Autocrine FGF feedback can establish distinct states of Nanog expression in pluripotent stem cells: a computational analysis.

BACKGROUND: The maintenance of stem cell pluripotency is controlled by a core cluster of transcription factors, NANOG, OCT4 and SOX2 - genes that jointly regulate each other's expression. The expression of some of these genes, especially of Nanog, is heterogeneous in a population of undifferentiated stem cells in culture. Transient changes in expression levels, as well as heterogeneity of the population is not restricted to this core regulator, but involve a large number of other genes that include growth factors, transcription factors or signal transduction proteins. RESULTS: As the molecular mechanisms behind NANOG expression heterogeneity is not yet understood, we explore by computational modeling the core transcriptional regulatory circuit and its input from autocrine FGF signals that act through the MAP kinase cascade. We argue that instead of negative feedbacks within the core NANOG-OCT4-SOX2 transcriptional regulatory circuit, autocrine signaling loops such as the Esrrb - FGF - ERK feedback considered here are likely to generate distinct sub-states within the "ON" state of the core Nanog switch. Thus, the experimentally observed fluctuations in Nanog transcription levels are best explained as noise-induced transitions between negative feedback-generated sub-states. We also demonstrate that ERK phosphorilation is altered and being anti-correlated with fluctuating Nanog expression - in accord with model simulations. Our modeling approach assigns an empirically testable function to the transcriptional regulators Klf4 and Esrrb, and predict differential regulation of FGF family members. CONCLUSIONS: We argue that slow fluctuations in Nanog expression likely reflect individual cell-specific changes in parameters of an autocrine feedback loop, such as changes in ligand capture efficiency, receptor numbers or the presence of crosstalks within the MAPK signal transduction pathway. We proposed a model that operates with binding affinities of multiple transcriptional regulators of pluripotency, and the activity of an autocrine signaling pathway. The resulting model produces varied expression levels of several components of pluripotency regulation, largely consistent with empirical observations reported previously and in this present work.

TGF-ß-superfamily signaling regulates embryonic stem cell heterogeneity: self-renewal as a dynamic and regulated equilibrium.

Embryonic stem cells dynamically fluctuate between phenotypic states, as defined by expression levels of genes such as Nanog, while remaining pluripotent. The dynamic phenotype of stem cells is in part determined by gene expression control and dictated by various signaling pathways and transcriptional regulators. We sought to define the activities of two TGF-ß-related signaling pathways, bone morphogenetic protein (BMP) and Nodal signaling, in modulating mouse embryonic stem (ES) cell heterogeneity in undifferentiated culture conditions. Both BMP and Nodal signaling pathways were seen to be active in distinct Nanog subpopulations, with subtle quantitative differences in activity. Pharmacological and genetic modulation of BMP or Nodal signaling strongly influenced the heterogeneous state of undifferentiated ES cells, as assessed by dynamic expression of Nanog reporters. Inhibition of Nodal signaling enhanced BMP activity, which through the downstream target Id factors, enhanced the capacity of ES cells to remain in the Nanog-high epigenetic state. The combined inhibition of Nodal and BMP signaling resulted in the accumulation of Nanog-negative cells, even in the presence of LIF, uncovering a shared role for BMP and Nodal signaling in maintaining Nanog expression and repression of differentiation. These results demonstrate a complex requirement for both arms of TGF-ß-related signaling to influence the dynamic cellular phenotype of undifferentiated ES cells in serum-based media, and that differing subpopulations of ES cells in heterogeneous culture have distinct responses to these signaling pathways. Several pathways, including BMP, Nodal, and FGF signaling, have important regulatory function in defining the steady-state distribution of heterogeneity of stem cells.

Nodal signaling regulates the bone morphogenic protein pluripotency pathway in mouse embryonic stem cells.

Members of the transforming growth factor-beta 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-beta 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.