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• W2026946881 abstract "The extraembryonic endoderm is derived from inner cell mass cells of the blastocyst during early mouse embryogenesis. Formation of the extraembryonic endoderm, which later contributes to the yolk sac, appears to be a prerequisite for subsequent differentiation of the inner cell mass. While embryonic stem cells can be induced to differentiate into extraembryonic endoderm cells in vitro, the molecular mechanisms underlying this process are poorly understood. We used a promoter trap approach to search for genes that are expressed in embryonic stem cells and are highly up-regulated during differentiation to the extraembryonic endoderm fate. We showed that fibronectin fits this expression profile. Moreover we identified an enhancer in the 12th intron of the fibronectin locus that recapitulated the endogenous pattern of fibronectin expression. This enhancer carries Sox protein-binding sequences, and our analysis demonstrated that Sox7 and Sox17, which are highly expressed in the extraembryonic endoderm, were involved in enhancer activity. The extraembryonic endoderm is derived from inner cell mass cells of the blastocyst during early mouse embryogenesis. Formation of the extraembryonic endoderm, which later contributes to the yolk sac, appears to be a prerequisite for subsequent differentiation of the inner cell mass. While embryonic stem cells can be induced to differentiate into extraembryonic endoderm cells in vitro, the molecular mechanisms underlying this process are poorly understood. We used a promoter trap approach to search for genes that are expressed in embryonic stem cells and are highly up-regulated during differentiation to the extraembryonic endoderm fate. We showed that fibronectin fits this expression profile. Moreover we identified an enhancer in the 12th intron of the fibronectin locus that recapitulated the endogenous pattern of fibronectin expression. This enhancer carries Sox protein-binding sequences, and our analysis demonstrated that Sox7 and Sox17, which are highly expressed in the extraembryonic endoderm, were involved in enhancer activity. Understanding cell fate commitment and differentiation at the molecular level is a major area of interest in the field of developmental biology. The first overt differentiation step in mouse development occurs at about 3.5 days postcoitum when the blastomeres segregate into two distinct cell lineages to form the blastocyst stage embryo (1Hogan B. Beddington R. Costantini F. Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar). The trophectoderm, which contributes exclusively to extraembryonic tissues, forms the outer population of cells, while the inner cell mass (ICM), 1The abbreviations used are: ICM, inner cell mass; ES, embryonic stem; BAC, bacterial artificial chromosome; tk, thymidine kinase; IRES, internal ribosomal entry site; EF-1, elongation factor 1; HMG, high mobility group; E14, embryonic day 14; EC, embryonic carcinoma; Feec, fibronectin enhancer involved in extraembryonic endoderm cells; PE, parietal endoderm; VE, visceral endoderm; RA, retinoic acid. which is competent to differentiate into all embryonic tissue types, comprises the inner population of cells. Although extraembryonic tissues such as the placenta, amnion, and yolk sac are not part of the fetus proper, these tissues play pivotal roles in development, including nourishment and protection of the fetus within the uterus as well as embryonic axis formation by extraembryonic endoderm cells at early stages. While substantial portions of extraembryonic tissues are derived from the trophectoderm, the ICM also contributes significantly to extraembryonic tissues. For example, the extraembryonic or primitive endoderm, which gives rise to portions of the yolk sac, is derived from the ICM. This differentiation step can be mimicked in vitro by culturing ICM-derived embryonic stem (ES) cells in the absence of a cytokine leukemia inhibitory factor (1Hogan B. Beddington R. Costantini F. Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar, 2Robertson E.J. Robertson E.J. Teratocarcinomas and Embryonic Stem Cells. IRL Press, Oxford, UK1987: 71-122Google Scholar), allowing systematic analysis of the gene regulation involved in this process. Recently Niwa et al. (3Niwa H. Miyazaki J. Smith A. Nat. Genet. 