F9 Stem Cells Research Articles

The Tight-Junction Protein Claudin-6 Induces Epithelial Differentiation from Mouse F9 and Embryonic Stem Cells

During epithelialization, cell adhesions and polarity must be established to maintain tissue assemblies and separate the biological compartments in the body. However, the molecular basis of epithelial morphogenesis, in particular, a role of cell adhesion molecules in epithelial differentiation from stem cells, remains unclear. Here, we show that the stable and conditional expression of a tight-junction protein, claudin-6 (Cldn6), triggers epithelial morphogenesis in mouse F9 stem cells. We also demonstrate that Cldn6 induces the expression of other tight-junction and microvillus molecules including Cldn7, occludin, ZO-1α+, and ezrin/radixin/moesin-binding phosphoprotein50. These events were inhibited by attenuation of Cldn6 using RNA interference or the C-terminal half of Clostridium Perfringens enterotoxin. Furthermore, similar results were obtained in mouse embryonic stem cells. Thus, we have uncovered that the Cldn6 functions as a novel cue to induce epithelial differentiation.

Epigenetic Regulatory Mechanisms Distinguish Retinoic Acid-mediated Transcriptional Responses in Stem Cells and Fibroblasts

Retinoic acid (RA), a vitamin A metabolite, regulates transcription by binding to RA receptor (RAR) and retinoid X receptor (RXR) heterodimers. This transcriptional response is determined by receptor interactions with transcriptional regulators and chromatin modifying proteins. We compared transcriptional responses of three RA target genes (Hoxa1, Cyp26a1, RARbeta(2)) in primary embryo fibroblasts (mouse embryonic fibroblasts), immortalized fibroblasts (Balb/c3T3), and F9 teratocarcinoma stem cells. Hoxa1 and Cyp26a1 transcripts are not expressed, but RARbeta(2) transcripts are induced by RA in mouse embryonic fibroblasts and Balb/c3T3 cells. Retinoid receptors (RARgamma, RXRalpha), coactivators (pCIP (NCOA3, SRC3)), and p300 and RNA polymerase II are recruited only to the RARbeta(2) RA response element (RARE) in Balb/c3T3, whereas these proteins are recruited to RAREs of all three genes by RA in F9 cells. In F9, RA reduces polycomb (PcG) protein Suz12 and the associated H3K27me3 repressive epigenetic modification at the RAREs of all three genes. In contrast, in Balb/c3T3 cells cultured in the +/-RA, Suz12 is not associated with the Hoxa1, RARbeta(2), and Cyp26a1 RAREs, whereas slow levels of the H3K27me3 mark are seen at these RAREs. Thus, Suz12 is not required for gene repression in the absence of RA. Even though the Hoxa1 RARE and proximal promoter show high levels of H3K9,K14 acetylation in Balb/c3T3, the Hoxa1 gene is not transcriptionally activated by RA. In Balb/c3T3, CpG islands are methylated in the Cyp26a1 promoter region but not in the Hoxa1 promoter or in these promoters in F9 cells. We have delineated the complex mechanisms that control RA-mediated transcription in fibroblasts versus stem cells.

The truncated Hoxa1 protein interacts with Hoxa1 and Pbx1 in stem cells

Hox genes contain a homeobox encoding a 60-amino acid DNA binding sequence. The Hoxa1 gene (Hox1.6, ERA1) encodes two alternatively spliced mRNAs that encode distinct proteins, one with the homeodomain (Hoxa1-993), and another protein lacking this domain (Hoxa1-399). The functions of Hoxa1-399 are unknown. We detected Hoxa1-993 and Hoxa1-399 by immunoprecipitation using Hoxa1 antibodies. To assess whether Hoxa1-399 functions in cellular differentiation, we analyzed Hoxb1, a Hoxa1 target gene. Hoxa1-993 and its cofactor, Pbx1, bind to the Hoxb1 SOct-R3 promoter to transcriptionally activate a luciferase reporter. Results from F9 stem cells that stably express ectopic Hoxa1-399 (the F9-399 line) show that Hoxa1-399 reduces this transcriptional activation. Gel shift assays demonstrate that Hoxa1-399 reduces Hoxa1-993/Pbx1 binding to the Hoxb1 SOct-R3 region. GST pull-down experiments suggest that Hoxa1-399, Hoxa1-993, and Pbx1 form a trimer. However, the F9-399 line exhibits no differences in RA-induced proliferation arrest or endogenous Hoxb1, Pbx1, Hoxa5, Cyp26a1, GATA4, or Meis mRNA levels when compared to F9 wild-type.

