The two notable characteristics of

The two notable characteristics of mESCs are their capacity to self-renew and differentiate into all embryonic lineages (Niwa et al., 1998). In mESCs, pluripotency is maintained by a core network of regulatory transcription factors, including Pou5f1, Sox2, and Nanog (Kashyap et al., 2009; Kim et al., 2008; Marson et al., 2008; Navarro et al., 2012); 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 (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 (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 (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 Senexin A (BRE) containing several PSMAD1/5 DNA-binding sites isolated from the Id1 promoter to drive GFP expression (Korchynskyi and ten Dijke, 2002; 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.

Results

Discussion
A recent study reported the absence of Bmp4 and Id1 in (embryonic day) E3.5 ICMs and a high transient upregulation in E4.5 epiblasts, followed by downregulation of Bmp4 and Id3 expression during the next 6 days of the derivation of mESCs and their further maintenance in 2i (Boroviak et al., 2014). We now show this in real-time using BRE:gfp blastocysts to derive mESCs. Moreover, we demonstrated that BMP-SMAD signaling is not functionally implicated in self-renewal, in agreement with studies that have mapped genome-wide the genes that are directly regulated by SMAD1/5 (Chen et al., 2008; Fei et al., 2010). They showed that the genes regulated by SMAD1/5 were involved in fate determination, rather than self-renewal. Here, we provide functional evidence that SMAD1/5 are not necessary for mESC self-renewal in either naive (serum) or ground (2i) state.
Specific levels of DNA methylation and associated enzymes have been associated with the different pluripotency states (ground, naive, primed) (Habibi et al., 2013; Hackett et al., 2013; Smallwood et al., 2014), as well as with different levels of GFP in Nanog:gfp naive mESCs (Ficz et al., 2013). This reflects faithfully the rapid loss of genomic DNA methylation that the embryo undergoes in vivo during pre-implantation development, and the gain of DNA methylation during the transition between ICM and epiblast (Smith et al., 2012). Therefore, it is perhaps not surprising that the machinery to regulate rapid switches in genomic DNA methylation is present in pluripotent stem cells derived from ICM and epiblast. A role for BMP-SMAD signaling in LIF-dependent conversion between EpiSCs and ESCs has been reported (Onishi et al., 2014), but the association with changes in DNA methylation between EpiSCs and ESCs remains to be investigated.
Finally, it has been suggested that the epigenetic variation observed in pluripotent cells is stochastic and results in a diversity of predispositions to acquire specific cell fates when the cells are triggered to differentiate (Lee et al., 2014). Our data provide evidence that the cellular diversity of both serum and 2i mESCs regarding DNA methylation and associated enzymes is not a stochastic process as previously thought, but is in fact regulated by cell-cell signaling interactions involving the BMP-SMAD signaling pathway.