2000; 24: 372-376Crossref PubMed Scopus (2926) Google Scholar) demonstrated that although the major role of Oct-3/4 in ES cells is to maintain pluripotency, elevated Oct-3/4 levels provide a key signal for differentiation of ES cells into extraembryonic endoderm. However, the molecular mechanisms underlying this differentiation remain unknown at present. To investigate the molecular basis of extraembryonic endoderm differentiation, we generated a promoter-trapped mouse ES clone in which expression of the pGTIRESβgeopA reporter gene (4Mountford P. Zevnik B. Duwel A. Nicholis J. Li M. Dani C. Robertson M. Chambers I. Smith A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4303-4307Crossref PubMed Scopus (284) Google Scholar) is highly up-regulated during differentiation of ES cells into extraembryonic endoderm. The reporter gene integrated into the fibronectin locus, which was previously reported to be expressed in the extraembryonic endoderm (5Zetter B.R. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 2324-2328Crossref PubMed Scopus (67) Google Scholar, 6Wartiovaara J. Leivo I. Vaheri A. Dev. Biol. 1979; 69: 247-257Crossref PubMed Scopus (167) Google Scholar, 7Thorsteinsdottir S. Anat. Rec. 1992; 232: 141-149Crossref PubMed Scopus (52) Google Scholar, 8George E.L. Georges-Labouesse E.N. Patel-King R.S. Rayburn H. Hynes R.O. Development. 1993; 119: 1079-1091Crossref PubMed Google Scholar). Wartiovaara et al. (6Wartiovaara J. Leivo I. Vaheri A. Dev. Biol. 1979; 69: 247-257Crossref PubMed Scopus (167) Google Scholar) have shown that fibronectin is first detected between the cells of the inner cell mass in late blastocyst stage embryos with the onset of expression coinciding with the appearance of extraembryonic endoderm cells. However, since the trophectoderm also produces fibronectin, it is unclear which cell type is the major source of fibronectin in Reichert's membrane. Our data clearly demonstrated that it is the extraembryonic endoderm, and not the trophectoderm, that represents the major source of fibronectin production. Moreover we identified a regulatory enhancer in intron 12 of the fibronectin gene that, when linked to a reporter gene, recapitulated endogenous expression during differentiation of ES cells to extraembryonic endoderm. We also showed that Sox7 and Sox17, whose expression is restricted to the extraembryonic endoderm in mouse early embryos (9Kanai-Azuma M. Kanai Y. Gad J.M. Tajima Y. Taya C. Kurohmatsu M. Sanai Y. Yonekawa H. Yazaki K. Tam P.P. Hayashi Y. Development. 2002; 129: 2367-2379Crossref PubMed Google Scholar, 10Murakami A. Shen H. Ishida S. Dickson C. J. Biol. Chem. 2004; 279: 28564-28573Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 11Niimi T. Hayashi Y. Futaki S. Sekiguchi K. J. Biol. Chem. 2004; 279: 38055-38061Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), were able to transcriptionally activate this enhancer. Plasmid Construction—The pGTIRESβgeopA reporter gene used for the promoter trap has been described by Mountford et al. (4Mountford P. Zevnik B. Duwel A. Nicholis J. Li M. Dani C. Robertson M. Chambers I. Smith A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4303-4307Crossref PubMed Scopus (284) Google Scholar). To construct genomic libraries carrying the fibronectin gene, a bacterial artificial chromosome (BAC) (GenBank™ accession number AC091456) carrying the entire fibronectin gene (67 kb) as well as 5′ (86 kb) and 3′ (51 kb) flanking sequences was prepared and digested with Sau3AI or HpaII. Subsequently Sau3AI-digested DNA was subcloned into the BamHI site of the tk-puro-Venus reporter plasmid in which the tk promoter (12Luckow B. Schutz G. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar) and cDNA encoding for a fusion protein (13Miyagi S. Saito T. Mizutani K. Masuyama N. Gotoh Y. Iwama A. Nakauchi H. Masui S. Niwa H. Nishimoto M. Muramatsu M. Okuda A. Mol. Cell. Biol. 2004; 24: 4207-4220Crossref PubMed Scopus (68) Google Scholar) of puromycin-detoxifying enzyme and Venus fluorescent protein (14Nagai T. Ibata K. Park E.S. Kubota M. Mikoshiba K. Miyawaki A. Nat. Biotech. 