Journal: PLoS ONE

Article Title: Global Chromatin Architecture Reflects Pluripotency and Lineage Commitment in the Early Mouse Embryo

doi: 10.1371/journal.pone.0010531

Figure Lengend Snippet: Left panels show fluorescence microscopy of physical sections (70 nm thickness) of E3.5 blastocysts immuno-labelled for Nanog (A, epiblast), Gata6 (B, primitive endoderm) and Cdx2 (C, polar and mural trophectoderm). Although auto-fluorescence was detected in the nucleolus of some cells, this does not impact the ability to distinguish between nuclei that are positive or negative for Cdx2/Gata6. Positive cells are indicated by arrows in each image. Panels in the second column show low magnification mass-sensitive image of a positive cell. Merged phosphorus and nitrogen maps of the area indicated in the mass-sensitive images are shown at two magnifications in the right-side panels. 10 nm chromatin fibres are prevalent in epiblast (arrows) whereas blocks of compact chromatin (arrowheads) with fewer dispersed 10 nm fibres are observed in extra-embryonic progenitor cells. (D), Fluorescence images of DAPI stained nuclei of epiblast (left) and trophoblast (right) cells of a E3.5 blastocyst (scale bar represents 5 µm). (E), Chromatin compaction was quantified by measuring the distribution of chromatin cluster size in each cell type (see for details). PE and polar TE cells contain larger chromatin clusters than EPI cells, suggesting that lineage-restricted cells have a more condensed chromatin architecture than pluripotent cells.

Article Snippet: Permeabilized embryos were labeled with mouse anti-Cdx2 (1∶200, Biogenex CDX2-88), rabbit anti-Cdx2 (1∶10) , mouse anti-Oct4 (1∶100, Santa Cruz C-10), rabbit anti-Nanog (1∶200, ReproCell) and goat anti-Gata6 (1∶200, R&D) for 2 hours at RT.

Techniques: Fluorescence, Microscopy, Staining