms in this process. On the basis of recent studies (24Fujikura J. Yamato E. Yonemura S. Hosoda K. Masui S. Nakao K. Miyazaki Ji J. Niwa H. Genes Dev. 2002; 16: 784-789Crossref PubMed Scopus (406) Google Scholar) and the results presented here, we also propose an integrative model of the regulatory network that governs the establishment and maturation of the VE. Plasmid Constructs—The 2.3-kb rat HNF1 cDNA, the 1.6 kb rat vHNF1-A, and mouse vHNF1-B cDNAs were cloned in the pCB6 vector (25Brewer C.B. Methods Cell Biol. 1994; 43: 233-244Crossref PubMed Scopus (84) Google Scholar) containing the cytomegalovirus promoter and the polyadenylation site of the bovine growth hormone at the BglII site. All the reporter plasmids contain the bacterial gene encoding chloramphenicol acetyltransferase (CAT). palb-CAT –386/+4 (26Ott M.O. Sperling L. Herbomel P. Yaniv M. Weiss M.C. EMBO J. 1984; 3: 2505-2510Crossref PubMed Scopus (89) Google Scholar) is a construct containing a promoter derived from the rat albumin promoter driving the CAT reporter gene. pHAF-CAT contains the –1023 to +33 bp region of the mouse Afp promoter (27Molne M. Houart C. Szpirer J. Szpirer C. Nucleic Acids Res. 1989; 17: 3447-3457Crossref PubMed Scopus (20) Google Scholar). pHNF4α-CAT contains the –572 to +25 region of the mouse Hnf4α promoter (28Taraviras S. Monaghan A.P. Schutz G. Kelsey G. Mech. Dev. 1994; 48: 67-79Crossref PubMed Scopus (143) Google Scholar) and was generated by PCR using mouse genomic DNA and the primers forward (TTCAGCTGGAGCAACCAAGAGATATC) and reverse (TCCCTTCTCTGCCTTCCTCTC). The PCR product was cloned in pGEM-Teasy (Promega), verified by sequencing, and subsequently subcloned into the pBLCAT6 (29Boshart M. Kluppel M. Schmidt A. Schutz G. Luckow B. Gene (Amst.). 1992; 110: 129-130Crossref PubMed Scopus (230) Google Scholar). The HNF1 binding sites within these plasmids and their localization within the original promoters of the genes tested are as follows: for the AFP reporter, two sites at –131 and –67, respectively (30Feuerman M. Godbout R. Ingram R. Tilghman S. Mol. Cell Biol. 1989; 9: 4204-4212Crossref PubMed Scopus (90) Google Scholar); for the HNF4α reporter, a site at –98 (28Taraviras S. Monaghan A.P. Schutz G. Kelsey G. Mech. Dev. 1994; 48: 67-79Crossref PubMed Scopus (143) Google Scholar, 31Zhong W. Mirkovitch J. Darnell Jr., J.E. Mol. Cell Biol. 1994; 14: 7276-7284Crossref PubMed Scopus (92) Google Scholar); and for the albumin reporter, a site at –60 (32Cereghini S. Blumenfeld M. Yaniv M. Genes Dev. 1988; 2: 957-974Crossref PubMed Scopus (226) Google Scholar). HNF1, vHNF1-A, and vHNF1-B cDNAs were cloned in the β-actin-puro expression vector at the BamHI site (33Cheng A.M. Saxton T.M. Sakai R. Kulkarni S. Mbamalu G. Vogel W. Tortorice C.G. Cardiff R.D. Cross J.C. Muller W.J. Pawson T. Cell. 1998; 95: 793-803Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Cell Culture, Transfections, and ES Cell Electroporation—Human epithelial cervical tumor cells (C33) and human embryonic kidney cells (HEK 293) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Transient transfections were carried out by the calcium phosphate co-precipitation procedure as described by Power and Cereghini (34Power S.C. Cereghini S. Mol. Cell Biol. 1996; 16: 778-791Crossref PubMed Google Scholar). Cells were transfected with increasing amounts of expression vectors (1–200 ng), 2 μg of the reporter plasmid containing the promoter of interest driving the CAT reporter gene, and 0.6 μg of pRSV-β-galactosidase as an internal control for transfection. Total amount of expression vector was equalized with the pCB6 plasmid. β-Galactosidase activity was measured by the standard colorimetric method, and CAT assays were performed as described by Seed and Sheen (35Seed B. Sheen J. Gene (Amst.). 1988; 67: 271-277Crossref PubMed Scopus (830) Google Scholar). Each measurement was repeated at least four times with different plasmid preparations. ES cells were cultured and differentiated as described by Barbacci et al. (12Barbacci E. Reber M. Ott M. Breillat C. Huetz F. Cereghini S. Development. 1999; 126: 4795-4805Crossref PubMed Google Scholar). 