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β-Catenin signaling contributes to stemness and regulates early differentiation in murine embryonic stem cells
FEBS Letters 581 (2007) 5247–5254
b-Catenin signaling contributes to stemness and regulates early differentiation in murine embryonic stem cells Roman Antona, Hans A. Kestlerb,c, Michael Ku¨hla,* b
a Institute for Biochemistry and Molecular Biology, Ulm University, D-89069 Ulm, Germany University Hospital, Internal Medicine I, Ulm University, Robert-Koch-Straße 8, D-89081 Ulm, Germany c Institute for Neural Information Processing, Ulm University, 89069 Ulm, D-89081 Ulm, Germany
Received 1 August 2007; revised 19 September 2007; accepted 5 October 2007 Available online 15 October 2007 Edited by Lukas Huber
Abstract ES cells can self-renew while preserving pluripotency and are able to differentiate into many cell types. In both processes, different signal transduction pathways are implicated, including the Wnt/b-catenin pathway, which we here further analyzed. We found that a loss of b-catenin in ES cells leads to altered expression of stem cell marker genes. TCF/b-catenin reporter gene assays indicate that undifferentiated murine ES cells are capable of reacting to LiCl and Wnt3a but not Wnt5a stimulation, but have low endogenous TCF/b-catenin activity. Oct-3/4, nanog and Wnt11 were able to repress TCF/b-catenin transcriptional activity. During differentiation, activation of the Wnt/b-catenin pathway influences formation of mesoderm and cardiomyocytes in a time and dose dependent manner. Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: ES cells; Wnt signaling; Stemness; b-Catenin
1. Introduction Stemness is a unique feature of stem cells defining their property to be developmentally paused, while retaining an unconfined ability to differentiate and self-renew. Initial clarification of the transcriptional network underlying stemness in murine ES cells identified key transcription factors like Oct3/4, Nanog and Sox2 as well as extrinsic stimuli for stem cell maintenance [1]. The Wnt-pathway consists of a well-studied ‘‘canonical’’ part, defining a signaling cascade responsible for the accumulation and nuclear translocation of b-catenin, and ‘‘non-canonical’’, b-catenin independent branches [2]. Several publications support a role of Wnt/b-catenin signaling during stem cell maintenance although the precise mode of action is not yet clear. Wnt3a conditioned media or BIO, an inhibitor of the Wnt signaling mediator GSK3, maintained mouse and human stem cell identity [3]. Hao et al. [4] suggested that Wnts inhibit differentiation via upregulation of Stat3, while Ogawa et al. [5] indicate that Wnt3a conditioned medium had no effect on Stat3 phosphorylation and it was suggested that there is no direct cross-reactivity between the Wnt and the Lif-pathway but rather a synergistic effect of both factors. Several Wnts were implicated to support stem cell propagation [4,6] of which * Corresponding author. Fax: +49 731 500 23277. E-mail address: [email protected] (M. Ku¨hl).
Wnt3a can also perpetuate self-renewal of hematopoietic stem cells [7,8]. However, purified recombinant Wnt3a protein did not suffice to abolish differentiation of mouse and human ES cells [5,6,9]. Furthermore, Wnt signaling has been shown to regulate differentiation of murine ES cells or during embryonic development such as cardiogenesis [10]. In this study, we further investigated the contribution of b-catenin signaling for stem cell maintenance and early differentiation of ES cells. Our data suggest that the influence of Wnt signaling on ES cells depends on the time point of the treatment and the activating substances used.
2. Materials and methods Cell culture experiments, reporter gene assays, FACS analysis, immunfluorescence and semi-quantitative RT-PCR experiments were performed using standard procedures. A detailed description of experiments including a list of used primers is provided within Supplementary information.
3. Results and discussion 3.1. Characterization of b-catenin / ES cells To further analyse the role of b-catenin in stem cell maintenance we characterized b-catenin deficient murine ES cells generated in the E14.1 cell line [11]. RT-PCR analysis confirmed that b-catenin / cells do not express detectable RNA levels for b-catenin (Fig. 1A). These cells can be cultured on feeder cells (Fig. 1B) and on gelatine coated dishes (Fig. 1C) and are positive for alkaline phosphatase (Fig. 1C). Both, wildtype as well as bcatenin / ES cells express several Wnt ligands, Frizzled receptors as well as transcription factors of the TCF/LEF family (Fig. 1D). Immunofluorescence studies (Fig. 1E) indicate that b-catenin is found predominantly at the membrane of undifferentiated wildtype ES cells. Diffuse staining of b-catenin, however, can also be found throughout the cytoplasm and the nucleus. We confirmed this finding by making co-culture experiments of wildtype and b-catenin / ES cells showing that the staining throughout the cytoplasm and the nucleus does not represent background staining (Fig. 1F). In contrast, the stem cell marker Oct-3/4 is found exclusively in the nucleus (Fig. 1E). 3.2. b-Catenin contributes to regulation of stemness marker genes in murine ES cells If Wnt/b-catenin signaling contributes to stemness, ES cells should be able to react with b-catenin – TCF/LEF – mediated
0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.10.012
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Fig. 1. Characterization of b-catenin deficient murine embryonic stem cells. (A) RT-PCR analysis indicates that b-catenin / cells do not express b-catenin. (B) Wildtype E14.1 and b-catenin / ES cells grow on feeder layer cells (FL). (C) Wildtype ES cells as well as b-catenin / cells show alkaline phosphatase staining. (D) RT-PCR analysis to monitor components of the canonical Wnt-pathway as well as expression of stemness marker genes in wildtype and b-catenin knock-out ES cells. HPRT was used as loading control for RT-PCR analyses. RT-PCR panels were assembled from different experiments but from identical experiments for individual genes. (E) Intracellular localization of Oct-3/4 and b-catenin in wildtype E14.1 ES cells as visualized through immunfluorescence (IF). DAPI counterstaining (DAPI) and an overlay are shown as well. (F) Immunfluorescence of Oct3/4 and b-catenin in co-cultures of wildtype E14.1 and b-catenin / ES cells.
activation of target genes upon pathway stimulation. Both Wnt3a and Wnt5a have been implicated in supporting stemness [3,4,6]. We therefore treated ES cells with different doses of purified, recombinant Wnt3a, Wnt5a or LiCl. LiCl is an inhibitor of GSK-3 and is widely used to activate the Wnt/bcatenin pathway in cell culture and embryogenesis [12]. Both Wnt3a as well as LiCl (Fig. 2A) resulted in a dose dependent activation of the pathway using TOPFlash reporter gene assays as a read out. In contrast, Wnt5a treatment of ES cells did not result in an activation of the pathway. Thus, two different Wnt ligands, both previously reported to support stemness of ES cells, clearly have different effects on activation of the Wnt/b-catenin pathway. Next we compared the expression of genes previously linked to pluripotency, self-renewal and stemness of ES cells in wildtype and b-catenin / ES cells. The loss of b-catenin results in a strong downregulation of Rex-1, dppa-4 and dppa-5,
whereas Oct-6 was upregulated (Fig. 1D). Other marker genes for stemness were slightly but reproducible downregulated such as nanog, lefty-1, lefty-2, Klf-2 or Sal1 whereas we could not detect any effect on the expression levels of Id1, Id2, Id3, STAT-3, Sox2 or Oct-3/4. These data further support the idea that b-catenin is involved in regulating the expression of stemness marker genes but likely is not required for stem cell maintenance since Oct-3/4, nanog, Sox2 or Id1-3 were still expressed. It needs to be mentioned though that these results are only based on a single cell line and ideally these results should be confirmed by use of other, independently generated b-catenin deficient ES cell lines. These experiments could also exclude the possibility that the expression of individual genes is altered during longer culture conditions in a particular cell line. Interestingly, b-catenin knock-out ES cells show an increase in Oct6 expression which might also contribute for the strong reduction in Rex-1 mRNA [13]. Mentionable is also
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Fig. 2. Activation of the Wnt/b-catenin pathway in murine ES cell. (A) Murine ES cells were transfected with TOPFlash reporter gene and treated with Wnt3a, Wnt5a or LiCl at indicated concentrations for 24 h. At least three independent measurements per time point were performed. (B) Untreated E14.1 ES cells show only low TOPFlash activity if at all. n = 18. Data were normalized to FOPFlash reporter. Oct-3/4, nanog and Wnt11 can reduce LiCl induces TOPFlash activity in ES cells (n = 13) or stabilized b-catenin induced TOPFlash activity in HEK293 cells (n = 6–9). Data were normalized to TOPFlash values in untreated cells. (C) siRNA against Wnt11 reduces endogenous Wnt11 RNA levels. RT-PCR panel was assembled from the same experiment. In transfection experiments Wnt11 siRNA increases TCF/b-catenin mediated TOPFlash activity (n = 8). siRNA against Wnt5a was not effective. HPRT was used as internal control for RT-PCR experiments. siRNA against Wnt11 did not change FOPFlash activity as control (n = 5). Data were normalized to TOPFlash or FOPFlash values, respectively, in control siRNA cells. Error bars indicate S.E. of the mean.
