It is now recognized that extensive expression heterogeneities among cells precede the emergence of lineages in the early mammalian embryo. To establish a map of pluripotent epiblast (EPI) versus primitive endoderm (PrE) lineage segregation within the inner cell mass (ICM) of the mouse blastocyst, we characterised the gene expression profiles of individual ICM cells. Clustering analysis of the transcriptomes of 66 cells demonstrated that initially they are non-distinguishable. Early in the segregation, lineage-specific marker expression exhibited no apparent correlation, and a hierarchical relationship was established only in the late blastocyst. Fgf4 exhibited a bimodal expression at the earliest stage analysed, and in its absence, the differentiation of PrE and EPI was halted, indicating that Fgf4 drives, and is required for, ICM lineage segregation. These data lead us to propose a model where stochastic cell-to-cell expression heterogeneity followed by signal reinforcement underlies ICM lineage segregation by antagonistically separating equivalent cells.

SUMMARY The first lineage choice in mammalian embryogenesis is that between the trophectoderm, which gives rise to the trophoblast of the placenta, and the inner cell mass, from which derive the embryo proper and the yolk sac. The establishment of these lineages is preceded by the inside-versus-outside positioning of cells in the early embryo and stochastic expression of key transcription factors, which is then resolved into lineage-restricted expression. The regulatory inputs that drive this restriction and how they relate to cell position are largely unknown. Here we show an unsuspected role of Notch signaling in regulating trophectoderm-specific expression of Cdx2 in cooperation with TEAD4. Notch activity is restricted to outer cells and is able to influence positional allocation of blastomeres, mediating preferential localization to the trophectoderm. Our results show that multiple signaling inputs at preimplantation stages specify the first embryonic lineages.

Summary The pluripotent epiblast (EPI) is the founder tissue of almost all somatic cells. EPI and primitive endoderm (PrE) progenitors arise from the inner cell mass (ICM) of the blastocyst stage embryo. The EPI lineage is distinctly identified by its expression of pluripotency-associated factors. Many of these factors have been reported to exhibit dynamic fluctuations of expression in embryonic stem cell cultures. Whether these fluctuations correlating with ICM fate choice occur in vivo remains an open question. Using single-cell resolution quantitative imaging of a Nanog transcriptional reporter, we noted an irreversible commitment to EPI/PrE lineages in vivo. A period of apoptosis occurred concomitantly with ICM cell fate choice, followed by a burst of EPI-specific cell proliferation. Transitions were occasionally observed from PrE-to-EPI, but not vice versa, suggesting that they might be regulated and not stochastic. We propose that the rapid timescale of early embryonic development prevents frequent fluctuations in cell fate.

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.

Development normally occurs similarly in all individuals within an isogenic population, but mutations often affect the fate of individual organisms differently1-4. This phenomenon, known as partial penetrance, has been observed in diverse developmental systems. However, it remains unclear how the underlying genetic network specifies the set of possible alternative fates and how the relative frequencies of these fates evolve5-8. Here, we identify a stochastic cell fate determination process that operates in Bacillus subtilis sporulation mutants and show how it allows genetic control of the penetrance of multiple fates. Mutations in an inter-compartmental signaling process generate a set of discrete alternative fates not observed in wild-type cells, including rare formation of two viable “twin” spores, rather than one within a single cell. By genetically modulating chromosome replication and septation, we could systematically tune the penetrance of each mutant fate. Furthermore, signaling and replication perturbations synergize to dramatically increase the penetrance of twin sporulation. These results suggest a potential pathway for developmental evolution between monosporulation and twin sporulation through states of intermediate twin penetrance. Furthermore, time-lapse microscopy of twin sporulation in wild-type Clostridium oceanicum showed a strong resemblance to twin sporulation in these B. subtilis mutants9,10. Together the results suggest that noise can facilitate developmental evolution by enabling the initial expression of discrete morphological traits at low penetrance, and allowing their stabilization by gradual adjustment of genetic parameters.