Retinoic acid receptor γ activates receptor tyrosine kinase Tie1 gene transcription through transcription factor GATA4 in F9 stem cells

The retinoic acid receptors (RARs) alpha, beta2, and gamma regulate specific subsets of target genes during all-trans retinoic acid (RA) induced differentiation of F9 teratocarcinoma stem cells. The Tie1 gene exhibited reduced expression in RA-treated F9 RARgamma-/- cells as compared to wild-type (WT) by microarray analysis. Our goal was to analyze the Tie1 gene, which encodes a surface receptor tyrosine kinase expressed in the hematovascular system. We assessed Tie1, Tie2, Flk1, Runx1, Peg/Mest2, and angiopoietin-1 and 2 mRNA levels and Tie1 promoter activity. We showed that RARgamma, but not RARalpha or RARbeta2, is required for Tie1 promoter activation by RA. Treatment with a RARgamma selective agonist plus a retinoid X receptor agonist (LGD1069) increased Tie1 mRNA levels by 11- +/- 2.5-fold 48 hours after RA addition in F9 WT, but not in F9 RARgamma-/- cells, by quantitative reverse transcription polymerase chain reaction. Multiple putative GATA elements were identified in the Tie1 proximal promoter. RA increased GATA4 transcripts by 12- +/- 1-fold in F9 WT at 48 hours, but not in F9 RARgamma-/- cells. In addition, transfection of a GATA4 expression vector increased Tie1 promoter/luciferase activity in both RA-treated F9 WT and RARgamma-/- cells. Tie1 promoter deletion analyses indicated that a region of the promoter that possessed multiple GATA sites mediated the RA-associated Tie1 transcriptional increase. Our results indicate that GATA4 plays a role in the RA/RARgamma-associated transcriptional activation of the Tie1 promoter. An understanding of RAR specificity in RA signaling should result in insights into hematopoietic stem cell signaling and potentially in improved therapies for several human diseases.

Roles for Rho/ROCK and Vinculin in Parietal Endoderm Migration

The first cell migration event in the mouse embryo is the movement of parietal endoderm cells from the surface of the inner cell mass facing the blastocoel cavity to line the inner surface of the trophectoderm. F9 embryoid bodies provide an in vitro model for this event. They have an inner core of undifferentiated stem cells surrounded by an outer visceral endoderm layer. When plated on a laminin coated substrate, visceral endoderm transitions to parietal endoderm and migrates onto the dish, away from the attached embryoid body. We now show that this outgrowth contains abundant focal complexes and focal adhesions, as well as lamellipodia and filopodia. Treatment with the ROCK inhibitor Y-27632 promotes a 2-fold increase in outgrowth, and a transition from focal adhesions and associated stress fibers, to focal complexes and a decrease in stress fibers. ROCK inhibition also leads to an increase in lamellipodia. Inhibition of RhoA by transfection of a vector encoding C3 transferase, direct administration of the C3 enzyme, or transfection of a vector encoding p190 Rho GTPase Activating Protein also promotes outgrowth and an apparent transition from focal adhesions to focal complexes. Parietal endoderm outgrowth generated using vinculin-deficient F9 stem cells migrates 2-fold further than wild type cultures, but this outgrowth retains the morphology of wild type parietal endoderm, including focal adhesions and stress fibers. Addition of Y-27632 to vinculin-null outgrowth cultures further stimulates migration an additional 2-fold, supporting the conclusion that Rho/ROCK and vinculin regulate parietal endoderm outgrowth by distinct pathways.

Mouse Mix gene is activated early during differentiation of ES and F9 stem cells and induces endoderm in frog embryos.

