The distinct epigenetic and molecular profile of PDGFR subpo

The distinct epigenetic and molecular profile of PDGFRα+ subpopulations was confirmed also by their developmental potential (Figure 5 and Table S1). The double-positive tnf alpha inhibitor could still colonize the epiblast while PrE-primed cells exclusively contributed to PrE derivatives. Again, these in vivo experiments confirmed that PDGFRα+ cells closely represent the PrE precursors. The comparative transcriptome analysis with epiblast-primed cells and with e/cXEN showed that PDGFRα+ subpopulations differentially express genes associated with core/naive pluripotency, and with JAK-STAT, WNT, and FGF signaling pathways (Figure S4). Unexpectedly, PCA, hierarchical clustering, and GSEA with previously available datasets, revealed that globally PDGFRα+ cells resemble more naive ESCs (Figures 6B and 6C; Tables S3, S4, and S5). When compared with single cells obtained from early embryos, PrE-primed cells, as their epiblast counterpart, clustered with cells from the EB-MB stage (∼E3.5–E4.0), further demonstrating that PDGFRα+ steady states mirror the pre-implantation developmental window (Figures 6D and S6A). The mechanisms involved in the regulation of the heterogeneity in vitro (Figure 7) confirmed previous studies in early development. The percentage of PDGFRα+ cells was influenced by: JAK/STAT signaling, shown to support the expansion of PrE in pre-implantation development (Morgani and Brickman, 2015); FGF signaling, known to control the segregation of PrE and epiblast in the ICM (Yamanaka et al., 2010) and amount of DNA methylation, is a dispensable mechanism for the growth of extraembryonic lineages (Sakaue et al., 2010). By contrast, absence of PDGFRα, necessary for the derivation of eXEN (Artus et al., 2010) and cXEN (Cho et al., 2012), did not alter the abundance of PDGFRα+ cells in vitro. Together, these results confirm that PDGFRα+ cells are the in vitro equivalent of PrE precursors. This model, which relies on the endogenous heterogeneous expression of PDGFRα, should facilitate and enable studies to gain insights in the factors regulating the early segregation of these different cell types within the ICM and to unravel the mechanisms involved in the different imprinting of embryonic and extraembryonic tissues (Hudson et al., 2010). Future studies are needed to determine whether PrE-primed cells recapitulate the imprinting associated with extraembryonic tissues (i.e., paternal imprinting of X chromosome) and whether a similar PrE-primed state is also present in human ESC cultures.
Experimental Procedures
Author Contributions
Acknowledgments We thank the late Vik Van Duppen for FACS experiments, Rob Van Rossom and the KU LEUVEN FACS CORE for sorting, Zhiyong Zhang and Liesbeth Vermeire from InfraMouse for chimera production, and Kristel Eggermont for help with image acquisition/processing. We thank Dr. Hadjantonakis and Dr. Niakan for the FGF4/PDGFRα ESC lines and Dr. Morrison for the Sox17GFP/+ line. We thank Dr. Kian Koh and Joris Vande Velde for dot blots. The work was supported by grants obtained from FWO (G.0832) and KU Leuven (EIW-B4855-EF/05/11 and ETH-C1900-PF to C.M.V.; EME-C2161-GOA/11/012 to C.M.V./A.Z.; C14/16/078 to F.LL.), Hercules Foundation (ZW09/03 to A.Z.) and by the BELSPO-IUAP-DEVREPAIR grant (to C.M.V./S.M.C.d.S.L./A.Z.).
Introduction Adult murine macrophages, in contrast to most other adult hematopoietic cells which renew from hematopoietic stem cells (HSCs) (Gekas et al., 2005), can derive from all three temporally and spatially distinct hematopoietic waves arising in the mouse embryo. A first wave occurs between E7.0 and E7.5 in the blood islands of the yolk sac (YS), producing Myb-independent nucleated erythrocytes, megakaryocytes, and macrophages (Mucenski et al., 1991). From E8.25, a second wave of hematopoietic cells emerge in the YS producing erythromyeloid progenitors (EMPs) (Palis et al., 1999, 2001), via a Runx1-dependent endothelial to hematopoietic transition (EHT) (Chen et al., 2009), that are capable of monocyte, macrophage, granulocyte, megakaryocyte, and erythrocyte differentiation. The presence of some kit+ EMPs (Schulz et al., 2012) and CD11 bF4/80 monocytes in the fetal liver of Myb−/− mouse embryos (Gomez Perdiguero et al., 2015) suggests that EMP-derived monocytes and macrophages can develop independently of the transcription factor Myb. The third wave, from E10.5, consists of HSCs that are generated in the aorto-gonado-mesonephros (AGM) region of the embryo through Runx1-dependent EHT (Chen et al., 2009). HSCs depend on the transcription factor Myb for their maintenance and self-renewal (Lieu and Reddy, 2009; Schulz et al., 2012). While Myb is differentially required for different macrophage populations, primitive erythrocytes are the only cells that still arise in Runx1−/− (Okada et al., 1998; Okuda et al., 1996; Wang et al., 1996) or Spi1−/− (Pu.1) embryos (Scott et al., 1994). Murine YS-derived macrophages and fetal monocytes seed most tissues before HSC-derived definitive hematopoiesis (Ginhoux et al., 2010; Hoeffel et al., 2015; Kierdorf et al., 2013; Gomez Perdiguero et al., 2015; Schulz et al., 2012; Sheng et al., 2015) where, with the exception of dermal (Tamoutounour et al., 2013), gut (Bain et al., 2014), and a fraction of cardiac macrophages (Epelman et al., 2014), they self-renew throughout life with minor contribution from adult blood monocytes (Epelman et al., 2014; Hashimoto et al., 2013).