Nodal Activin signaling is an

Nodal/Activin signaling is an evolutionarily conserved signal transduction pathway, and plays critical roles in the maintenance and differentiation of hESCs. Nodal and Activin (Activin A and Activin B [Thompson et al., 2004]) are members of the transforming growth factor beta (TGFβ) superfamily, and bind to heteromeric complexes between type I (Alk4, Alk5, and Alk7) and type II (ActRIIB) activin receptors, which in turn activate Smad2/3 to regulate the expressions of various target genes (Schier, 2003; Valdimarsdottir and Mummery, 2005; Payne et al., 2011). Moderate activation of Nodal/Activin signaling is essential for the maintenance of pluripotency in hESCs (Beattie et al., 2005; James et al., 2005; Vallier et al., 2005). A small amount (e.g.,10ng/mL) of Activin A or TGFβ1 is an essential ingredient in the chemically defined culture media that are formulated for routine maintenance of hESCs under feeder-free conditions (Amit et al., 2004; Brons et al., 2007; Ludwig et al., 2006). Inhibition of Nodal/Activin signaling with small molecule inhibitors of Alk4, Alk5, and Alk7, such as SB431542 (Callahan et al., 2002; Inman et al., 2002) or LY364947 (Sawyer et al., 2003; Peng et al., 2005), downregulates the expressions of pluripotency regulators NANOG and OCT4 (POU5F1) in hESCs (James et al., 2005). Concomitantly, inhibition of Nodal/Activin signaling directs hESCs toward the neuroectoderm (NE) lineage, and upregulates the expression of NE marker genes, such as PAX6, GBX2, and NFH (Wassarman et al., 1997; Camus et al., 2006; Smith et al., 2008; Patani et al., 2009; Chng et al., 2010; Hu et al., 2010; Zhou et al., 2010). NE differentiation can be promoted by a pharmacological inhibitor of Nodal/Activin signaling, such as SB431542, or by ectopic expression of Nodal antagonists, such as Lefty2 and Cer-S (Smith et al., 2008; Patani et al., 2009), whereas it is inhibited by overexpression of NODAL (Vallier et al., 2004). On the other hand, a high level (e.g., 50ng/mL) of Activin A promotes the differentiation of hESCs into the definitive prostaglandin endoperoxide synthase (DE) lineage, which is characterized by the expression of SOX17, CXCR4, and FOXA2 (D\'Amour et al., 2005; Borowiak et al., 2009; Mayhew and Wells, 2010; Sulzbacher et al., 2009; Wang et al., 2011). Thus, the level of Nodal/Activin signaling imposes critical impacts on hESCs, and regulates three distinct developmental states: a low level or absence of signaling for the NE lineage, an intermediate level for the maintenance of pluripotency, and a high level for the DE lineage. However, the mechanisms of how and when each developmental state is determined by a specific level of Nodal/Activin signaling are still not well-understood. For example, questions like how long hESCs need to be exposed to a high level of Activin A to become committed toward the DE lineage, and at what point hESCs lose competence to undertake the DE lineage when Nodal/Activin signaling is inhibited, have not been addressed. In the present study, we conducted a series of experiments to gain insight into the regulation of competence and commitment towards the DE lineage in hESCs. We found that the duration of exposure to a high level of Activin A had a cumulative effect on the activation of the DE marker genes up to 6days of culture. When hESCs were exposed to Activin A for 4days, the inhibition of Nodal/Activin signaling was no longer able to downregulate the expression of DE markers, suggesting that hESCs had already become committed to the DE lineage. We also showed that the commitment toward DE was associated with the loss of competence toward NE. To investigate the nature of the competence toward DE, we tested 5 different pharmacological inhibitors of Nodal/Activin signaling. While all of them strongly inhibited Nodal/Activin signaling, only LY364947 was fully reversible, with the property to be washed way without impairing activation of Nodal/Activin signaling in response to Activin A treatment. By taking advantage of the reversible nature of LY364947, we determined that 2days, but not 1day, of Nodal/Activin signaling inhibition was detrimental to the DE competence. Finally, we showed that the downregulation of POU5F1 coincided with the loss of DE competence in response to Nodal/Activin signaling inhibition, raising the possibility that this key pluripotency regulator plays a critical role in maintaining the competence toward DE in hESCs.