br Experimental Procedures br Author Contributions br Acknow

Experimental Procedures
Author Contributions
Acknowledgments We are grateful for the technical assistance of Maria Ensthaler and the support by the “Core facility Cell Sorting.” We also thank the Research Core Unit Transcriptomics of Hannover Medical School for generating and processing the microarray raw data. This work was funded by grants to R.Z. including the German Research Foundation (DFG; including grants: Cluster of Excellence REBIRTH DFG EXC62/3 and ZW64/4-1), the German Ministry for Education and Science (BMBF; including grants: 13N12606 and 13N14086), StemBANCC (support from the Innovative Medicines Initiative joint undertaking under grant 115439-2, whose resources are composed of financial contribution from the European Union [FP7/2007-2013] and EFPIA companies\' in-kind contribution), and TECHNOBEAT (European Union H2020 grant 668724). H.K. was supported by Hannover Medical School Internal Program (HiLF) and by Joachim Herz Stiftung.
Introduction Totipotency is the capacity to form an entire organism, including embryonic and extraembryonic tissues. In mouse, totipotency lasts from fertilization at embryonic day (E)0 until the morula stage (∼E2.5). Loss of totipotency, early in pre-implantation development, is accompanied by segregation of the first lineage: the outer trophectoderm (TE) that separates from the inner cell mass (ICM). At implantation (∼E4.5), the ICM further generates two distinct layers: the epiblast and the primitive endoderm (PrE, also known as hypoblast) (Arnold and Robertson, 2009). At this stage, lineage identities are dictated by the expression of specific transcription factors (TFs). The pluripotent epiblast fate is induced by the expression of Oct4, Nanog, and Sox2 (Wicklow et al., 2014; Yamanaka et al., 2010); the segregated PrE layer is positive for Oct4, Gata4, Gata6, Sox7, and Sox17, whereas the order a01 of the TE express Cdx2 (Artus et al., 2011; Plusa et al., 2008). At earlier stages, these determinants are not specific: in the morula, embryonic and extraembryonic TFs are co-expressed in all blastomeres (Bessonnard et al., 2014; Dietrich and Hiiragi, 2007; Guo et al., 2010; Ohnishi et al., 2014; Schrode et al., 2014). Proceeding with development, the epiblast forms all embryonic tissues but also the extraembryonic mesoderm of the visceral yolk sac, the chorion, the allantois, and the amnion. The PrE subsequently gives rise to the parietal endoderm (PE) of the transient parietal yolk sac and the visceral endoderm (VE). The VE consists of embryonic and extraembryonic VE. The extraembryonic VE, together with extraembryonic mesoderm, forms the visceral yolk sac, while the embryonic VE is necessary for correct anterior-posterior patterning of the embryo. In addition, recent findings suggest that embryonic VE also contributes to the gut (Kwon et al., 2008). The TE forms trophoblast giant cells, the extraembryonic ectoderm and its derivatives, the ectoplacental cone, and the chorionic ectoderm. TE is necessary for implantation of the conceptus and exchange of products between the maternal and fetal circulation. Mouse embryonic stem cell (ESC) lines are derived from the ICM of developing blastocysts at ∼E3.5 (Evans and Kaufman, 1981; Martin, 1981). ESC lines capture many features of the epiblast and are defined as pluripotent because they can differentiate into the three definitive germ layers of the embryo when injected in recipient blastocysts or aggregated with morulas. In addition, pluripotent ESC lines can also generate trophoblast (Hayashi et al., 2010) and PrE cell types in vitro (i.e., extraembryonic endodermal cells [XENs]) (Kunath et al., 2005; Niakan et al., 2013), aside from cells of the three germ layers of the embryo. There is also evidence that ESCs rarely contribute to extraembryonic lineages in vivo (Beddington and Robertson, 1989). Taken together, these data indicate that ESC cultures contain precursors of extraembryonic lineages.