Calcineurin-NFAT Signaling Critically Regulates Early Lineage Specification in Mouse Embryonic Stem Cells and Embryos

Cell Stem Cell

Article Calcineurin-NFAT Signaling Critically Regulates Early Lineage Specification in Mouse Embryonic Stem Cells and Embryos Xiang Li,1,2,5 Lili Zhu,1,2,5 Acong Yang,1,3 Jiangwei Lin,4 Fan Tang,1,3 Shibo Jin,1 Zhe Wei,1,2 Jinsong Li,4 and Ying Jin1,3,* 1Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences/Shanghai JiaoTong University School of Medicine, Shanghai, 200025, China 2Graduate School of Chinese Academy of Sciences, Beijing, 100000, China 3Shanghai Stem Cell Institute, Shanghai JiaoTong University School of Medicine, Shanghai, 200025, China 4Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China 5These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.stem.2010.11.027

SUMMARY

Self-renewal and pluripotency are hallmarks of embryonic stem cells (ESCs). However, the signaling pathways that trigger their transition from selfrenewal to differentiation remain elusive. Here, we report that calcineurin-NFAT signaling is both necessary and sufficient to switch ESCs from an undifferentiated state to lineage-specific cells and that the inhibition of this pathway can maintain long-term ESC self-renewal independent of leukemia inhibitory factor. Mechanistically, this pathway converges with the Erk1/2 pathway to regulate Src expression and promote the epithelial-mesenchymal transition (EMT), a process required for lineage specification in response to differentiation stimuli. Furthermore, calcineurin-NFAT signaling is activated when the earliest differentiation event occurs in mouse embryos, and its inhibition disrupts extraembryonic lineage development. Collectively, our results demonstrate that the NFAT and Erk1/2 cascades form a signaling switch for early lineage segregation in mouse ESCs and provide significant insights into the regulation of the balance between ESC selfrenewal and early lineage specification. INTRODUCTION During early mouse development, lineage specification begins at embryonic day 2.5 (E2.5) in 8-cell embryos. The first lineage decision leads to the establishment of the inner cell mass (ICM) and the trophectoderm at the blastocyst stage. The second lineage decision gives rise to the epiblast and the primitive endoderm when the latter delaminates from the ICM (Rossant, 2007; Rossant et al., 2003). The epiblast contains pluripotent cells that generate the three germ layers as well as germ cells. Embryonic stem cells (ESCs), derived from the preimplantation blastocyst, have the potential to differentiate into all cell types 46 Cell Stem Cell 8, 46–58, January 7, 2011 ª2011 Elsevier Inc.

of an organism and to grow indefinitely in culture (Martin, 1981; Smith, 2001). Mouse ESCs can be maintained in an undifferentiated self-renewal state in the presence of leukemia inhibitory factor (LIF) and either bone morphogenetic protein 4 (BMP4) or serum without feeder cells (Ying et al., 2003). Withdrawal of LIF results in extensive ESC differentiation with downregulation of the pluripotency-associated core transcription factors Oct4, Sox2, and Nanog (Kim et al., 2008). Recently, maintaining ESCs at a ground state of self-renewal in the absence of LIF and serum was reported via two inhibitors (2i) of fibroblast growth factor/extracellular signal-related kinase 1/2 (Fgf/Erk1/2) and glycogen synthase kinase 3 (GSK3) (Ying et al., 2008). Despite these major advances, the molecular basis for ESCs to transit from the state of self-renewal to early differentiation has not been fully elucidated. The Fgf/Mek/Erk1/2 pathway is considered important for the formation of the first two extraembryonic lineages (trophectoderm and primitive endoderm) in early murine development and for ESC differentiation in vitro (Chazaud et al., 2006; Lu et al., 2008; Nichols et al., 2009; Yamanaka et al., 2010). We were interested in the question of whether there are additional signaling pathways critical for the early lineage specification and, if so, how they may be integrated to orchestrate early development. To address this question, we employed the piggyBac (PB) transposon, which randomly inserts into a host genome and disrupts gene function (Ding et al., 2005), and then selected transfected ESC colonies with undifferentiated ESC morphology after LIF withdrawal. One gene that was identified through this approach was Cnb1, also known as Ppp3r1, which encodes calcium binding B (CnB), a subunit of calcineurin. Calcineurin is a Ca2+ influx-activated serine/threoninespecific phosphatase composed of the CnA and CnB subunits (Crabtree, 1999; Crabtree and Schreiber, 2009). Three genes (Ppp3ca, Ppp3cb, and Ppp3cc) encode three members of the catalytic CnA subunit, whereas two members of the regulatory CnB unit of calcineurin are products of two genes (Ppp3r1 and Ppp3r2). Calcineurin dephosphorylates cytoplasmic NFAT (nuclear factor of activated T cell, the products of four NFATc genes, NFATc1-c4) to promote translocation of NFAT to the nucleus, where NFAT and its usual partner AP1 (Fos/Jun, substrates of Erk1/2) bind target promoters to control the