the fact that FGF5 was found to be upregulated, indicating together with the loss of Rex-1 and the reduction of Gbx2, that b-catenin / ES cells exhibit a primitive ectoderm like or epiblast like [14] gene expression pattern. To investigate whether this effect of b-catenin might be mediated through transcription factors of the TCF/LEF family [2], the endogenous TCF-mediated gene expression in ES cells was measured by comparing endogenous TOPFlash activity with a FOPFlash reporter, which contains no functional TCF/LEF binding sites [15]. In several experiments, we could detect only a minimal TCF/b-catenin mediated reporter activity if at all (Fig. 2B). This observation is in agreement with published data by others [5,16–19] that could not demonstrate any or only low TOPflash activity in ES cells. We therefore hypothesize that the effect of b-catenin on stemness marker gene expression might be mediated at least in part by other factors than TCF/LEF. One might argue that a loss of b-catenin results in defects in cadherin mediated cell–cell adhesion. However, it has been shown that plakoglobin can compensate the loss of b-catenin in cell adhesion in F9 teratocarcinoma stem cells [20]. b-Catenin has been shown to be localized in the nucleus
of undifferentiated ES cells [21,22] and our finding of a diffuse staining of b-catenin throughout the cytoplasm and the nucleus of stem cells is in agreement with these reports. b-Catenin has recently been shown to interact with transcription factors other than TCF/LEF, i.e, KLF-4 [23], Sox-2 [24] and just recently, Oct-3/4 [21], none of which can be monitored through the TOPflash reporter. In particular, Takao et al. just recently demonstrated that b-catenin can interact with Oct-3/4 and that this interaction is likely involved in regulating the nanog gene [21]. Consistently, Cao et al. demonstrated that the corresponding Xenopus Oct homologs Oct-25 and Oct-60 repress TCF/b-catenin signaling in early Xenopus embryos [25]. 3.3. Oct-3/4, nanog and Wnt11 act as negative regulators of Wnt signaling We therefore analyzed whether well-known regulators of stemness in murine ES cells, Oct-3/4 and nanog, or a known antagonist of Wnt/b-catenin signaling, Wnt-11 [26], might act as negative regulators of the Wnt/b-catenin pathway. Oct-3/4, nanog and Wnt11 were able to inhibit LiCl or b-catenin mediated activation of the Wnt-pathway in E14.1 cells or in human
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embryonic kidney cells (HEK 293 cells), respectively (Fig. 2B). As Wnt11 is able to repress TOPFlash activity in both cell types and as Wnt11 has been shown to be a repressor of b-catenin signaling in P19 cells, we next investigated whether Wnt11 contributes to the low TCF/b-catenin activity in undifferentiated ES cells. To test this hypothesis we transfected undifferentiated ES cells with commercially available siRNA directed against Wnt11 leading to a downregulation of Wnt11 RNA (Fig. 2C). Measuring TCF/b-catenin mediated gene activity, we observed an increase in TOPFlash activity (Fig. 2C) indicating that Wnt11 functions as a repressor of Wnt/b-catenin signaling in ES cells. siRNA against Wnt-5A however was not effective. 3.4. Treatment of ES cells with LiCl results in delayed differentiation In a second line of experiments we investigated the influence of activating the Wnt/b-catenin pathway on differentiation of ES cells. Treatment of embryoid bodies with LiCl from days 0 to 4 of differentiation resulted in a prolonged expression of Oct-3/4, nanog, Rex-1, dppa-4, dppa-5 in three out of four experiments in comparison to untreated controls (Fig. 3). At the same time, expression of the mesodermal marker gene Brachyury [27,28] is also extended, suggesting that differentiation of these embryoid bodies is delayed. We next analysed the
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expression of differentiation marker genes of the cardiac lineage. Whereas we could not observe a significant change in expression for Nkx2.5, other marker genes showed a delay or a reduction in expression (Tbx-5, Mef2C, Isl-1, cardiac actin or a-MHC). In summary, these data indicate, that treatment of ES cells with LiCl leads to a delay in differentiation. 3.5. LiCl treatment influences onset of Brachyury expression in embryoid bodies To monitor differentiation in embryoid bodies on a cellular level we made use of an ES cell line in which GFP was knocked into one allele of the Brachyury locus and marks mesodermal cells [28]. Treatment of embryoid bodies derived from this cell line with LiCl from days 0 to 4 of differentiation resulted in a severe reduction of GFP-positive cells at day 5 (Fig. 4A) which was quantified by FACS analyses (Fig. 4B). To monitor whether activation of the Wnt/b-catenin pathway results in a delay of differentiation we performed time course analyses of GFP-expressing cells. Whereas untreated embryoid bodies of Bry-GFP cells showed a maximum of GFP expression at day 5, LiCl (days 0–4) treated embryoid bodies reached their maximum at day 7 (Fig. 4C). Recent reports suggested that Brachyury is a positive target gene of the Wnt/b-catenin pathway [29,30]. In a next step, we therefore analyzed the influence of shorter LiCl treatment on GFP expression in Bry-GFP cells.
Fig. 3. Activation of the Wnt/b-catenin pathway by LiCl in differentiating embryoid bodies prolongs expression of stemness genes and delays the expression of differentiation marker genes. The expression of marker genes in differentiating embryoid bodies was analyzed by RT-PCR at days 2, 4, 6, 8 and 10 of differentiation and compared between untreated (left) and LiCl treated embryoid bodies (right). RT-PCR panel was assembled from different experiments but from identical experiments for individual genes.
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Fig. 4. Activation of the Wnt/b-catenin pathway delays differentiation in embryoid bodies. Brachyury-GFP (Bry-GFP) knock-in ES cells were used to monitor differentiation. (A) LiCl treatment from days 0 to 4 of differentiation reduces the expression GFP in day 5 embryoid bodies. (B) Quantification of GFP-expressing Bry-GFP ES cells by FACS at day 2 and 5 of differentiation in untreated and LiCl treated (days 0–4) ES cells. (C) Kinetics of GFP expression in Bry-GFP cells is altered by different timeframes of LiCl incubation. (D) Quantitative analysis of GFP expression in Bry-GFP cells using days of maximal GFP expression (upper panel, n = 14) and the location in time of the center of gravity (CoG) of the area under the curve (lower panel, n = 13). Statistical evaluation was done with a two-sided paired exact Wilcoxon rank sum test. CGR-8-MHC-GFP cells were used to monitor formation of terminally differentiated cardiomyocytes as indicated by a contractile phenotype (n = 4) (E) or cardiac MHC driven GFP expression (F) after treatment with LiCl from days 0 to 4 in comparison to untreated controls. In F a representative view is given. Error bars indicate S.E. of the mean.
