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. 2021 Oct 1;12:5771. doi: 10.1038/s41467-021-26065-6

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a WT ESC and ESC lines with MLL3/4 amino acid substitutions were differentiated into PGCLC. PGCLC were quantified as the percentage of CD15⁺CD61⁺ cells within d4 EB. Barplots: mean percentage of PGCLC ± SD from at least four biological replicates. P-values were calculated using two-sided Wilcoxon tests. b UMAP plot showing cell clusters identified within WT and dCD d4 EB according to scRNA-seq data. Cluster identity was determined by differential expression analysis between clusters and using specific tissue markers found in E8.25 mouse embryos⁴². c Same UMAP plot as in b, with WT and dCD cells represented by red and blue dots, respectively. d Percentage of WT and dCD cells found within the PGCLC cluster shown in c. e Cluster distribution of WT (left) and dCD (right) Prdm1orDppa3⁺/Klf4^- cells. f Transcript levels of all PGCLC genes in WT or dCD Prdm1orDppa3⁺/Klf4^- cells located within the indicated d4 EB clusters. Dots represent cells and the dashed line shows the mean transcript level of the PGCLC genes for all WT and dCD cells except those within the PGCLC cluster. g Expression of the PGCLC genes linked (n = 216) or not (n = 170) to at least one PGCLC enhancer within WT and dCD Prdm1orDppa3 + /Klf4- cells. P-values were calculated using paired two-sided student t-tests. The horizontal lines in the boxplots indicate the median, the boxes indicate the first and third quartiles, and the whiskers the ± 1.5 × interquartile range. h R1 WT, Otx2^−/−, dCD, and dCD Otx2^−/− ESC were differentiated into PGCLC. PGCLC were quantified as the proportion of CD15⁺CD61⁺ cells within d4 EB. dCD Otx2^−/− differentiations were performed in biological triplicates using two different clonal lines (n = 3 × 2). Other PGCLC measurements were performed in biological triplicates. Barplots: mean percentage of PGCLC ± SD. P-values were calculated using two-sided Wilcoxon tests. i Percentage of CpG methylation for Group I and II PGCLC enhancers in R1 Otx2^−/− and dCD Otx2^−/− d2 EpiLC. P-values were calculated using paired two-sided Wilcoxon tests. Scales: percentage of methylated CpGs.