Cell Stem Cell NFAT Pathway Regulates Early Lineage Specification

expression of genes with diverse functions. Although NFAT proteins were first recognized for their central role in T lymphocyte activation, they have since been demonstrated to orchestrate diverse developmental programs, including nervous, cardiovascular, hematopoietic, and muscular-skeletal development, as well as to maintain the quiescent state of stem cells in skin (Chin et al., 1998; Clipstone and Crabtree, 1992; Graef et al., 2001, 2003; Molkentin et al., 1998; Mu¨ller et al., 2009; Stankunas et al., 1999). Recently, an intrinsic role of calcineurin signaling in keratinocyte tumor suppression was reported (Wu et al., 2010). Mice with disrupted Cnb1 gene, or NFATc1 gene, or both NFATc3 and NFATc4 genes, die around E11 to E14 (de la Pompa et al., 1998; Graef et al., 2001). However, the expression and function of calcineurin-NFAT signaling in ESCs, as well as in early embryonic development at the periimplantation stage, have remained unnoticed, possibly because of genetic redundancy among family members of the calcineurin-NFAT signaling pathway. The advent of pharmacologic inhibitors of NFAT translocation has greatly facilitated our understanding of the functions of calcineurin-NFAT signaling. The specificity of these inhibitors for calcineurin, and potentially NFAT, was established based upon the nearly identical phenotypes observed for Cnb1 null mice, NFATc3/c4 double null mice, and embryos of mothers given the inhibitor at E7.5 to E8.5 (Crabtree and Olson, 2002; Graef et al., 2001). Here, utilizing the inhibitors and genetic approaches, we demonstrate that calcineurin-NFAT signaling is both necessary and sufficient to trigger lineage commitment through the upregulation of Src expression to promote epithelial-mesenchymal transition (EMT) in mouse ESCs. RESULTS Calcineurin-NFAT Signaling Is Required for Multilineage Differentiation of ESCs A PB plasmid (PGK-Neo) and a PBase expression plasmid (Act-PBase) were coelectroporated into ESCs and transfectants were selected with G418 in the presence of LIF and serum, as described (Ding et al., 2005). Approximately 1000 G418-resistant colonies were obtained from each 10 cm dish. Colonies that maintained an undifferentiated morphology in the absence of LIF after several passages in culture were expanded. Integration sites were analyzed with inverse PCR. Among these sites, genes were identified that potentially associate with the Ras-MAPK, Akt, and calcineurin signaling pathways, such as Grb10, Ptpn21, Inpp4b, and Ppp3r1 (Table S1 available online). We were particularly interested in the Ppp3r1 gene (Figure S1A), because it encodes the regulatory subunit of calcineurin, and the role of calcineurin-NFAT signaling in ESCs has not been reported. The disruption of Ppp3r1 expression by PB insertion and its role in the prevention of ESCs from LIF withdrawal-induced differentiation were validated by quantitative real-time RT-PCR (qRT-PCR) analysis of the levels of expression of Ppp3r1 and other marker genes in both control and Ppp3r1-disrupted ESCs (Figure S1B). Subsequently, to determine the function of the calcineurin-NFAT pathway, we treated ESCs with cyclosporine A (CsA), a specific inhibitor of calcineurin (Clipstone and Crabtree, 1992), and examined cell morphology and the

expression of the marker genes in the presence and absence of LIF. As expected, the removal of LIF resulted in extensively differentiated cell morphology, increased transcript levels for various lineage markers (including Fgf5, Cdx2, Dab2, Nestin, T, and Mixl1), and downregulation of pluripotency markers such as Oct4, Nanog, and Rex1 (Figures 1A and 1B). Moreover, at the protein level, nuclear staining of Nanog was dramatically reduced after LIF withdrawal (Figure 1C). All these LIF withdrawal-induced changes were efficiently abolished by CsA treatment (Figures 1A–1C), although CsA-treated ESCs displayed a relatively reduced growth rate (Figure S1C). In contrast, CsA did not affect the LIF withdrawal-induced reduction in the level of phosphorylated Stat3, although it did block the LIF withdrawal-induced decrease in total Stat3 protein levels (Figure S1D). The inhibitory effect of CsA on calcineurin-NFAT signaling under such conditions was verified by a reporter assay (Figure S1E). Furthermore, FK506 (the unrelated calcineurin inhibitor) and the NFAT-selective inhibitory peptide VIVIT (Yu et al., 2007) significantly attenuated ESC differentiation induced by LIF removal (Figures S1F–S1I). A similar effect was also observed when RNA interference (RNAi) targeting Ppp3r1 was introduced (Figure 1D; Figure S1J), suggesting that the attenuation was calcineurin-NFAT signaling dependent. In addition, CsA could block neural differentiation of ESC-derived epiblast stem cells as well as ESC differentiation in the presence of retinoic acid (RA) and during embryoid body (EB) formation (Figures S1K–S1N). Collectively, our findings demonstrate that calcineurin-NFAT signaling is crucial for ESC differentiation in response to various differentiation stimuli. We next sought to determine whether, in the absence of LIF, CsA could maintain ESCs in an undifferentiated state indefinitely. After 40 days, ESCs cultured under such conditions displayed an undifferentiated morphology. After withdrawal of CsA, they retained a normal differentiation potential and formed EBs with differentiated cells expressing markers of all three germ layers (Figures 1E and 1F). Teratomas exhibiting cells of the three germ layers were also detected when these cells were injected into nude mice (Figure 1G). Finally, the ESCs re-entered embryonic development in the chimeric mice (Figure 1H) when CsA-expanded ESCs carrying a histone 2B-GFP gene were injected into mouse blastocysts. Therefore, the inhibition of the calcineurin-NFAT pathway can sustain ESC properties for a long period in the absence of LIF. Finally, we cultured CsA-treated ESCs in the serum-free condition in the absence of LIF (Ying et al., 2008). Similar to 2i, CsA or CsA plus the GSK3 inhibitor (CHIR) could maintain the expression of Oct4 and Nanog as well as a compact undifferentiated morphology (Figures S1O and S1P). Moreover, the combination of CsA and CHIR could sustain a cell growth rate comparable to that observed with 2i treatment (Figure S1Q). Calcineurin-NFAT Signaling Is Activated upon ESC Differentiation We then examined the expression patterns of calcineurin and NFAT in mouse ESCs and their progeny. The transcriptional levels of the calcineurin subunits increased when ESCs were induced into differentiation by LIF withdrawal (Figure 2A). Published microarray data (Ivanova et al., 2006) showed low NFATc1/c2 expression and abundant NFATc3/c4 expression Cell Stem Cell 8, 46–58, January 7, 2011 ª2011 Elsevier Inc. 47