10 μg of HNF1-βactin-puro vector or vHNF1-βactin-puro vector were linearized after digestion with SfiI restriction enzyme and electroporated into 1 × 107 vHnf1–/– ES cells (clone 9), resuspended in 800 μl of Dulbecco's modified Eagle's medium without fetal calf serum at 180 V and 1050 microfarads. Transfected ES cells were grown 5 days in Dulbecco's modified Eagle's medium containing 2 μg/ml puromycin, and roughly 30 puromycin-resistant clones for each electroporation were picked for amplification. Protein Extracts and Gel Mobility Shift Assays—Gel retardation assays were performed as described by Cereghini (32Cereghini S. Blumenfeld M. Yaniv M. Genes Dev. 1988; 2: 957-974Crossref PubMed Scopus (226) Google Scholar). The probes used encompassed the HNF1 binding site of the albumin promoter (proximal element probe) (32Cereghini S. Blumenfeld M. Yaniv M. Genes Dev. 1988; 2: 957-974Crossref PubMed Scopus (226) Google Scholar) and that of the Hnf4α promoter (4FP1 probe) (31Zhong W. Mirkovitch J. Darnell Jr., J.E. Mol. Cell Biol. 1994; 14: 7276-7284Crossref PubMed Scopus (92) Google Scholar). RNA and Protein Analysis—Screening of the ES clones was carried out by both gel retardation assay and RT-PCR. vHNF1 and HNF1 ES clones were differentiated into EBs as previously described (12Barbacci E. Reber M. Ott M. Breillat C. Huetz F. Cereghini S. Development. 1999; 126: 4795-4805Crossref PubMed Google Scholar). Total RNA and nuclear protein extractions from ES cells and EBs, gel retardation assay, and first strand cDNA synthesis were performed as described by Cereghini (11Cereghini S. Ott M.O. Power S. Maury M. Development. 1992; 116: 783-797PubMed Google Scholar). 2 μl of the reverse transcriptase reaction were amplified in a 25-μl PCR reaction. The number of cycles for amplification of each PCR product was determined to be in the linear phase of amplification. PCR products were electrophoresed on 1.8% agarose gel, transferred to nylon membrane (NX; Amersham Biosciences), and hybridized with the corresponding internal probes. RNA levels were normalized by the expression of GAPDH. The forward (f) and reverse (r) primer sequences, chosen in different exons, were as follows: rat/mouse HNF1 f, AGCACCCTTGCCAGCCTC; rat HNF1 f, GATGGCTCTGAGGTGTCT; mouse HNF1 r, GACACCTCAGAGCCATCC; mouse vHNF1 f, TGGTGTCCAAGCTCACGTC; mouse vHNF1 r, CAGTCGGAGGACACCTGCTC; HNF3γ f, ATGCTGGGCTCAGTGAAGAT; HNF3γ r, CAAGCTTGGGAAGGTGGGC; HNF4α1 f, ACCCAGCCTACACCACC; HNF4α1 r, CTTCCTTCTTCATGCCAGCCC; HNF6 f, AACTCCCAGCAAGGACTTCC; HNF6 r, TGCCCTGAATTACTTCCATGGC; HEX f, GTGTACGAGCCCACGCCG; HEX r, TCACTGAGCTGCAACATCTTG; Nodal f, GGAGTTTCATCCTACCAACC; Nodal r, CTGCCATGCCACGGTCGC; IHH f, ACGTGCATTGCTCTGTCAAG; IHH r, CTGGAAAGCTCTCAGCCGGAFP; AFP f, GGATAGCTTCCACGTTAGATTCC, AFP r, TGTTGCCTGGAGGTTTCGGGATCC; albumin f, TGAACTGGCTGACTGCTGTG; albumin r, CATCCTTGGCCTCAGCATAG; αAT-1 f, TGCAATCAGCCTATACCGGGA; αAT-1 r, TCAACTGCAGCTCACTGTCTG; TTR f, AGTCCTGGATGCTGTCCGAG; TTR r, TTCCTGAGCTGCTAACACGG; GAPDH f, TCCAGTATGACTCCACTCAC, GAPDH r, ACCTTGCCCACAGCCTGG. AFP Staining and Histological Analysis—EBs were fixed for 2 min at room temperature in 3% phosphate-buffered saline (PBS)-formalde-hyde and permeabilized for 15 min in cold methanol. After three washings in PBS, EBs were incubated for 30 min in containing 10% fetal calf serum and bovine serum The was incubated at at a of in containing fetal calf bovine serum and After three washings in PBS, EBs were incubated for 1 at room temperature with the used at a of EBs were in PBS, with a medium and by were performed as described by Barbacci et al. (12Barbacci E. Reber M. Ott M. Breillat C. Huetz F. Cereghini S. Development. 1999; 126: 4795-4805Crossref PubMed Google Scholar). of the by of ES cells into EBs results in the generation of of the three embryonic in a process that is of early day cells at the of the EBs into primitive which subsequently to visceral and endoderm by day This is by processes formation and the development of a of early embryonic cell Thus, this is to of early including the process of VE formation, as as to the of a of a gene S. S. J. P. J. Dev. Biol. 2002; Google Scholar). Using this we have that vHNF1 is required for the activation of members of the family including HNF1, and HNF4α1 as as a of genes encoding serum either by vHNF1, or both (12Barbacci E. Reber M. Ott M. Breillat C. Huetz F. Cereghini S. Development. 1999; 126: 4795-4805Crossref PubMed Google Scholar). In this study, we have examined additional potential target genes of vHNF1 to a more of this regulatory RNA was from the the (clone and the vHnf1–/– mutant (clone ES cells differentiated into EBs at specific corresponding to of VE formation and The vHnf1–/– was derived from the The with the ES cell with to VE differentiation (12Barbacci E. Reber M. Ott M. Breillat C. Huetz F. Cereghini S. Development. 