br Results br Discussion In

Results
Discussion In this study, we investigated the mode of developmental commitment and competence toward the DE and NE lineages in hESCs in response to changes in Nodal/Activin signaling activity. Four days of enhanced Nodal/Activin signaling was sufficient to commit hESCs toward the DE lineage, as the inhibition of signaling afterward could no longer suppress the expression of DE genes. Concomitantly, competence toward the NE lineage was significantly impaired by prior treatment with high Activin A for 4days. Thus, the commitment to DE was associated with the loss of competence to NE, suggesting that the developmental potential of hESCs is critically altered after 4days of enhanced Nodal/Activin signaling. Interestingly, the key regulators of developmental pluripotency, POU5F1 and NANOG, were still robustly expressed after 4days of Activin A treatment, indicating that the loss of NE competence was not due to lack of expression of these pluripotency regulators. However, it is possible that DE-specific genes that were activated by 4days of Activin A treatment, namely SOX17, could impose inhibitory effects on the pluripotency regulators and the expression of NE-specific genes. A study showed that POU5F1 has a dual function by working with both SOX2 and SOX17 to maintain pluripotency and to promote the mesendoderm lineage, respectively (Stefanovic et al., 2009). POU5F1 binds to SOX17, and both together occupy the promoter of NCT-501 genes, such as HEX and SOX17 itself. Elevation of SOX17 by enhanced Nodal/Activin signaling may deprive POU5F1 from operating with SOX2, which is involved in both pluripotency maintenance as well as NE development (Avilion et al., 2003; Li et al., 2005; Zappone et al., 2000; Papanayotou et al., 2008; Archer et al., 2011; Thomson et al., 2011). Another possibility is that the expression levels or epigenetic status of genes important for NE differentiation may be altered after 4days of enhanced Nodal/Activin signaling. For example, Nodal/Activin signaling, along with NANOG and POU5F1, repress the expression of SIP1 (Smad-interacting protein 1; also known as ZEB2), which is essential for NE differentiation (Chng et al., 2010). It is possible that the extended enhancement of Nodal/Activin signaling may result in permanent interference with the expression of NE regulators, such as SIP1, thereby inhibiting competence toward NE. A moderate level 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). In the present study, hESCs were maintained in a chemically defined medium, mTeSR1, which contains TGFβ1 to activate Nodal/Activin signaling (Ludwig et al., 2006). Our basal medium itself was not supplemented with any growth factors to activate Nodal/Activin signaling. Nonetheless, a low level of Nodal/Activin signaling was likely to be active in the basal medium, owing to the endogenous expression of NODAL in hESCs (Vallier et al., 2004). This endogenous low level of Nodal/Activin signaling may be sufficient to sustain the expression of pluripotency regulators POU5F1 for up to 4days and NANOG for up to 2days (Figure 4B). This is in striking contrast to the basal medium supplemented with SB431542, which downregulated POU5F1 and NANOG much more rapidly (Figure 4B). However, the level of Nodal/Activin signaling in the basal medium was possibly too low to prevent the appearance of NE features, specifically the expression of PAX6, as it was gradually upregulated in the basal medium alone (Figure 1B). PAX6 has been shown to act as the NE fate determinant in hESCs, as its expression is both necessary and sufficient to induce NE differentiation (Zhang et al., 2010). Regardless, the other NE markers, GBX2 and NFH, were not upregulated in the basal medium even after 8days of culture, raising the possibility that expression of PAX6 alone is not enough to activate these NE markers under this culture condition.