Abstract
Activation of the FGF-ERK pathway is necessary for naïve 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 self-renewal 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.
Highlights
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 [1,2,3]
Heterozygous ES cell clones were first screened for reduction of exon 3 alleles by quantitative PCR and putative targeted lines were confirmed by Southern blotting (Figure 1B)
We have found that ES cells compensate for the loss of ERK2 by hyper-phosphorylation of the remaining ERK1 isozyme without an increase in Erk1 transcription or translation
Summary
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 [1,2,3]. 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]
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