Fig. 1: Exit from primed pluripotency is associated with mechano-osmotic remodelling of the nucleus.
From: Mechano-osmotic signals control chromatin state and fate transitions in pluripotent stem cells
a, Representative images (from five embryos) and quantification of nuclear volume in human preimplantation stage embryos stained for DAPI, Gata6, Nanog and LaminB1. b, Nuclear volume of Gata6-high cells in the ICM (scale bars, 50 and 5 µm; n = 20 (Gata high), 16 (Nanog high) nuclei pooled across 5 embryos; mean ± s.d.; Mann–Whitney). c, Representative images of human preimplantation stage embryos stained for DAPI, Nanog and F-Actin (phalloidin). Note actin-rich bleb-like structures with corresponding nuclear deformation (scale bars, 20 and 10 µm; images representative of 3 embryos). d, Representative images and quantification of nuclear volume and phosphorylated p38 (p-p38) in Gata6-positive and Gata6-negative ICM cells from human blastoids generated from naive hiPS cells stained for Gata6, Oct3/4 and pp38 (scale bar, 100 µm; n = 6 blastoids representing 33 (Gata6 high) and 76 (Gata6 low) nuclei for volumes and 26 (Gata6 high) and 126 (Gata6 low) for p-p38 intensity, respectively; paired t-test). e, Representative top views (x–y), 3D reconstructions and cross-sections (z), of Sox2-GFP-tagged hiPS cells undergoing ectodermal differentiation for the indicated timepoints (representative of 3 independent experiments; scale bars, 15 µm). f, Quantification of nuclear height from hiPS cells undergoing ectodermal differentiation for the indicated timepoints (n = 3 independent experiments with 360 (t = 0), 591 (t = 8), 656 (t = 24), 1,086 (t = 48) total nuclei/timepoint; Kruskal–Wallis/Dunn’s). g, Quantification of nuclear volume from hiPS cells undergoing trilineage differentiation for the indicated timepoints (n = 3 independent experiments with 903, 946, 1,771 and 1,636 (ectoderm 0, 8, 24 and 48 h, respectively); 334, 437, 816 and 741 (mesoderm 0, 8, 24 and 48 h, respectively); and 393, 416, 709 and 835 (endoderm 0, 8, 24 and 48 h, respectively) total nuclei/condition; Kruskal–Wallis/Dunn’s). h, Representative immunofluorescence images of Sox2-GFP-tagged hiPS cells on 2D micropatterns treated with BMP4 for the indicated timepoints and stained for Brachyury and Nanog. Note radial pattern of differentiation at 48 h (scale bars, 100 µm; n = 3 independent experiments). i,j, Representative snapshots (i) and quantification of nuclear deformation and Sox2 intensity dynamics (j) from live imaging videos of mosaic micropatterns with Sox2-GFP and LaminB1-RFP hiPS cells treated with BMP4 for the indicated timepoints. Note transient compaction of colony, accompanied by nuclear deformation before appearance of radial differentiation pattern (scale bars, 100 µm; n = 9 gastruloids pooled across 4 independent experiments; mean ± s.d.). k,l, Representative immunofluorescence images (k) and quantification (l) of LaminB1-RFP-tagged hiPS cells on 2D micropatterns treated with BMP4 for the indicated timepoints of maximal colony compaction and stained for p-p38 and YAP. Note transient YAP and p38 activation at pattern centres as well as sustained YAP and p38 activation within edge cells (scale bars, 100 µm; n = 9 (9 h)/10 (rest) 2D gastruloids pooled across 3 independent experiments with 1,278, 1,918 and 3,203 (6 h centre, mid and edge, respectively); 1,249, 1,881 and 3,135 (7 h centre, mid and edge, respectively); 1,388, 2,155 and 3,590 (8 h centre, mid and edge, respectively); 1,363, 2,054 and 3,421 (9 h centre, mid and edge, respectively); and 1,260, 1,898 and 3,750 (10 h centre, mid and edge, respectively) total nuclei/condition; minimum-to-maximum box plots show 75th, 50th and 25th percentiles; ANOVA/Dunnett’s). a.u., arbitrary units; diff, differentiation; nuc, nucleus; cyto, cytoplasm.
