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• W2068001725 abstract "The Nkx2–5 homeodomain protein plays a key role in cardiomyogenesis. Ectopic expression in frog and zebrafish embryos results in an enlarged myocardium; however, expression of Nkx2–5 in fibroblasts was not able to trigger the development of beating cardiac muscle. In order to examine the ability of Nkx2–5 to modulate endogenous cardiac specific gene expression in cells undergoing early stages of differentiation, P19 cell lines overexpressing Nkx2–5 were differentiated in the absence of Me2SO. Nkx2–5 expression induced cardiomyogenesis in these cultures aggregated without Me2SO. During differentiation into cardiac muscle, Nkx2–5 expression resulted in the activation of myocyte enhancer factor 2C (MEF2C), but not MEF2A, -B, or -D. In order to compare the abilities of Nkx2–5 and MEF2C to induce cellular differentiation, P19 cells overexpressing MEF2C were aggregated in the absence of Me2SO. Similar to Nkx2–5, MEF2C expression initiated cardiomyogenesis, resulting in the up-regulation of Brachyury T, bone morphogenetic protein-4, Nkx2–5, GATA-4, cardiac α-actin, and myosin heavy chain expression. These findings indicate the presence of a positive regulatory network between Nkx2–5 and MEF2C and show that both factors can direct early stages of cell differentiation into a cardiomyogenic pathway. The Nkx2–5 homeodomain protein plays a key role in cardiomyogenesis. Ectopic expression in frog and zebrafish embryos results in an enlarged myocardium; however, expression of Nkx2–5 in fibroblasts was not able to trigger the development of beating cardiac muscle. In order to examine the ability of Nkx2–5 to modulate endogenous cardiac specific gene expression in cells undergoing early stages of differentiation, P19 cell lines overexpressing Nkx2–5 were differentiated in the absence of Me2SO. Nkx2–5 expression induced cardiomyogenesis in these cultures aggregated without Me2SO. During differentiation into cardiac muscle, Nkx2–5 expression resulted in the activation of myocyte enhancer factor 2C (MEF2C), but not MEF2A, -B, or -D. In order to compare the abilities of Nkx2–5 and MEF2C to induce cellular differentiation, P19 cells overexpressing MEF2C were aggregated in the absence of Me2SO. Similar to Nkx2–5, MEF2C expression initiated cardiomyogenesis, resulting in the up-regulation of Brachyury T, bone morphogenetic protein-4, Nkx2–5, GATA-4, cardiac α-actin, and myosin heavy chain expression. These findings indicate the presence of a positive regulatory network between Nkx2–5 and MEF2C and show that both factors can direct early stages of cell differentiation into a cardiomyogenic pathway. The NK-2 class homeobox gene product Nkx2–5/Csx plays a key role in cardiac muscle development (1Fishman M.C. Olson E.N. Cell. 1997; 91: 153-156Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 2Harvey R.P. Dev. Biol. 1996; 178: 203-216Crossref PubMed Scopus (496) Google Scholar, 3Mohun T. Sparrow D. Curr. Opin. Genet. Dev. 1997; 7: 628-633Crossref PubMed Scopus (38) Google Scholar). Nkx2–5 is the mouse homologue of the Drosophila gene tinman (4Komuro I. Izumo S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8145-8149Crossref PubMed Scopus (464) Google Scholar, 5Lints T.J. Parsons L.M. Hartley L. Lyons I. Harvey R.P. Development. 1993; 119: 419-431Crossref PubMed Google Scholar), which is essential for specification of heart muscle progenitors in the fly (6Bodmer R. Development. 