These experiments indicate that a treatment from days 0 to 2 also leads to a delay in Bry expression, whereas a treatment from day 2 to 4 results in a slightly earlier expression of Brachyury (Fig. 4C). These data indicate that the delay in ES cell differentiation triggered through LiCl treatment can only be observed when treatment starts with ES cells that are still in the undifferentiated state and, vice versa, that treatment is not effective when differentiation has already started. During the time window when the endogenous Brachyury locus is open starting at day 3, however, LiCl treatment results in an activation of the gene which is in agreement with the above mentioned earlier reports. Determining the maximum of GFP expression or the location in time of the center of gravity for the area under the curve indicates that treatment of LiCl significantly alters the velocity of differentiation (Fig. 4D). To investigate the effect of LiCl on terminal differentiation we analysed the formation of a contractile phenotype in differentiating embryoid bodies. For this purpose we used CGR-8 ES cells with a cardiac specific MHC-GFP reporter, that give rise to GFP-positive cardiomyocytes [31]. Treatment of embryoid bodies with LiCl during days 0–4 leads to a significant delay in formation of a contractile phenotype (Fig. 4E) and reduced number of GFP-positive cells (Fig. 4F). Furthermore, the potential to differentiate into cardiomyocytes is reduced as less embryoid bodies finally show contractile foci that are in addition smaller in size. These b-catenin gain of function data were derived through treating embryoid bodies with LiCl. Although one can not exclude the possibility that LiCl has additional effects other than inhibiting GSK3, we would like to conclude that these effects were due to a stabilization and accumulation of b-catenin for
several reasons. First, we received complementary results for the b-catenin loss of function situation and the LiCl treatment. Second, LiCl treatment of murine ES cells results in an upregulation of a TCF/LEF driven reporter as does treatment with Wnt3a. Third, it has been shown recently that stable transfection of murine ES cells with b-catenin results in a prolonged expression of stemness marker genes upon LIF withdrawal [21]. Fourth, treatment of ES cells with Wnt3a conditioned media maintained expression of Oct-3/4, Sox2 and Rex-1 for several days [5]. Finally, our new data provided here complement recent publications suggesting a role for b-catenin signaling in murine ES cells [3–6]. Based on our here provided data and those of others we conclude that b-catenin signaling supports stemness. 3.6. Wnt treatment inhibits or activates cardiomyocyte formation depending on treatment time We next analyzed whether purified Wnt3a has the same effect on cardiomyocyte formation as has LiCl. As in case of LiCl, Wnt3A treatment (100 ng/ml, days 0–4) resulted in a dramatic decrease of contracting embryoid bodies (Fig. 5A). Again we dissected this four day time window and treated ES cells from days 0 to 2 and from days 2 to 4. These experiments revealed, that treatment from days 2 to 4 also results in an inhibition of cardiomyocyte formation whereas treatment from days 0 to 2 promotes cardiomyocyte formation (Fig. 5A) indicating a biphasic influence of Wnt3a. As the supportive role of Wnt3a on cardiogenesis (days 0–2) could not have been anticipated from the comparable experiments using LiCl as an activator of b-catenin signaling (see Fig. 4) we also investigated the influence of Wnt3a treatment on Brachyury
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Fig. 5. (A) Treatment of Bry-GFP knock in ES cells with Wnt3a (100 ng/ml) from days 0 to 4 or days 2 to 4 reduces formation of a contractile phenotype at day 10, whereas treatment from days 0 to 2 enhanced cardiomyocyte formation. Data of nine representative experiments as well as the average are shown. (B) Treatment of Bry-GFP knock in murine ES cells with Wnt3a (100 ng/ml) slightly increases number of Bry-positive cells at day 4 (n = 16) at all tested conditions. (C) This effect is more clearly seen when selecting differentiation experiments in which the number of Bry-positive cells at day 4 was below 50% (n = 9, low velocity). (D) This effect could not be seen in experiments with a high number of GFP-positive cells at day 4 (n = 7, high velocity). (E) Treatment with a higher concentration of Wnt3a (400 ng/ml) between day 0 and 2 results in a modest decrease of Brypositive cells at day 4 (n = 9). Error bars indicate S.E. of the mean.
expression. In a set of 16 independent experiments, we treated differentiating embryoid bodies with Wnt3a and measured the number of Bry-positive cells by FACS. Taking all these experiments into account we did not observe a relevant influence of Wnt3a on mesoderm induction (Fig. 5B). When separating the aforementioned set of data into slowly differentiating cultures and faster developing cultures (less or more than 50% GFP-positive cells at day 4), we indeed observed a positive influence of Wnt3a on Bry expression in slow cultures for all tested conditions at day 4 of differentiation (Fig. 5C), while this effect could not be seen in experiments that already reached high levels of Bry-positive cells at day 4 (Fig. 5D). Furthermore, using higher concentrations of Wnt3a (400 ng/ml) rather led to an inhibition of Brachyury induction (Fig. 5E), which indicates a dose dependent effect of Wnt3a. These data suggest a biphasic function of b-catenin signaling during cardiomyogenesis as also indicated recently [22,32]. If time, however, plays an important role in this context, than
the velocity of differentiation of ES cells in a particular experiment is a critical aspect. When comparing the maxima of Brachyury expression in our study and in different papers one can find values ranging between day 3 and 8 [22,28,32]. Based on published [22,32,33] and our data, we conclude, that Wnt3a treatment enhances cardiogenesis before or during the onset of Brachyury expression but inhibits cardiogenesis once a significant number of Brachyury positive cells have been generated. Similarly, Ueno et al. [32] suggested that Wnt/b-catenin signaling supports cardiogenesis before gastrulation but inhibits cardiogenesis after onset of gastrulation. If Wnt3a supports formation of Bry-positive cells in all tested conditions (days 0– 2, 0–4 and 2–4), why does treatment with Wnt3a between day 2 and 4 in our hands inhibit cardiomyocyte formation? An answer might be provided by Kattman et al. [34] who observed that belated mesodermal cells inherit the cardiovascular potential, while an earlier population does not. Thus exogenous activation of the Wnt-pathway from days 2 to 4, which accelerates
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mesoderm formation could for example promote the generation of cell identities of the early population with the low cardiovascular potential. The different effects between initial LiCl and Wnt3a treatment on mesoderm and cardiomyocyte formation could reflect different doses of b-catenin signaling triggered by the different stimuli, different kinetics with respect to onset of b-catenin signaling upon treatment or even a more complex response of the Wnt signaling network [2] after Wnt3a stimulation in contrast to inhibition of GSK-3b upon LiCl treatment. Furthermore it may be possible that both stimuli differentially regulate the assembly of an TCF/b-catenin mediated co-activator/repressor complex [35]. Also one can not exclude Wnt-pathway independent effects of the LiCl treatment as a reason for the different effects. These alternatives need further investigations in the future. In summary we have shown in this study that b-catenin contributes to maintenance of the stemness network in murine ES cells. Our findings also support the idea that the contribution of b-catenin to stemness likely is mediated at least in part through other factors than TCF/LEF like Oct-3/4. During early differentiation of murine embryonic stem cells the effect of Wnt/b-catenin signaling is likely time and dose dependent. Acknowledgements: We thank W. Birchmeier (Berlin), J. Huelsken (Lausanne), H.J. Fehling (Ulm) and R. Lee (Boston) for providing us with the different ES cell lines used in this study. We thank R.T. Moon for providing SuperTOPFlash and SuperFOPFlash plasmids. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 451, Tp B13) and Landesforschungsschwerpunkt Baden-Wu¨rttemberg ‘‘Molecular mechanisms of stemness’’ to MK. Research in the group of HAK was funded in part by a Grant through the ‘‘Stifterverband fu¨r die Deutsche Wissenschaft’’ (Forschungsdozentur Bioinformatik) and by the Deutsche Forschungsgemeinschaft (SFB 518, Project C05).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2007. 10.012. References [1] Boiani, M. and Scholer, H.R. (2005) Regulatory networks in embryo-derived pluripotent stem cells. Nat. Rev. Mol. Cell Biol. 6, 872–884. [2] Kestler, H.A. and Kuhl, M. (in press). From individual Wnt pathways towards a Wnt signalling network. Phil. Trans. R. Soc. B. [3] Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. and Brivanlou, A.H. (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med. 10, 55– 63. [4] Hao, J., Li, T.G., Qi, X., Zhao, D.F. and Zhao, G.Q. (2006) WNT/beta-catenin pathway up-regulates Stat3 and converges on LIF to prevent differentiation of mouse embryonic stem cells. Dev. Biol. 290, 81–91. [5] Ogawa, K., Nishinakamura, R., Iwamatsu, Y., Shimosato, D. and Niwa, H. (2006) Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells. Biochem. Biophys. Res. Commun. 343, 159–166. [6] Singla, D.K., Schneider, D.J., LeWinter, M.M. and Sobel, B.E. (2006) wnt3a but not wnt11 supports self-renewal of embryonic stem cells. Biochem. Biophys. Res. Commun. 345, 789–795.
