ERK2 Suppresses Self-Renewal Capacity of Embryonic Stem Cells, but Is Not Required for Multi-Lineage Commitment

but Is Not Required for Multi-Lineage Commitment. PLoS ONE 8(4): e60907. doi:10.1371/journal.pone.0060907 ERK2 Suppresses Self-Renewal Capacity of Embryonic Stem Cells, but Is Not Required for Multi-Lineage Commitment William B. Hamilton 0 1 2 Keisuke Kaji 0 1 2 Tilo Kunath 0 1 2 Qiang Wu, National University of Singapore, Singapore 0 Current address: The Danish Stem Cell Center (DanStem), University of Copenhagen , Copenhagen , Denmark 1 Funding: WBH was supported by a studentship from the School of Biological Sciences, University of Edinburgh, and the work was funded by the Scottish Funding Council and Parkinson's UK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 MRC Centre for Regenerative Medicine, University of Edinburgh , Edinburgh , United Kingdom Activation of the FGF-ERK pathway is necessary for nave mouse embryonic stem (ES) cells to exit self-renewal and commit to early differentiated lineages. Here we show that genetic ablation of Erk2, the predominant ERK isozyme expressed in ES cells, results in hyper-phosphorylation of ERK1, but an overall decrease in total ERK activity as judged by substrate phosphorylation and immediate-early gene (IEG) induction. Normal induction of this subset of canonical ERK targets, as well as p90RSK phosphorylation, was rescued by transgenic expression of either ERK1 or ERK2 indicating a degree of functional redundancy. In contrast to previously published work, Erk2-null ES cells exhibited no detectable defect in lineage specification to any of the three germ layers when induced to differentiate in either embryoid bodies or in defined neural induction conditions. However, under self-renewing conditions Erk2-null ES cells express increased levels of the pluripotency-associated transcripts, Nanog and Tbx3, a decrease in Nanog-GFP heterogeneity, and exhibit enhanced selfrenewal in colony forming assays. Transgenic add-back of ERK2 is capable of restoring normal pluripotent gene expression and self-renewal capacity. We show that ERK2 contributes to the destabilization of ES cell self-renewal by reducing expression of pluripotency genes, such as Nanog, but is not specifically required for the early stages of germ layer specification. - Pluripotent mouse embryonic stem (ES) cells are immortal, karyotypically stable, cell lines derived from the inner cell mass (ICM) or early epiblast of pre-implantation embryos [13]. They maintain many of the characteristics of their in vivo counterparts even after prolonged periods of culture, not least of which is the ability to generate differentiated cell types of all three germ layers as well as contribute to the germ line [4]. The efficient derivation and maintenance of ES cells is heavily dependent on genetic background [5,6], and supported by the combined activities of leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP) signaling [7]. Both genetic studies [8,9] and whole transcriptome analysis [10] have provided a wealth of information as to the factors that maintain ES cells in an undifferentiated state, and the signaling networks that regulate their expression [11]. Our current understanding is that the combined activities of a core network consisting of the transcription factors OCT4, SOX2 and Nanog act to maintain ES cell identity by inhibiting the expression of lineage affiliated genes [12] and supporting a pluripotent epigenetic signature [10]. Although much is now known about the molecular requirements for efficient ES cell self-renewal, the means by which cells exit this state and acquire the competence to respond to differentiation cues is less well understood. Genetic studies have shown that fibroblast growth factor (FGF) signaling through GRB2-RAS is essential for peri-implantation mouse embryogenesis and the formation of the primitive endoderm lineage [1316]. The ES cell, and presumably ICM/epiblast expression of Fgf4 is driven by a ternary complex consisting of OCT4, SOX2 and the Fgf4 distal enhancer [17], and acts as a major auto-inductive cue for ES cell differentiation, but is largely dispensable for proliferation [18,19]. Moreover, it has been shown that the combined pharmacological inhibition of FGF-ERK and glycogen synthase kinase 3 (GSK3) signaling pathways promotes long-term selfrenewal of ES cells in the absence of both LIF and BMP4 [7]. This dual inhibitor (2i) culture condition permitted the derivation of ES cell lines from previously refractory genetic backgrounds such as CBA [5], and non-obese diabetic (NOD) mice [20], and importantly the establishment of authentic rat ES cells [21,22]. Because ERK2 is the predominant ERK enzyme expressed in ES cells, and because of its early embryonic phenotype [23,24], we sought to determine the effect of Erk2 genetic ablation on FGF signaling, self-renewal and lineage specification. Here we present evidence that ERK2 is necessary for maximal FGF-ERK signaling and its deletion results in a modest, but consistent increase in selfrenewal capacity and reduction in heterogeneity of Nanog expression in self-renewing culture conditions. In contrast to our previous work which indicated a requirement for ERK2 in both neural and mesodermal specification [18], we found that our new Erk22/2 ES cells did not exhibit any significant defects in germ layer specification when induced to differentiate. Either ERK1 or ERK2 could rescue the FGF signaling defects in Erk22/2 cells suggesting these enzymes share some functional redundancy in mouse ES cells. We propose that total combined ERK activity impacts on ES cell self-renewal by reducing pluripotent gene expression and that ERK1 and ERK2 share overlapping functions during the earliest stages of ES cell differentiation. Targeting Erk2 in Mouse ES Cells To disrupt Erk2 function, germ-line competent 129/Ola ES cells (E14Ju) were depleted for exon 3 of the Erk2 gene by two round of homologous recombination (Figure 1A). This strategy removes the region coding for kinase sub-domains V and VI, essential for ERK2 function, and has been previously shown to create a protein-null Erk2 allele [23,25]. Heterozygous ES cell clones were first screened for reduction of exon 3 alleles by quantitative PCR (qPCR) and putative targeted lines were confirmed by Southern blotting (Figure 1B). Two heterozygous Erk2+/2 ES cell clones (D2 and D7) were subjected to a second round of homologous recombination and clones were first screened for complete loss of exon 3 by qPCR, and further confirmed by Southern blotting (Figure 1C). All clones were karyotyped following clonal expansion and retained normal ploidy (Figure S1). Two Erk2-null clones, D7N2 and D2N4, each derived from independent heterozygous clones, D7 and D2, respectively, were selected for further analysis. To test whether the targeted mutation generated the predicted null allele, Erk2 expression was assessed by qRT-PCR and ERK2 protein by western blotting. (...truncated)