1993; 118: 719-729Crossref PubMed Google Scholar). Both tinman and Nkx2–5 are expressed in the heart lineage as a result of signals from decapentaplegic and bone morphogenetic protein (BMP) 1The abbreviations used are: BMP, bone morphogenetic protein; MEF2, myocyte enhancer factor 2; PCR, polymerase chain reaction; RT-PCR, reverse transcription PCR; kb, kilobase pair(s). signaling, which are members of the TGFβ superfamily (2Harvey R.P. Dev. Biol. 1996; 178: 203-216Crossref PubMed Scopus (496) Google Scholar, 7Frasch M. Nature. 1995; 374: 464-467Crossref PubMed Scopus (374) Google Scholar, 8Schultheiss T.M. Burch J.B. Lassar A.B. Genes Dev. 1997; 11: 451-462Crossref PubMed Scopus (587) Google Scholar). Mice lacking Nkx2–5 still form a beating linear heart tube, in which most myogenic genes are expressed, but the mice die due to defective heart looping (9Lyons I. Parsons L.M. Hartley L. Li R. Andrews J.E. Robb L. Harvey R.P. Genes Dev. 1995; 9: 1654-1666Crossref PubMed Scopus (962) Google Scholar, 10Zou Y.M. Evans S. Chen J. Kuo H.C. Harvey R.P. Chien K.R. Development. 1997; 124: 793-804Crossref PubMed Google Scholar, 11Biben C. Harvey R.P. Genes Dev. 1997; 11: 1357-1369Crossref PubMed Scopus (271) Google Scholar). Ectopic expression of Nkx2–5 in frog and zebrafish embryos results in an enlarged myocardium, suggesting that Nkx2–5 recruits additional cells into the heart from the heart morphogenetic field (12Chen J.N. Fishman M.C. Development. 1996; 122: 3809-3816Crossref PubMed Google Scholar,13Cleaver O.B. Patterson K.D. Krieg P.A. Development. 1996; 122: 3549-3556Crossref PubMed Google Scholar). However, expression of Nkx2–5 in fibroblasts was not able to trigger the development of beating cardiac muscle (12Chen J.N. Fishman M.C. Development. 1996; 122: 3809-3816Crossref PubMed Google Scholar, 13Cleaver O.B. Patterson K.D. Krieg P.A. Development. 1996; 122: 3549-3556Crossref PubMed Google Scholar). The MEF2 family of transcription factors has been shown to play a critical role in the cell type-specific transcription of genes in cardiac, skeletal, and smooth muscle cells (14Molkentin J.D. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9366-9373Crossref PubMed Scopus (375) Google Scholar, 15Olson E.N. Perry M. Schulz R.A. Dev. Biol. 1995; 172: 2-14Crossref PubMed Scopus (317) Google Scholar) as well as in brain and neuronal cells (16Black B.L. Ligon K.L. Zhang Y. Olson E.N. J. Biol. Chem. 1996; 271: 26659-26663Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 17Leifer D. Golden J. Kowall N.W. Neuroscience. 1994; 63: 1067-1079Crossref PubMed Scopus (80) Google Scholar, 18Lyons G.E. Micales B.K. Schwarz J. Martin J.F. Olson E.N. J. Neurosci. 1995; 15: 5727-5738Crossref PubMed Google Scholar, 19Mao Z. Nadal-Ginard B. J. Biol. Chem. 1996; 271: 14371-14375Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 20Leifer D. Krainc D. Yu Y.T. McDermott J. Breitbart R.E. Heng J. Neve R.L. Kosofsky B. Nadal-Ginard B. Lipton S.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1546-1550Crossref PubMed Scopus (187) Google Scholar, 21Schulz R.A. Chromey C. Lu M.F. Zhao B. Olson E.N. Oncogene. 1996; 12: 1827-1831PubMed Google Scholar). There are four vertebrate MEF2 family members, MEF2A, -B, -C, and -D (14Molkentin J.D. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9366-9373Crossref PubMed Scopus (375) Google Scholar). They contain a conserved MADS box/MEF2 domain at their N termini, which is both necessary and sufficient for dimerization and DNA binding to an AT-rich MEF2 binding site. Drosophila lacking the single D-mef2gene are deficient in cardiac, skeletal, and smooth muscle development, indicating an essential role for D-MEF2 in the development of these tissues (22Lilly B. Zhao B. Ranganayakulu G. Paterson B.M. Schulz R.A. Olson E.N. Science. 