Cell Stem Cell NFAT Pathway Regulates Early Lineage Specification

Figure 1. Calcineurin-NFAT Signaling Is Required for Multilineage Differentiation of ESCs (A) Phase contrast images of CGR8 ESCs grown for 3.5 days under the indicated conditions. Scale bars represent 100 mm. (B) qRT-PCR analysis of gene expression levels in cells described in (A). The mRNA level in ESCs cultured with LIF was set at 1.0. Data are shown as the mean ± SD (n = 3). (C) Confocal images of ESCs grown under the indicated conditions and incubated with Nanog antibody. Scale bars represent 50 mm. (D) qRT-PCR analysis of marker gene expression levels in CGR8 ESCs cultured in the presence or absence of LIF, the control oligonucleotide, and the Ppp3r1 oligonucleotide. The mRNA level in ESCs cultured with LIF and the control oligonucleotide was set at 1.0. Data are shown as the mean ± SD (n = 3). (E) RT-PCR analysis of marker genes in EBs derived from CsA-expanded ESCs. Gapdh was used as a loading control. (F) Immunofluorescence staining of EBs after they adhered to culture dishes. Cells were stained with antibodies against Sox17 (endoderm), Nestin (ectoderm), and Flk1 (mesoderm). Scale bars represent 50 mm. (G) H&E staining of teratomas from ESCs grown in medium containing CsA (15 mM) and subcutaneously injected into nude mice. Scale bars represent 50 mm. (H) A chimeric embryo at day E18.5 produced from CsA-expanded ESCs, expressing a histone 2B-GFP fusion protein. See also Figure S1 and Table S1.

in mouse ESCs (Figure 2B). Results from our western blot analysis indicated that the steady-state level of NFATc4 proteins increased markedly after ESC differentiation, whereas the NFATc3 level remained consistently high (Figure 2C). The specificity of NFATc3 and NFATc4 antibodies was verified by RNAi-specific expression knockdown (Figures S2A and S2B). Notably, NFATc3 was primarily found in the cytoplasm of undifferentiated ESCs but moved to the nucleus after LIF withdrawal 48 Cell Stem Cell 8, 46–58, January 7, 2011 ª2011 Elsevier Inc.

(Figure 2D), indicative of NFATc3 activation upon ESC differentiation. In line with this observation, the activity of a luciferase reporter containing three tandem copies of the murine Il2 promoter element, a prototypical NFAT:AP-1 composite (Macia´n et al., 2000), was significantly upregulated after the removal of LIF (Figure 2E). In later experiments of this study, we primarily focused on NFATc3 because of its high expression level in ESCs and rapid activation upon differentiation.

Cell Stem Cell NFAT Pathway Regulates Early Lineage Specification

Figure 2. Calcineurin-NFAT Signaling Is Activated during ESC Differentiation (A) qRT-PCR analysis of calcineurin subunit mRNA levels in ESCs after LIF withdrawal. The mRNA level in ESCs cultured with LIF was set at 1.0. Data are shown as the mean ± SD (n = 3). (B) Expression of NFATc1-4 at different days after RA induction. The microarray expression data were obtained from the literature (Ivanova et al., 2006). (C) Western blot analysis of NFATc3 and NFATc4 steadystate levels in ESCs after LIF withdrawal. Tubulin was used as a control. (D) Subcellular localization of NFATc3 in ESCs 3.5 days after LIF withdrawal was detected by immunostaining. DAPI was used to visualize the nucleus. Scale bars represent 25 mm. (E) Activation of the NFAT:AP-1 reporter in ESCs upon LIF removal. The activity in the cells cultured with LIF was set at 1.0. Data are shown as the mean ± SD (n = 3). See also Figure S2.

suggesting the existence of trophoblastic cell types in the tumor. RT-PCR analysis showed that levels of trophoblast and primitive endoderm markers were markedly higher in teratomas derived from CA-NFATc3-expressing cells than from control cells (Figure 3E). Therefore, the activation of calcineurin-NFAT signaling is sufficient to induce ESCs into early differentiated lineages.

Activation of Calcineurin-NFAT Signaling Triggers an ESC Transition from Self-Renewal to Differentiation To address whether calcineurin-NFAT signaling acts as a permissive signal or if its activation is sufficient to initiate ESC differentiation, we overexpressed constitutively active forms of calcineurin (DCnA) or NFATc1-4 (CA-NFATc1-4) in ESCs. Strikingly, in the presence of LIF, forced expression of CA-NFATc1-4 rapidly induced the differentiated cell morphology (Figure 3A), substantially reduced the mRNA levels of pluripotency genes (Oct4, Nanog, and Rex1), and enhanced the expression of differentiation markers, especially those of the trophectoderm (Cdx2, Hand1) and the primitive endoderm (Gata6, Ihh) (Figure 3B); DCnA induced relatively weaker differentiation than did CANFATc1-4 (Figures 3C). After injecting ESCs overexpressing CA-NFATc3 into immunodeficient mice, teratomas containing regional hemorrhages were subsequently detected (Figure 3D),

