Fig 2: fPSCs possess similar chromatin states of E6.5 epiblasts.a Heatmaps showing H3K4me3/H3K27me3 enrichment at all promoters of genes (± 2.5 kb) in naïve mESCs, EpiLCs, EpiSCs, fPSCs, E6.5 epiblast and recently reported RSCs, FTW-ESCs as well as FS-AXR. The intensity of H3K4me3/H3K27me3 was normalized as RPKM. The data of H3K4me3/H3K27me3 for naïve mESCs, EpiLCs, EpiSCs, RSCs, FTW-ESCs, FS-AXR, and E6.5 epiblast were obtained from the published resources.42–44,56,57 b Average plot showing H3K4me3 enrichment at all loci of super-bivalent, housekeeping, and bivalent genes in naïve mESCs, EpiLCs, EpiSCs, RSCs, FTW-ESCs, FS-AXR, fPSCs, and E6.5 epiblast. The H3K4me3 enrichment at super bivalent and bivalent genes was normalized to the average intensity of housekeeping genes in each cell line. c Scatter plots displayed the enrichment of H3K4me3 and H3K27me3 of 3992 bivalent genes in different cell types. The average H3K4me3 enrichment of housekeeping genes (HK. ave) was shown for each of cell types. The blue lane indicate an arbitrary cutoff of H3K27me3 for each stage (normalized RPKM > 1). Super-bivalent and bivalent genes were shown as red and gray, respectively. The number of super-bivalent genes (right top) was shown in naïve mESCs, EpiLCs, EpiSCs, RSCs, fPSCs, FTW-ESCs, FS-AXR, and E6.5 epiblasts. d UCSC Genome Browser views showing H3K4me3 enrichment at the locus of super-bivalent genes (Hoxa cluster, Gata4, and Pax2) and housekeeping gene (Rpn1) in mESCs, EpiLCs, EpiSCs, RSCs, FTW-ESCs, FS-AXR, fPSCs, and E6.5 epiblast. e Hierarchical clustering analysis of DNA methylation for naïve mESCs, EpiLCs, fPSCs, EpiSCs and E5.5/6.5/7.5 epiblasts.58–61 f Smoothed scatter plot of genome-wide DNA methylation between EpiLCs or fPSCs and E5.5, or E6.5 epiblasts. Density of data pointed the ranges from blue (low) to green (intermediate) and yellow (high). g Genome Browser views showing the DNA methylation and H3K4me3/H3K27me3 enrichment at the promoters of selected formative markers (Otx2, Zic5) in naïve mESCs, EpiLCs, and EpiSCs, as well as fPSCs and E6.5 epiblasts. h Snapshots of DNA methylation and enrichment of H3K4me3 and H3K27me3 at the promoters of selected germ-layer markers (Pax6, Foxf1 and Foxa2) and Oct4 in naïve mESCs, EpiLCs, and EpiSCs, as well as fPSCs and E6.5 epiblasts.

Fig 3: Otx2 expression in mouse and human lens epitheliums with or without UVB irradiation. (A) Otx2 expression in lens epithelial cells (LECs) with (UV) or without UVB irradiation (Cont) was confirmed by RT-qPCR. (B) Mouse lens sagittal sections from in vivo UVB irradiation experiments containing LECs of the surface and transitional zones were stained with anti-Otx2 antibody or normal IgG as a negative control, and fluorescent images were captured by LSM 710 confocal microscopy. e, LECs; r, retinal cells. Scale bars: 100 µm. (C) Individual fluorescence intensities of cells in Otx2 staining of five randomly selected images were quantified and their lens epithelia (surface area) and transitional zones were compared. The number of quantified cells is indicated. The similar results were obtained in three independent experiments. (D) Human lens epitheliums from six patients were collected and subjected to the UVB irradiation experiment. Otx2 expression was analyzed by RT-qPCR and quantified. Average of three measurements was recorded for each half. For the plots in C and D, all data points are indicated as dots and medians are indicated as horizontal bars connected by a solid diagonal line between the samples. Dashed lines in D indicate expressional differences in the median of each patient pair measured by RT-qPCR. **P<0.01, ***P<0.001.

Fig 4: Epithelial-mesenchymal transition (EMT) in Otx2-OE lens epithelial cells. (A) Otx2-overexpressing cells (Otx2-OE) and control cells (Cont) were stained with anti-fibronectin or anti-α-SMA antibodies (green) and with the nuclear stain DAPI (blue). Scale bars: 20 µm. (B) The fluorescent intensities of anti-fibronectin and anti-α-SMA signals per cell area in five randomly selected images were measured and quantified. Similar results were obtained in three independent experiments. Data points of measurement are indicated as dots and medians are indicated by horizontal bars. (C) TGFβ2 mRNA expression in Otx2-OE cells was analyzed by RT-qPCR. (D) Phosphorylation of Smad 2 (p-SMAD2) and 3 (p-SMAD3) was analyzed by western blot. Smad2/3 and β-actin expression in each sample were used for loading controls in western blotting. (E,F) mRNA expression of α-SMA (E) and γ-SMA (F) in Otx2-OE cells were analyzed by RT-qPCR. **P<0.01, ***P<0.001.

Fig 5: Membrane-Tension-Mediated Endocytosis Promotes ERK Activation during Early Differentiation(A) Fluorescent Western blot for ERK, pERK, and histone3 in ESCs at different time points during exit from naive pluripotency.(B) Time course of the ratio of pERK levels (normalized to corresponding histone 3 levels) in WT and iEZR_CA cells during exit from naive pluripotency (N = 6).(C) Representative single z plane image of WT, iEZR_CA, and iEZR_CA T24 treated with 3 µM BI-D1870, an RSK-inhibitor (resulting in ERK activation) immunostained for Nanog and Otx2.(D) Quantification of Nanog and Otx2 expression in WT, iEZR_CA, iEZR_CA+BI-D1870 T24 cells, normalized to WT mean levels (N = 3).(E) Schematic of the FRET ERK sensor used to measure ERK activation at early endosomes (Palamidessi et al., 2019).(F) Left: time-lapse of a representative cell expressing the FRET ERK sensor and exiting pluripotency at 6 h (top, the cell is still round) and 18 h 20 min (bottom, the cell is spread). Right: time course of the FRET ratio for the cell is shown at left.(G) Quantification of the FRET ratio in ESCs exiting naive pluripotency; time is normalized to the time of spreading (means ± SEMs, n = 10, N = 3). Inset: FRET ratio for round and spread cells in the same dataset.(H) Mean FRET ratio for fixed ESCs and T24 cells in WT and iEZR_CA transfected or not with Rab5a (N = 3).(I) Schematic of the proposed mechanism for how membrane tension regulates signaling during exit from naive pluripotency. The graphical data represent means ± SDs. p values calculated using Welch’s unpaired Student’s t test (unless specified otherwise) and indicated in the figure. Scale bars represent 10 µm.