1995; 267: 688-693Crossref PubMed Scopus (426) Google Scholar, 23Bour B.A. O'Brien M.A. Lockwood W.L. Goldstein E.S. Bodmer R. Taghert P.H. Abmayr S.M. Nguyen H.T. Genes Dev. 1995; 9: 730-741Crossref PubMed Scopus (363) Google Scholar, 24Ranganayakulu G. Zhao B. Dokidis A. Molkentin J.D. Olson E.N. Schulz R.A. Dev. Biol. 1995; 171: 169-181Crossref PubMed Scopus (178) Google Scholar). However, mice lacking MEF2B showed no phenotype, whereas mice lacking MEF2C were deficient in cardiac looping, dying around embryonic day 10 (25Lin Q. Schwarz J. Bucana C. Olson E.N. Science. 1997; 276: 1404-1407Crossref PubMed Scopus (786) Google Scholar). Consequently, the role of MEF2 family members in murine muscle development is more difficult to assess due to possible functional redundancy. Promoter analysis has shown that MEF2 sites mediate the expression of several muscle-specific genes in cardiac muscle, including cardiac myosin light chain 2, cardiac troponin T, muscle creatine kinase, and α-myosin heavy chain (26Iannello R.C. Mar J.H. Ordahl C.P. J. Biol. Chem. 1991; 266: 3309-3316Abstract Full Text PDF PubMed Google Scholar, 27Molkentin J.D. Markham B.E. J. Biol. Chem. 1993; 268: 19512-19520Abstract Full Text PDF PubMed Google Scholar, 28Navankasattusas S. Zhu H. Garcia A.V. Evans S.M. Chien K.R. Mol. Cell. Biol. 1992; 12: 1469-1479Crossref PubMed Scopus (86) Google Scholar, 29Amacher S.L. Buskin J.N. Hauschka S.D. Mol. Cell. Biol. 1993; 13: 2753-2764Crossref PubMed Scopus (135) Google Scholar). The first MEF2 family member to be expressed during mouse development is MEF2C, which is found on embryonic day 7.5 in cells of the cardiac mesoderm (30Edmondson D.G. Lyons G.E. Martin J.F. Olson E.N. Development. 1994; 120: 1251-1263Crossref PubMed Google Scholar). Mice lacking MEF2C were deficient in the expression of a subset of cardiac specific genes, including atrial natriuretic factor, cardiac α-actin, α-myosin heavy chain, and the basic helix-loop-helix factor dHAND. However, other cardiac muscle genes such as MLC2v andMLC2a were expressed normally (25Lin Q. Schwarz J. Bucana C. Olson E.N. Science. 1997; 276: 1404-1407Crossref PubMed Scopus (786) Google Scholar). Since MEF2B was up-regulated in MEF2C mutant mice, it is likely that MEF2B may partially substitute for MEF2C activity. The ability of MEF2 family members to induce muscle development in tissue culture is controversial. One study documented that MEF2A initiates skeletal myogenesis in fibroblasts (31Kaushal S. Schneider J.W. Nadal-Ginard B. Mahdavi V. Science. 1994; 266: 1236-1240Crossref PubMed Scopus (197) Google Scholar), but these results were not confirmed by others (32Molkentin J.D. Black B.L. Martin J.F. Olson E.N. Cell. 1995; 83: 1125-1136Abstract Full Text PDF PubMed Scopus (708) Google Scholar). The zinc finger transcription factor GATA-4 also plays a key role in cardiac muscle development (3Mohun T. Sparrow D. Curr. Opin. Genet. Dev. 1997; 7: 628-633Crossref PubMed Scopus (38) Google Scholar, 33Fishman M.C. Chien K.R. Development. 1997; 124: 2099-2117Crossref PubMed Google Scholar, 34Evans T. Trends Cardiovasc. Med. 1997; 7: 75-83Crossref PubMed Scopus (62) Google Scholar). GATA-4 is expressed in the precardiac mesoderm at 7.5 days postcoitum and in the endocardial and myocardial layers of the heart tube (35Heikinheimo M. Scandrett J.M. Wilson D.B. Dev. Biol. 1994; 164: 361-373Crossref PubMed Scopus (250) Google Scholar). GATA-4 can regulate a number of cardiac structural genes, such as α-myosin heavy chain, cardiac troponin-C, atrial natriuretic factor, and brain natriuretic peptide (36Grepin C. Dagnino L. Robitaille L. Haberstroh L. Antakly T. Nemer M. Mol. Cell. Biol. 1994; 14: 3115-3129Crossref PubMed Scopus (249) Google Scholar, 37Molkentin J.D. Kalvakolanu D.V. Markham B.E. Mol. Cell. Biol. 1994; 14: 4947-4957Crossref PubMed Google