NFAT Directly Activates Src Expression To identify the downstream molecules regulated by calcineurin-NFAT signaling, we examined the expression of a repertoire of genes while calcineurin-NFAT signaling was either activated or inhibited. We uncovered 306 candidate targets, including molecules involved in cell migration and focal adhesions (Figures S3A and S3B; Table S2). To find direct NFAT targets, the promoter sequences of these candidate genes were examined for binding sites of NFAT and AP-1, the common cofactor of NFAT (Macia´n et al., 2001; Sanna et al., 2005). The consensus NFAT and AP-1 binding sites were found upstream of the nonreceptor tyrosine kinase Src (also known as c-Src) gene, and these elements were well conserved (Figure S3C). AP1 is required for Src expression (Jiao et al., 2008; Kumagai et al., 2004), whereas the involvement of NFAT in Src expression has not been reported. The results of qRT-PCR analysis showed that the tetracycline (Tc)-inducible expression of a constitutively active NFATc3 significantly elevated Src transcript levels in an induction-time-dependent manner (Figure 4A), and the transient overexpression of CA-NFATc3 also increased Src protein levels (Figure 4B). Moreover, the transient expression of DCnA or CA-NFATc1-4 substantially upregulated Src transcript levels (Figure S3D), comparable to the effect of activated Ras (Figure S3E). Strikingly, the activation of Src expression and kinase activity by LIF withdrawal was completely abolished by CsA treatment (Figure 4C; Figure S3F), indicating Cell Stem Cell 8, 46–58, January 7, 2011 ª2011 Elsevier Inc. 49

Cell Stem Cell NFAT Pathway Regulates Early Lineage Specification

Figure 3. Activation of Calcineurin-NFAT Signaling Triggers ESC Transition from Self-Renewal to Lineage Commitment (A) Morphological changes of ESCs induced by the transient overexpression of the constitutively active form of calcineurin (DCnA) or the constitutively active form of NFATc1-4 (CA-NFATc1-4). CGR8 ESCs were grown under selection for 3 days after transfection. Scale bars represent 50 mm. (B and C) qRT-PCR analysis of marker expression levels in cells described in (A). The mRNA level in cells transfected with the control vector was set at 1.0. Data are shown as the mean ± SD (n = 3). (D) The images of teratomas derived from control cells or CA-NFATc3 transiently expressing cells. (E) RT-PCR analysis of gene expression of three teratomas from control cells and hemorrhagic teratomas from CA-NFATc3 transiently expressing cells.

a regulatory role for calcineurin-NFAT signaling in endogenous Src expression. To test whether NFAT directly regulates Src expression, electrophoretic mobility shift assays (EMSAs) were conducted with a Src oligo probe (containing the NFAT consensus sequence from the Src promoter), an Il2 control probe, and nuclear extracts from ESCs overexpressing CA-NFATc3 (Figure 4D). NFATc3-containing DNA-protein complexes were identified by the appearance of a super-shifted band when NFATc3 antibody was included in the incubation mixture, which was identical to the super-shifted band observed with the control Il2 probe. To test the functional significance of the NFAT binding site for Src expression, we performed luciferase reporter assays and verified critical roles for NFAT and AP-1 binding sites in Src transcription in response to NFATc3 (Figure 4E). Finally, chromatin immunoprecipitation (ChIP) assays showed an enrichment of NFATc3 at the Src gene, but not at the Cdx2 regulatory region during ESC differen50 Cell Stem Cell 8, 46–58, January 7, 2011 ª2011 Elsevier Inc.

tiation induced by either LIF withdrawal or CA-NFATc3 overexpression (Figure 4F). These observations revealed that endogenous NFATc3 or ectopically expressed NFATc3 binds to the Src regulatory sequence in differentiating ESCs but not in undifferentiated ESCs. Src Is Required for Calcineurin-NFAT-Induced ESC Differentiation We next examined whether Src could initiate ESC differentiation. Transient expression of a sustained active form of Src (Src F) (Bra´bek et al., 2004) resulted in the rapid appearance of the differentiated cell morphology (Figure 5A) with the simultaneous downregulation of pluripotency markers and the activation of differentiation genes. Markers robustly activated by Src F included trophectoderm markers (Cdx2, Hand1, Prl2c2, and Psx1) followed by primitive endoderm lineage markers, such as Gata6 (Figure 5B), which is similar to the expression pattern

Cell Stem Cell NFAT Pathway Regulates Early Lineage Specification

Figure 4. NFAT Directly Activates Src Expression (A) qRT-PCR analysis of dynamic expression patterns of Src after the induction of CA-NFATc3 expression by removing Tc (0.5 mM) in iNFATc3ES cells. The relative mRNA values in control cells were set at 1.0. Data are shown as the mean ± SD (n = 3). (B) Western blot analysis of Src protein levels after transient overexpression of CA-NFATc3. Tubulin was used as a control. The number indicates the relative density of specific bands from the western blot measured by densitometer and normalized by the density of Tubulin (n = 3). (C) qRT-PCR analysis of Src mRNA levels in ESCs cultured under the indicated conditions for 4 days. Data are shown as the mean ± SD (n = 3). (D) EMSAs via the Src or Il2 probe and the nuclear extract of E14T ESCs transiently overexpressing CA-NFATc3 for 2 days. (E) Luciferase assays with the wild-type (wt) Src promoter luciferase reporter with or without mutations in the NFAT (NFATm) or AP-1 (AP-1 m) element in ESCs. The activity of wt Src promoter reporter cotransfected with empty vector was set at 1.0. Data are shown as the mean ± SD (n = 3). (F) ChIP assays demonstrating the capacity of NFATc3 to bind to the Src upstream fragment during ESC differentiation induced by LIF withdrawal or Tc-induced CA-NFATc3 expression. See also Figure S3 and Table S2.