2002; 20: 87-90Crossref PubMed Scopus (2197) Google Scholar) were subcloned into the BamHI/SpeI site and SpeI/NotI sites of Bluescript II KS+, respectively. HpaII-digested DNA was inserted into the ClaI site of the vector. To construct the Feec1-tk-β-geo reporter gene, the splice acceptor domain of the Engrailed gene and the internal ribosomal entry site (IRES) sequence were removed from pGTIRESβgeopA reporter plasmid (4Mountford P. Zevnik B. Duwel A. Nicholis J. Li M. Dani C. Robertson M. Chambers I. Smith A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4303-4307Crossref PubMed Scopus (284) Google Scholar), and a fibronectin enhancer involved in extraembryonic endoderm cells (Feec) 1 DNA fragment obtained by PCR was subcloned as a SalI/BamHI fragment 5′ of the tk promoter. To generate tk-puro-Venus reporter constructs bearing wild type and deletion mutants of Feec1, the SalI/BamHI PCR products shown in Fig. 5B were individually subcloned into the puro-Venus reporter plasmid together with the tk promoter. For construction of puro-Venus reporter genes bearing wild type, nucleotide substitution mutants of Feec1 shown in Fig. 7A,or canonical B1 repetitive sequence, we used our previously established method (15Okuda A. Imagawa M. Maeda Y. Sakai M. Muramatsu M. J. Biol. Chem. 1989; 264: 16919-16926Abstract Full Text PDF PubMed Google Scholar) using overlapping oligonucleotides that carry a portion (from 20 to 30 nucleotides) of the target DNA regions. For nucleotide substitution mutants, underlined nucleotides in Fig. 7A were converted to non-complementary ones, i.e. G, A, T, and C were changed to T, C, G, and A, respectively. Construction of the EF-1-puro-Venus reporter plasmid was described previously (13Miyagi S. Saito T. Mizutani K. Masuyama N. Gotoh Y. Iwama A. Nakauchi H. Masui S. Niwa H. Nishimoto M. Muramatsu M. Okuda A. Mol. Cell. Biol. 2004; 24: 4207-4220Crossref PubMed Scopus (68) Google Scholar). For constructing Sox expression vectors, the entire coding regions of Sox2, Sox7, and Sox17 were recovered as Asp718/BamHI fragments by PCR in which Asp718 sites were at the initiating ATG codons. These were subcloned into an expression vector bearing a FLAG tag sequence, which was generated by introducing specific oligonucleotides into the pHβApr-1 expression vector (16Gunning P. Leavitt J. Muscat G. Ng S.-Y. Kedes L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4831-4835Crossref PubMed Scopus (667) Google Scholar). To construct the Sox17 HMG-Engrailed expression vector, the aminoterminal portion of Sox17 (amino acids 1–164) bearing the HMG domain (17Kanai Y. Kanai-Azuma M. Noce T. Saido T.C. Shiroishi T. Hayashi Y. Yazaki K. J. Cell Biol. 1996; 133: 667-681Crossref PubMed Scopus (189) Google Scholar) and the Drosophila Engrailed repressor domain (amino acids 2–298) (18Jaynes J.B. O'Farrell P.H. EMBO J. 1991; 10: 1427-1433Crossref PubMed Scopus (182) Google Scholar) were amplified by PCR and subcloned together into the pHβApr-1 expression vector. To construct the Feec1-tk-Luc reporter plasmid, the Feec1 regulatory region was amplified by PCR as an Asp718/XhoI fragment and was subcloned into the tk-Luc reporter plasmid.Fig. 7Identification of the core regulatory sequence of the Feec1 enhancer.A, the SRY element, as delineated by the analysis shown in Fig. 5B, plays a crucial role in Feec1 activity. Putative transcription factor binding sites are underlined. Mutations were generated at these sites as described under “Experimental Procedures,” and enhancer activity indexes of the wild-type and Feec1 mutants were calculated as in Fig. 5. B, canonical B1 repetitive elements do not function as an enhancer element in the extraembryonic endoderm. The tk-puro-Venus reporter plasmid bearing the Feec1 or B1 repetitive element or no canonical regulatory region was individually introduced into differentiated ZHTc6 cells, and puromycin-resistant colonies were obtained. Enhancer activity indexes of the Feec1 and B1 element were calculated as in Fig. 5. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Culture and Transfection of ES Cells and Other Cells—E14 ES cells were cultured as described previously (19Nishimoto M. Fukushima A. Okuda A. Muramatsu M. Mol. Cell. Biol. 1999; 19: 5453-5465Crossref PubMed Scopus (311) Google Scholar). The pGTIRESβgeopA reporter plasmid was introduced into ES cells by electroporation according to the method described by Thomas and Capecchi (20Thomas K.R. Capecchi M.R. Cell. 1987; 51: 503-512Abstract Full Text PDF PubMed Scopus (1832) Google Scholar). After selection with G418, the drug-resistant clones were picked and expanded. These cells were stained for LacZ to examine the level and pattern of reporter gene expression. ZHBTc4 and ZHTc6 ES cells were cultured as described by Niwa et al. (3Niwa H. Miyazaki J. Smith A. Nat. Genet. 2000; 24: 372-376Crossref PubMed Scopus (2926) Google Scholar) and were differentiated into trophectoderm and extraembryonic endoderm, respectively. F9 embryonic carcinoma (EC) cells and COS cells were cultured as described previously (21Tomioka M. Nishimoto M. Miyagi S. Katayanagi T. Fukui N. Niwa H. Muramatsu M. Okuda A. Nucleic Acids Res. 2002; 30: 3202-3213Crossref PubMed Scopus (249) Google Scholar). Introduction of plasmid DNAs into ZHBTc4, ZHTc6, and F9 cells was done by lipofection using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen), while the conventional calcium phosphate method was used to introduce DNA into COS cells. In Vitro Differentiation of ES Cells and F9 EC Cells—E14 ES cells were induced to differentiate in vitro essentially as described by Robertson (2Robertson E.J. Robertson E.J. Teratocarcinomas and Embryonic Stem Cells. IRL Press, Oxford, UK1987: 71-122Google Scholar). In brief, embryoid bodies were formed on bacteria grade plates in the absence of leukemia inhibitory factor for 4 days. These cells were further cultured to form cystic embryoid bodies or replated on tissue culture grade plates. F9 EC cells were induced to differentiate with 0.5 μm all-trans-retinoic acid alone or together with 1 mm dibutyryl cAMP as described by Hogan et al. (1Hogan B. Beddington R. Costantini F. Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar). Determination of the Integration Site of pGTIRESβgeopA Reporter Gene—Genomic DNA was prepared from ES cell clone 183, which has extraembryonic endoderm-specific reporter gene expression (see text for details). The DNA was cut with NcoI, which is present in the reporter gene (4Mountford P. Zevnik B. Duwel A. Nicholis J. Li M. Dani C. Robertson M. Chambers I. Smith A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4303-4307Crossref PubMed Scopus (284) Google Scholar), and BspHI, which is present in the chromosomal integration site of the reporter gene by Southern blot analysis (data not shown, see Fig. 3A for details). The genomic DNA was then circularized with T4 DNA ligase and used as template for PCR to identify the junction between the reporter gene and chromosomal DNA. The primers used for PCR were as follows: 5′-GATGATAAGCTTGCCACAACCATG-3′ and 5′-ACCCGCACGCCATACAGTCCTCTT-3′. The PCR product was directly used for sequencing analysis. RNA Preparation and RNase Mapping Analysis—RNA was prepared from ZHBTc4 and ZHTc6 ES cells using the RNeasy midikit from Qiagen. RNA was also prepared from undifferentiated as well as differentiated F9 cells and was used to examine gene expression levels by RNase protection as described previously (19Nishimoto M. Fukushima A. Okuda A. Muramatsu M. Mol. Cell. Biol. 1999; 19: 5453-5465Crossref PubMed Scopus (311) Google Scholar). Probes to the following sequences were used: fibronectin, nucleotides 4081–4291; hepatocyte nuclear factor 3β, nucleotides 377–604; Rex-1, nucleotides 75–352; Mash2, nucleotides 190–408; and β-actin, nucleotides 903–1023. Reverse Transcription-PCR Analysis—RNA was prepared as above from wild-type E14 ES cells and ES cell clone 183 (see text for details) and used to produce first strand complementary DNA by Powerscript reverse transcriptase from BD Biosciences. The cDNA was used for PCR analysis to detect the presence of wild-type fibronectin RNA and fibronectin fused to IRES-β-geo RNA derived from pGTIRESβgeopA. The following primers were used: for wild-type fibronectin RNA: Primer A, 5′-CTCCAGGCGTGGAATACACTTACACCATCC-3′; and Primer B, 5′-GATCAGCATGGACCACTTCTTCCAGAGAGG-3′; for fibronectin RNA fused to IRES-β-geo RNA: Primer C, 5′-AACCCTGACACTGGAGTGCTTACTGTCTCC-3′; and Primer D, 5′-TTCTCTAGAGTCCAGATCTTCCGGGTACCG-3′. Reporter Assay with Puro-Venus Vector—The puro-Venus reporter gene