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.

Embryonic stem cells (ESCs) possess the capacity to proliferate indefinitely in an undifferentiated state and to differentiate into various cell types in an organism. However, the critical question of how self-renewal and differentiation are precisely regulated in ESCs is not entirely understood at present. Here, we report the essential role of Tbx3, a pluripotency-related transcription factor of the T-box gene family, for both the maintenance of self-renewal of mouse ESCs and for their differentiation into extraembryonic endoderm (ExEn). We show that Tbx3 is highly expressed in ExEn cells in addition to undifferentiated ESCs. Knockdown of Tbx3 expression using tetracycline-regulated Tbx3 siRNA resulted in the attenuation of ESC self-renewal ability and aberrant differentiation processes, including reduced ExEn differentiation but enhanced ectoderm and trophectoderm differentiation. Conversely, inducible forced expression of Tbx3 triggered ExEn lineage commitment. Mechanistically, Tbx3 directly activated the expression of Gata6, an essential regulator of ExEn. Interestingly, Tbx3 modulated H3K27me3 modification and the association of the PRC2 complex with the promoter region of Gata6. Taken together, the results of this study revealed a previously unappreciated role of a pluripotency factor in ExEn differentiation. Additionally, our data reveal that Tbx3 may function through direct binding and epigenetic modification of histones on the Gata6 promoter to maintain the ExEn differentiation potential of ESCs.The mouse blastocyst contains a trophectodermal epithelium surrounding an inner cell mass that later gives rise to primitive endoderm and epiblast cells. The primitive endoderm will develop into extraembryonic endoderm (ExEn), 2 which ultimately contributes to the yolk sac, whereas the epiblast contains pluripotent cells that generate all of the cell types of the three embryonic germ layers (ectoderm, endoderm, and mesoderm) and some extraembryonic tissues (1, 2). Mouse embryonic stem cells (ESCs) derived from the inner cell mass of blastocyst-stage embryos retain the capacity for unlimited self-renewal and their full developmental potential when cultured in vitro in the presence of leukemia inhibitory factor (LIF). Withdrawal of LIF leads to the disruption of ESC selfrenewal and extensive differentiation into various lineages. Understanding the mechanisms governing self-renewal and early lineage commitment is an essential step toward the future utilization of ESC derivatives in clinical applications. The unique properties of ESCs are conferred by a set of pluripotency-associated transcription factors, including Oct4, Sox2, and Nanog (3-6). Recent studies of the transcriptome and protein interactome of ESCs have further revealed the key role of Oct4/Sox2/Nanog-centered transcriptional and protein interaction networks in controlling ESC identity (7-11). However, the precise molecular mechanisms through which ESCs determine the cell fate between self-renewal and lineage commitment remain largely unknown. In additi...