Nodal Signaling Regulates the Bone Morphogenic Protein Pluripotency Pathway in Mouse Embryonic Stem Cells

Members of the transforming growth factor-β superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-β pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling.     

Regulation of developmental competence and commitment towards the definitive endoderm lineage in human embryonic stem cells

Human embryonic stem cells (hESCs) can self-renew and become all three germ layers. Nodal/Activin signaling specifies developmental status in hESCs: moderate Nodal/Activin signaling maintains pluripotency, while enhancement and inhibition promote definitive endoderm (DE) and neuroectoderm (NE) development, respectively. However, how modulation of Nodal/Activin signaling influences developmental competence and commitment toward specific lineages is still unclear. Here, we showed that enhancement of Nodal/Activin signaling for 4 days was necessary and sufficient to upregulate DE markers, while it diminished the upregulation of NE markers by inhibition of Nodal/Activin signaling. This suggests that after 4 days of enhanced Nodal/Activin signaling, hESCs are committed to the DE lineage and have lost competence toward the NE lineage. In contrast, inhibition of Nodal/Activin signaling using LY364947 for 2 days was sufficient to impair competence toward the DE lineage, although cells were still able to activate LEFTY1 and NODAL, direct targets of Nodal/Activin signaling. Expression analyses indicated that the levels of pluripotency regulators NANOG and POU5F1 were significantly diminished by 2 days of LY364947 treatment, although the expression of NANOG, but not POU5F1, was restored immediately upon Activin A treatment. Thus, downregulation of POU5F1 coincided with the abrogation of DE competence caused by inhibition of Nodal/Activin signaling.