br Experimental Procedures br Author Contributions

Experimental Procedures
Author Contributions
Acknowledgments
Introduction Mouse embryonic stem tnf alpha inhibitor (mESCs) are cells derived from inner cell mass (ICM) that self-renew indefinitely in culture and can give rise to all somatic cell types both in vitro and in vivo (Evans and Kaufman, 1981; Martin, 1981; Murry and Keller, 2008). The balance between self-renewal and differentiation is maintained by a milieu of transcription factors (TFs) (Ivanova et al., 2006; Thomson et al., 2011), epigenetic modifiers (Ang et al., 2011; Liang and Zhang, 2013; Loh et al., 2007), and signaling cascades (Lee et al., 2012a; Niwa et al., 2009). Many ESC-specific TFs such as Nanog (Chambers et al., 2007), Rex1 (Toyooka et al., 2008), Klf4 (Niwa et al., 2009; Toyooka et al., 2008), and Tbx3 (Niwa et al., 2009) are heterogeneously expressed in a population of mESCs (Faddah et al., 2013; MacArthur et al., 2012). These factors display low and high protein expression levels (Chambers et al., 2007; Toyooka et al., 2008). In the absence of some of these TFs (like Nanog), mESCs can maintain self-renewal and pluripotency (Chambers et al., 2007). Expression of TFs like Nanog, Sox2, Esrrb, Klf4, and Tbx3 is controlled by external signaling cues (Niwa et al., 2009). Wnt signaling pathway has been widely studied in development (Nusse and Varmus, 2012), plays important roles in maintaining pluripotency (ten Berge et al., 2011; Ying et al., 2008), and is important for acquisition of pluripotency during induced pluripotent stem cell (iPSC) reprogramming (Ho et al., 2013; Marson et al., 2008a). Downstream of Wnt ligand/receptor binding, the inhibition of GSK3β is necessary for stabilization of β-CATENIN (Nusse and Varmus, 2012). Inhibition of GSK3β and Mapk signaling with small molecules maintains a naive pluripotent state in mESCs (Sato et al., 2004; Ying et al., 2008). Wnt signaling stabilizes the mESC state in limiting amounts of LIF (Ogawa et al., 2006). Wnt3a prevents mESC differentiation from a naive to an epiblast-like or primed state (ten Berge et al., 2011). Apart from its role in maintenance of pluripotency, Wnt signaling is also required for differentiation of mESCs into early mesoderm derivatives (Gadue et al., 2006). Specifically, Wnt3 and Wnt8a are necessary for mesoderm differentiation (Kemp et al., 2005; Lindsley et al., 2006). During in vivo development, Wnt signaling is required for formation of primitive streak and mesoderm (Arnold et al., 2000; Martin and Kimelman, 2010; Yamaguchi et al., 1999). In summary, Wnt signaling is required both for stabilization of an undifferentiated pluripotent state and for promoting early mesoderm differentiation. Expression of Tbx3 is regulated by Wnt signaling in mESCs (Kelly et al., 2011; Price et al., 2013) and cancer (Renard et al., 2007). Tbx3, the only member of its subfamily expressed in the ICM, is required for maintenance of ESC state (Chapman et al., 1996; Han et al., 2010; Ivanova et al., 2006). Tbx3 maintains self-renewal of mESCs in absence of LIF, a property shared by Nanog and Klf4 (Han et al., 2010). Tbx3 overexpressed with Oct4, Sox2, and Klf4 during iPSC-reprogramming improves germline competence of fully reprogrammed iPSCs (Han et al., 2010). Tbx3 overexpression also improves the efficiency of cell fusion-based reprogramming (Han et al., 2010). However, in vivo, Tbx3 null embryos survive until E10.5 (Davenport et al., 2003), beyond the blastocyst stage. Many ESC-specific TFs like Oct4, Nanog, Sox2 (Mendjan et al., 2014; Thomson et al., 2011), and Sall4 (Lim et al., 2008) play important roles during early differentiation in addition to their roles in repressing differentiation of ESCs (Loh and Lim, 2011). Tbx3 promotes the formation of the mesendoderm lineage and is expressed both in ESCs and in vivo during primitive streak formation (Kartikasari et al., 2013; Weidgang et al., 2013). Furthermore, Tbx3 is required for generation of extraembryonic endoderm cells (Lu et al. 2011; Rugg-Gunn et al., 2010) and for opening up of Eomes promoter during differentiation to definitive endoderm (Kartikasari et al., 2013). However, the mechanisms by which Tbx3 regulates transition from a stable pluripotent state into differentiated progenitors remain unclear. Here, we explore how Tbx3 modulates the response of extracellular signaling and maintains balance between mESC self-renewal and differentiation. We isolate an alternate and stable pluripotent state in the absence of Tbx3 and identify Dppa3 (Stella/PGC7) as a direct downstream target of Tbx3. We present a model in which the Wnt/Tbx3/Dppa3 signaling-TF axis controls specification of mESCs into mesoderm lineage.