Panagiotis Xenopoulos

Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages

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 characterized 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.

Notch and Hippo Converge on Cdx2 to Specify the Trophectoderm Lineage in the Mouse Blastocyst

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 is derived 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.

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 extraembryonic 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 extraembryonic trophectoderm (TE) lineage from the inner cell mass (ICM); the second occurs within the ICM and involves the segregation of the extraembryonic 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. genesis 51:219–233.

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

Summary 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.

A bright single-cell resolution live imaging reporter of Notch signaling in the mouse

BackgroundLive imaging provides an essential methodology for understanding complex and dynamic cell behaviors and their underlying molecular mechanisms. Genetically-encoded reporter expressing mouse strains are an important tool for use in live imaging experiments. Such reporter strains can be engineered by placing cis-regulatory elements of interest to direct the expression of desired reporter genes. If these cis-regulatory elements are downstream targets, and thus activated as a consequence of signaling pathway activation, such reporters can provide read-outs of the signaling status of a cell. The Notch signaling pathway is an evolutionary conserved pathway operating in multiple developmental processes as well as being the basis for several congenital diseases. The transcription factor CBF1 is a central evolutionarily conserved component of the Notch signaling pathway. It binds the active form of the Notch receptor (NICD) and subsequently binds to cis-regulatory regions (CBF1 binding sites) in the promoters of Notch responsive genes. In this way, CBF1 binding sites represent a good target for the design of a Notch signaling reporter.ResultsTo generate a single-cell resolution Notch signaling reporter, we used a CBF responsive element to direct the expression of a nuclear-localized fluorescent protein. To do this, we linked 4 copies of a consensus CBF1 binding site to the basal simian virus 40 (SV40) promoter, placed this cassette in front of a fluorescent protein fusion comprising human histone H2B linked to the yellow fluorescent protein (YFP) Venus, one of the brightest available YFPs. We used the CBF:H2B-Venus construct to generate both transgenic embryonic mouse stem (ES) cell lines and a strain of transgenic mice that would report Notch signaling activity.ConclusionBy using multiple CBF1 binding sites together with a subcellular-localized, genetically-encoded fluorescent protein, H2B-Venus, we have generated a transgenic strain of mice that faithfully recapitulates Notch signaling at single-cell resolution. This is the first mouse reporter strain in which individual cells transducing a Notch signal can be visualized. The improved resolution of this reporter makes it ideal for live imaging developmental processes regulated by the Notch signaling pathway as well as a short-term lineage tracer of Notch expressing cells due to the perdurance of the fluorescent reporter. Taken together, the CBF:H2B-Venus mouse strain is a unique tool to study and understand the morphogenetic events regulated by the Notch signaling pathway.

Cell Lineage Allocation Within the Inner Cell Mass of the Mouse Blastocyst

At the time of implantation, the early mouse embryo consists of three distinct cell lineages: the epiblast (EPI), primitive endoderm (PrE), and trophectoderm (TE). Here we will focus on the EPI and PrE cell lineages, which arise within the inner cell mass (ICM) of the blastocyst. Though still poorly understood, our current understanding of the mechanisms underlying this lineage allocation will be discussed. It was originally thought that lineage choice was strictly controlled by the position of a cell within the ICM. However, it is now believed that the EPI and PrE lineages are defined both by their position and by the expression of lineage-specific transcription factors. Interestingly, these lineage-specific transcription factors are initially co-expressed in early ICM cells, suggesting an initial multi-lineage priming state. Thereafter, lineage-specific transcription factors display a mutually exclusive salt-and-pepper distribution that reflects cell specification of the EPI or PrE fates. Later on, lineage segregation and likely commitment are completed with the sequestration of PrE cells to the surface of the ICM, which lies at the blastocyst cavity roof. We discuss recent advances that have focused on elucidating how the salt-and-pepper pattern is established and then resolved within the ICM, leading to the correct apposition of cell lineages in preparation for implantation.

Live Imaging Fluorescent Proteins in Early Mouse Embryos

Mouse embryonic development comprises highly dynamic and coordinated events that drive key cell lineage specification and morphogenetic events. These processes involve cellular behaviors including proliferation, migration, apoptosis, and differentiation, each of which is regulated both spatially and temporally. Live imaging of developing embryos provides an essential tool to investigate these coordinated processes in three-dimensional space over time. For this purpose, the development and application of genetically encoded fluorescent protein (FP) reporters has accelerated over the past decade allowing for the high-resolution visualization of developmental progression. Ongoing efforts are aimed at generating improved reporters, where spectrally distinct as well as novel FPs whose optical properties can be photomodulated, are exploited for live imaging of mouse embryos. Moreover, subcellular tags in combination with using FPs allow for the visualization of multiple subcellular characteristics, such as cell position and cell morphology, in living embryos. Here, we review recent advances in the application of FPs for live imaging in the early mouse embryo, as well as some of the methods used for ex utero embryo development that facilitate on-stage time-lapse specimen visualization.

Live Imaging, Identifying, and Tracking Single Cells in Complex Populations In Vivo and Ex Vivo

Advances in optical imaging technologies combined with the use of genetically encoded fluorescent proteins have enabled the visualization of stem cells over extensive periods of time in vivo and ex vivo. The generation of genetically encoded fluorescent protein reporters that are fused with subcellularly localized proteins, such as human histone H2B, has made it possible to direct fluorescent protein reporters to specific subcellular structures and identify single cells in complex populations. This facilitates the visualization of cellular behaviors such as division, movement, and apoptosis at a single-cell resolution and, in principle, allows the prospective and retrospective tracking towards determining the lineage of each cell.

Quantitative analyses for elucidating mechanisms of cell fate commitment in the mouse blastocyst

In recent years we have witnessed a shift from qualitative image analysis towards higher resolution, quantitative analyses of imaging data in developmental biology. This shift has been fueled by technological advances in both imaging and analysis software. We have recently developed a tool for accurate, semi-automated nuclear segmentation of imaging data from early mouse embryos and embryonic stem cells. We have applied this software to the study of the first lineage decisions that take place during mouse development and established analysis pipelines for both static and time-lapse imaging experiments. In this paper we summarize the conclusions from these studies to illustrate how quantitative, single-cell level analysis of imaging data can unveil biological processes that cannot be revealed by traditional qualitative studies.

Stem Cells from Early Mammalian Embryos

Early mammalian embryonic development is characterized by the existence of pluripotent or multipotent cell populations that are present for limited periods of time during lineage specification and cellular differentiation. In addition to the pluripotent embryonic stem cell lines that can be derived from the epiblast lineage of the inner cell mass of the blastocyst, stem cell lines have now been derived from the trophectoderm layer, the primitive endoderm, and the postimplantation epiblast, as well as from the primordial germ cells. These stem cell lines generally have the developmental potential and characteristics corresponding to their tissue of origin and the conditions for their derivation reflect the intrinsic genetic programs of lineage specification and differentiation. The extensive investigation of mouse embryo-derived stem cell lines has paved the way for therapeutic applications of stem cells. The derivation of pluripotent stem cells from human embryos as well as the development of induced pluripotent stem cell lines from non-embryo sources brings widespread application of stem cell-based therapies closer to reality.