In frog and zebrafish, the Mix/Bix family of paired type homeodomain proteins play key roles in specification and differentiation of mesendoderm. However, in mouse, only a single Mix gene (mMix) has been identified to date and its function is unknown. We have analyzed the expression of mouse Mix RNA and protein in embryos, embryoid bodies formed from embryonic stem cells and F9 teratocarcinoma cells, as well as several differentiated cell types. Expression in embryoid bodies in culture mirrors that in embryos, where Mix is transcribed transiently in primitive (visceral) endoderm (VE) and in nascent mesoderm. In F9 cells induced by retinoic acid to differentiate to VE, mMix is coordinately expressed with three other endodermal transcription factors, well before AFP, and its protein product is localized to the nucleus. In a subpopulation of nascent mesodermal cells from embryonic stem cell embryoid bodies, mMix is coexpressed with Brachyury. Intriguingly, mMix mRNA is detected in a population (T+Flk1+) of cells which may contain hemangioblasts, before the onset of hematopoiesis and activation of hematopoietic markers. In vitro and in vivo, mMix expression in nascent mesoderm is rapidly down-regulated and becomes undetectable in differentiated cell types. In the region of the developing gut, mMix expression is confined to the mesoderm of mid- and hindgut but is absent from definitive endoderm. Injection of mouse mMix RNA into early frog embryos results in axial truncation of developing tadpoles and, in animal cap assays, mMix alone is sufficient to activate expression of several endodermal (but not mesodermal) markers. Although these observations do not exclude a possible cell-autonomous function for mMix in mesendodermal progenitor cells, they do suggest an additional, non-cell autonomous role in nascent mesoderm in the formation and/or patterning of adjacent definitive endoderm.

Retinoic acid induces parietal endoderm but not primitive endoderm and visceral endoderm differentiation in F9 teratocarcinoma stem cells with a targeted deletion of the Rex-1 (Zfp-42) gene

Cultured murine F9 teratocarcinoma stem cells resemble pluripotent stem cells of the inner cell mass of the mouse blastocyst and, depending upon their treatment, can be induced to differentiate along the primitive endoderm, the parietal endoderm (PE), or the visceral endoderm (VE) pathway. The Rex-1 gene encodes a zinc finger family transcription factor which is expressed at high levels in undifferentiated F9 stem cells, embryonic stem cells, and in other types of stem cells. To examine whether the Rex-1 protein plays a role in F9 cell differentiation, homologous recombination was employed to generate F9 cell lines which lack both alleles of Rex-1. F9 wild type cells in monolayer culture require both retinoic acid and cyclic AMP analogs to differentiate into PE, whereas the F9 Rex-1 −/− cells differentiate into PE, as assessed by several molecular markers, including thrombomodulin and laminin B1, in the presence of RA alone. The F9 Rex-1 −/− cells do not completely differentiate into VE after RA treatment in aggregate culture; they do not express alpha-fetoprotein, a definitive marker of VE differentiation. These results indicate that the Rex-1 transcription factor regulates the differentiation of F9 stem cells along several distinct cell lineages found in the early embryo.

Phosphorylation of the β1 Integrin Cytoplasmic Domain: Toward an Understanding of Function and Mechanism

As F9 stem cells differentiate into parietal endoderm they form focal adhesion sites. There is a concomitant decrease in the level of phosphorylation of S785 in the cytoplasmic domain of the β1 integrin subunit. Previous transfection studies demonstrate that site-specific mutations at this residue, mimicking different phosphorylation states, can alter the subcellular localization of the subunit in differentiating F9 cells. We now extend these observations in an attempt to substantiate the function of β1 phosphorylation and determine how the phosphorylation levels are regulated. We show that treatment of parietal endoderm with okadaic acid induces an increase in β1 phosphorylation and selective loss of β1 from focal adhesion sites. Using a PCR approach, we identify two phosphatases expressed in parietal endoderm, including PP2A. Using a crosslinking approach, where antibodies are added to live cells, we show that the catalytic subunit of PP2A co-immunoprecipitates with β1. Immunocytochemistry shows PP2A colocalizing to focal adhesion sites with β1. In addition integrin-linked kinase (ILK) co-immunoprecipitates with β1 in parietal endoderm and localizes to focal adhesion sites. Okadaic acid treatment significantly decreases the level of ILK associated with β1. A possible role for regulated β1 phosphorylation in cell migration is discussed.