Scholar, 38Ip H.S. Wilson D.B. Heikinheimo M. Tang Z. Ting C.N. Simon M.C. Leiden J.M. Parmacek M.S. Mol. Cell. Biol. 1994; 14: 7517-7526Crossref PubMed Scopus (165) Google Scholar, 39Thuerauf D.J. Hanford D.S. Glembotski C.C. J. Biol. Chem. 1994; 269: 17772-17775Abstract Full Text PDF PubMed Google Scholar, 40Durocher D. Charron F. Warren R. Schwartz R.J. Nemer M. EMBO J. 1997; 16: 5687-5696Crossref PubMed Scopus (551) Google Scholar). Mice lacking GATA-4 develop cardiomyocytes, which express cardiac muscle-specific genes. However, these mice die early due to defective morphogenetic movements required for the formation of the linear cardiac tube (41Molkentin J.D. Lin Q. Duncan S.A. Olson E.N. Genes Dev. 1997; 11: 1061-1072Crossref PubMed Scopus (961) Google Scholar, 42Kuo C.T. Morrisey E.E. Anandappa R. Sigrist K. Lu M.M. Parmacek M.S. Soudais C. Leiden J.M. Genes Dev. 1997; 11: 1048-1060Crossref PubMed Scopus (872) Google Scholar). We have analyzed the ability of both Nkx2–5 and MEF2C to activate endogenous cardiac muscle-specific gene expression in murine P19 embryonal carcinoma cells. The differentiation of these pluripotent stem cells is initiated by cellular aggregation in the presence of differentiating agents and emulates the biochemical and morphological processes that occur during early embryonic development (43McBurney M.W. Int. J. Dev. Biol. 1993; 37: 135-140PubMed Google Scholar, 44Rudnicki M.A. McBurney M.W. Robertson E.J. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. IRL Press, Oxford1987: 19-49Google Scholar). Aggregation of P19 cells in the absence of differentiating agents activates the expression of the mesoderm marker, Brachyury T (45Vidricaire G. Jardine K. McBurney M.W. Development. 1994; 120: 115-122PubMed Google Scholar), but few of the cells continue to differentiate. P19 cells treated with retinoic acid differentiate into various neuroectodermal derivatives, including neurons, astrocytes, and glia (44Rudnicki M.A. McBurney M.W. Robertson E.J. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. IRL Press, Oxford1987: 19-49Google Scholar, 46Jones-Villeneuve E.M. McBurney M.W. Rogers K.A. Kalnins V.I. J. Cell Biol. 1982; 94: 253-262Crossref PubMed Scopus (680) Google Scholar). P19-derived neurons express the neurogenic basic helix-loop-helix transcription factor MASH1 (47Johnson J.E. Zimmerman K. Saito T. Anderson D.J. Development. 1992; 114: 75-87PubMed Google Scholar) and MEF2C (16Black B.L. Ligon K.L. Zhang Y. Olson E.N. J. Biol. Chem. 1996; 271: 26659-26663Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), which can physically interact to synergistically activate transcription. P19 cells aggregated in the presence of Me2SO may differentiate into cardiac and skeletal muscle along with other mesodermal and endodermal cell types (48Edwards M.K. Harris J.F. McBurney M.W. Mol. Cell. Biol. 1983; 3: 2280-2286Crossref PubMed Scopus (141) Google Scholar). The resulting cardiomyocytes are embryonic in nature and first appear at day 6 following Me2SO treatment. Semiquantitative RT-PCR analysis has shown that GATA-4 is first expressed on day 3 of differentiation and is followed by Nkx2–5 on day 4 of differentiation (49Grepin C. Nemer G. Nemer M. Development. 1997; 124: 2387-2395Crossref PubMed Google Scholar). MEF2C appears to be expressed constitutively but is up-regulated at day 6. The rationale for examining the ability of stem cells to differentiate in the presence of exogenous transcription factors is to provide the factor of interest with an environment similar to that of the developing embryo. Therefore, the presence or absence of tissue-restricted components within the host cells does not limit the function of the transcription factor. In addition, sufficient material can be obtained for subsequent analysis. For example, P19 cells overexpressing either GATA-4 (49Grepin C. Nemer G. Nemer M. Development. 