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Previous Article | Next Article 
Molecular and Cellular Biology, October 2006, p. 7539-7549, Vol. 26, No. 20
0270-7306/06/$08.00+0 doi:10.1128/MCB.00508-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Pathology, University of Florida College of Medicine, Gainesville, Florida 32610,1 Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka, Japan2
Received 22 March 2006/ Returned for modification 5 May 2006/ Accepted 1 August 2006
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ABSTRACT |
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INTRODUCTION |
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Primitive endoderm is the first differentiated cell type to arise from the inner cell mass (ICM) in blastocysts during mammalian embryogenesis. Derivation of primitive endoderm from the ICM can be recapitulated by forming ES cell aggregates in suspension culture (10). As observed in the ICM during development, primitive endoderm differentiation is restricted to the outermost layer of the ES cell aggregates, and the rest of the inner cells retain their pluripotency in the presence of LIF (25, 31). Our previous study identified that the Nanog gene was selectively repressed in the outermost layer of ES cell aggregates in the presence of LIF, and it identified that Nanog repression was essential for primitive endoderm differentiation in the outermost layer (13). However, it is largely undetermined what external cues and intracellular signals cause this repression.
Previous studies using gene targeting suggested that several signaling molecules may be involved in primitive endoderm specification. Disruption of FGFR1 or FGFR2, which are family members of receptor tyrosine kinases, inhibited primitive endoderm differentiation (2, 11). In addition, when fibroblast growth factor (FGF) signaling was blocked by the overexpression of a dominant-negative mutant of FGFR2 or by the FGF receptor (FGFR)-specific inhibitor SU5042, the formation of the primitive endoderm layer was abolished (8, 17, 18). These studies strongly suggest that the FGF-FGFR interaction is pivotal for primitive endoderm specification. Of interest, the phosphatidylinositol 3-kinase (PI3K)-Akt pathway rather than Erk signaling was predominately affected by the dominant-negative mutant of FGFR (8). Furthermore, the addition of a PI3K inhibitor to ES cell aggregates reduced primitive endoderm differentiation (8). These studies imply that the PI3K-Akt pathway may play a role in FGF-mediated primitive endoderm differentiation.
Another signaling pathway involved in primitive endoderm differentiation is the Ras-Erk pathway. Targeted disruption of Grb2, which links phosphotyrosine to intracellular signaling pathways, abrogated the formation of primitive endoderm in blastocysts. This phenotype was rescued by the expression of a Grb2-Sos1 fusion protein, which activated the downstream Ras-Erk pathway (9). Further, another study showed that introducing an active mutant of Ras into murine ES cells resulted in primitive endoderm differentiation (39). These studies suggest that the Grb2/Sos/Ras/Erk pathway plays an essential role in primitive endoderm specification. Thus, it is still controversial which intracellular signaling pathway is predominately involved in primitive endoderm specification. Moreover, none of the previous studies clearly demonstrated how these signaling pathways affected Nanog gene expression.
In the present study, we have attempted to reveal the intracellular signaling pathways controlling Nanog gene expression during ES cell differentiation into primitive endoderm. Using various pharmacologically active small molecules as well as genetic or biochemical modifications of signaling molecules, we determined that Nanog gene repression was primarily mediated by the Grb2/Mek pathway.
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MATERIALS AND METHODS |
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Flow cytometry analysis. A single-cell suspension was prepared from aggregates of ES cells by treating them with 0.05% trypsin (GIBCO-BRL). Dissociated cells were separated by FACS Sort (Becton Dickinson, Franklin Lakes, NJ). Data analysis was performed by using CellQuest Acquisition software (Becton Dickinson).
Whole-mount X-Gal staining. Cells were fixed in solution containing 2% formaldehyde, 0.2% glutaraldehyde, 5 mM EGTA, 2 mM MgCl2, and 0.02% NP-40 for 10 min at room temperature. After washing twice with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST), the samples were stained in 5-bromo-4-chloro-3-indolyl-D-galactoside (X-Gal) mix [containing 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2, 10% dimethyl sulfoxide, 0.01% sodium deoxycholate, 0.02% NP-40, and 1 mg/ml X-Gal] for 8 to 12 h at 37°C and then rinsed in PBST. Poststain fix was performed in 3.7% formaldehyde at 4°C overnight and followed by paraffin embedding. Sections (7 µm) were counterstained by eosin Y.
Plasmid construction and transfection.