Activation of a frizzled-2/beta-adrenergic receptor chimera promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via Galphao and Galphat.

The frizzled gene family of putative Wnt receptors encodes proteins that have a seven-transmembrane-spanning motif characteristic of G protein-linked receptors, though no loss-of-function studies have demonstrated a requirement for G proteins for Frizzled signaling. We engineered a Frizzled-2 chimera responsive to beta-adrenergic agonist by using the ligand-binding domains of the beta(2)-adrenergic receptor. The expectation was that the chimera would be sensitive both to drug-mediated activation and blockade, thereby circumventing the problem of purifying soluble and active Wnt ligand to activate Frizzled. Expression of the chimera in zebrafish embryos demonstrated isoproterenol (ISO)-stimulated, propranolol-sensitive calcium transients, thereby confirming the beta-adrenergic nature of Wnt signaling by the chimeric receptor. Because F9 embryonic teratocarcinoma cells form primitive endoderm after stable transfection of Frizzled-2 chimera and stimulation with ISO, they were subject to depletion of G protein subunits. ISO stimulation of endoderm formation of F9 stem cells expressing the chimeric receptor was blocked by pertussis toxin and by oligodeoxynucleotide antisense to Galphao, Galphat2, and Gbeta2. Our results demonstrate the requirement of two pertussis toxin-sensitive G proteins, Galphao and Galphat, for signaling by the Frizzled-2 receptor.

Activation of Rat Frizzled-1 Promotes Wnt Signaling and Differentiation of Mouse F9 Teratocarcinoma Cells via Pathways That Require Gαq and Gαo Function

The frizzled gene family of putative Wnt receptors encodes proteins that have a seven transmembrane-spanning motif characteristic of G-protein-linked receptors, although no loss-of-function studies have demonstrated a requirement for G-proteins for Wnt signaling by the gene product of frizzled-1. Medium conditioned by mouse F9 teratocarcinoma stem cells stably transfected to express either Xenopus Wnt-5a or Wnt-8 was used to test primitive endoderm formation of F9 stem cells. F9 stem cells expressing the rat Frizzled-1 receptors demonstrated endoderm formation in response to conditioned medium containing Wnt-8 but not to medium containing Wnt-5a. Primitive endoderm formation stimulated by Wnt-8 acting on the rat Frizzled-1 receptor was blocked by treatment with pertussis toxin by depletion of either Galpha(o) or Galpha(q) via antisense oligodeoxynucleotides, as well as by inhibitors of protein kinase C (bisindoylmaleimide) and of mitogen-activated protein kinase kinase (PD98059). Our results demonstrate the requirement for G-protein subunits Galpha(o) (a pertussis toxin substrate) and Galpha(q) for signaling by Frizzled-1, and an obligate role for the protein kinase C (likely mediated through stimulation of Galpha(q)) and mitogen-activated protein kinase network at the level of mitogen-activated protein kinase kinase.

Modulation of α6/β1 Integrin Expression during Differentiation of F9 Murine Embryonal Carcinoma Cells to Parietal Endoderm

In vitrodifferentiation of the murine embryonal carcinoma (EC) cell line F9 parallels that of the early blastocyst, where visceral (VE) and parietal endoderm (PE) diverge from a common precursor, the primitive endoderm. This differentiation pathway is induced by retinoic acid (RA) and dibutyryl cyclicAMP (dcAMP) and is accompanied by progressive and dramatic changes in cell morphology and functions. Within 7 days of treatment the cells organize their cytoskeleton and synthesize large amounts of extracellular matrix proteins, becoming fully differentiated migratory cells; all these changes are likely to involve integrins expression and organization. We have investigated the changes in β1 integrin expression, its maturation, and organization on the cell surface in association with α6, during the transition from undifferentiated F9 stem cells to migrating PE cells. By Western blotting and immunoprecipitation we showed a gradual decrease in the amount of the β1 subunit on the cell surface and a parallel progressive accumulation of immature protein, indicating that the control of β1 expression during F9 cells differentiation occurs first at posttranslational level and then at the level of transcription. Moreover, the induction of differentiation produces a marked decrease of α6B and its association to a high molecular weight protein, while α6A level increases. By immunofluorescence we found that upon differentiation there is a relocation of the β1 and α6B integrin subunits from cell–cell contacts to focal contacts where they colocalize with vinculin. On the contrary α6A, weakly present in F9 stem cells, is present in the focal contacts of PE cells and along the stress fibers. We suggest different roles for the two α6 isoforms.