5253 [7] Reya, T. et al. (2003) A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423, 409–414. [8] Willert, K., Brown, J.D., Danenberg, E., Duncan, A.W., Weissman, I.L., Reya, T., Yates 3rd, J.R. and Nusse, R. (2003) Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448–452. [9] Dravid, G., Ye, Z., Hammond, H., Chen, G., Pyle, A., Donovan, P., Yu, X. and Cheng, L. (2005) Defining the role of Wnt/betacatenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells. Stem Cells 23, 1489–1501. [10] Tzahor, E. (2007) Wnt/beta-catenin signaling and cardiogenesis: timing does matter. Dev. Cell 13, 10–13. [11] Huelsken, J., Vogel, R., Brinkmann, V., Erdmann, B., Birchmeier, C. and Birchmeier, W. (2000) Requirement for beta-catenin in anterior–posterior axis formation in mice. J. Cell Biol. 148, 567–578. [12] Klein, P.S. and Melton, D.A. (1996) A molecular mechanism for the effect of lithium on development. Proc. Natl. Acad. Sci. USA 93, 8455–8459. [13] Ben-Shushan, E., Thompson, J.R., Gudas, L.J. and Bergman, Y. (1998) Rex-1, a gene encoding a transcription factor expressed in the early embryo, is regulated via Oct-3/4 and Oct-6 binding to an octamer site and a novel protein, Rox-1, binding to an adjacent site. Mol. Cell Biol. 18, 1866–1878. [14] Pelton, T.A., Sharma, S., Schulz, T.C., Rathjen, J. and Rathjen, P.D. (2002) Transient pluripotent cell populations during primitive ectoderm formation: correlation of in vivo and in vitro pluripotent cell development. J. Cell Sci. 115, 329–339. [15] Veeman, M.T., Axelrod, J.D. and Moon, R.T. (2003) A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev. Cell 5, 367–377. [16] Kielman, M.F. et al. (2002) Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signaling. Nat. Genet 32, 594–605. [17] Watanabe, S., Umehara, H., Murayama, K., Okabe, M., Kimura, T. and Nakano, T. (2006) Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells. Oncogene 25, 2697–2707. [18] Pereira, L., Yi, F. and Merrill, B.J. (2006) Repression of Nanog gene transcription by Tcf3 limits embryonic stem cell self-renewal. Mol. Cell Biol. 26, 7479–7491. [19] Smits, R. et al. (1999) Apc1638T: a mouse model delineating critical domains of the adenomatous polyposis coli protein involved in tumorigenesis and development. Genes Dev. 13, 1309–1321. [20] Fukunaga, Y., Liu, H., Shimizu, M., Komiya, S., Kawasuji, M. and Nagafuchi, A. (2005) Defining the roles of beta-catenin and plakoglobin in cell–cell adhesion: isolation of beta-catenin/plakoglobin-deficient F9 cells. Cell Struct. Funct. 30, 25–34. [21] Takao, Y., Yokota, T. and Koide, H. (2007) Beta-catenin upregulates Nanog expression through interaction with Oct-3/4 in embryonic stem cells. Biochem. Biophys. Res. Commun. 353, 699–705. [22] Naito, A.T., Shiojima, I., Akazawa, H., Hidaka, K., Morisaki, T., Kikuchi, A. and Komuro, I. (2006) Developmental stage-specific biphasic roles of Wnt/beta-catenin signaling in cardiomyogenesis and hematopoiesis. Proc. Natl. Acad. Sci. USA 103, 19812–19817. [23] Zhang, W. et al. (2006) Novel cross talk of Kruppel-like factor 4 and beta-catenin regulates normal intestinal homeostasis and tumor repression. Mol. Cell Biol. 26, 2055–2064. [24] Mansukhani, A., Ambrosetti, D., Holmes, G., Cornivelli, L. and Basilico, C. (2005) Sox2 induction by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation. J. Cell Biol. 168, 1065–1076. [25] Cao, Y., Siegel, D., Donow, C., Knochel, S., Yuan, L. and Knochel, W. (2007) POU-V factors antagonizematernal VegT activity and beta-catenin signaling in Xenopus embryos. EMBO J. 26, 2942–2954. [26] Maye, P., Zheng, J., Li, L. and Wu, D. (2004) Multiple mechanisms for Wnt11-mediated repression of the canonical Wnt signaling pathway. J. Biol. Chem. 279, 24659–24665. [27] Wilkinson, D.G., Bhatt, S. and Hermann, B.G. (1990) Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 343, 657–659. [28] Fehling, H.J., Lacaud, G., Kubo, A., Kennedy, M., Robertson, S., Keller, G. and Kouskoff, V. (2003) Tracking mesoderm
5254
[29]
[30]
[31]
[32]
R. Anton et al. / FEBS Letters 581 (2007) 5247–5254 induction and its specification to the hemangioblast during embryonic stem cell differentiation. Development 130, 4217–4227. Yamaguchi, T.P., Takada, S., Yoshikawa, Y., Wu, N. and McMahon, A.P. (1999) T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification. Genes Dev. 13, 3185– 3190. Galceran, J., Hsu, S.C. and Grosschedl, R. (2001) Rescue of a Wnt mutation by an activated form of LEF-1: regulation of maintenance but not initiation of Brachyury expression. Proc. Natl. Acad. Sci. USA 98, 8668–8673. Takahashi, T., Lord, B., Schulze, P.C., Fryer, R.M., Sarang, S.S., Gullans, S.R. and Lee, R.T. (2003) Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes. Circulation 107, 1912–1916. Ueno, S. et al. (2007) Biphasic role for Wnt/{beta}-catenin signaling in cardiac specification in zebrafish and embryonic stem cells. Proc. Natl. Acad. Sci. USA 104, 9685–9690.
