Self-renewal and pluripotency of mouse embryonic stem (ES) cells are controlled by a complex transcriptional regulatory network (TRN) which is rich in positive feedback loops. A number of key components of this TRN, including Nanog, show strong temporal expression fluctuations at the single cell level, although the precise molecular basis for this variability remains unknown. In this talk I will discuss recent work which uses a genetic complementation strategy to investigate genome-wide mRNA expression changes during transient periods of Nanog down-regulation. Nanog removal triggers widespread changes in gene expression in ES cells. However, we found that significant early changes in gene expression were reversible upon re-induction of Nanog, indicating that ES cells initially adopt a flexible “primed” state. Nevertheless, these changes rapidly become consolidated irreversible fate decisions in the continued absence of Nanog. Using high-throughput single cell transcriptional profiling we observed that the early molecular changes are both stochastic and reversible at the single cell level. Since positive feedback commonly gives rise to phenotypic variability, we also sought to determine the role of feedback in regulating ES cell heterogeneity and commitment. Analysis of the structure of the ES cell TRN revealed that Nanog acts as a feedback “linchpin”: in its presence positive feedback loops are active and the extended TRN is self-sustaining; while in its absence feedback loops are weakened, the extended TRN is no longer self-sustaining and pluripotency is gradually lost until a critical “point-of-no-return” is reached. Consequently, fluctuations in Nanog expression levels transiently activate different sub-networks in the ES cell TRN, driving transitions between a (Nanog expressing) feedback-rich, robust, self-perpetuating pluripotent state and a (Nanog-diminished), feedback-depleted, differentiation-sensitive state. Taken together, our results indicate that Nanog- dependent feedback loops play a central role in controlling both early fate decisions at the single cell level and cell-cell variability in ES cell populations.
