Chromatin remodeling and <i>Xist</i> expression in ROSI-derived embryos.

<p>A) Left panel: Representative image of a decondensed nucleus of a mature mouse spermatozoon stained with H3.1/2 (green) and anti-centromere antibody (ACA) (red), to illustrate the limited presence of histones in association with pericentromeric chromatin in mature mouse sperm, as shown previously by others [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006358#pgen.1006358.ref024" target="_blank">24</a>]. Right panels: Representative image of immunolocalization of H3K9me3 (green) in chromosome spreads of prometaphase-arrested mouse zygotes obtained by ICSI (as represented by the drawing, n = 3 zygotes). The condensed chromosomes of paternal and maternal origin are cutouts from the whole zygote images (paternal chromosome set on top; maternal below). DNA is counterstained with DAPI (blue). B) Left panel: Representative image of a nucleus of a mouse round spermatid immunostained for H3K9me3 (green), to illustrate the enrichment of H3K9me3 on the chomocenter (encircled) and adjacent sex chromosome (arrowhead) as described previously [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006358#pgen.1006358.ref004" target="_blank">4</a>]. Right panels: Representative image of immunolocalization of H3K9me3 (green) in chromosome spreads of prometaphase-arrested zygotes obtained by ROSI (as represented by the drawing, n = 20 zygotes). The condensed chromosomes of paternal and maternal origin are cutouts from the whole zygote images (paternal chromosome set on top; maternal below). The X chromosome is indicated by a white dashed square box, whose identity is inferred from the known enrichment of the round spermatid X chromosome for this marker [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006358#pgen.1006358.ref005" target="_blank">5</a>]. C) Representative image of immunofluorescence analysis for H3K9me3 (green) on chromosome spreads of a representative prometaphase-arrested 2-cell stage embryo obtained by ROSI (as indicated by the drawing on the left, n = 2). Each blastomere from the same embryo is cutout into separate images (1^st blastomere on top; 2^nd blastomere below). The X chromosome is indicated by a white dashed square box. <i>Xist</i> DNA FISH (left panel) was performed on the same chromosome spreads represented on the left. Square boxes on the right are blowups of each corresponding boxed area containing one X chromosome (1^st blastomere, one X is not visible) or two X chromosomes (2^nd blastomere). D) Representative images of <i>Xist</i> RNA FISH on a ROSI-derived 4-cell stage embryo on the left (n = 5 ROSI embryos), and a 4-cell stage embryo derived by <i>in vivo</i> fertilization (n = 4). Dashed red square boxes are blowups of each corresponding boxed area. E) Representative image of <i>Xist</i> RNA FISH on a ROSI-derived 8-cell stage embryo on the left (n = 5 ROSI embryos), and an 8-cell stage embryo derived by <i>in vivo</i> fertilization. Dashed red square boxes are blowups of each corresponding boxed area.