[33] Lindsley, R.C., Gill, J.G., Kyba, M., Murphy, T.L. and Murphy, K.M. (2006) Canonical Wnt signaling is required for development of embryonic stem cell-derived mesoderm. Development 133, 3787–3796. [34] Kattman, S.J., Huber, T.L. and Keller, G.M. (2006) Multipotent flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages. Dev. Cell 11, 723–732. [35] Miyabayashi, T., Teo, J.L., Yamamoto, M., McMillan, M., Nguyen, C. and Kahn, M. (2007) Wnt/beta-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency. Proc. Natl. Acad. Sci. USA 104, 5668–5673.
b-Catenin signaling contributes to stemness and regulates early differentiation in murine embryonic stem cells Roman Antona, Hans A. Kestlerb,c, Michael Ku¨hla,* b
a Institute for Biochemistry and Molecular Biology, Ulm University, D-89069 Ulm, Germany University Hospital, Internal Medicine I, Ulm University, Robert-Koch-Straße 8, D-89081 Ulm, Germany c Institute for Neural Information Processing, Ulm University, 89069 Ulm, D-89081 Ulm, Germany
Received 1 August 2007; revised 19 September 2007; accepted 5 October 2007 Available online 15 October 2007 Edited by Lukas Huber
Abstract ES cells can self-renew while preserving pluripotency and are able to differentiate into many cell types. In both processes, different signal transduction pathways are implicated, including the Wnt/b-catenin pathway, which we here further analyzed. We found that a loss of b-catenin in ES cells leads to altered expression of stem cell marker genes. TCF/b-catenin reporter gene assays indicate that undifferentiated murine ES cells are capable of reacting to LiCl and Wnt3a but not Wnt5a stimulation, but have low endogenous TCF/b-catenin activity. Oct-3/4, nanog and Wnt11 were able to repress TCF/b-catenin transcriptional activity. During differentiation, activation of the Wnt/b-catenin pathway influences formation of mesoderm and cardiomyocytes in a time and dose dependent manner. Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: ES cells; Wnt signaling; Stemness; b-Catenin
1. Introduction Stemness is a unique feature of stem cells defining their property to be developmentally paused, while retaining an unconfined ability to differentiate and self-renew. Initial clarification of the transcriptional network underlying stemness in murine ES cells identified key transcription factors like Oct3/4, Nanog and Sox2 as well as extrinsic stimuli for stem cell maintenance [1]. The Wnt-pathway consists of a well-studied ‘‘canonical’’ part, defining a signaling cascade responsible for the accumulation and nuclear translocation of b-catenin, and ‘‘non-canonical’’, b-catenin independent branches [2]. Several publications support a role of Wnt/b-catenin signaling during stem cell maintenance although the precise mode of action is not yet clear. Wnt3a conditioned media or BIO, an inhibitor of the Wnt signaling mediator GSK3, maintained mouse and human stem cell identity [3]. Hao et al. [4] suggested that Wnts inhibit differentiation via upregulation of Stat3, while Ogawa et al. [5] indicate that Wnt3a conditioned medium had no effect on Stat3 phosphorylation and it was suggested that there is no direct cross-reactivity between the Wnt and the Lif-pathway but rather a synergistic effect of both factors. Several Wnts were implicated to support stem cell propagation [4,6] of which * Corresponding author. Fax: +49 731 500 23277. E-mail address: [email protected] (M. Ku¨hl).
Wnt3a can also perpetuate self-renewal of hematopoietic stem cells [7,8]. However, purified recombinant Wnt3a protein did not suffice to abolish differentiation of mouse and human ES cells [5,6,9]. Furthermore, Wnt signaling has been shown to regulate differentiation of murine ES cells or during embryonic development such as cardiogenesis [10]. In this study, we further investigated the contribution of b-catenin signaling for stem cell maintenance and early differentiation of ES cells. Our data suggest that the influence of Wnt signaling on ES cells depends on the time point of the treatment and the activating substances used.
2. Materials and methods Cell culture experiments, reporter gene assays, FACS analysis, immunfluorescence and semi-quantitative RT-PCR experiments were performed using standard procedures. A detailed description of experiments including a list of used primers is provided within Supplementary information.
3. Results and discussion 3.1. Characterization of b-catenin / ES cells To further analyse the role of b-catenin in stem cell maintenance we characterized b-catenin deficient murine ES cells generated in the E14.1 cell line [11]. RT-PCR analysis confirmed that b-catenin / cells do not express detectable RNA levels for b-catenin (Fig. 1A). These cells can be cultured on feeder cells (Fig. 1B) and on gelatine coated dishes (Fig. 1C) and are positive for alkaline phosphatase (Fig. 1C). Both, wildtype as well as bcatenin / ES cells express several Wnt ligands, Frizzled receptors as well as transcription factors of the TCF/LEF family (Fig. 1D). Immunofluorescence studies (Fig. 1E) indicate that b-catenin is found predominantly at the membrane of undifferentiated wildtype ES cells. Diffuse staining of b-catenin, however, can also be found throughout the cytoplasm and the nucleus. We confirmed this finding by making co-culture experiments of wildtype and b-catenin / ES cells showing that the staining throughout the cytoplasm and the nucleus does not represent background staining (Fig. 1F). In contrast, the stem cell marker Oct-3/4 is found exclusively in the nucleus (Fig. 1E). 3.2. b-Catenin contributes to regulation of stemness marker genes in murine ES cells If Wnt/b-catenin signaling contributes to stemness, ES cells should be able to react with b-catenin – TCF/LEF – mediated
0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.10.012
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Fig. 1. Characterization of b-catenin deficient murine embryonic stem cells. (A) RT-PCR analysis indicates that b-catenin / cells do not express b-catenin. (B) Wildtype E14.1 and b-catenin / ES cells grow on feeder layer cells (FL). (C) Wildtype ES cells as well as b-catenin / cells show alkaline phosphatase staining. (D) RT-PCR analysis to monitor components of the canonical Wnt-pathway as well as expression of stemness marker genes in wildtype and b-catenin knock-out ES cells. HPRT was used as loading control for RT-PCR analyses. RT-PCR panels were assembled from different experiments but from identical experiments for individual genes. (E) Intracellular localization of Oct-3/4 and b-catenin in wildtype E14.1 ES cells as visualized through immunfluorescence (IF). DAPI counterstaining (DAPI) and an overlay are shown as well. (F) Immunfluorescence of Oct3/4 and b-catenin in co-cultures of wildtype E14.1 and b-catenin / ES cells.
activation of target genes upon pathway stimulation. Both Wnt3a and Wnt5a have been implicated in supporting stemness [3,4,6]. We therefore treated ES cells with different doses of purified, recombinant Wnt3a, Wnt5a or LiCl. LiCl is an inhibitor of GSK-3 and is widely used to activate the Wnt/bcatenin pathway in cell culture and embryogenesis [12]. Both Wnt3a as well as LiCl (Fig. 2A) resulted in a dose dependent activation of the pathway using TOPFlash reporter gene assays as a read out. In contrast, Wnt5a treatment of ES cells did not result in an activation of the pathway. Thus, two different Wnt ligands, both previously reported to support stemness of ES cells, clearly have different effects on activation of the Wnt/b-catenin pathway. Next we compared the expression of genes previously linked to pluripotency, self-renewal and stemness of ES cells in wildtype and b-catenin / ES cells. The loss of b-catenin results in a strong downregulation of Rex-1, dppa-4 and dppa-5,
whereas Oct-6 was upregulated (Fig. 1D). Other marker genes for stemness were slightly but reproducible downregulated such as nanog, lefty-1, lefty-2, Klf-2 or Sal1 whereas we could not detect any effect on the expression levels of Id1, Id2, Id3, STAT-3, Sox2 or Oct-3/4. These data further support the idea that b-catenin is involved in regulating the expression of stemness marker genes but likely is not required for stem cell maintenance since Oct-3/4, nanog, Sox2 or Id1-3 were still expressed. It needs to be mentioned though that these results are only based on a single cell line and ideally these results should be confirmed by use of other, independently generated b-catenin deficient ES cell lines. These experiments could also exclude the possibility that the expression of individual genes is altered during longer culture conditions in a particular cell line. Interestingly, b-catenin knock-out ES cells show an increase in Oct6 expression which might also contribute for the strong reduction in Rex-1 mRNA [13]. Mentionable is also
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Fig. 2. Activation of the Wnt/b-catenin pathway in murine ES cell. (A) Murine ES cells were transfected with TOPFlash reporter gene and treated with Wnt3a, Wnt5a or LiCl at indicated concentrations for 24 h. At least three independent measurements per time point were performed. (B) Untreated E14.1 ES cells show only low TOPFlash activity if at all. n = 18. Data were normalized to FOPFlash reporter. Oct-3/4, nanog and Wnt11 can reduce LiCl induces TOPFlash activity in ES cells (n = 13) or stabilized b-catenin induced TOPFlash activity in HEK293 cells (n = 6–9). Data were normalized to TOPFlash values in untreated cells. (C) siRNA against Wnt11 reduces endogenous Wnt11 RNA levels. RT-PCR panel was assembled from the same experiment. In transfection experiments Wnt11 siRNA increases TCF/b-catenin mediated TOPFlash activity (n = 8). siRNA against Wnt5a was not effective. HPRT was used as internal control for RT-PCR experiments. siRNA against Wnt11 did not change FOPFlash activity as control (n = 5). Data were normalized to TOPFlash or FOPFlash values, respectively, in control siRNA cells. Error bars indicate S.E. of the mean.
