Nanog-dependent feedback loops regulate murine embryonic stem cell heterogeneity

Abstract

A number of key regulators of mouse embryonic stem (ES) cell identity, including the transcription factor Nanog, show strong expression fluctuations at the single cell level. The molecular basis for these fluctuations is unknown. Here we used a genetic complementation strategy to investigate expression changes during transient periods of Nanog downregulation. Employing an integrated approach, that includes high-throughput single cell transcriptional profiling and mathematical modelling, we found that early molecular changes subsequent to Nanog loss are stochastic and reversible. However, analysis also revealed that Nanog loss severely compromises the self-sustaining feedback structure of the ES cell regulatory network. Consequently, these nascent changes soon become consolidated to committed fate decisions in the prolonged absence of Nanog. Consistent with this, we found that exogenous regulation of Nanog-dependent feedback control mechanisms produced more a homogeneous ES cell population. Taken together our results indicate that Nanog-dependent feedback loops have a role in controlling both ES cell fate decisions and population variability.

Modeling heterogeneity in the pluripotent state: A promising strategy for improving the efficiency and fidelity of stem cell differentiation

“…It has been suggested that different topologies of pluripotent gene regulatory networks maintain the heterogeneous molecular states characteristic of individual PSCs 60. Recently, different studies have addressed this important question by integrating single cell expression and perturbation data for identifying the underlying PGRNs 4, 16, 22, 61, which are involved in the stabilization of the pluripotent state and lineage commitment 62, 63. In most of these studies, topological analyses of PGRNs have identified feed forward loops (FFLs) linked to regulation of the balance between self‐renewal and differentiation.…”

“…activation and inhibition, respectively – have great information processing potential, allowing the generation of alternative output solutions. The feedback structure of the PGRNs have been reported to be partially destroyed, with main variations in the loop between Nanog and other pluripotency factors, leading to a transient activation of different motifs that trigger differentiation 16. Incoherent FFLs have been inferred in mESCs subpopulations linking Oct4 and Nanog with their gene targets, suggesting an antagonistic interaction between these TFs for the regulation of pluripotency and self‐renewal 4.…”

“…However, in general, more complex circuit architectures underlie this mechanism, especially when antagonistic groups contain more than one gene product. Positive feedback loops, known as positive circuits 93, generalize the role of toggle switches in reprogramming or differentiation, including several pluripotency regulators 4, 16, 22, which regulate the balance between self‐renewal and cell fate commitment. The presence of positive circuits in a gene regulatory network is a necessary condition for the existence of multiple attractors – i.e.…”

“…Intense research in this field has allowed the construction of PGRNs, and the identification of network motifs regulating the transitions among different cell subpopulations in the heterogeneous continuum expression landscape 4, 16, 22, 61, 62, 63. So far, the identification of simple network motifs – i.e.…”

“…The differential expression patterns of some pluripotency regulators in different subpopulations have been linked to fine tuning the balance between self‐renewal and cell fate commitment 7, 14, 15, 16, 17. The mechanisms underlying gene expression heterogeneity in the pluripotent state are still not well understood, and evidence suggests that gene fluctuations could arise from stochastic noise in gene expression regulatory mechanisms 18, 19, 20, 21.…”