There is evidence that pluripotency of mouse embryonic stem (ES) cells is associated with the activity of a network of transcription factors with Sox2, Oct4, and Nanog at the core. Using fluorescent reporters for the expression of Nanog, we observed that a population of ES cells is best described by a dynamic distribution of Nanog expression characterized by two peaks defined by high (HN) and low (LN) Nanog expression. Typically, the LN state is 5%–20% of the total population, depending on the culture conditions. Modelling of the activity of Nanog reveals that a simple network of Oct4/Sox2 and Nanog activity can account for the observed distribution and its properties as long as the transcriptional activity is tuned by transcriptional noise. The model also predicts that the LN state is unstable, something that is born out experimentally. While in this state, cells can differentiate. We suggest that transcriptional fluctuations in Nanog expression are an essential element of the pluripotent state and that the function of Sox2, Oct4, and Nanog is to act as a network that promotes and maintains transcriptional noise to interfere with the differentiation signals. Embryonic stem (ES) cells are a pluripotent cell population derived from early mammalian embryos. An intrinsic feature of ES cells is their phenotypic heterogeneity: they display promiscuous activation of lineage-specific genes and exhibit a fluctuating flow of differentiating cells. A gene regulatory network (GRN) centred around the transcription factors Sox2, Oct4, and Nanog is essential for the establishment and the maintenance of the pluripotent state. Previous studies had suggested that ES cells can reversibly change their state of Nanog expression without losing pluripotency. Here, we extend these studies by quantifying and monitoring the expression of Nanog in a Nanog-GFP reporter cell line. We show that Nanog levels undergo slow, random fluctuations in ES cells, giving rise to heterogeneous cell populations. We identify two states, one stable, characterized by high levels of expression (HN), and another with low levels of Nanog expression (LN), which is highly unstable. While in the LN state, cells are more likely to differentiate depending on the culture medium. Mathematical modelling shows that a simple excitable system driven by transcriptional noise can account for the observed distributions and behaviours in gene expression. Our study suggests that rather than a discrete state dependent on the fixed expression of a small set of genes, pluripotency is best represented by a state of dynamic heterogeneity of a population driven by transcriptional noise, and that the function of the gene regulatory network centred around Nanog might be to generate dynamic heterogeneities at the population level.

The transcription factor Oct4 is required in vitro for establishment and maintenance of embryonic stem cells and for reprogramming somatic cells to pluripotency. In vivo, it prevents the ectopic differentiation of early embryos into trophoblast. Here, we further explore the role of Oct4 in blastocyst formation and specification of epiblast versus primitive endoderm lineages using conditional genetic deletion. Experiments involving mouse embryos deficient for both maternal and zygotic Oct4 suggest that it is dispensable for zygote formation, early cleavage and activation of Nanog expression. Nanog protein is significantly elevated in the presumptive inner cell mass of Oct4 null embryos, suggesting an unexpected role for Oct4 in attenuating the level of Nanog, which might be significant for priming differentiation during epiblast maturation. Induced deletion of Oct4 during the morula to blastocyst transition disrupts the ability of inner cell mass cells to adopt lineage-specific identity and acquire the molecular profile characteristic of either epiblast or primitive endoderm. Sox17, a marker of primitive endoderm, is not detected following prolonged culture of such embryos, but can be rescued by provision of exogenous FGF4. Interestingly, functional primitive endoderm can be rescued in Oct4-deficient embryos in embryonic stem cell complementation assays, but only if the host embryos are at the pre-blastocyst stage. We conclude that cell fate decisions within the inner cell mass are dependent upon Oct4 and that Oct4 is not cell-autonomously required for the differentiation of primitive endoderm derivatives, as long as an appropriate developmental environment is established.

Segmentation is a fundamental problem that dominates the success of microscopic image analysis. In almost 25 years of cell detection software development, there is still no single piece of commercial software that works well in practice when applied to early mouse embryo or stem cell image data. To address this need, we developed MINS (modular interactive nuclear segmentation) as a MATLAB/C++-based segmentation tool tailored for counting cells and fluorescent intensity measurements of 2D and 3D image data. Our aim was to develop a tool that is accurate and efficient yet straightforward and user friendly. The MINS pipeline comprises three major cascaded modules: detection, segmentation, and cell position classification. An extensive evaluation of MINS on both 2D and 3D images, and comparison to related tools, reveals improvements in segmentation accuracy and usability. Thus, its accuracy and ease of use will allow MINS to be implemented for routine single-cell-level image analyses.