the fact that FGF5 was found to be upregulated, indicating together with the loss of Rex-1 and the reduction of Gbx2, that b-catenin / ES cells exhibit a primitive ectoderm like or epiblast like [14] gene expression pattern. To investigate whether this effect of b-catenin might be mediated through transcription factors of the TCF/LEF family [2], the endogenous TCF-mediated gene expression in ES cells was measured by comparing endogenous TOPFlash activity with a FOPFlash reporter, which contains no functional TCF/LEF binding sites [15]. In several experiments, we could detect only a minimal TCF/b-catenin mediated reporter activity if at all (Fig. 2B). This observation is in agreement with published data by others [5,16–19] that could not demonstrate any or only low TOPflash activity in ES cells. We therefore hypothesize that the effect of b-catenin on stemness marker gene expression might be mediated at least in part by other factors than TCF/LEF. One might argue that a loss of b-catenin results in defects in cadherin mediated cell–cell adhesion. However, it has been shown that plakoglobin can compensate the loss of b-catenin in cell adhesion in F9 teratocarcinoma stem cells [20]. b-Catenin has been shown to be localized in the nucleus
of undifferentiated ES cells [21,22] and our finding of a diffuse staining of b-catenin throughout the cytoplasm and the nucleus of stem cells is in agreement with these reports. b-Catenin has recently been shown to interact with transcription factors other than TCF/LEF, i.e, KLF-4 [23], Sox-2 [24] and just recently, Oct-3/4 [21], none of which can be monitored through the TOPflash reporter. In particular, Takao et al. just recently demonstrated that b-catenin can interact with Oct-3/4 and that this interaction is likely involved in regulating the nanog gene [21]. Consistently, Cao et al. demonstrated that the corresponding Xenopus Oct homologs Oct-25 and Oct-60 repress TCF/b-catenin signaling in early Xenopus embryos [25]. 3.3. Oct-3/4, nanog and Wnt11 act as negative regulators of Wnt signaling We therefore analyzed whether well-known regulators of stemness in murine ES cells, Oct-3/4 and nanog, or a known antagonist of Wnt/b-catenin signaling, Wnt-11 [26], might act as negative regulators of the Wnt/b-catenin pathway. Oct-3/4, nanog and Wnt11 were able to inhibit LiCl or b-catenin mediated activation of the Wnt-pathway in E14.1 cells or in human
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embryonic kidney cells (HEK 293 cells), respectively (Fig. 2B). As Wnt11 is able to repress TOPFlash activity in both cell types and as Wnt11 has been shown to be a repressor of b-catenin signaling in P19 cells, we next investigated whether Wnt11 contributes to the low TCF/b-catenin activity in undifferentiated ES cells. To test this hypothesis we transfected undifferentiated ES cells with commercially available siRNA directed against Wnt11 leading to a downregulation of Wnt11 RNA (Fig. 2C). Measuring TCF/b-catenin mediated gene activity, we observed an increase in TOPFlash activity (Fig. 2C) indicating that Wnt11 functions as a repressor of Wnt/b-catenin signaling in ES cells. siRNA against Wnt-5A however was not effective. 3.4. Treatment of ES cells with LiCl results in delayed differentiation In a second line of experiments we investigated the influence of activating the Wnt/b-catenin pathway on differentiation of ES cells. Treatment of embryoid bodies with LiCl from days 0 to 4 of differentiation resulted in a prolonged expression of Oct-3/4, nanog, Rex-1, dppa-4, dppa-5 in three out of four experiments in comparison to untreated controls (Fig. 3). At the same time, expression of the mesodermal marker gene Brachyury [27,28] is also extended, suggesting that differentiation of these embryoid bodies is delayed. We next analysed the
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expression of differentiation marker genes of the cardiac lineage. Whereas we could not observe a significant change in expression for Nkx2.5, other marker genes showed a delay or a reduction in expression (Tbx-5, Mef2C, Isl-1, cardiac actin or a-MHC). In summary, these data indicate, that treatment of ES cells with LiCl leads to a delay in differentiation. 3.5. LiCl treatment influences onset of Brachyury expression in embryoid bodies To monitor differentiation in embryoid bodies on a cellular level we made use of an ES cell line in which GFP was knocked into one allele of the Brachyury locus and marks mesodermal cells [28]. Treatment of embryoid bodies derived from this cell line with LiCl from days 0 to 4 of differentiation resulted in a severe reduction of GFP-positive cells at day 5 (Fig. 4A) which was quantified by FACS analyses (Fig. 4B). To monitor whether activation of the Wnt/b-catenin pathway results in a delay of differentiation we performed time course analyses of GFP-expressing cells. Whereas untreated embryoid bodies of Bry-GFP cells showed a maximum of GFP expression at day 5, LiCl (days 0–4) treated embryoid bodies reached their maximum at day 7 (Fig. 4C). Recent reports suggested that Brachyury is a positive target gene of the Wnt/b-catenin pathway [29,30]. In a next step, we therefore analyzed the influence of shorter LiCl treatment on GFP expression in Bry-GFP cells.
Fig. 3. Activation of the Wnt/b-catenin pathway by LiCl in differentiating embryoid bodies prolongs expression of stemness genes and delays the expression of differentiation marker genes. The expression of marker genes in differentiating embryoid bodies was analyzed by RT-PCR at days 2, 4, 6, 8 and 10 of differentiation and compared between untreated (left) and LiCl treated embryoid bodies (right). RT-PCR panel was assembled from different experiments but from identical experiments for individual genes.
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Fig. 4. Activation of the Wnt/b-catenin pathway delays differentiation in embryoid bodies. Brachyury-GFP (Bry-GFP) knock-in ES cells were used to monitor differentiation. (A) LiCl treatment from days 0 to 4 of differentiation reduces the expression GFP in day 5 embryoid bodies. (B) Quantification of GFP-expressing Bry-GFP ES cells by FACS at day 2 and 5 of differentiation in untreated and LiCl treated (days 0–4) ES cells. (C) Kinetics of GFP expression in Bry-GFP cells is altered by different timeframes of LiCl incubation. (D) Quantitative analysis of GFP expression in Bry-GFP cells using days of maximal GFP expression (upper panel, n = 14) and the location in time of the center of gravity (CoG) of the area under the curve (lower panel, n = 13). Statistical evaluation was done with a two-sided paired exact Wilcoxon rank sum test. CGR-8-MHC-GFP cells were used to monitor formation of terminally differentiated cardiomyocytes as indicated by a contractile phenotype (n = 4) (E) or cardiac MHC driven GFP expression (F) after treatment with LiCl from days 0 to 4 in comparison to untreated controls. In F a representative view is given. Error bars indicate S.E. of the mean.