1997; 124: 2387-2395Crossref PubMed Google Scholar) or MyoD (50Skerjanc I.S. Slack R.S. McBurney M.W. Mol. Cell. Biol. 1994; 1464: 8451-8459Crossref Scopus (70) Google Scholar) differentiate into cardiac muscle or skeletal muscle, respectively, when aggregated in the absence of Me2SO. Using this model system, we report that both Nkx2–5 and MEF2C initiated the development of cardiac muscle when overexpressed in cells aggregated in the absence of Me2SO. The DNA construct PGK-MEF2C contains the phosphoglycerate kinase (pgk-1) promoter (51Adra C.N. Boer P.H. McBurney M.W. Gene (Amst.). 1987; 60: 65-74Crossref PubMed Scopus (308) Google Scholar) driving the coding region of human MEF2C (20Leifer D. Krainc D. Yu Y.T. McDermott J. Breitbart R.E. Heng J. Neve R.L. Kosofsky B. Nadal-Ginard B. Lipton S.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1546-1550Crossref PubMed Scopus (187) Google Scholar). This isoform of MEF2C binds DNA and activates transcription. The construct PGK-Nkx2–5 contains thepgk-1 promoter driving a 1.6-kb EcoRI fragment containing the complete open reading frame of mouse Nkx2–5 cDNA (5Lints T.J. Parsons L.M. Hartley L. Lyons I. Harvey R.P. Development. 1993; 119: 419-431Crossref PubMed Google Scholar). The construct PGK-Puro contains the pgk-1 promoter driving the gene encoding puromycin resistance, as described (50Skerjanc I.S. Slack R.S. McBurney M.W. Mol. Cell. Biol. 1994; 1464: 8451-8459Crossref Scopus (70) Google Scholar). The construct PGK-LacZ contains the pgk-1 promoter driving the gene encoding β-galactosidase. PGK-vector DNA is a plasmid containing the pgk-1 promoter alone. P19 embryonal carcinoma cells were cultured as described (44Rudnicki M.A. McBurney M.W. Robertson E.J. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. IRL Press, Oxford1987: 19-49Google Scholar) with the modification that 5% Cosmic calf serum (HyClone, Logan, Utah) and 5% fetal bovine serum (CanSera, Rexdale, Ontario, Canada) were used to supplement the α-minimal essential medium. Stable cell lines expressing Nkx2–5 (5Lints T.J. Parsons L.M. Hartley L. Lyons I. Harvey R.P. Development. 1993; 119: 419-431Crossref PubMed Google Scholar) were isolated following transfection of P19 cells with 8 μg of PGK-Nkx2–5, 2.5 μg of B17 (52McBurney M.W. Fournier S. Schmidt-Kastner P.K. Jardine K. Craig J. Somat. Cell Mol. Genet. 1994; 20: 529-540Crossref PubMed Scopus (13) Google Scholar), 1 μg of PGK-LacZ, and 1 μg of PGK-Puro, as described previously (50Skerjanc I.S. Slack R.S. McBurney M.W. Mol. Cell. Biol. 1994; 1464: 8451-8459Crossref Scopus (70) Google Scholar). Control P19 cells were isolated by transfection with the same complement of plasmids except that the 8 μg of PGK-Nkx2–5 plasmid was replaced by PGK-vector DNA. Cells were selected for 1 week in 2 μg/ml puromycin. Three cell lines that expressed high levels of Nkx2–5 were termed P19(Nkx2–5) cells. Transfected cells expressing low levels of Nkx2–5 behaved similarly to P19 cells and were not pursued further. Stable cell lines expressing MEF2C were isolated in a similar fashion to P19(Nkx2–5) cells. P19 cells were transfected with 6.5 μg of a plasmid containing PGK-MEF2C or 6.5 μg of PGK-vector alone, 2.5 μg of B17, 1 μg of PGK-LacZ, and 1 μg of PGK-Puro. All experiments reported were performed at least twice with at least two cell lines, with similar results. Fig. 1shows immunofluorescence from P19(Nkx2–5) clone 1, and Figs.3 B, 4, 5, and 6 show data from P19(MEF2C) clone 1.Figure 3MEF2C induces cardiac muscle development in cells