Heterogeneities in Nanog Expression Drive Stable Commitment to Pluripotency in the Mouse Blastocyst

SummaryThe 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 mammalian embryonic development prevents fluctuations in cell fate

A Rapid and Efficient 2D/3D Nuclear Segmentation Method for Analysis of Early Mouse Embryo and Stem Cell Image Data

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

Partial penetrance facilitates developmental evolution in bacteria

Development normally occurs similarly in all individuals within an isogenic population, but mutations often affect the fates of individual organisms differently. 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 evolve. 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 intercompartmental signalling 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 can systematically tune the penetrance of each mutant fate. Furthermore, signalling and replication perturbations synergize to significantly 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 shows a strong resemblance to twin sporulation in these B. subtilis mutants. 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

Blocking Chromosome Translocation during Sporulation of Bacillus subtilis Can Result in Prespore-Specific Activation of σ(G) That Is Independent of σ(E) and of Engulfment

Formation of spores by Bacillus subtilis is characterized by cell compartment-specific gene expression directed by four RNA polymerase σ factors, which are activated in the order σ(F)-σ(E)-σ(G)-σ(K). Of these, σ(G) becomes active in the prespore upon completion of engulfment of the prespore by the mother cell. Transcription of the gene encoding σ(G), spoIIIG, is directed in the prespore by RNA polymerase containing σ(F) but also requires the activity of σ(E) in the mother cell. When first formed, σ(G) is not active. Its activation requires expression of additional σ(E)-directed genes, including the genes required for completion of engulfment. Here we report conditions in which σ(G) becomes active in the prespore in the absence of σ(E) activity and of completion of engulfment. The conditions are (i) having an spoIIIE mutation, so that only the origin-proximal 30% of the chromosome is translocated into the prespore, and (ii) placing spoIIIG in an origin-proximal location on the chromosome. The main function of the σ(E)-directed regulation appears to be to coordinate σ(G) activation with the completion of engulfment, not to control the level of σ(G) activity. It seems plausible that the role of σ(E) in σ(G) activation is to reverse some inhibitory signal (or signals) in the engulfed prespore, a signal that is not present in the spoIIIE mutant background. It is not clear what the direct activator of σ(G) in the prespore is. Competition for core RNA polymerase between σ(F) and σ(G) is unlikely to be of major importance

Expression of the σF-Directed csfB Locus Prevents Premature Appearance of σG Activity during Sporulation of Bacillus subtilis▿

During sporulation, σG becomes active in the prespore upon the completion of engulfment. We show that the inactivation of the σF-directed csfB locus resulted in premature activation of σG. CsfB exerted control distinct from but overlapping with that exerted by LonA to prevent inappropriate σG activation. The artificial induction of csfB severely compromised spore formation

Loss of Compartmentalization of σE Activity Need Not Prevent Formation of Spores by Bacillus subtilis▿ †

Compartmentalization of the activities of RNA polymerase sigma factors is a hallmark of formation of spores by Bacillus subtilis. It is initiated soon after the asymmetrically located sporulation division takes place with the activation of σF in the smaller cell, the prespore. σF then directs a signal via the membrane protease SpoIIGA to activate σE in the larger mother cell by processing of pro-σE. Here, we show that σE can be activated in the prespore with little effect on sporulation efficiency, implying that complete compartmentalization of σE activity is not essential for spore formation. σE activity in the prespore can be obtained by inducing transcription in the prespore of spoIIGA or of sigE*, which encodes a constitutively active form of σE, but not of spoIIGB, which encodes pro-σE. We infer that σE compartmentalization is partially attributed to a competition between the compartments for the activation signaling protein SpoIIR. Normally, SpoIIGA is predominantly located in the mother cell and as a consequence confines σE activation to it. In addition, we find that CsfB, previously shown to inhibit σG, is independently inhibiting σE activity in the prespore. CsfB thus appears to serve a gatekeeper function in blocking the action of two sigma factors in the prespore: it prevents σG from becoming active before completion of engulfment and helps prevent σE from becoming active at all

Anatomy of a blastocyst: Cell behaviors driving cell fate choice and morphogenesis in the early mouse embryo

The preimplantation period of mouse early embryonic development is devoted to the specification of two extra-embryonic tissues and their spatial segregation from the pluripotent epiblast. During this period two cell fate decisions are made while cells gradually lose their totipotency. The first fate decision involves the segregation of the extra-embryonic trophectoderm (TE) lineage from the inner cell mass (ICM); the second occurs within the ICM and involves the segregation of the extra-embryonic primitive endoderm (PrE) lineage from the pluripotent epiblast (EPI) lineage, which eventually gives rise to the embryo proper. Multiple determinants, such as differential cellular properties, signaling cues and the activity of transcriptional regulators, influence lineage choice in the early embryo. Here, we provide an overview of our current understanding of the mechanisms governing these cell fate decisions ensuring proper lineage allocation and segregation, while at the same time providing the embryo with an inherent flexibility to adjust when perturbed