induced by activated NFAT. To determine whether Src is essential for NFATc3- or LIF withdrawal-initiated ESC differentiation, PP2 (Hamadi et al., 2009), a specific Src inhibitor, was used. Strikingly, PP2 efficiently abolished ESC differentiation induced by CA-NFATc3 overexpression or LIF withdrawal, as determined by cell morphology and marker expression (Figures 5C–5F). Interestingly, CsA and PP2 combined treatment sustained a faster cell growth rate than CsA treatment alone (Figure S1C). In addition to PP2, SKI-1, another Src inhibitor, also abrogated LIF withdrawal-induced differentiation (Figures S4A and S4B). Specifically, the knockdown of Src by specific RNAi also markedly attenuated NFATc3- or LIF withdrawalinduced differentiation (Figure 5G; Figure S4D), whereas the depletion of another Src family kinase, Lck, did not block differentiation (Figures S4C and S4D). Our results indicate that Src, like NFAT, is both necessary and sufficient for ESC differentiation. NFAT-Src-Mediated EMT Is an Essential Step for Lineage Specification Src is closely associated with EMT, and many EMT inducers (Tgfb, Fgf family members, Ras-Erk1/2) also induce ESC differentiation (Guarino, 2010). We therefore asked whether EMT was an essential step for ESC differentiation. In addition to the

induction of ESC differentiation, Src F overexpression led to the rapid appearance of typical EMT characteristics (Mandal et al., 2008; Thiery et al., 2009), including the activation of EMT markers such as Igf2, SIP1, Ncad, and Snai1, as well as upregulated matrix metalloproteinases (MMPs), which are critical components of EMT (Figure 6A; Cavallaro and Christofori, 2004). The redistribution of E-cadherin, an epithelial cell marker, was also observed from the membrane to the cytoplasm in Src F-expressing cells (Figure 6B). As upstream activators of Src, both NFAT and Ras also increased expression of the EMT markers (Figures S5A and S5B). Furthermore, E-cadherin redistribution was observed upon either overexpressing active NFATc3 or withdrawing LIF (Figures S5C and S5D). To define the relationship between EMT and ESC lineage commitment, the kinetics of gene expression were examined. After active NFATc3 induction, the elevation of EMT genes (on day 1) occurred prior to the activation of lineage markers (on day 1.5) and the downregulation of pluripotency genes (on day 3) (Figure 6C), suggesting that the EMT process precedes ESC differentiation. Furthermore, the broad-spectrum MMP inhibitor GM6001, known to prevent EMT (Tan et al., 2010), blocked NFATc3- induced ESC differentiation (Figures 6D and 6E) and partially blocked LIF withdrawal-induced ESC differentiation (Figures S5E and S5F). The calcineurin inhibitor CsA and Cell Stem Cell 8, 46–58, January 7, 2011 ª2011 Elsevier Inc. 51

Cell Stem Cell NFAT Pathway Regulates Early Lineage Specification

Figure 5. Src Is Required for Calcineurin-NFAT-Induced ESC Differentiation (A) Morphological changes in ESCs induced by the constitutively active form of Src (Src F). CGR8 ESCs were grown under selection for 3 days after transient transfection. Scale bars represent 50 mm. (B) qRT-PCR analysis of marker expression levels in cells described in (A). The expression level in cells transfected with the control vector was set at 1.0. Data are shown as the mean ± SD (n = 3). (C) Blockade of NFATc3-mediated ESC differentiation by Src inhibitor PP2. Phase contrast images of iNFATc3ES cells cultured under the indicated conditions are shown. CA-NFATc3 was induced by removing Tc (0.5 mM) for 3.5 days in iNFATc3 ESCs. Scale bars represent 100 mm. (D) qRT-PCR analysis of marker expression levels in cells described in (C). The expression level in control cells without PP2 was set at 1.0. Data are shown as the mean ± SD (n = 3). (E) Abolishment of LIF withdrawal-induced ESC differentiation by PP2. Phase contrast images of CGR8 ESCs cultured under the indicated conditions for 3 days are shown. Scale bars represent 100 mm. (F) qRT-PCR analysis of marker expression levels in cells described in (E). The relative mRNA level in cells cultured with LIF was set at 1.0. Data are shown as the mean ± SD (n = 3). (G) Blockade of NFATc3-mediated ESC differentiation by Src knockdown via RNAi. qRT-PCR analysis of marker expression levels in cells in the presence or absence of Src siRNAs. Expression of CA-NFATc3 was induced by removing Tc (0.5 mM) for 2 days in iNFATc3ES cells. The level in control cells with control-siRNA was set at 1.0. Data are shown as the mean ± SD (n = 3). See also Figure S4.

the Src inhibitor PP2 also efficiently abrogated the activation of EMT markers caused by LIF withdrawal in a dose-dependent manner (Figures S5G and S5H), indicating that these inhibitors 52 Cell Stem Cell 8, 46–58, January 7, 2011 ª2011 Elsevier Inc.

may maintain ESC self-renewal through the inhibition of EMT. Taken together, our data indicate that EMT is an essential step required for differentiation events.

