LETTER doi:10.1038/nature11918 PRC1 coordinates timing of sexual differentiation of female primordial germ cells Shihori Yokobayashi 1 {, Ching-Yeu Liang 1,2 , Hubertus Kohler 1 , Peter Nestorov 1,2 , Zichuan Liu 1 , Miguel Vidal 3 , Maarten van Lohuizen 4 , Tim C. Roloff 1 & Antoine H. F. M. Peters 1,2 In mammals, sex differentiation of primordial germ cells (PGCs) is determined by extrinsic cues from the environment 1 . In mouse female PGCs, expression of stimulated by retinoic acid gene 8 (Stra8) and meiosis are induced in response to retinoic acid provided from the mesonephroi 2–5 . Given the widespread role of retinoic acid signalling during development 6,7 , the molecular mechanisms that enable PGCs to express Stra8 and enter meiosis in a timely manner are unknown 2,8 . Here we identify gene-dosage-dependent roles in PGC development for Ring1 and Rnf2, two central components of the Polycomb repressive complex 1 (PRC1) 9,10 . Both paralogues are essential for PGC development between days 10.5 and 11.5 of gesta- tion. Rnf2 is subsequently required in female PGCs to maintain high levels of Oct4 (also known as Pou5f1) and Nanog expression 11 , and to prevent premature induction of meiotic gene expression and entry into meiotic prophase. Chemical inhibition of retinoic acid signal- ling partially suppresses precocious Oct4 downregulation and Stra8 activation in Rnf2-deficient female PGCs. Chromatin immunopre- cipitation analyses show that Stra8 is a direct target of PRC1 and PRC2 in PGCs. These data demonstrate the importance of PRC1 gene dosage in PGC development and in coordinating the timing of sex differentiation of female PGCs by antagonizing extrinsic retinoic acid signalling. In mammalian somatic cells, PRC1 and PRC2 proteins are trans- criptional repressors that function in large multiprotein complexes and that modify chromatin by mono-ubiquitinating histone H2A at lysine 119 (H2AK119u1) and trimethylating histone H3 at lysine 27 (H3K27me3), respectively 9,12 . At day 12.5 of embryonic development (E12.5), in PGCs marked by Cdh1 (E-cadherin) staining 13 , we obser- ved nuclear localization of PRC1 components Rnf2 (also known as Ring1B), Mel18 (also known as Pcgf2) and Rybp (Fig. 1a and Supplementary Fig. 1) as well as a robust H2AK119u1 signal, suggest- ing the presence of catalytically active PRC1 complexes (Fig. 1a). To 1 Friedrich Miescher Institute for Biomedical Research (FMI), Maulbeerstrasse 66, CH-4058 Basel, Switzerland. 2 Faculty of Sciences, University of Basel, CH-4056 Basel, Switzerland. 3 Centro de Investigaciones Biolo ´ gicas, Consejo Superior de Investigaciones Cientı ´ficas (CSIC), 28040 Madrid, Spain. 4 Division of Molecular Genetics and Centre for Biomedical Genetics, the Netherlands Cancer Institute (NKI), 1066 CX Amsterdam, the Netherlands. {Present address: Department of Reprogramming Science, Center for iPS Cell Research and Application, Kyoto University, 53 Kawahara-cho, Shogoin Yoshida, Sakyo-ku, Kyoto 606-8507, Japan. DNA Cdh1 e c Number of Oct4 + PGCs per embryo Female Rnf2 + Female Rnf2 cko Male Rnf2 + Male Rnf2 cko 0 2,000 4,000 6,000 8,000 10,000 12,000 E10.5 E11.5 E12.0 E12.5 E13.5 * ** Female Male b Rnf2 + Rnf2 cko Oct4 Rnf2 Nanog E12.5 0 1 0 1 0 5 ** ** f Rnf2 + Rnf2 Δ Female Male Female Male Female Male * * Rnf2 + Rnf2 cko Rnf2 H2AK119u1 * * * * * * * * * * * * * * * * * * * * * 0.2 0.4 0.6 0.8 1.0 Rnf2 + Rnf2 cko Female E12.5 E12.5 E11.5 Fraction of high GFP PGCs (22) (15) (16) (12) (18) (9) (20) (12) * * ** * + Rnf2: Female Δ + Male Δ + Female Δ + Male Δ Number of cells Rnf2 + Oct4 + Rnf2 + Oct4 – Rnf2 – Oct4 + Rnf2 – Oct4 – Number of Cdh1 + PGCs per gonadal area (E12.5) d 0 10 20 5 15 Rnf2 + Rnf2 cko Male Rnf2 + Rnf2 cko Female (161)(152) (101)(160) (167)(178)(179) (101) (77) (30) * a 50 37 25 12 0 10 0 10 1 10 2 10 3 10 4 GFP intensity 50 37 25 12 0 10 0 10 1 10 2 10 3 10 4 Figure 1 | Rnf2 regulates PGC development and Oct4 and Nanog expression in Rnf2 cko female gonads. a, Immunofluorescence staining of H2AK119u1, Rnf2 and Cdh1 with DAPI (49,6-diamidino-2-phenylindole) in Rnf2 1 and Rnf2 cko gonadal sections from E12.5 female embryos. Arrowheads denote Rnf2 1 PGCs and asterisks denote Rnf2 D PGCs. Scale bars, 10 mm. b, Immunofluorescence staining of Oct4 in E13.5 Rnf2 1 and Rnf2 cko whole gonads and mesonephroi. Scale bars, 300 mm. c, Average number of Oct4- positive cells in whole gonads at E10.5–E13.5. Error bars indicate 1s.d. n 5 2–12. *P , 0.005; **P , 1.0 3 10 25 (Student’s t-test). d, Classification of Cdh1-positive PGCs according to Rnf2 and Oct4 protein levels in Rnf2 1 and Rnf2 cko E12.5 gonads. y axis represents the number of PGCs that were normalized to areas analysed (10,000 mm 2 ). Numbers in brackets denote number of PGCs scored per embryo. *P , 1.0 3 10 28 (chi-squared test). e, Representative histograms showing Oct4(DPE)–GFP signals in PGCs from female Rnf2 1 and Rnf2 cko E12.5 gonads. Boxplots showing the ratios of PGCs with high GFP intensity (.10 3 , enclosed by dashed line in histogram) over all GFP-positive cells (enclosed by solid line) in different embryos. Numbers in brackets denote number of embryos analysed. *P , 0.05; **P , 0.005 (Student’s t-test). f, Representative qRT–PCR data of Rnf2, Oct4 and Nanog in Rnf2 1 and Rnf2 D PGCs (normalized to Tbp). Error bars indicate 1s.d. of 2–3 technical replicates. *P , 0.05; **P , 0.01 (Student’s t-test). 236 | NATURE | VOL 495 | 14 MARCH 2013 Macmillan Publishers Limited. All rights reserved ©2013