Fig 2: Gene expression analysis reveals two PGCLC subpopulations Top panel shows representative contour plots of FACS analysis of PGCLC induction, without or with Dox, in PGCLC d5. The number indicates the percentage of gated germ cells identified by CD61 and SSEA1 signal. Bottom panel indicates the percentages of XGFP+ (green box) or XGFP- (red box) cells. For the +Dox condition, percentages of XGFP+ and XGFP- cells originating from SSEA1+/CD61+ gate are shown. Shown are contour plots gated on live cells.PCA of gene expression dynamics during PGCLC differentiation. n = Top 500 most variable genes excluding X-chromosomal genes. Axes indicate the variance. Arrows indicate hypothetical trajectory. Shapes indicate the clones (A11 = square, E9 = rhombus).Quantitative RT–PCR of selected differentially expressed early germ cell marker genes in ESCs, EpiLCs, XGFP+ PGCLCs and XGFP- PGCLCs during the differentiation time course. Each point represents a separate biological replicate, originating from either clone A or clone E. n = 3 biological replicates. The numbers above the bars indicate P-values (two-sample unpaired Wilcoxon–Mann–Whitney test with R defaults).Expression levels (normalized DEseq2 counts) of selected differentially expressed meiosis marker genes in ESCs, EpiLCs, XGFP+ PGCLCs and XGFP- PGCLCs during the differentiation time course. Genes with FDR < 0.001 were considered significantly differentially expressed. Points indicate expression of individual biological replicates.Representative images showing the X-activity reporter status in colonies formed by ESCs, XGFP+ PGCLCs and XGFP- PGCLCs after 7 days of culture in 2i/LIF on immortalized mouse embryonic fibroblasts. BF, bright field. Scale bars = 200 µm.FACS analysis showing the X-reporter status of the indicated cell types after 7 days of culture in 2i/LIF on immortalized mouse embryonic fibroblasts. Numbers indicate the percentage of cells falling in the corresponding gate. Histograms come from XTomato+ gated cells depleted of immortalized mouse embryonic fibroblasts.

Fig 3: A tailor-made system to trace X-chromosome inactivation and reactivation dynamics during PGCLC induction The left panel shows representative contour plots of FACS analysis of PGCLC induction, without or with Dox, in PGCLC d4 induced from EpiLC d2. The right panel shows PGCLC d5 induced from EpiLC d4. The number indicates the percentage of gated germ cells identified by CD61 and SSEA1 signal. Shown are contour plots gated on live cells.Bar plots showing XGFP percentages from CD61+ SSEA1+ PGCLCs. Each dot represents a separate induction (n = 3), performed in two biological clones.Quantitative RT–PCR of XGFP and XTomato reporters throughout the differentiation timeline, normalized to embryonic stem cells. Horizontal line indicates mean fold change for three separate inductions, using two different clones.Immunolabelling with antibodies against SOX2 (yellow), XGFP (green) and H3K27me3 (red) in EpiLCs, PGCLCs d1 and PGCLCs d5. Images show representative groups of cells showing H3K27me3 enrichment on the Xmus. The white squares represent the position of the magnified region at the right. Cells were counterstained with DAPI (grey). Dashed line indicates SOX2+/H3K27me3- cells. Continuous line indicates SOX2+/H3K27me3+ cells. Scale bar, 50 and 10 µm for the magnified region.Barplots indicating the percentage of cells having H3K27me3 accumulation. PGCLC d1 and PGCLC d5 H3K27me3 percentages are calculated from SOX2-positive cells. On top of the bars, the total cell number analysed from n = 3 separate inductions, using two biological clones, is indicated.Barplots indicating the percentage of H3K27me3 accumulation separated by XGFP- and XGFP+ cells. PGCLC d1 and PGCLC d5 H3K27me3 percentages are calculated from SOX2-positive cells. On top of the bars, the total cell number analysed from n = 3 separate inductions, using two biological clones is indicated.

Fig 4: Gene expression analysis reveals two PGCLC subpopulations PCA of gene expression dynamics during PGCLC differentiation. Four biological replicates are shown. n = top 500 most variable genes. PGCLCs were sorted for SSEA1 and CD61 expression and further divided into XGFP+ and XGFP-. Axes indicate the variance. Arrows indicate hypothetical trajectory. Shapes indicate the biological clone (clone A11 = square, clone E9 = rhombus).PCA of gene expression dynamics compared to in vivo samples from Nagaoka et al (2020). n = top 500 most variable genes, calculated including in vivo samples. Grey arrow indicates putative developmental trajectory. Shapes indicate the replicates (clone A11 = square, clone E9 = rhombus, in vivo samples = circle).MA plot of differential gene expression changes between XGFP- and XGFP+ PGCLCs as determined by RNA-seq. Log2-mean expression (log2-normalized counts from DESeq2) on the X-axis and the log2-fold change on the Y-axis are shown. Significantly upregulated and downregulated genes are highlighted in red and green respectively. False discovery rate (FDR) < 0.001. Non-significant genes with log2-mean expression between 0 and 0.2 were removed for easier plot visualization.Expression levels (normalized DEseq2 counts) of selected differentially expressed genes between XGFP- and XGFP+ PGCLCs during the differentiation time course. Genes with FDR < 0.001 were considered significantly differentially expressed. Points indicate expression of individual biological replicates.Heatmap of RNA-seq normalized counts showing the Z-score across PGCLC induction time points of 31 manually selected and manually ordered marker genes belonging to the categories reported on the side.Selected GO terms enriched in XGFP- PGCLCs and XGFP+ PGCLCs.FACS analysis of cell cycle using DAPI. Numbers indicate the percentage of cells in G1, S and G2/M respectively.Alkaline phosphatase staining for ESCs, XGFP+ PGCLCs and XGFP- PGCLCs grown for 7 days in 2i/LIF medium on immortalized mouse embryonic fibroblasts.Barplot indicating the absolute numbers of alkaline phosphatase (AP)-positive colonies in each cell type after 7 days of culture in 2i/LIF medium on immortalized mouse embryonic fibroblasts. Y-axis is in square root scale (sqrt) for better plot visualization. Each white dot represents one technical replicate. Source data are available online for this figure.