The Grb2 cDNA was PCR amplified by LA-Taq polymerase (TAKARA Bio, Otsu, Japan) using cDNAs prepared from wild-type ES cells as a template. Primer sequences, which contain BamHI and NheI restriction sites, are the following: sense, 5'-CGGGATCATGGAAGCCATCGCCAAA; antisense, 5'-CTAGCTAGCTTAGACGTTCCGGTTCACTG. The cDNA fragment was cloned into pCAG-IRES-hygromycin vector. The resultant vector was transfected into Grb2 null ES cells using Fugene 6 (Roche, Indianapolis, IN), and colonies constitutively expressing Grb2 were selected by hygromycin B (100 mg/ml) (Invitrogen, Carlsbad, CA). Plasmids containing the Mek1 constitutively active mutant (
N3 S218E, S222D) and the kinase-dead mutant (K97M) were kindly provided by N. Ahn (20). The coding region was shuttled into NotI and MluI sites of pCAG-hygromycin vector. The expression vectors were transfected by Fugene 6 (Roche), and stable colonies were selected by hygromycin B (100 µg/ml) (Invitrogen, Carlsbad, CA).
Reverse transcription-PCR (RT-PCR). Total RNA was extracted by using an RNA aqueous kit (Ambion Inc., Austin, TX). The cDNA was synthesized by using a SuperScript II first-strand synthesis system with oligo(dT) (GIBCO-BRL). PCR was performed by using Taq DNA polymerase (Eppendorf, Westbury, NY). For each gene, the DNA primers were originated from different exons to ensure that the PCR product represents the specific mRNA species and not genomic DNA (primer sequences are available on request).
Western blotting. Cells were lysed in radioimmunoprecipitation assay buffer, and 10 µg of total proteins was separated with sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The following were used as primary antibodies: Nanog, AB5731 (Chemicon); Oct3/4, C-10; Sox2, H-65; and phosphotyrosine, PY20 (Santa Cruz, Santa Cruz, CA); phospho-Erk1/2, E10 (#9106); phospho-Akt, #9271; and phospho-SAPK/Jnk, #9251 (Cell signaling, Beverly, MA). Peroxidase-conjugated immunoglobulin G (Santa Cruz) was used as the secondary antibody, followed by enhanced chemiluminescence (ECL) detection (Amersham, Piscataway, NJ).
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RESULTS |
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To monitor primitive endoderm differentiation, we used transgenic ES cells carrying a green fluorescent protein (GFP) reporter under the control of the 
-fetoprotein promoter (Afp-GFP ES cells) (13). As we previously reported, when these ES cells were aggregated, only the outermost layer became GFP positive. In contrast, within 48 h after sodium vanadate treatment, GFP-positive cells appeared in the inner core of the aggregates in addition to the outer layer (Fig. 1A). The percentage of total GFP-positive cells in the aggregates was evaluated by flow cytometry analysis after the cells were dissociated into a suspension of single cells. Without sodium vanadate treatment, GFP-positive cells appeared only in the outermost layer and accounted for 2.2% (±0.2% [standard deviation]) of the total cells, whereas in sodium vanadate-treated cells, the percentage of GFP-positive cells reached a high of 86.7% (±1.9% standard deviations) of the population when the maximum dose (100 µM) was added (Fig. 1B). Figure 1C demonstrates that sodium vanadate increases protein tyrosine phosphorylation in a dose-dependent manner.
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Effects of sodium vanadate on gene expression of pluripotency and differentiation markers: time course and dose responses. To further explore the mechanisms by which sodium vanadate represses Nanog, we examined mRNA expression levels of Nanog and other pluripotency-related genes as well as primitive endoderm marker genes within 32 h after sodium vanadate treatment. As shown in Fig. 2A, Nanog mRNA levels significantly decreased within 24 h after sodium vanadate treatment (25 µM). Previously, putative downstream target genes of Nanog were reported based on a predicted Nanog binding consensus sequence (23). Zfp42/Rex1, which is suggested to be positively regulated by Nanog, was also downregulated upon sodium vanadate treatment. In contrast, Gata6, which is suggested to be negatively regulated by Nanog, was upregulated at the same time point that Nanog mRNA decreased. Other primitive endoderm marker genes such as Gata4 and Bmp2 mRNA increased following Gata6 expression (Fig. 2A).
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Oct3/4 and Sox2 are critical transcription factors that maintain the pluripotency of ES cells (3). Increasing evidence suggests that both factors regulate Nanog gene transcription by binding to the proximal promoter region of the Nanog gene (3, 16, 30). In our study, the mRNA levels of the Oct3/4 gene were not affected by sodium vanadate treatment throughout the time course (Fig. 2A). Sox2 mRNA started decreasing at a later time point (32 h) after Nanog was already repressed (Fig. 2A). Furthermore, the protein expression levels of Oct3/4 and Sox2 did not decrease within 48 h after treatment, whereas Nanog protein significantly decreased at this time point (Fig. 2C). These data indicate that sodium vanadate primarily suppresses Nanog expression among pluripotency-related genes and that Nanog repression is not likely a consequence of a loss of Oct3/4 and Sox2 expression in ES cells.