aggregated without Me2SO. P19 (A andC) and P19(MEF2C) (B) cells were aggregated with (C) and without (A and B) Me2SO. Cells were fixed in methanol on day 9 and stained with MF20 (magnification, × 40). The total number of cardiomyocytes was counted from each condition, averaged, and depicted graphically (D). Error bars represent S.E. (n = 3–5).View Large Image Figure ViewerDownload (PPT)Figure 4MEF2C up-regulates the expression of cardiac α-actin and mesoderm-patterning factors. P19 and P19(MEF2C) cells were differentiated with and without Me2SO (DMSO), and total RNA was harvested on day 0, 6, and 9 of differentiation. Northern analysis was performed on 6 μg of RNA with probes for MEF2C (A), cardiac α-actin (B), Brachyury T (C), BMP-4 (D), and 18 S (E, loading standard). The approximate sizes of the bands are indicated relative to the position of 18 or 28 S rRNA, measured as 6.3 or 2.4 kb, respectively.View Large Image Figure ViewerDownload (PPT)Figure 5MEF2C up-regulates the expression of Nkx2–5 and GATA-4. Semiquantitative RT-PCR was used to examine the level of expression of GATA-4 (A), Nkx2–5 (B), and tubulin (C), in P19 and P19(MEF2C) cultures aggregated without Me2SO on days 0, 6, and 9 of differentiation.View Large Image Figure ViewerDownload (PPT)Figure 6MEF2C directs early stages of P19 cell differentiation into a cardiomyogenic pathway. P19 and P19(MEF2C) cells were differentiated without Me2SO and total RNA was harvested during a time course of differentiation. Northern analysis was performed on 6 μg of RNA with probes from MEF2C (A), Brachyury T (B), BMP-4 (C), and cardiac α-actin (D, loading standard).View Large Image Figure ViewerDownload (PPT) Differentiation was initiated by plating 5 × 105cells into 60-mm bacterial dishes in the presence or absence of 0.8% Me2SO. Cells were cultured as aggregates for 4 days and then plated in tissue culture dishes and harvested for RNA or fixed for immunofluorescence at the time indicated. P19, P19(Nkx2–5), and P19(MEF2C) cells were plated on day 4 of differentiation onto gelatin coated coverslips. For identifying myosin heavy chain (53Bader D. Masaki T. Fischman D.A. J. Cell Biol. 1982; 95: 763-770Crossref PubMed Scopus (796) Google Scholar), cells were fixed in methanol at −20 °C and reacted with antibody as described (50Skerjanc I.S. Slack R.S. McBurney M.W. Mol. Cell. Biol. 1994; 1464: 8451-8459Crossref Scopus (70) Google Scholar). Immunofluorescence was visualized with a Zeiss Axioskop microscope. Images were captured with a Sony 3CCD color video camera; processed using Northern Exposure, Adobe photoshop, and Corel Draw software; and printed with a dye sublimation phaser 450 Tektronic printer. Total RNA was isolated by the lithium chloride/urea extraction method, and 6 μg were examined by Northern blot analysis as described previously (50Skerjanc I.S. Slack R.S. McBurney M.W. Mol. Cell. Biol. 1994; 1464: 8451-8459Crossref Scopus (70) Google Scholar). The probes used were a 600-base pair PstI fragment from the human cardiac α-actin last exon (54Rudnicki M.A. Jackowski G. Saggin L. McBurney M.W. Dev. Biol. 1990; 138: 348-358Crossref PubMed Scopus (89) Google Scholar), a 1.5-kb HindIII/XbaI fragment of MEF2C cDNA (55Martin J.F. Schwarz J.J. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 5282-5286Crossref PubMed Scopus (220) Google Scholar), a 1.55-kb XhoI/BamHI fragment of MEF2A cDNA (56Yu Y.T. Breitbart R.E. Smoot L.B. Lee Y. Mahdavi V. Nadal-Ginard B. Genes Dev. 1992; 6: 1783-1798Crossref PubMed Scopus (385) Google Scholar), a 1.55-kbXhoI/BamHI fragment of MEF2B cDNA (57Molkentin J.D. Firulli A.B. Black B.L. Martin J.F. Hustad C.M. Copeland N. Jenkins N. Lyons G. Olson E.N. Mol. Cell. Biol. 1996; 16: 3814-3824Crossref PubMed Scopus (105) Google Scholar), a 1.5-kb XhoI/BamHI fragment of MEF2D cDNA (58Martin J.F. Miano J.M. Hustad C.M. Copeland N.G. Jenkins N.A. Olson E.N. Mol. Cell. Biol. 1994; 14: 1647-1656Crossref PubMed Scopus (188) Google Scholar), a 1-kb HindIII/BamHI fragment of mouse BMP-4, a 2.4-kb XbaI fragment of GATA-4 (36Grepin C. Dagnino L. Robitaille L. Haberstroh L. Antakly T. Nemer M. Mol. Cell. Biol. 1994; 14: 3115-3129Crossref PubMed Scopus (249) Google Scholar), a 1.6-kbEcoRI fragment of Nkx2–5 cDNA (5Lints T.J. Parsons L.M. Hartley L. Lyons I. Harvey R.P. Development. 1993; 119: 419-431Crossref PubMed Google Scholar), and a 1.6-kbEcoRI/BamHI fragment of the mouse Brachyury T cDNA (59Herrmann B.G. Labeit S. Poustka A. King T.R. Lehrach H. Nature. 1990; 343: 617-622Crossref PubMed Scopus (681) Google Scholar). Northern blots were visualized with a Molecular Dynamics PhosphorImager SI and quantitated with ImageQuant software. Averages and S.E. values were calculated and reported. Total cellular RNA was extracted with TRIzol Reagent according to the manufacturer (Life Technologies, Burlington, Ontario, Canada) and treated with DNase I, amplification grade, at a concentration of 1 unit/μg RNA. The first strand cDNA synthesis was performed using Superscript II RNase H− reverse transcriptase according to the manufacturer (Life Technologies) with 1 μg of total RNA. PlatinumTaq DNA Polymerase (Life Technologies) was used to perform the PCR with 25 cycles: 94 °C for 1 min, 55–72 °C for 2 min, depending on the melting temperature of the primers, and 72 °C for 2 min. The amount of first strand reaction added to the PCR was titrated for each set of primers, and the quantity used was chosen in the middle of the linear range. Products were detected by Southern blot analysis with a probe from the corresponding cDNA. Negative controls performed with all RT-PCR experiments included a water control for the PCR and a water control for the complete RT-PCR. RNA was examined for genomic contamination after the DNase treatment by performing the RT-PCR in the absence of Superscript II RNase H− reverse transcriptase. The following pairs of primers were used: 5′-tccatccacgtcggccaggct-3′ and 5′-gtagggctcaaccacagcagt-3′ for tubulin, with an annealing temperature of 61 °C; 5′-actctggaggcgagatggg-3′ and 5′-ctcggcattacgacgccacag-3′ for GATA-4 (41Molkentin J.D. Lin Q. Duncan S.A. Olson E.N. Genes Dev. 1997; 11: 1061-1072Crossref PubMed Scopus (961) Google Scholar), with an annealing temperature of 72 °C; and 5′-cctctagagcagagctgcgcgcggagatg-3′ and 5′-ggtggcttccgtcgccgccgtgc-3′ for Nkx2–5 (5Lints T.J. Parsons L.M. Hartley L. Lyons I. Harvey R.P. Development. 1993; 119: 419-431Crossref PubMed Google Scholar), with an annealing temperature of 72 °C. In order to examine the activity of Nkx2–5 in the context of differentiating stem cells, three P19 cell lines that stably express high levels of Nkx2–5 were isolated and termed P19(Nkx2–5) cells. These cells continued to express the stem cell marker Oct-3 (60Okamoto K. Okazawa H. Okuda A. Sakai M. Muramatsu M. Hamada H. Cell. 1990; 60: 461-472Abstract Full Text PDF PubMed Scopus (620) Google Scholar) when grown as monolayer cultures, indicating that they retain a stem cell phenotype (data not shown) similar to that observed for P19(MyoD) cells (50Skerjanc I.S. Slack R.S. McBurney M.W. Mol. Cell. Biol. 1994; 1464: 8451-8459Crossref Scopus (70) Google Scholar). To examine whether expression of Nkx2–5 in P19 cells can induce differentiation into cardiac muscle, P19 and P19(Nkx2–5) cells were aggregated for 4 days without Me2SO and fixed on day 6. The amount of cardiac muscle was quantitated by counting cardiomyocytes in cultures that