encodes a fusion protein of puromycin-detoxifying enzyme and Venus fluorescent protein (for details see Ref. 13Miyagi S. Saito T. Mizutani K. Masuyama N. Gotoh Y. Iwama A. Nakauchi H. Masui S. Niwa H. Nishimoto M. Muramatsu M. Okuda A. Mol. Cell. Biol. 2004; 24: 4207-4220Crossref PubMed Scopus (68) Google Scholar). The reporter can be used for both transient and stable transfection assays with puromycin detoxifying activity used for the stable transformant analysis as shown in Figs. 5, 6B, and 7, while transient transfection analysis can be done by monitoring the level of Venus fluorescence protein as shown in Fig. 6D. Identification of a Fibronectin Regulatory Region That Functions in the Extraembryonic Endoderm—Puro-Venus reporter plasmid libraries were constructed by inserting a small piece of genomic DNA from the fibronectin locus. These libraries were then introduced by lipofection into ZHTc6 cells that had been differentiated into extraembryonic endoderm cells, and puromycin-resistant colonies were obtained. After expansion, genomic DNA was recovered from these cells, and PCR was performed with the reverse primer from the Bluescript II KS+ vector sequence and a primer recognizing a portion of the tk promoter sequence. PCR products derived from HpaII and Sau3AI libraries were digested with SalI/BamHI and SalI/Sau3AI, respectively, and subcloned into the tk-puro-Venus reporter gene. The constructed plasmids were again transfected into the differentiated ZHTc6 cells as above to examine the enhancer activities of the subcloned PCR fragments. Luciferase Reporter Assay—COS cells in 10-cm dishes were transfected by the calcium phosphate method as described previously (21Tomioka M. Nishimoto M. Miyagi S. Katayanagi T. Fukui N. Niwa H. Muramatsu M. Okuda A. Nucleic Acids Res. 2002; 30: 3202-3213Crossref PubMed Scopus (249) Google Scholar) with 2 μg of the Feec1-tk-Luc reporter plasmid and the indicated amounts of expression vectors. The total amount of DNA was adjusted to 12 μg with pUC18. At 48 h post-transfection, the transcription level was determined by the dual luciferase method according to the manufacturer's instructions (Promega). Gel Shift Assay—The Sox7 or Sox17 expression vector or empty vector was introduced into COS cells by the calcium phosphate method as described above. Preparation of whole cell extracts and subsequent gel shift analysis were done as described previously (21Tomioka M. Nishimoto M. Miyagi S. Katayanagi T. Fukui N. Niwa H. Muramatsu M. Okuda A. Nucleic Acids Res. 2002; 30: 3202-3213Crossref PubMed Scopus (249) Google Scholar). The sequences used for the analysis were as follows: wild type, 5′-TGTCGTTCCAGGACAGCCAGGGCTATAACAAAGGAACCCTGTCTCGAAACCACCCCCAAA-3′; mutant, 5′-TGTCGTTCCAGGACAGCCAGGGCTATCCACCCTGAACCCTGTCTCGAAACCACCCCCAAA-3′. The underlined portion and bold portion indicate the wild-type and mutagenized Sox-binding sequence, respectively. The probe sequences represent part of the Feec1 regulatory sequence and contain two potential transcription factor binding sites (γ and ϵ) in addition to the Sox binding site (δ). However, the γ and ϵ sites were mutagenized to simplify the analysis. Identification of a Gene Whose Expression Level Is Highly Up-regulated during Differentiation of ES Cells into Extraembryonic Endoderm Cells—To elucidate the molecular mechanism of extraembryonic endoderm differentiation from ES cells, we searched for genes whose expression is highly up-regulated during this process. To this end, we randomly integrated the pGTIRESβgeopA reporter gene encoding a fusion protein of β-galactosidase and neomycin-detoxifying enzyme (4Mountford P. Zevnik B. Duwel A. Nicholis J. Li M. Dani C. Robertson M. Chambers I. Smith A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4303-4307Crossref PubMed Scopus (284) Google Scholar) into the genome of ES cells by electroporation. Although cells were cultured in conditions to maintain stem cell state, a small subpopulation of ES cells adopted extraembryonic endodermal fate due to spontaneous differentiation, particularly at the periphery of colonies. Since much higher levels of gene expression are required to turn on LacZ+ than to confer G418 resistance, we picked ES cell clones that were mostly LacZ-negative but had