The notion that the differentiated state of a cell population is determined simply by expression of specific marker genes is changing. In this work, the authors reveal that a pluripotent cell population comprises cells with temporal fluctuations in the expression of Nanog.

SummarySegmentation 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 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.

The activation-induced cytidine deaminase enzyme (AID) is required for somatic hyper-mutation and class switch recombination at the immunoglobulin locus1. In GC-B cells, AID is highly expressed, with inherent mutator activity that helps generate antibody diversity2. However, AID may also regulate gene expression epigenetically by directly deaminating 5-methylcytosine (5mC) 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 promoting pluripotency in somatic nuclei following fusion with embryonic stem cells (ESCs). We tested directly whether AID regulates epigenetic memory, by comparing the relative ability of cells lacking AID to reprogram from a differentiated cell type to an induced pluripotent stem cell (iPSC). 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 the pluripotent state. The genome of AID-null cells remains hyper-methylated in reprogramming cells, and hyper-methylated genes associated with pluripotency fail to be stably up-regulated, 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.