quantitative-analysis-protein-expression-to-study-lineage

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

synthesis-characterization-self-assembled-metal-organic-framework

Synthesis and Characterization of Self-Assembled Metal-Organic Framework Monolayers Using Polymer-Coated Particles

Metal-organic frameworks (MOFs) are materials with potential applications in fields such as gas adsorption and separation, catalysis, and biomedicine. ...

Sloan Kettering Institute

University of California, San Diego

We investigate the feasibility of numerically calculating morphology-dependent resonance (MDR) peaks. To do so, one has to calculate the scattering intensities numerically and determine how difficult it is to numerically predict the position and the magnitude of the MDR peaks. However, at present, in practice it is impossible to calculate MDR peaks with a personal computer because so much computing time is required. Therefore the surface values of the Debye potential and its derivative for a homogeneous sphere are obtained from Mie's analytical solution and then used in integral equations to give the scattering intensities at a specific position of infinity by numerical integrations. It is shown that if a sufficient number of surface elements are used, the MDR peaks are exactly calculated for a homogeneous sphere with refractive index of 1.5, 1.4, and 1.3 up to a size parameter of 20. One can conjecture the number of finite and boundary elements necessary to numerically compute accurate scattering intensities. It should be also noted that the number of surface elements necessary for exact integration shows peaks similar to MDR peaks with respect to the size parameter. Therefore one will need many more elements at the size parameter at which the MDR occurs.

Feasibility of calculating morphology-dependent resonance peaks by numerical methods.

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.

Cell lineage allocation within the inner cell mass of the mouse blastocyst.

The emergence of pluripotent epiblast (EPI) and primitive endoderm (PrE) lineages within the inner cell mass (ICM) of the mouse blastocyst involves initial co-expression of lineage-associated markers followed by mutual exclusion and salt-and-pepper distribution of lineage-biased cells. Precisely how EPI and PrE cell fate commitment occurs is not entirely clear; however, previous studies in mice have implicated FGF/ERK signaling in this process. Here, we investigated the phenotype resulting from zygotic and maternal/zygotic inactivation of Fgf4. Fgf4 heterozygous blastocysts exhibited increased numbers of NANOG-positive EPI cells and reduced numbers of GATA6-positive PrE cells, suggesting that FGF signaling is tightly regulated to ensure specification of the appropriate numbers of cells for each lineage. Although the size of the ICM was unaffected in Fgf4 null mutant embryos, it entirely lacked a PrE layer and exclusively comprised NANOG-expressing cells at the time of implantation. An initial period of widespread EPI and PrE marker co-expression was however established even in the absence of FGF4. Thus, Fgf4 mutant embryos initiated the PrE program but exhibited defects in its restriction phase, when lineage bias is acquired. Consistent with this, XEN cells could be derived from Fgf4 mutant embryos in which PrE had been restored and these cells appeared indistinguishable from wild-type cells. Sustained exogenous FGF failed to rescue the mutant phenotype. Instead, depending on concentration, we noted no effect or conversion of all ICM cells to GATA6-positive PrE. We propose that heterogeneities in the availability of FGF produce the salt-and-pepper distribution of lineage-biased cells.

FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse.

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.

Live imaging, identifying, and tracking single cells in complex populations in vivo and ex vivo.

At the end of the preimplantation period, the inner cell mass (ICM) of the mouse blastocyst is composed of two distinct cell lineages, the pluripotent epiblast (EPI) and the primitive endoderm (PrE). The current model for their formation involves initial co-expression of lineage-specific markers followed by mutual-exclusive expression resulting in a salt-and-pepper distribution of lineage precursors within the ICM. Subsequent to lineage commitment, cell rearrangements and selective apoptosis are thought to be key processes driving and refining the emergence of two spatially distinct compartments. Here, we have addressed a role for Platelet Derived Growth Factor (PDGF) signaling in the regulation of programmed cell death during early mouse embryonic development. By combining genetic and pharmacological approaches, we demonstrate that embryos lacking PDGF activity exhibited caspase-dependent selective apoptosis of PrE cells. Modulating PDGF activity did not affect lineage commitment or cell sorting, suggesting that PDGF is involved in the fine-tuning of patterning information. Our results also indicate that PDGF and fibroblast growth factor (FGF) tyrosine kinase receptors exert distinct and non-overlapping functions in PrE formation. Taken together, these data uncover an early role of PDGF signaling in PrE cell survival at the time when PrE and EPI cells are segregated.

PDGF signaling is required for primitive endoderm cell survival in the inner cell mass of the mouse blastocyst.

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.

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

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 rapid and efficient 2D/3D nuclear segmentation method for analysis of early mouse embryo and stem cell image data.

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 mammalian embryonic development prevents fluctuations in cell fate.

Minjung Kang

Developmental Biology Program

Sloan Kettering Institute

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

Synthesis and Characterization of Self-Assembled Metal-Organic Framework Monolayers Using Polymer-Coated Particles

Minjung Kang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Developmental Biology Program

Sloan Kettering Institute

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

Synthesis and Characterization of Self-Assembled Metal-Organic Framework Monolayers Using Polymer-Coated Particles

Developmental Biology Program