Cell Stem Cell NFAT Pathway Regulates Early Lineage Specification

Figure 6. NFAT-Src-Mediated EMT Is an Essential Step for Lineage Specification (A) qRT-PCR analysis of EMT-related markers in CGR8 ESCs transiently overexpressing Src F. The value in cells transfected with the control vector was set at 1.0. Data are shown as the mean ± SD (n = 3). (B) Redistribution of E-cadherin from the membrane to the cytoplasm stimulated by the inducible expression of Src F. Immunostaining of E-cadherin was conducted in iSrcES cells after removing Tc for 3 days. Scale bars represent 25 mm. (C) The time course of marker gene expression after inducible expression of NFATc3 in ESCs for different days. The qRT-PCR analysis was conducted to determine the gene expression level and the value in control cells was set at 1.0. Data are shown as the mean ± SD (n = 3). (D) Blockade of NFATc3-mediated ESC differentiation by the broad-spectrum MMP inhibitor GM6001. Phase contrast images of iNFATc3ES cells cultured under the indicated conditions for 3 days are shown. Scale bars represent 100 mm. (E) qRT-PCR analysis of marker expression levels in cells described in (D). The value in control cells was set at 1.0. Data are shown as the mean ± SD (n = 3). See also Figure S5.

The Calcineurin-NFAT Signaling Cascade Is Indispensable for Early Embryo Development Finally, we investigated the expression and possible role for NFAT in mouse embryos prior to implantation. Intriguingly, immunofluorescence staining of mouse embryos revealed that NFATc3 was primarily localized to the cytoplasm in ICM cells at E3.5 and the epiblast cells at E4.5, which is the same as in undifferentiated ESCs. However, NFATc3 was detected in the nucleus of the differentiated trophectoderm of the blastocyst. As a control, Oct4 was exclusively found in the nucleus of epiblast cells (Figure 7A). This expression pattern indicates that NFATc3 was inactive in undifferentiated cells and became activated upon the earliest lineage commitment. Thus, the differential activation of NFAT signaling is clearly identifiable among the earliest lineages during mouse embryonic development. To test whether this calcineurin-NFAT pathway is functionally relevant to early lineage segregation, CsA was used to treat 8-cell mouse embryos, which markedly increased the percent-

age of embryos that stopped development at the morula stage and attenuated the percentage of embryos developing into the blastocoele formation (Figure 7B). Confocal immunofluorescence analysis showed that embryos treated with CsA contained few Cdx2-positive trophectoderm cells, and the majority of cells expressed Oct4 (Figure 7C). Moreover, NFATc3 was detected in the nucleus of trophectoderm cells in control embryos and in the cytoplasm of CsA-treated embryos (Figure S6A). These observations indicate that the inhibition of calcineurin-NFAT signaling blocks embryo development at the morula stage. To test whether the effect of CsA on trophectoderm formation is reversible, 8-cell embryos were treated with CsA for 1.5 days and then cultured for an additional 1.5 days in the control medium. Interestingly, embryo development resumed, and numerous Cdx2positive cells appeared in the developing blastocyst after CsA withdrawal. Moreover, CsA treatment after culture of the 8-cell embryo in the control medium for 1.5 days, when the trophectoderm is thought to have already formed, did not disturb Cell Stem Cell 8, 46–58, January 7, 2011 ª2011 Elsevier Inc. 53

Cell Stem Cell NFAT Pathway Regulates Early Lineage Specification

Figure 7. Calcineurin-NFAT Signaling Is Indispensable for Early Mouse Embryonic Development (A) Optical sections of the mouse embryo immunofluorescently stained with NFATc3 (red) and Oct4 (green) antibodies. Scale bars represent 25 mm. (B) The effect of CsA and PP2 on the kinetics of blastocoele formation. Mouse embryos were treated from 8-cell stage (E2.5) and observed for the presence of blastocoeles after 1.5 days. (C) Confocal images of embryos grown from the 8-cell stage (E2.5) for 1.5 days in control medium or in the medium containing CsA (2.5 mM). Embryos were immunostained with antibodies against Oct4 (green) and Cdx2 (red), respectively. Scale bars represent 25 mm. (D) The effect of PP2 on the trophectoderm development. Embryos from the 8-cell stage were treated with or without PP2 for 1.5 days and were immunostained as in (C). Scale bars represent 25 mm. (E) A bar chart showing the percentage of cell numbers of the ICM and the trophectoderm of embryos cultured under the conditions described in (D). Data are shown as the mean ± SD. (F) The effect of CsA and PP2 on the primitive endoderm development. Confocal images of embryos grown from E3.25 stage for 1.5 days in control medium or in medium containing CsA (1.75 mM) or PP2 (10 mM) are shown. Embryos were immunostained with antibodies against Nanog (green) and Gata4 (red), and nuclei were counterstained with DAPI. Scale bars represent 25 mm. (G) Bar chart showing the percentage of cell numbers of the epiblast (green) and primitive endoderm (red) of embryos cultured in the conditions described in (F). Data are shown as the mean ± SD. (H) A proposed model for the signaling circuit involved in ESC differentiation. See also Figure S6.

trophectoderm development evidently (Figure S6B). Therefore, the blockage of embryos at the morula stage might be due to the inability of morula cells to differentiate into trophectoderm cells when calcineurin-NFAT signaling is inhibited. We also 54 Cell Stem Cell 8, 46–58, January 7, 2011 ª2011 Elsevier Inc.

