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: A tailor-made system to trace X-chromosome inactivation and reactivation dynamics during PGCLC induction Schematic representation of the features implemented in the XRep cell line. A hybrid background in which cells carry one X chromosome from M.m. musculus (Xmus) and one from M.m. castaneus (Xcas). The cell line carries an rtTA under the control of the Rosa26 locus and piggyBac transposon-based vectors with doxycycline (Dox)-responsive promoters driving the expression of Prdm14, Blimp1 and Tfap2c. The Xmus carries a GFP reporter and a truncation of the Tsix transcript while the Xcas carries a tdTomato reporter.Overview of the adapted PGCLC differentiation timeline. Stages of the culture system are shown.Representative FACS data of primordial germ cell-specific surface markers CD61 and SSEA1 in ESCs, EpiLCs d4 and PGCLCs d5. Numbers indicate the percentages of SSEA1+/CD61+ gated cells over time. Shown are contour plots gated on live cells.Immunostaining of PGCLCs d5 cryosections for SOX2 (magenta) and TFAP2C (cyan). Barplot indicates the quantification of SOX2+ cells, TFAP2C+ cells and SOX2+/TFAP2C+. n = 1,150 cells, from n = 3 separate inductions, using two biological clones. The white squares represent the position of the magnified region at the bottom. Scale bar, 50 µm and 10 µm for the magnified region.Representative culture showing the X-activity reporter during PGCLC induction. Images for bright field (BF), XGFP and XTomato were taken for ESCs, EpiLC d4 and PGCLC d5. Scale bar, 50 µm.Representative FACS data showing XGFP (left) and XTomato (right) distribution during PGCLC induction. Numbers indicate the percentage of cells gated according to the XGFP and XTomato status (grey = X-inactive, green/red = X-active). Dashed line indicates the transition from X-active to X-inactive according to XGFP levels. XGFP and XTomato percentages in ESCs, EpiLCs and d1 full bodies are calculated from the entire cell population, while in PGCLC d2 to PGCLC d5 are calculated from SSEA1+/CD61+ PGCLCs as indicated. Shown are histograms gated on live cells.