were stained by immunofluorescence with the anti-myosin heavy chain antibody, MF20 (53Bader D. Masaki T. Fischman D.A. J. Cell Biol. 1982; 95: 763-770Crossref PubMed Scopus (796) Google Scholar). Aggregation of P19(Nkx2–5) cells in the absence of Me2SO resulted in the differentiation of abundant cardiac muscle (30–60% of total cells, Table I). This is demonstrated by the presence of cardiomyocytes expressing myosin heavy chain in P19(Nkx2–5) cells (Fig. 1 D), compared with control cell cultures (Fig. 1 B).Table IComparison of the cell types produced after aggregation of various P19 cell linesCell line and sourceDifferentiated cell types producedDrug requirementExtent of differentiated cell typeTime required for differentiation%daysP19(MEF2C) (this report)CardiacNone1.5–3aThe percentage of differentiated cells was calculated by dividing the total number of cardiomyocytes counted in 15 representative fields by the total number of cells, estimated from counting Hoechst-stained nuclei, for at least two cell lines in at least two experiments.6P19(Nkx2–5) (this report)CardiacNone30–60aThe percentage of differentiated cells was calculated by dividing the total number of cardiomyocytes counted in 15 representative fields by the total number of cells, estimated from counting Hoechst-stained nuclei, for at least two cell lines in at least two experiments.6P19(MyoD) (50Skerjanc I.S. Slack R.S. McBurney M.W. Mol. Cell. Biol. 1994; 1464: 8451-8459Crossref Scopus (70) Google Scholar)SkeletalNone∼306P19(GATA-4) (49Grepin C. Nemer G. Nemer M. Development. 1997; 124: 2387-2395Crossref PubMed Google Scholar)CardiacNone10–256P19 (44Rudnicki M.A. McBurney M.W. Robertson E.J. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. IRL Press, Oxford1987: 19-49Google Scholar)CardiacMe2SO∼156SkeletalMe2SO<59a The percentage of differentiated cells was calculated by dividing the total number of cardiomyocytes counted in 15 representative fields by the total number of cells, estimated from counting Hoechst-stained nuclei, for at least two cell lines in at least two experiments. Open table in a new tab Total mRNA was isolated from P19 and P19(Nkx2–5) cultures on days 0 and 6 of differentiation in the absence of Me2SO and subjected to Northern blot analysis. High levels of Nkx2–5 were expressed on both day 0 and day 6 in P19(Nkx2–5) cultures (Fig.2 A, lanes 3–8) but not in P19 control cultures (Fig. 2 A,lanes 1 and 2). The formation of abundant Nkx2–5-induced cardiac muscle is indicated by high levels of cardiac α-actin expression present on day 6 of differentiation in P19(Nkx2–5) cultures (Fig. 2 B, lanes 4, 6, 8) but not in P19 control cultures (Fig. 2 B, lane 2). The MEF2 family of transcription factors has been shown to bind MEF2 sites present in muscle-specific genes and activate their expression (14Molkentin J.D. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9366-9373Crossref PubMed Scopus (375) Google Scholar). The expression patterns of the four MEF2 family members were examined in P19 and P19(Nkx2–5) cultures aggregated in the absence of Me2SO. Of the four factors, only MEF2C demonstrated a 9 ± 3-fold increase in expression (n = 3) in P19(Nkx2–5) cultures on day 6 (Fig. 2 E, lanes 4, 6, and 8), compared with P19 control cultures (Fig. 2 E, lane 2). MEF2A showed a slight 0.32 ± 0.07-fold increase (n = 3) in the expression of the upper transcript on day 6 compared with day 0 in P19(Nkx2–5) cultures (Fig. 2 C,lanes 4," @default.
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• W2068001725 title "Myocyte Enhancer Factor 2C and Nkx2–5 Up-regulate Each Other's Expression and Initiate Cardiomyogenesis in P19 Cells" @default.
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