examined the effect of the Src inhibitor, PP2, on trophectoderm formation of early mouse embryos. PP2 treatment partially blocked embryos at the morula stage (Figure 7B), and in embryos developing into blastocysts, PP2 treatment significantly reduced Cdx2-positive trophectoderm cells and enhanced the Oct4-positive ICM cells compared to control embryos (Figures 7D and 7E). Finally, to determine whether calcineurin-NFAT signaling was also required for formation of the primitive endoderm, embryos of E3.25 were treated with CsA or PP2 and costained for Nanog and Gata4. Immunofluorescence staining revealed that CsA or PP2 treatment eliminated Gata4-positive primitive endoderm cells and increased the Nanog-positive cell number (Figures 7F and 7G), implicating an indispensable role of the signaling pathway

Cell Stem Cell NFAT Pathway Regulates Early Lineage Specification

for the segregation of the primitive endoderm from the ICM. Taken together, these data suggest that the calcineurin-NFATSrc signaling cascade is activated and indispensable for extraembryonic development in early mouse embryos. DISCUSSION Our data establish a model whereby calcineurin-NFAT signaling collaborates with Ras-Erk-AP-1 to activate Src, promote EMT, and initiate lineage specification in mouse ESCs (Figure 7H). We demonstrate that the calcineurin-NFAT-Src cascade critically regulates the transition of ESCs from self-renewal to lineage commitment, having a similar role to Ras-Erk1/2 signaling, which was previously shown to be crucial for ESC differentiation (Kunath et al., 2007; Nichols et al., 2009). Interestingly, the activation of Ras-Erk1/2 and calcineurin-NFAT signaling resulted in nearly identical gene expression profiles in mouse ESCs (Figure 3B; Figure S4E). Moreover, we found that the two pathways were mutually dependent as shown by the fact that the blockade of either one significantly attenuated the ESC differentiation phenotypes induced by the other one (Figures S4F–S4I), which is in line with a previous study conducted in cardiomyocytes (Sanna et al., 2005). The cooperation that was discovered between Ras-Erk1/2 and calcineurin-NFAT signaling provides new insights into early ESC differentiation events; these two distinct signaling pathways could be integrated to precisely regulate ESC fates, depending upon whether both pathways are concomitantly activated and which distinct sets of target genes are activated. Mechanistically, Ras-Erk1/2 augments NFAT transcriptional activity through the activation of AP-1, which complexes with NFAT at their coregulated genes (Macia´n et al., 2001). Importantly, we uncovered Src as one of these genes, which functionally phenocopied both NFATc3 and Ras in ESCs. Moreover, Src inhibition abrogated NFAT- or Ras-triggered ESC differentiation (Figures 5C and 5D; Figures S4J and S4K). The involvement of Src family kinases in the regulation of ESC self-renewal and differentiation has been previously studied; individual members of the Src family play distinct and possibly opposite roles in the control of ESC fates (Meyn et al., 2005; Meyn and Smithgall, 2009). One Src family member, c-Yes, was reported to be important for ESC self-renewal (Annere´n et al., 2004), whereas Src was found to activate primitive ectoderm formation in mouse ESCs (Meyn and Smithgall, 2009). The latter finding is consistent with our observation that the expression of constitutively active Src induces ESC differentiation. Therefore, we propose that calcineurin-NFAT and Ras-Erk1/2 signaling pathways converge to regulate Src expression and that Src might be a critical mediator for both signaling pathways. Nevertheless, we do not rule out other possible mechanisms for ESC differentiation induced by these two important signaling pathways. The molecular mechanism by which Src regulates ESC differentiation remains unknown. In this study, we propose that the promotion of EMT might account for the role of Src in ESC differentiation. EMT regulates multiple critical processes during early development, and development cannot proceed past the blastula stage without EMT (Thiery and Sleeman, 2006). Recently, the involvement of EMT in ESC differentiation has been described (Eastham et al., 2007; Spencer et al., 2007), although

its role in ESC differentiation is not well defined. It is not clear whether EMT is essential or merely a concomitant event during differentiation. Our study indicates that differentiation stimuli induce EMT prior to the differentiation processes and that inhibition of EMT blocks ESC differentiation, thus placing EMT as an early and essential step in lineage specification. In addition, we found that the GSK3b inhibitor CHIR99021 suppressed the expression of EMT-related genes after LIF withdrawal (Figures S5I and S5J) in a manner similar to the calcineurin-NFAT, RasErk1/2, Src, and MMP inhibitors. GSK3b regulates focal adhesion kinase (FAK) (Bianchi et al., 2005; Kobayashi et al., 2006), an important player in EMT, which might explain the effect of GSK3b inhibitors in suppressing ESC differentiation (Ullmann et al., 2008; Ying et al., 2008). Furthermore, some ESC-specific transcription factors were found to bind promoters of EMTrelated genes (Figure S5K; Chen et al., 2008). Interestingly, EMT inhibition was recently found to promote somatic cell reprogramming (Lin et al., 2009). Therefore, EMT appears to be essential for ESC differentiation, and its inhibition may promote differentiated cells to revert to a pluripotent state. Another important advance of this study is the discovery that calcineurin-NFAT signaling is activated during the first differentiation event in the preimplantation embryo and is essential for early embryo development. We show that the subcellular localization of NFATc3 proteins is different between ICM and trophectoderm cells of the blastocyst, although they are detected in both cell types. The NFAT activity is tightly regulated by phosphorylation and dephosphorylation; NFAT is activated by calcineurin-dependent dephosphorylation, which stimulates NFAT translocation from the cytoplasm into the nucleus. Therefore, our data showing that NFAT localizes to the nucleus of the trophectoderm suggest that NFAT is activated in the trophectoderm. Moreover, the reversible and selective effect of CsA on the trophectoderm formation at the restricted stage, as well as the inhibitory effect of CsA and PP2 on the primitive formation, argues for the specific and essential role of the calcineurinNFAT-Src cascade in extraembryonic lineage specification during early embryonic development. In summary, this study reveals a role for a well-characterized signaling pathway in ESC differentiation and early embryonic development. The identification of calcineurin-NFAT signaling as one of the initial triggers of the ESC exit from self-renewal, and of Src as a key player downstream of NFAT and Erk1/2 in ESC early fate determination, provides new insights into how the self-renewal of pluripotent stem cells is orchestrated. Improved understanding of the role that EMT plays in ESC differentiation also opens up additional avenues for developing more efficient platforms for ESC programming and somatic cell reprogramming.