The Involvement of Tissue-Type Plasminogen Activator in Parietal Endoderm Outgrowth

When F9 stem cells are treated in suspension with retinoic acid, they differentiate into embryoid bodies (EBs) consisting of an inner core of undifferentiated stem cells surrounded by an outer layer of visceral endoderm (VE). When these EBs are plated onto a fibronectin (FN)-coated substrate, VE-derived parietal endoderm (PE) cells migrate onto the substrate. It has been suggested that increased levels of tPA associated with the emerging PE cells may help mediate PE outgrowth. We now show that goat anti-human tPA, an anticatalytic antibody that crossreacts with mouse tPA, and a panel of serine protease inhibitors partially inhibit PE outgrowth. Extracellular matrix (ECM) degradation analysis demonstrates that PE cell-mediated degradation of [ 3H]proline-labeled ECM is time- and cell concentration-dependent. A serine protease inhibitor reduced the extent of degradation, suggesting that tPA might play a role in PE outgrowth by cleaving the ECM. In support of this contention, we demonstrate that incubation of purified FN with conditioned medium plus plasminogen results in FN proteolysis. The degradation of FN is blocked by either serine protease inhibitors or goat anti-human tPA. Our data suggest that enhanced production of tPA during PE outgrowth may facilitate the migratory behavior of PE cells by mediating the degradation of ECM components such as FN.

Regulation of Ribosomal RNA Gene Transcription during Retinoic Acid-Induced Differentiation of Mouse Teratocarcinoma Cells

We have examined the mechanism of regulation of rRNA synthesis in mouse F9 teratocarcinoma cells that were induced to differentiate by retinoic acid and dibutyryl cAMP. Ribosomal RNA (rRNA) synthesis was significantly reduced during differentiation of F9 cells into parietal endoderm cells. Nuclear run-on assay revealed that the rRNA gene transcription rates were reduced in differentiated cells, and this phenomenon could be mimicked by in vitrotranscription assay using nuclear extracts prepared from F9 stem and F9 parietal endoderm cells. Analysis of the DNA-binding activities of two RNA polymerase I (pol I) transcription factors E 1BF/Ku and UBF revealed decreased affinity for their cognate recognition sequences. Immunoblot analysis showed a marked reduction in the amounts of E 1BF/Ku and UBF in the differentiated cells. Analysis of the steady-state RNA levels for the smaller subunit of E 1BF/Ku and for UBF in differentiating F9 cells revealed decreased mRNA synthesis and increase in message level for the differentiation-specific marker laminin B1 with progression of the differentiated status of the cells. This study has demonstrated that differentiation of mouse F9 teratocarcinoma cells into parietal endoderm cells leads to diminished rRNA synthesis, which may be mediated by reduced DNA-binding activities and amounts of at least two pol I transcription factors.

In vitro induction of differentiation by ginsenoides in F9 teratocarcinoma cells.

