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

摘要

Highlights ? BMP-SMAD signaling in mESCs is more prominent in naive than ground state ? BMP-SMAD signaling is dispensable for pluripotency in mESCs ? BMP-SMAD signaling facilitates lineage priming in mESCs ? BMP-SMAD signaling regulates Dnmt3b and hence levels of DNA methylation Summary Naive mouse embryonic stem cells (mESCs) are in a metastable state and fluctuate between inner cell mass- and epiblast-like phenotypes. Here, we show transient activation of the BMP-SMAD signaling pathway in mESCs containing a BMP-SMAD responsive reporter transgene. Activation of the BMP-SMAD reporter transgene in naive mESCs correlated with lower levels of genomic DNA methylation, high expression of 5-methylcytosine hydroxylases Tet1/2 and low levels of DNA methyltransferases Dnmt3a/b . Moreover, naive mESCs, in which the BMP-SMAD reporter transgene was activated, showed higher resistance to differentiation. Using double Smad1;Smad5 knockout mESCs, we showed that BMP-SMAD signaling is dispensable for self-renewal in both naive and ground state. These mutant mESCs were still pluripotent, but they exhibited higher levels of DNA methylation than their wild-type counterparts and had a higher propensity to differentiate. We showed that BMP-SMAD signaling modulates lineage priming in mESCs, by transiently regulating the enzymatic machinery responsible for DNA methylation. Graphical Abstract Figure options Download full-size image Download as PowerPoint slide prs.rt("abs_end"); Introduction Culture 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 ( Marks et?al., 2012 and Tesar et?al., 2007 ). 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 ( Sasai et?al., 2013 and Trott and Martinez Arias, 2013 ). 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 ( Marks et?al., 2012 , Nichols et?al., 2009 and Ying et?al., 2003 ). 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 ( Graf and Stadtfeld, 2008 , Hanna et?al., 2009 and Hayashi et?al., 2008 ). How the metastable transcriptional and epigenetic diversity of cultured mESCs is regulated and maintained has remained elusive.