Grb2 deficiency abrogated the effects of sodium vanadate on Nanog repression. Grb2 has an SH2 domain that specifically binds to a peptide motif containing a phosphotyrosine. Thus, it works as an adaptor protein, linking the signals received by receptor tyrosine kinases, such as FGF receptors, to their downstream targets. Grb2 is also known to be essential to the formation of the primitive endoderm layer both in vivo in the ICM of a blastocyst stage embryo and in vitro during ES cell aggregation (9). We therefore examined whether Grb2 was involved in sodium vanadate-induced Nanog repression. We treated both wild-type and Grb2 null ES cells with sodium vanadate (25 to 100 µM) and compared their morphological changes and gene expression patterns by RT-PCR. In wild-type ES cells, the cells were enlarged and dissociated in suspension culture. In contrast to wild-type ES cells, there were no obvious morphological changes in Grb2 null ES cells treated with sodium vanadate (Fig. 3A). In addition, Nanog expression was not affected by sodium vanadate in Grb2 null ES cells, and there was no sign of differentiation towards the primitive endoderm lineage. In wild-type ES cells, Nanog and Zfp42 genes were repressed, while Gata6, along with other primitive endoderm marker genes, was upregulated upon sodium vanadate treatment (Fig. 3B). To further confirm the role of Grb2 in sodium vanadate-mediated Nanog repression, we added back a Grb2 expression vector into the Grb2 null ES cells and established stable clones. In the Grb2 add-back ES cells, Nanog and Zfp42 genes were now repressed and Gata6 was upregulated upon sodium vanadate treatment (Fig. 3C). These data indicate that sodium vanadate-induced Nanog repression and primitive endoderm differentiation are mediated through Grb2.
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Cell aggregation-induced Nanog repression was also blocked by Mek inhibition. We next examined whether Mek activation was required to repress Nanog during ES cell aggregation. Nanog ß-geo ES cells were cultured in suspension and were supplemented with a Mek inhibitor (PD98059). While the ES cells in the vehicle control (Fig. 5A) repressed Nanog in the outermost layer of the aggregates, the ES cell aggregates cultured in the presence of a Mek inhibitor formed no such layer (Fig. 5B). These data indicate that Mek activity is required to repress Nanog during ES cell aggregation.
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The phenotypes resulting from either a targeted disruption of FGFR or from introducing a dominant-negative FGFR into ES cells have revealed the role of FGF signaling in primitive endoderm formation (2, 8, 11). We blocked the kinase activity of FGFR using SU5402, which binds to the ATP binding site of FGFR (24), and found that our results were similar to those obtained when we used a Mek inhibitor, in that Nanog was not repressed in the outermost layer (Fig. 5E). These data imply the activation of Mek during ES cell aggregation is mediated through the FGF receptor.
Of interest, addition of SU5402 increased Nanog expression even when ES cells were maintained on gelatin-coated dishes. When undifferentiated Nanog ß-geo ES cells were maintained on gelatin-coated culture dishes in the presence of LIF, ES cells with reduced Nanog expression were detected sparsely using X-Gal staining (Fig. 6A, control). In order to further examine the roles of FGF and FGFR in Nanog repression, we added SU5402 to the maintenance culture of Nanog ß-geo ES cells for 48 h. The SU5402-treated ES cells were more homogeneously stained with X-Gal. In addition, SU5402 treatment increased Nanog mRNA expression by approximately 1.6-fold (Fig. 6). Addition of FGF1 or FGF2 into the culture medium did not significantly alter Nanog expression in ES cells.
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N3 S218E, S222D) (20). As shown in Fig. 7A, constitutive activation of Mek1 resulted in primitive endoderm differentiation of ES cells, and this was visualized by monitoring GFP expression in Afp-GFP ES cells. The enzymatic activity of this constitutive active mutant of Mek is known to be inhibited by PD98059 (1). The endoderm promoting effects of the active Mek mutant were then abrogated in the presence of PD98059. Moreover, the Mek 1 kinase-dead mutant, K97M (20), did not promote primitive endoderm differentiation. Using the same Mek mutants, we also monitored Nanog gene expression by X-Gal staining of Nanog ß-geo ES cells (Fig. 7B). We confirmed that constitutive activation of Mek 1 leads to Nanog repression in ES cells.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The effects of sodium vanadate on Nanog repression and primitive endoderm differentiation were completely abrogated by Grb2 deficiency (Fig. 3). This clearly demonstrates that the sodium vanadate-induced tyrosine phosphorylation signal to repress Nanog is transmitted via Grb2. Grb2 is an adaptor molecule with an SH2 domain that specifically binds to a peptide motif containing a phosphotyrosine. This motif links Grb2 to downstream signaling cascades, in particular to the Sos/Ras/Raf/Mek/Erk pathway (6, 29). Among the various kinase inhibitors tested, only the Mek inhibitor selectively blocked the effects of sodium vanadate on Nanog repression (Fig. 4B and C). Moreover, transfection of a constitutively active form of Mek mutant repressed Nanog and led to primitive endoderm differentiation (Fig. 7). These data indicate that tyrosine phosphorylation induces Nanog repression through the Grb2/Sos/Ras/Raf/Mek/Erk pathway. During preparation of this report, Chazaud et al. demonstrated that Grb2 deficiency abrogated Nanog repression in the ICM of blastocysts in vivo (7).