The maintenance of pluripotency in mouse embryonic stem cells (mESCs) relies on the activity of a transcriptional network that is fuelled by the activity of three transcription factors (Nanog, Oct4 and Sox2) and balanced by the repressive activity of Tcf3. Extracellular signals modulate the activity of the network and regulate the differentiation capacity of the cells. Wnt/β-catenin signaling has emerged as a significant potentiator of pluripotency: increases in the levels of β-catenin regulate the activity of Oct4 and Nanog, and enhance pluripotency. A recent report shows that β-catenin achieves some of these effects by modulating the activity of Tcf3, and that this effect does not require its transcriptional activation domain. Here, we show that during self-renewal there is negligible transcriptional activity of β-catenin and that this is due to its tight association with membranes, where we find it in a complex with Oct4 and E-cadherin. Differentiation triggers a burst of Wnt/β-catenin transcriptional activity that coincides with the disassembly of the complex. Our results establish that β-catenin, but not its transcriptional activity, is central to pluripotency acting through a β-catenin/Oct4 complex.

Fibroblasts deficient in the activation-induced cytidine deaminase (AID) enzyme are shown to fail to stabilize in the pluripotent state, despite initiating the expression of pluripotency genes. Todd Evans and colleagues studied whether activation-induced cytidine deaminase enzyme (AID) regulates epigenetic memory during reprogramming of somatic cells as induced pluripotent stem (iPS) cells. Using fibroblasts deficient for AID, the authors observed that the mutant cells were transiently hyper-responsive to the reprogramming process. But although they initiate expression of pluripotency genes, cells lacking AID fail to stabilize the pluripotent state. The authors propose that AID regulates this late step to stabilize pluripotency by removing epigenetic memory to promote expression of secondary pluripotency network genes. The activation-induced cytidine deaminase (AID; also known as AICDA) enzyme is required for somatic hypermutation and class switch recombination at the immunoglobulin locus1. In germinal-centre B cells, AID is highly expressed, and has an inherent mutator activity that helps generate antibody diversity2. However, AID may also regulate gene expression epigenetically by directly deaminating 5-methylcytosine in concert with base-excision repair to exchange cytosine3. This pathway promotes gene demethylation, thereby removing epigenetic memory. For example, AID promotes active demethylation of the genome in primordial germ cells4. However, different studies have suggested either a requirement5 or a lack of function6 for AID in promoting pluripotency in somatic nuclei after fusion with embryonic stem cells. Here we tested directly whether AID regulates epigenetic memory by comparing the relative ability of cells lacking AID to reprogram from a differentiated murine cell type to an induced pluripotent stem cell. We show that Aid-null cells are transiently hyper-responsive to the reprogramming process. Although they initiate expression of pluripotency genes, they fail to stabilize in the pluripotent state. The genome of Aid-null cells remains hypermethylated in reprogramming cells, and hypermethylated genes associated with pluripotency fail to be stably upregulated, including many MYC target genes. Recent studies identified a late step of reprogramming associated with methylation status7, and implicated a secondary set of pluripotency network components8. AID regulates this late step, removing epigenetic memory to stabilize the pluripotent state.

Notch receptors act as ligand-dependent membrane-tethered transcription factors with a prominent role in binary cell fate decisions during development, which is conserved across species. In addition there is increasing evidence for other functions of Notch, particularly in connection with Wnt signalling: Notch is able to modulate the activity of Armadillo/ß-catenin, the effector of Wnt signalling, in a manner that is independent of its transcriptional activity. Here we explore the mechanism of this interaction in the epithelium of the Drosophila imaginal discs and find that it is mediated by the ligand-independent endocytosis and traffic of the Notch receptor. Our results show that Notch associates with Armadillo near the adherens junctions and that it is rapidly endocytosed promoting the traffic of an activated form of Armadillo into endosomal compartments, where it may be degraded. As Notch has the ability to interact with and downregulate activated forms of Armadillo, it is possible that in vivo Notch regulates the transcriptionally competent pool of Armadillo. These interactions reveal a previously unknown activity of Notch, which serves to buffer the function of activated Armadillo and might underlie some of its transcription-independent effects. Establishment of the correct shape and pattern of tissues within an organism requires the integration of molecular information present in signalling and transcriptional networks and demands delicate exchanges and balances of their activities. A large body of experimental work has revealed close correlations in the activities of two pathways: Notch and Wnt, which suggest the existence of multiple links between them. Notch signalling relies in part upon the activity of the Notch protein, a membrane-bound receptor with a transcription factor domain that can be released from the membrane by proteolytic cleavage. On the other hand Wnt proteins are ligands that trigger changes in the activity of ß-catenin, which is called Armadillo in the fruit fly Drosophila melanogaster. In this study we uncover a previously unknown activity for Notch: endocytosis and trafficking of full length Notch, which targets Armadillo for degradation. This activity of Notch is independent of its ligands, Delta and Serrate, and of its downstream effector, the transcription factor Suppressor of Hairless. We further show that in the absence of Notch, which has been shown to act as a tumor suppressor in mammals, expression of an activated form of Armadillo causes tissue overgrowth and changes in the polarity of cells. Our results suggest that Drosophila Notch can promote the degradation of activated forms of Armadillo and may buffer cells against fluctuations in Wnt signalling activity.