The aim of this study was to determine the ability of the ginsenosides, extracts of Panax ginseng C.A. Meyer, to cause differentiation of F9 teratocarcinoma stem cells as a model system. F9 stem cells cultured in the presence of the ginsenosides together with dibutyryl cyclic AMP (dbcAMP) became parietal endoderm-like cells. Moreover, the expression of differentiation marker genes, such as laminin B1 and type IV collagen, was increased after treatment with the ginsenosides. Among the various purified ginsenosides, Rh1 and Rh2 were the most effective at causing differentiation of F9 cells. Since ginsenosides and glucocorticoid hormone have similar chemical structures, we examined the possibility of the involvement of a glucocorticoid receptor (GR) in the differentiation process induced by the ginsenosides. According to Southwestern blot analysis, a 94 kDa protein regarded as a GR was detected in F9 cells cultured in the medium containing the ginsenosides Rh1 or Rh2. In addition, F9 stem cells treated with the ginsenosides Rh1 or Rh2 and with RU486, a glucocorticoid antagonist with a high affinity for the GR, did not differentiate into endoderm cells morphologically, and the expression of laminin B1 gene was not induced in these cells. In a gel mobility shift assay, protein factors capable of binding to the glucocorticoid responsive element (GRE) specifically were detected in nuclear extracts of the ginsenoside-treated F9 cells. Moreover, overexpression of GR by cotransfection of GR expression vector and GRE-luciferase vector enhanced the transactivation activity of GRE promoter in the presence of ginsenosides Rh1 or Rh2 and was further augmented by dbcAMP. In addition, ginsenosides Rh1 and Rh2 bound to a GR assessed by whole-cell binding assay, even though the specific binding affinity was weaker compared to dexamethasone. Based on these data, we suggest that the ginsenosides Rh1 and Rh2 cause the differentiation of F9 cells and the effects of ginsenosides might be exerted via binding with a GR or its analogous nuclear receptor.

Morphogen-induced Decline in G Triggers F9 Teratocarcinoma Stem Cell Progression via Phospholipase C and Mitogen-activated Protein Kinase

The linkage between Gialpha2 and morphogen-induced promotion of F9 embryonic teratocarcinoma stem (F9 stem) cells to primitive endoderm was explored using probes of the mitogen-activated protein (MAP) kinase network. The morphogen-induced decline in Gialpha2 is shown to trigger activation of phospholipase C, thereby activating protein kinase C, MAP kinase, and cell progression to primitive endoderm. In the absence of retinoic acid, reduction-of-function mutants (Gialpha2-deficient) display the effects of morphogen, i.e. activation of phospholipase C, protein kinase C, MAP kinase, and progression to primitive endoderm. Gain-of-function mutants (expressing the Q205L activating-mutation of Gialpha2) displayed no activation of phospholipase C, protein kinase C, MAP kinase and no progression to primitive endoderm, even in the presence of retinoic acid. Selective inhibitors of protein kinase C, like the gain-of-function mutations, effectively block morphogen-induced progression to primitive endoderm. Morphogen triggers F9 stem cell progression by triggering Gialpha2 loss and thereby activation of downstream elements, including protein kinase C and MAP kinase.

Induction, not associated with host-cell re-activation of damaged plasmid DNA, of damaged-DNA-recognition proteins by retinoic acid and dibutyryl cyclic AMP in mammalian cells

Our previous studies [Chao (1992) Biochem. J. 282, 203-207; C.C.-K. Chao, unpublished work] has suggested a correlation between the levels of constitutive UV-damaged-DNA-recognitionproteins (UVDRP) and cellular DNA repair in different cell types. In the present study, UVDRP were induced in F9 and NIH3T3 cells by 0.1 microM retinoic acid (RA) and 1 mM dibutyryl cyclic AMP (dbcAMP), which is sufficient to induce differentiation in murine F9 stem cells. The induction of UVDRP in F9 and NIH3T3 cells was optimized after 6 and 2 days incubation with RA/dbcAMP respectively. Since NIH3T3 cells were not induced to differentiate by RA/dbcAMP, the upregulation of the UVDRP in mammalian cells would thus seem not to be mediated directly by differentiation. Using a plasmid re-activation assay to estimate DNA repair, we did not find a correlation between DNA repair and UVDRP in RA/dbcAMP-treated cells. The results suggest that UVDRP may have a function other than, or in addition to, its role in DNA repair.

The Role of Phosphorylation in Modulating β1Integrin Localization

The β1-integrin subunit localizes to focal contacts during F9 teratocarcinoma stem cell differentiation to parietal endoderm. Concomitantly, this integrin subunit becomes dephosphorylated at serine 790, the only serine in the well-conserved cytoplasmic domain of β1. We hypothesized that this dephosphorylation is required for this subunit to move to a focal contact. To test this, we transfected F9 stem cells with distinct cDNAs encoding three forms of chicken β1: the wildtype, and two site-specific mutants possessing amino acid substitutions at residue 790 which included methionine and aspartate. The negatively charged aspartate was selected to mimic the phosphorylated serine found at position 790 in the wildtype protein but in a form that cannot be dephosphorylated upon differentiation. The chicken β1subunits heterodimerized with endogenous mouse α subunits and, using a chicken β1-specific monoclonal antibody, we examined movements of these chimeric integrins to focal contacts using immunofluorescence microscopy. The chimeric integrins possessing either the wildtype or methionine mutant β1subunits localized to focal contacts upon F9 differentiation. In contrast, the aspartate chimeric integrins failed to localize. These data suggest that the dephosphorylation of serine 790 is required for the β1subunit to localize to focal contacts during F9 differentiation.