These experiments indicate that a treatment from days 0 to 2 also leads to a delay in Bry expression, whereas a treatment from day 2 to 4 results in a slightly earlier expression of Brachyury (Fig. 4C). These data indicate that the delay in ES cell differentiation triggered through LiCl treatment can only be observed when treatment starts with ES cells that are still in the undifferentiated state and, vice versa, that treatment is not effective when differentiation has already started. During the time window when the endogenous Brachyury locus is open starting at day 3, however, LiCl treatment results in an activation of the gene which is in agreement with the above mentioned earlier reports. Determining the maximum of GFP expression or the location in time of the center of gravity for the area under the curve indicates that treatment of LiCl significantly alters the velocity of differentiation (Fig. 4D). To investigate the effect of LiCl on terminal differentiation we analysed the formation of a contractile phenotype in differentiating embryoid bodies. For this purpose we used CGR-8 ES cells with a cardiac specific MHC-GFP reporter, that give rise to GFP-positive cardiomyocytes [31]. Treatment of embryoid bodies with LiCl during days 0–4 leads to a significant delay in formation of a contractile phenotype (Fig. 4E) and reduced number of GFP-positive cells (Fig. 4F). Furthermore, the potential to differentiate into cardiomyocytes is reduced as less embryoid bodies finally show contractile foci that are in addition smaller in size. These b-catenin gain of function data were derived through treating embryoid bodies with LiCl. Although one can not exclude the possibility that LiCl has additional effects other than inhibiting GSK3, we would like to conclude that these effects were due to a stabilization and accumulation of b-catenin for
several reasons. First, we received complementary results for the b-catenin loss of function situation and the LiCl treatment. Second, LiCl treatment of murine ES cells results in an upregulation of a TCF/LEF driven reporter as does treatment with Wnt3a. Third, it has been shown recently that stable transfection of murine ES cells with b-catenin results in a prolonged expression of stemness marker genes upon LIF withdrawal [21]. Fourth, treatment of ES cells with Wnt3a conditioned media maintained expression of Oct-3/4, Sox2 and Rex-1 for several days [5]. Finally, our new data provided here complement recent publications suggesting a role for b-catenin signaling in murine ES cells [3–6]. Based on our here provided data and those of others we conclude that b-catenin signaling supports stemness. 3.6. Wnt treatment inhibits or activates cardiomyocyte formation depending on treatment time We next analyzed whether purified Wnt3a has the same effect on cardiomyocyte formation as has LiCl. As in case of LiCl, Wnt3A treatment (100 ng/ml, days 0–4) resulted in a dramatic decrease of contracting embryoid bodies (Fig. 5A). Again we dissected this four day time window and treated ES cells from days 0 to 2 and from days 2 to 4. These experiments revealed, that treatment from days 2 to 4 also results in an inhibition of cardiomyocyte formation whereas treatment from days 0 to 2 promotes cardiomyocyte formation (Fig. 5A) indicating a biphasic influence of Wnt3a. As the supportive role of Wnt3a on cardiogenesis (days 0–2) could not have been anticipated from the comparable experiments using LiCl as an activator of b-catenin signaling (see Fig. 4) we also investigated the influence of Wnt3a treatment on Brachyury
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Fig. 5. (A) Treatment of Bry-GFP knock in ES cells with Wnt3a (100 ng/ml) from days 0 to 4 or days 2 to 4 reduces formation of a contractile phenotype at day 10, whereas treatment from days 0 to 2 enhanced cardiomyocyte formation. Data of nine representative experiments as well as the average are shown. (B) Treatment of Bry-GFP knock in murine ES cells with Wnt3a (100 ng/ml) slightly increases number of Bry-positive cells at day 4 (n = 16) at all tested conditions. (C) This effect is more clearly seen when selecting differentiation experiments in which the number of Bry-positive cells at day 4 was below 50% (n = 9, low velocity). (D) This effect could not be seen in experiments with a high number of GFP-positive cells at day 4 (n = 7, high velocity). (E) Treatment with a higher concentration of Wnt3a (400 ng/ml) between day 0 and 2 results in a modest decrease of Brypositive cells at day 4 (n = 9). Error bars indicate S.E. of the mean.
expression. In a set of 16 independent experiments, we treated differentiating embryoid bodies with Wnt3a and measured the number of Bry-positive cells by FACS. Taking all these experiments into account we did not observe a relevant influence of Wnt3a on mesoderm induction (Fig. 5B). When separating the aforementioned set of data into slowly differentiating cultures and faster developing cultures (less or more than 50% GFP-positive cells at day 4), we indeed observed a positive influence of Wnt3a on Bry expression in slow cultures for all tested conditions at day 4 of differentiation (Fig. 5C), while this effect could not be seen in experiments that already reached high levels of Bry-positive cells at day 4 (Fig. 5D). Furthermore, using higher concentrations of Wnt3a (400 ng/ml) rather led to an inhibition of Brachyury induction (Fig. 5E), which indicates a dose dependent effect of Wnt3a. These data suggest a biphasic function of b-catenin signaling during cardiomyogenesis as also indicated recently [22,32]. If time, however, plays an important role in this context, than
the velocity of differentiation of ES cells in a particular experiment is a critical aspect. When comparing the maxima of Brachyury expression in our study and in different papers one can find values ranging between day 3 and 8 [22,28,32]. Based on published [22,32,33] and our data, we conclude, that Wnt3a treatment enhances cardiogenesis before or during the onset of Brachyury expression but inhibits cardiogenesis once a significant number of Brachyury positive cells have been generated. Similarly, Ueno et al. [32] suggested that Wnt/b-catenin signaling supports cardiogenesis before gastrulation but inhibits cardiogenesis after onset of gastrulation. If Wnt3a supports formation of Bry-positive cells in all tested conditions (days 0– 2, 0–4 and 2–4), why does treatment with Wnt3a between day 2 and 4 in our hands inhibit cardiomyocyte formation? An answer might be provided by Kattman et al. [34] who observed that belated mesodermal cells inherit the cardiovascular potential, while an earlier population does not. Thus exogenous activation of the Wnt-pathway from days 2 to 4, which accelerates
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mesoderm formation could for example promote the generation of cell identities of the early population with the low cardiovascular potential. The different effects between initial LiCl and Wnt3a treatment on mesoderm and cardiomyocyte formation could reflect different doses of b-catenin signaling triggered by the different stimuli, different kinetics with respect to onset of b-catenin signaling upon treatment or even a more complex response of the Wnt signaling network [2] after Wnt3a stimulation in contrast to inhibition of GSK-3b upon LiCl treatment. Furthermore it may be possible that both stimuli differentially regulate the assembly of an TCF/b-catenin mediated co-activator/repressor complex [35]. Also one can not exclude Wnt-pathway independent effects of the LiCl treatment as a reason for the different effects. These alternatives need further investigations in the future. In summary we have shown in this study that b-catenin contributes to maintenance of the stemness network in murine ES cells. Our findings also support the idea that the contribution of b-catenin to stemness likely is mediated at least in part through other factors than TCF/LEF like Oct-3/4. During early differentiation of murine embryonic stem cells the effect of Wnt/b-catenin signaling is likely time and dose dependent. Acknowledgements: We thank W. Birchmeier (Berlin), J. Huelsken (Lausanne), H.J. Fehling (Ulm) and R. Lee (Boston) for providing us with the different ES cell lines used in this study. We thank R.T. Moon for providing SuperTOPFlash and SuperFOPFlash plasmids. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 451, Tp B13) and Landesforschungsschwerpunkt Baden-Wu¨rttemberg ‘‘Molecular mechanisms of stemness’’ to MK. Research in the group of HAK was funded in part by a Grant through the ‘‘Stifterverband fu¨r die Deutsche Wissenschaft’’ (Forschungsdozentur Bioinformatik) and by the Deutsche Forschungsgemeinschaft (SFB 518, Project C05).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2007. 10.012. References [1] Boiani, M. and Scholer, H.R. (2005) Regulatory networks in embryo-derived pluripotent stem cells. Nat. Rev. Mol. Cell Biol. 6, 872–884. [2] Kestler, H.A. and Kuhl, M. (in press). From individual Wnt pathways towards a Wnt signalling network. Phil. Trans. R. Soc. B. [3] Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. and Brivanlou, A.H. (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med. 10, 55– 63. [4] Hao, J., Li, T.G., Qi, X., Zhao, D.F. and Zhao, G.Q. (2006) WNT/beta-catenin pathway up-regulates Stat3 and converges on LIF to prevent differentiation of mouse embryonic stem cells. Dev. Biol. 290, 81–91. [5] Ogawa, K., Nishinakamura, R., Iwamatsu, Y., Shimosato, D. and Niwa, H. (2006) Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells. Biochem. Biophys. Res. Commun. 343, 159–166. [6] Singla, D.K., Schneider, D.J., LeWinter, M.M. and Sobel, B.E. (2006) wnt3a but not wnt11 supports self-renewal of embryonic stem cells. Biochem. Biophys. Res. Commun. 345, 789–795.