In the present study, we demonstrated that the addition of a PI3K inhibitor was not able to block sodium vanadate-induced Nanog repression (Fig. 4B and C). Further, when ES cells were aggregated in the presence of the PI3K inhibitor, Nanog repression still occurred in the outermost layer of the aggregates (Fig. 5C). These data indicate that Nanog repression is not mediated by the PI3K-Akt pathway. However, the data did not exclude the role of the PI3K-Akt pathway in primitive endoderm differentiation. Indeed, when the PI3K inhibitor was added to the aggregates of Afp-GFP ES cells in conjunction with sodium vanadate, there was a partial inhibition of GFP-positive primitive endoderm cells compared to that of cells treated only with sodium vanadate (data not shown). A previous study showed that a constitutively active Akt enhanced the synthesis of laminin and collagen IV isotypes and promoted basement membrane formation (19). Collectively, Mek/Erk activation may specifically repress Nanog gene expression and initiate primitive endoderm differentiation, whereas PI3K/Akt activation may induce the production of extracellular matrix proteins to promote further primitive endoderm differentiation.
The previous studies indicate that the FGF-FGFR interaction is pivotal for primitive endoderm specification (2, 11, 17, 18). Indeed, addition of the FGFR inhibitor (SU5402) to ES cell aggregates almost completely blocked Nanog repression (Fig. 5). Further, addition of SU5402 increased Nanog expression even when ES cells were maintained on gelatin-coated dishes (Fig. 6). The SU5402-treated ES cells were more homogeneously positive for Nanog expression. Our data imply that the signals mediated through FGFR impose a continuous pressure on ES cells to repress Nanog, even when ES cells are maintained on gelatin-coated dishes in the presence of LIF. Although FGFR1 is expressed in undifferentiated ES cells (data not shown) (22), addition of FGF1 or FGF2 into the ES cell culture did not induce Nanog repression (Fig. 6). Moreover, mouse ES cells are also known to express FGF4 (36). This implies that a molecular mechanism exists in ES cells that attenuates the FGFR/Grb2/Mek signaling pathway while maintaining Nanog expression. Alternatively, a differential expression of other FGFRs in a subpopulation of ES cells may trigger a more distinct activation of Grb2/Mek, thereby resulting in Nanog repression. Elucidating such signals will be a necessary task in order to reveal the entire signaling cascade controlling Nanog repression.
The pluripotency of murine ES cells is dependent upon LIF-induced STAT3 activity when they are cultured on gelatin-coated dishes (21, 26, 32). Although LIF also activates the Mek/Erk pathway, this Mek/Erk activation is not essential for ES cell propagation. Moreover, inhibition of Mek by PD98059 rather enhanced the growth of undifferentiated ES cells (4). The present study demonstrated that PD98059 prevented Nanog repression, which explains, at least in part, how the drug supported ES cell self renewal in the previous study. In addition, another study has shown that the PI3K-Akt pathway played a pivotal role in suppressing Erk1/2 activation during ES cell maintenance and that the addition of LY294002 in ES cells enhanced Erk1/2 activation and ES cell differentiation (28). Indeed, we observed that the addition of LY294002 repressed Nanog in the inner cells of ES cell aggregates in the presence of LIF (Fig. 5C). We did not, however, detect any elevation in Erk1/2 phosphorylation after the addition of LY294002 in our cell aggregation culture (Fig. 4B). It should be noted that introducing a myristoylated active form of Akt into ES cells was sufficient to maintain the culture of undifferentiated ES cells in the absence of LIF, and this event was not through the inhibition of Erk activity (35). Collectively, it is still unclear how the PI3K-Akt pathway facilitates the self renewal of ES cells.
The present study elucidated a signaling pathway that specifically led to Nanog gene repression in ES cells. Modifying this signaling pathway by sodium vanadate or an active Mek mutant led ES cells to differentiate into primitive endoderm with high efficiency. By further dissecting the intracellular signaling pathways governing ES cell self renewal and differentiation, we will be able to develop novel methods to direct ES cell fate, making ES cells a promising source for future cell-based therapies.
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ACKNOWLEDGMENTS |
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This work was supported in part by National Institutes of Health grant DK59699 (to N.T.).
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FOOTNOTES |
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Published ahead of print on 14 August 2006.
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