BMP-SMAD Signaling Regulates Lineage Priming but Is Dispensable for Self-Renewal in Mouse Embryonic Stem Cells

摘要

class="head no_bottom_margin" id="sec1title">IntroductionCulture conditions affect features of mouse embryonic stem cells (mESCs), such as their proliferation, gene expression, epigenetic status, self-renewal, and capacity for multi-lineage differentiation (, ). In culture medium with fetal calf serum, naive mESCs grown on mouse embryonic fibroblast feeder cells (here abbreviated as “serum”) transit between inner cell mass (ICM)-like and epiblast-like pluripotency states (, ). However, when cultured in serum-free conditions with inhibitors of mitogen-activated protein kinase and glycogen synthase kinase 3 signaling, also called “2i” medium, mESCs become more homogeneous and adopt the more ICM-like or “ground” state (, , ). The observation that naive mESCs interconvert between pluripotent states while remaining uncommitted has raised the suggestion that such heterogeneity may allow the cells to respond differently to environmental cues. In agreement, subpopulations of naive mESCs show different potentials to differentiate (, , ). How the metastable transcriptional and epigenetic diversity of cultured mESCs is regulated and maintained has remained elusive.The two notable characteristics of mESCs are their capacity to self-renew and differentiate into all embryonic lineages (). In mESCs, pluripotency is maintained by a core network of regulatory transcription factors, including Pou5f1, Sox2, and Nanog (, , , ); the balance between self-renewal and differentiation is regulated by protein-encoding genes that include Id1 and Dusp9, both downstream targets of the bone morphogenetic protein (BMP) signaling pathway (href="#bib18" rid="bib18" class=" bibr popnode">Li and Chen, 2013). Moreover, it has been shown that both the BMP and TGFβ (via NODAL) SMAD-mediated signaling pathways are involved in maintaining heterogeneity of NANOG in naive mESCs (href="#bib5" rid="bib5" class=" bibr popnode">Galvin-Burgess et al., 2013). Conversely, NANOG may attenuate BMP signaling via a feedback loop that involves titration of phosphorylated (P)SMAD1 by direct NANOG-SMAD1 interaction (href="#bib32" rid="bib32" class=" bibr popnode">Suzuki et al., 2006). However, the functional role of BMP-SMAD signaling in the metastable state of naive pluripotency has not been investigated.Here, we report the derivation and characterization of transgenic mESCs that allow a real-time readout of SMAD-mediated BMP signaling activity. This transgenic BRE:gfp reporter mESC line expresses a well-characterized BMP responsive element (BRE) containing several PSMAD1/5 DNA-binding sites isolated from the Id1 promoter to drive GFP expression (href="#bib16" rid="bib16" class=" bibr popnode">Korchynskyi and ten Dijke, 2002, href="#bib21" rid="bib21" class=" bibr popnode">Monteiro et al., 2008). Activation of the BMP-SMAD reporter transgene was heterogeneous in serum mESCs (±50% GFP + cells) and 2i mESCs (±4% GFP + cells). By genetic abrogation of the core BMP pathway components SMAD1 and SMAD5, we demonstrated that BMP-SMAD signaling is dispensable for the maintenance and self-renewal of mESCs both in serum and 2i states, but that it regulates the levels of DNA methylation (via Dnmt3a/b and Tet1/2) and hence lineage priming in pluripotent mESCs.