expression at the periphery of colonies. Of 400 independent G418-resistant clones, one clone (clone 183) showed the expected expression profile (Fig. 1A) in marked contrast to the expression profile of a clone in which the reporter gene integrated into Rex-1 (Fig. 1B), which is expressed in pluripotent ES cells (22Ben-Shushan E. Thompson J.R. Gudas L.J. Bergmasn Y. Mol. Cell. Biol. 1998; 18: 1866-1878Crossref PubMed Scopus (219) Google Scholar). LacZ Reporter Gene Expression Profile in ES Cell Clone 183 during Differentiation into Extraembryonic Endoderm—Next cells from clone 183 were allowed to differentiate into embryoid bodies (2Robertson E.J. Robertson E.J. Teratocarcinomas and Embryonic Stem Cells. IRL Press, Oxford, UK1987: 71-122Google Scholar) and stained for β-galactosidase activity. As shown in Fig. 2, A and B, a simple embryoid body (day 4) shows LacZ expression in the outer extraembryonic endoderm layer but not in the inner, pluripotent ectodermal cells. In cystic embryoid bodies (day 8) LacZ staining was intensified (Fig. 2C), and when replated onto a tissue culture surface, strong LacZ staining pattern was observed in cells derived from the outer layer but not in undifferentiated, inner layer cells (Fig. 2D). The pGTIRESβgeopA Reporter Gene Integrated into the Fibronectin Locus in ES Cell Clone 183—To determine the chromosomal integration site of the pGTIRESβgeopA reporter gene in ES cell clone 183, we first isolated genomic DNA from these cells. Southern blot analysis using a mouse Engrailed probe to detect the pGTIRESβgeopA reporter gene (4Mountford P. Zevnik B. Duwel A. Nicholis J. Li M. Dani C. Robertson M. Chambers I. Smith A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4303-4307Crossref PubMed Scopus (284) Google Scholar) revealed that a BspHI site, which is at the initiating methionine codon of β-geo, was present at about 2.7 kb upstream of the NcoI site (see Fig. 3A for details). Therefore, we digested genomic ES cell DNA with BspHI/NcoI and circularized with T4 DNA ligase. Subsequently the integrated portion of genomic DNA was obtained by PCR (for details see “Experimental Procedures”). As depicted in Fig. 3A, sequencing analyses revealed that the reporter gene was integrated into the 23rd intron of the fibronectin gene. Moreover reverse transcription-PCR analyses confirmed that fibronectin mRNA and that from the reporter gene were fused in ES cell clone 183 but not in parental ES cells (Fig. 3B). Fibronectin Gene Expression Is Up-regulated during Differentiation of Pluripotent ES Cells to Extraembryonic Endodermal Cells—To determine whether the endogenous expression of fibronectin is extraembryonic endoderm-specific, we examined expression in two specialized ES cell lines (ZHTc6 and ZHBTc4) (3Niwa H. Miyazaki J. Smith A. Nat. Genet. 2000; 24: 372-376Crossref PubMed Scopus (2926) Google Scholar) and their differentiated derivatives. ZHTc6 ES cells can be maintained in a pluripotent state in the presence of tetracycline, and withdrawal of tetracycline from the culture medium leads to extraembryonic endoderm differentiation. In contrast, ZHBTc4 ES cells are maintained in a pluripotent state in the absence of tetracycline, and addition of tetracycline results in dedifferentiation to trophectoderm fate. We prepared RNA from these cells cultured in the presence or absence of tetracycline and performed RNase protection assays. As shown in Fig. 4 when cultured in the absence of tetracycline, fibronectin was indeed highly expressed in ZHTc6 cells, which adopt extraembryonic endoderm fate. We also examined the expression levels of Rex-1 (22Ben-Shushan E. Thompson J.R. Gudas L.J. Bergmasn Y. Mol. Cell. Biol. 1998; 18: 1866-1878Crossref PubMed Scopus (219) Google Scholar), hepatocyte nuclear factor 3β (23Farrington S.M. Belaoussoff M. Baron M.H. Mech. Dev. 1997; 62: 197-211Crossref PubMed Scopus (75) Google Scholar), and Mash2 (24Tanaka M. Gertsenstein M. Rossant J. Nagy A. Dev. Biol. 1997; 190: 55-65Crossref PubMed Scopus (185) Google Scholar) and confirmed that pluripotent/differentiated states of these ES cells are, as expected, regulated by tetracycline. Identification of an Enhancer Involved in Fibronectin Gene Expression in Extraembryonic Endodermal Cells—To identify the regulatory