1999; 126: 4795-4805Crossref PubMed Google Scholar) also We on the homeodomain transcription factor a of the family not HNF6 to in the differentiation of tissues derived from including the and cells of the F. Reber M. Cereghini S. D. P. T. G. F. Development. 2002; PubMed Google Scholar, P. S.M. J. C. G. F. P. M. D. Mol. Cell Biol. PubMed Scopus Google Scholar). the expression of which is as Hnf1, only at late stages of differentiation, is in the vHnf1–/– EBs as with or The of in of Hnf1 homozygous mutant mice S. L. M. Yaniv M. Polonsky K.S. M. 2001; PubMed Scopus Google Scholar), with this that either vHNF1 or HNF1 the expression of through an as binding site. In contrast, the expression level of the homeobox transcription factor first expressed in VE A. R.S. Development. 1998; 125: Google Scholar), and a of liver J.P. M. P. T. D. D. R.S. Development. Google Scholar), not differ between vHnf1–/– and The was for the expression of a protein present in the embryonic ectoderm and in the VE and in the specification of the primitive H. L. 1993; PubMed Scopus Google Scholar, K.S. A. B. Robertson E.J. Development. 1994; PubMed Google Scholar). We subsequently other regulatory to be in early functions of VE. We first examined is expressed in the VE of mouse embryos S.M. M. Baron M.H. Mech. Dev. PubMed Scopus Google Scholar) and to be in VE differentiation and in formation of in differentiated ES cells P. S. E. L. Mech. Dev. PubMed Scopus Google Scholar). the expression of expressed in the VE in ES EBs and similar to vHnf1 P. S. E. L. Mech. Dev. PubMed Scopus Google Scholar) was in the vHnf1–/– EBs at all stages of differentiation In contrast, the expression of which is at later stages of differentiation, was only in vHnf1–/– EBs at day whereas was expressed at all stages of differentiation in both vHnf1–/– and EBs not In addition, the expression of different of the including and and and was not affected in vHnf1-deficient EBs not we not in the expression levels of proteins 2 and and expressed in the VE and and in formation in EBs (18Coucouvanis E. Martin G.R. Development. 1999; 126: 535-546Crossref PubMed Google Scholar). Thus, in this context, the expression of in our vHnf1–/– EBs at least in the impaired as for other serum proteins as and (12Barbacci E. Reber M. Ott M. Breillat C. Huetz F. Cereghini S. Development. 1999; 126: 4795-4805Crossref PubMed Google Scholar), the expression of the and was absent or severely in vHnf1–/– EBs not These new in the regulatory network by vHNF1 that the the transcription factor HNF6 and the regulatory as as their which are markers of a functional VE. This be to the of vHNF1 isoforms and HNF1 in the differentiation of this cell as described by vHNF1-A and vHNF1-B studies using the albumin promoter as as a promoter have previously that vHNF1-A is a more activator than vHNF1-B (8Ringeisen F. Rey-Campos J. Yaniv M. J. Biol. Chem. 1993; 268: 25706-25711Abstract Full Text PDF PubMed Google Scholar). whether these two isoforms with to other promoters is We therefore this question in the of different genes in the VE We have chosen for this analysis promoters of both early and late markers of this cell all of vHNF1 and the Hnf4α promoter (28Taraviras S. Monaghan A.P. Schutz G. Kelsey G. Mech. Dev. 1994; 48: 67-79Crossref PubMed Scopus (143) Google Scholar, 31Zhong W. Mirkovitch J. Darnell Jr., J.E. Mol. Cell Biol. 1994; 14: 7276-7284Crossref PubMed Scopus (92) Google Scholar), the Afp promoter (30Feuerman M. Godbout R. Ingram R. Tilghman S. Mol. Cell Biol. 1989; 9: 4204-4212Crossref PubMed Scopus (90) Google Scholar), and the albumin promoter (32Cereghini S. Blumenfeld M. Yaniv M. Genes Dev. 1988; 2: 957-974Crossref PubMed Scopus (226) Google Scholar), each of which contains either one or two functional HNF1 binding sites embryonic kidney cells, which do not HNF1 or vHNF1 were transfected with increasing amounts of the pCB6 plasmid (25Brewer C.B. Methods Cell Biol. 1994; 43: 233-244Crossref PubMed Scopus (84) Google Scholar) containing vHNF1-A, vHNF1-B, and HNF1 In with (4Rey-Campos J. Chouard T. Yaniv M. Cereghini S. EMBO J. 1991; 10: 1445-1457Crossref PubMed Scopus (239) Google Scholar), HNF1