EXPERIMENTAL PROCEDURES Cell Culture and Differentiation E14T and CGR8 mouse ESCs (gift of Austin Smith) were grown as previously described (Li et al., 2010). To induce differentiation, CGR8 cells were cultured with 0.1 mM RA (Sigma). Tetracycline (Tc)-inducible iDCnAES, iNFATc3ES, and iSrcES cell lines were established with the Rosa-Tet system (Masui et al., 2005), and the cells were maintained in medium containing Tc (0.5 mM). Exogenous gene expression was induced after removal of Tc.

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Cell Stem Cell NFAT Pathway Regulates Early Lineage Specification

Luciferase Assays CGR8 ESCs were seeded at a density of 1.1 3 105 per well into 24-well tissueculture plates 24 hr before transfection. For NFAT:AP-1 reporter assays, LIF was removed on different days, as indicated in the text. To determine the endogenous activity, cells were transfected with reporter plasmids (500 ng) and vector pRL-TK (20 ng, Promega) as a control for the transfection efficiency via Lipofectamine 2000 (Invitrogen). To examine the effect of exogenously expressed factors on the reporters, control vector and CA-NFATc3 expression plasmids were cotransfected with reporters and pRL-TK. Cell extracts were prepared 48 hr after transfection. Luciferase activity was evaluated with the Dual-Luciferase Assay System (Promega) according to the manufacturer’s recommendations.

Statistical Analysis All values are shown as means ± SD. To determine the significance between groups, comparison was made with Student’s t test. For all statistical tests, the 0.05 confidence level was considered statistically significant. In all figures, * denotes p < 0.05 and ** denotes p < 0.01 in an unpaired Student’s t test. ACCESSION NUMBERS Microarray data are accessible at the GEO database under accession number GSE21378. SUPPLEMENTAL INFORMATION

ChIP Assays CGR8 ESCs were cross-linked by incubating cells on plates with 1% formaldehyde, and ChIP assays were carried out as described (Li et al., 2010). Ten percent of the total genomic DNA from the nuclear extract was used as the input. The primers used are provided in Table S3.

Supplemental Information includes Supplemental Experimental Procedures, six figures, and three tables and can be found with this article online at doi:10.1016/j.stem.2010.11.027. ACKNOWLEDGMENTS

EMSA Nuclear extracts isolated from E14T ESCs overexpressing CA-NFATc3 or the vector alone for 2 days were prepared, and EMSAs were performed as described previously (Yang et al., 2008). In brief, the oligonucleotide probes were synthesized and labeled with biotin at the 50 end of the forward oligonucleotide. For the competitive assay, an additional 200-fold molar excess of the unlabeled probe was added. For super-shift analysis, NFATc3 antibody (2 ml per reaction) was added. Probe sequences are provided in Table S3. RNAi and Oligonucleotides siRNAs were introduced into cells according to the manufacturer’s instructions. The oligonucleotide sequences for mouse Ppp3r1, Src, and Lck are provided in Table S3. The negative controls were obtained from Invitrogen (Stealth RNAi Negative Control Med GC). Western Blot Analysis Protein (30 mg) from whole ESC extracts was used for western blot analysis as described previously (Li et al., 2010). RT-PCR and qRT-PCR Analysis RT-PCR and qRT-PCR were conducted as previously described (Li et al., 2010), and the primers used are provided in Table S3. Generation of Teratomas For teratoma generation, 5 3 106 ESCs were harvested and injected intramuscularly into nude mice. 6 to 8 weeks later, teratomas were harvested and processed with hematoxylin and eosin staining.

The authors wish to thank Drs. H. Niwa, A. Rao, S. Miyatake, X. Cao, S. Hanks, T. Xu, and T. Takeya for generously providing plasmids; Drs. A. Smith and G. Daley for their mouse ESC lines, and Drs. A. Smith, R.D. McKinnon, D. Li, and A. Chong for critical reading of the manuscript. This study was supported by grants from the National Natural Science Foundation (91019929, 30911130361, and 31000625) and National High Technology Research and Development Program of China (2009CB941100, 2007CB947904, 2007CB948004, 2101CB945200, and 2007CB947101) and from Shanghai Science & Technology Developmental Foundations (08dj1400502 and 07DZ22919). The study was also supported by the Shanghai Leading Academic Discipline Project (S30201). Received: June 2, 2010 Revised: October 2, 2010 Accepted: October 25, 2010 Published: January 6, 2011 REFERENCES Annere´n, C., Cowan, C.A., and Melton, D.A. (2004). The Src family of tyrosine kinases is important for embryonic stem cell self-renewal. J. Biol. Chem. 279, 31590–31598. Bianchi, M., De Lucchini, S., Marin, O., Turner, D.L., Hanks, S.K., and VillaMoruzzi, E. (2005). Regulation of FAK Ser-722 phosphorylation and kinase activity by GSK3 and PP1 during cell spreading and migration. Biochem. J. 391, 359–370.

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