element responsible for extraembryonic endoderm expression of fibronectin, we first digested BAC DNA bearing the entire fibronectin gene as well as 5′ and 3′ flanking regions into small fragments using the 4-base cutter Sau3AI or HpaII. The digested DNA was inserted upstream of the tk promoter in the tk-puro-Venus reporter plasmid to generate plasmid libraries (for details see “Experimental Procedures”). These plasmid libraries were then introduced by lipofection into ZHTc6 cells, which had been cultured in the absence of tetracycline, and puromycin-resistant colonies were obtained. The BAC DNA was recovered by PCR amplification of genomic DNA from 84 independent puromycin-resistant colonies. These fragments were again individually inserted upstream of the tk promoter of the reporter plasmid and introduced into ZHTc6 cells, and puromycin-resistant colonies were obtained. In most cases, an equivalent number of puromycin-resistant colonies was obtained with BAC-transfected and control tk-puro-Venus reporter plasmid-transfected cells, indicating that the original puromycin resistance observed from the primary screen was not due to the inserted BAC DNA but was instead dependent on the site of reporter gene integration. However, in one case, significantly more resistant colonies were obtained (Fig. 5A), suggesting that the BAC DNA inserted in the reporter plasmid was involved in producing the resistant colonies. Sequencing analysis revealed that the BAC DNA carries a portion of intron 12 (1568 bp) of the fibronectin gene, and preliminary characterization demonstrated that this DNA fragment exerted its activity rather specifically in extraembryonic ectodermal cells (data not shown, but see below for details). We next delineated the regulatory region using a series of deletion mutants shown in Fig. 5B. We characterized their enhancer activities by counting the number of puromycin-resistant stable colonies, and these analyses allowed us to narrow down the regulatory region to 144 bp (the sequence is shown in Fig. 7A), and we termed this regulatory region Feec1. The Feec1 Enhancer Drives Expression Specifically in Extraembryonic Endoderm Cells—Next we examined whether the Feec1 enhancer can drive expression specifically in extraembryonic endoderm cells. We constructed a tk-β-geo reporter plasmid carrying the Feec1 enhancer and introduced it into ES cells by electroporation along with pSV2Neo since Feec1 alone might not be strong enough to produce G418-resistant colonies when the ES cells are in the pluripotent state. We obtained four independent ES cell clones in which both Feec1-tk-β-geo and the pSV2Neo reporter gene integrated into the genome. These clones were then allowed to differentiate, and reporter gene expression was examined at day 8 following replating on tissue culture dishes. We found that in all cases, LacZ reporter expression was detected in cells derived from outer layer cells but not in pluripotential inner cells (two representative examples are shown in Fig. 6A), although the signal was not as strong as that shown in Fig. 2D. Next we examined the activity of the Feec1 regulatory region in ZHTc6 and ZHBTc4 cells using the Feec1-puro-Venus reporter plasmid and found that, as expected, the Feec1 enhancer showed strong activity in ZHTc6 cells that had differentiated into endoderm-like cells (Fig. 6B). However, weaker activity was observed for Feec1 in ZHBTc4 ES cells that were maintained in pluripotent state. To obtain an independent line of evidence that the Feec1 functions as an extraembryonic endoderm-specific enhancer, we performed similar experiments using F9 EC cells. While extraembryonic endoderm cells are first comprised of primitive endoderm upon segregation from the embryonic ICM cells, the primitive endoderm differentiates further into parietal endoderm (PE) and" @default.
• W2026946881 created "2016-06-24" @default.
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• W2026946881 date "2005-02-01" @default.
• W2026946881 modified "2023-10-11" @default.
• W2026946881 title "Identification of an Enhancer That Controls Up-regulation of Fibronectin during Differentiation of Embryonic Stem Cells into Extraembryonic Endoderm" @default.
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