Regulation of DNA methyltransferase during differentiation of F9 mouse embryonal carcinoma cells

DNA becomes demethylated when F9 mouse embryonal carcinoma cells differentiate into parietal endoderm. DNA methyltransferase (DNA-MTase) activity decreased by 50% during 1 week of differentiation. The level of DNA-MTase mRNA was also diminished accordingly, but the transcription rate of the DNA-MTase gene measured by run-on transcription was essentially unchanged, indicating regulation of DNA-MTase expression at a posttranscriptional step. The decline of DNA-MTase mRNA paralleled that of histone H3 mRNA in accord with the notion that DNA-MTase is preferentially expressed in the S phase of the cell cycle. Since DNA-MTase expression decreases in parallel with DNA synthesis, DNA demethylation during differentiation of F9 cells appears not to be due to limited expression of DNA-MTase. However, the plasmid pAFP7000CAT, alpha-fetoprotein (AFP), which is strongly de novo methylated when transfected into F9 stem cells became only weakly methylated after transfection into the F9 parietal endoderm derivative P1, indicating that the activity of DNA-MTase within parietal endoderm cells is more strongly diminished than is apparent from measurements of mRNA amounts and of overall DNA-MTase activity in vitro. The discrepancy between DNA-MTase expression and its actual activity within the cell indicates the existence of a novel mechanism controlling the activity of DNA-MTase.

Targeted disruption of vinculin genes in F9 and embryonic stem cells changes cell morphology, adhesion, and locomotion.

Vinculin, a major constituent of focal adhesions and zonula adherens junctions, is thought to be involved in linking the microfilaments to areas of cell-substrate and cell-cell contacts. To test the role of vinculin in cell adhesion and motility, we used homologous recombination to generate F9 embryonal carcinoma and embryonic stem cell clones homozygous for a disrupted vinculin gene. When compared to wild-type cells, vinculin-mutant cells displayed a rounder morphology and a reduced ability to adhere and spread on plastic or fibronectin. Decreased adhesion of the mutant cells was associated with a reduction in lamellipodial extensions, as observed by time-lapse video microscopy. The locomotive activities of control F9 and the vinculin-null cells were compared in two assays. Loss of vinculin resulted in a 2.4-fold increase in cell motility. These results demonstrate an important role for vinculin in determining cell shape, adhesion, surface protrusive activity, and cell locomotion.

Constitutively active mutant GS alpha (G225T) and null-mutant G alpha i-2 (G203T) induce primitive endoderm from stem cells

In F9 teratocarcinoma stem cells, retinoic acid induces a primitive endoderm-like phenotype and a sharp decline in G alpha i-2, a response mimicked by expression of RNA antisense to G alpha i-2 in the absence of this morphogen (D. C. Watkins, G. L. Johnson, and C. C. Malbon. Science Wash. DC 258: 1373-1375, 1992). The role of the GS alpha/G alpha i-2 axis in cellular differentiation was explored. In the absence of retinoic acid, F9 stem cells stably expressing a constitutively active mutant of GS alpha (G225T) progressed to the primitive endoderm phenotype, as judged by morphological and differentiation markers, such as tissue plasminogen activator. Although elevated in cells expressing G225T GS alpha, adenosine 3',5'-cyclic monophosphate does not mimic retinoic acid action and alone fails to induce stem cells to primitive endoderm. In the absence of retinoic acid, expression of a null mutant of G alpha i-2 (G203T) also induced stem cells to primitive endoderm. These observations establish G proteins in the GS alpha/G alpha i-2 axis as a control point for regulating progression to primitive endoderm independent of adenylate cyclase, in the present study's model of early mouse development.