5253 [7] Reya, T. et al. (2003) A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423, 409–414. [8] Willert, K., Brown, J.D., Danenberg, E., Duncan, A.W., Weissman, I.L., Reya, T., Yates 3rd, J.R. and Nusse, R. (2003) Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448–452. [9] Dravid, G., Ye, Z., Hammond, H., Chen, G., Pyle, A., Donovan, P., Yu, X. and Cheng, L. (2005) Defining the role of Wnt/betacatenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells. Stem Cells 23, 1489–1501. [10] Tzahor, E. (2007) Wnt/beta-catenin signaling and cardiogenesis: timing does matter. Dev. Cell 13, 10–13. [11] Huelsken, J., Vogel, R., Brinkmann, V., Erdmann, B., Birchmeier, C. and Birchmeier, W. (2000) Requirement for beta-catenin in anterior–posterior axis formation in mice. J. Cell Biol. 148, 567–578. [12] Klein, P.S. and Melton, D.A. (1996) A molecular mechanism for the effect of lithium on development. Proc. Natl. Acad. Sci. USA 93, 8455–8459. [13] Ben-Shushan, E., Thompson, J.R., Gudas, L.J. and Bergman, Y. (1998) Rex-1, a gene encoding a transcription factor expressed in the early embryo, is regulated via Oct-3/4 and Oct-6 binding to an octamer site and a novel protein, Rox-1, binding to an adjacent site. Mol. Cell Biol. 18, 1866–1878. [14] Pelton, T.A., Sharma, S., Schulz, T.C., Rathjen, J. and Rathjen, P.D. (2002) Transient pluripotent cell populations during primitive ectoderm formation: correlation of in vivo and in vitro pluripotent cell development. J. Cell Sci. 115, 329–339. [15] Veeman, M.T., Axelrod, J.D. and Moon, R.T. (2003) A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev. Cell 5, 367–377. [16] Kielman, M.F. et al. (2002) Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signaling. Nat. Genet 32, 594–605. [17] Watanabe, S., Umehara, H., Murayama, K., Okabe, M., Kimura, T. and Nakano, T. (2006) Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells. Oncogene 25, 2697–2707. [18] Pereira, L., Yi, F. and Merrill, B.J. (2006) Repression of Nanog gene transcription by Tcf3 limits embryonic stem cell self-renewal. Mol. Cell Biol. 26, 7479–7491. [19] Smits, R. et al. (1999) Apc1638T: a mouse model delineating critical domains of the adenomatous polyposis coli protein involved in tumorigenesis and development. Genes Dev. 13, 1309–1321. [20] Fukunaga, Y., Liu, H., Shimizu, M., Komiya, S., Kawasuji, M. and Nagafuchi, A. (2005) Defining the roles of beta-catenin and plakoglobin in cell–cell adhesion: isolation of beta-catenin/plakoglobin-deficient F9 cells. Cell Struct. Funct. 30, 25–34. [21] Takao, Y., Yokota, T. and Koide, H. (2007) Beta-catenin upregulates Nanog expression through interaction with Oct-3/4 in embryonic stem cells. Biochem. Biophys. Res. Commun. 353, 699–705. [22] Naito, A.T., Shiojima, I., Akazawa, H., Hidaka, K., Morisaki, T., Kikuchi, A. and Komuro, I. (2006) Developmental stage-specific biphasic roles of Wnt/beta-catenin signaling in cardiomyogenesis and hematopoiesis. Proc. Natl. Acad. Sci. USA 103, 19812–19817. [23] Zhang, W. et al. (2006) Novel cross talk of Kruppel-like factor 4 and beta-catenin regulates normal intestinal homeostasis and tumor repression. Mol. Cell Biol. 26, 2055–2064. [24] Mansukhani, A., Ambrosetti, D., Holmes, G., Cornivelli, L. and Basilico, C. (2005) Sox2 induction by FGF and FGFR2 activating mutations inhibits Wnt signaling and osteoblast differentiation. J. Cell Biol. 168, 1065–1076. [25] Cao, Y., Siegel, D., Donow, C., Knochel, S., Yuan, L. and Knochel, W. (2007) POU-V factors antagonizematernal VegT activity and beta-catenin signaling in Xenopus embryos. EMBO J. 26, 2942–2954. [26] Maye, P., Zheng, J., Li, L. and Wu, D. (2004) Multiple mechanisms for Wnt11-mediated repression of the canonical Wnt signaling pathway. J. Biol. Chem. 279, 24659–24665. [27] Wilkinson, D.G., Bhatt, S. and Hermann, B.G. (1990) Expression pattern of the mouse T gene and its role in mesoderm formation. Nature 343, 657–659. [28] Fehling, H.J., Lacaud, G., Kubo, A., Kennedy, M., Robertson, S., Keller, G. and Kouskoff, V. (2003) Tracking mesoderm
5254
[29]
[30]
[31]
[32]
R. Anton et al. / FEBS Letters 581 (2007) 5247–5254 induction and its specification to the hemangioblast during embryonic stem cell differentiation. Development 130, 4217–4227. Yamaguchi, T.P., Takada, S., Yoshikawa, Y., Wu, N. and McMahon, A.P. (1999) T (Brachyury) is a direct target of Wnt3a during paraxial mesoderm specification. Genes Dev. 13, 3185– 3190. Galceran, J., Hsu, S.C. and Grosschedl, R. (2001) Rescue of a Wnt mutation by an activated form of LEF-1: regulation of maintenance but not initiation of Brachyury expression. Proc. Natl. Acad. Sci. USA 98, 8668–8673. Takahashi, T., Lord, B., Schulze, P.C., Fryer, R.M., Sarang, S.S., Gullans, S.R. and Lee, R.T. (2003) Ascorbic acid enhances differentiation of embryonic stem cells into cardiac myocytes. Circulation 107, 1912–1916. Ueno, S. et al. (2007) Biphasic role for Wnt/{beta}-catenin signaling in cardiac specification in zebrafish and embryonic stem cells. Proc. Natl. Acad. Sci. USA 104, 9685–9690.
[33] Lindsley, R.C., Gill, J.G., Kyba, M., Murphy, T.L. and Murphy, K.M. (2006) Canonical Wnt signaling is required for development of embryonic stem cell-derived mesoderm. Development 133, 3787–3796. [34] Kattman, S.J., Huber, T.L. and Keller, G.M. (2006) Multipotent flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages. Dev. Cell 11, 723–732. [35] Miyabayashi, T., Teo, J.L., Yamamoto, M., McMillan, M., Nguyen, C. and Kahn, M. (2007) Wnt/beta-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency. Proc. Natl. Acad. Sci. USA 104, 5668–5673.