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• W4281381837 abstract "Article23 May 2022Open Access Source DataTransparent process Controlled X-chromosome dynamics defines meiotic potential of female mouse in vitro germ cells Jacqueline Severino Jacqueline Severino orcid.org/0000-0001-6959-0342 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Moritz Bauer Moritz Bauer orcid.org/0000-0002-5937-8169 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Conceptualization, Data curation, Software, Formal analysis, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Tom Mattimoe Tom Mattimoe orcid.org/0000-0002-7174-6729 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Data curation, Formal analysis, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Niccolò Arecco Niccolò Arecco orcid.org/0000-0003-1760-3898 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Data curation, Software, Formal analysis, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Luca Cozzuto Luca Cozzuto orcid.org/0000-0003-3194-8892 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Software, Methodology Search for more papers by this author Patricia Lorden Patricia Lorden orcid.org/0000-0003-3400-2576 CNAG-CRG, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Contribution: ​Investigation Search for more papers by this author Norio Hamada Norio Hamada orcid.org/0000-0002-0906-8277 Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Contribution: Resources, Methodology Search for more papers by this author Yoshiaki Nosaka Yoshiaki Nosaka orcid.org/0000-0001-8395-1212 Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Contribution: Resources, Methodology Search for more papers by this author So I Nagaoka So I Nagaoka orcid.org/0000-0002-2377-4936 Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Contribution: Resources, Methodology Search for more papers by this author Pauline Audergon Pauline Audergon orcid.org/0000-0002-1624-7388 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Methodology Search for more papers by this author Antonio Tarruell Antonio Tarruell orcid.org/0000-0002-2664-9700 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Methodology Search for more papers by this author Holger Heyn Holger Heyn orcid.org/0000-0002-3276-1889 CNAG-CRG, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Contribution: Resources, Supervision, Methodology Search for more papers by this author Katsuhiko Hayashi Katsuhiko Hayashi orcid.org/0000-0002-2479-2941 Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Contribution: Resources, Supervision, Methodology Search for more papers by this author Mitinori Saitou Mitinori Saitou orcid.org/0000-0002-2895-6798 Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Contribution: Resources, Supervision, Methodology Search for more papers by this author Bernhard Payer Corresponding Author Bernhard Payer [email protected] orcid.org/0000-0002-4694-2082 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Jacqueline Severino Jacqueline Severino orcid.org/0000-0001-6959-0342 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Search for more papers by this author Moritz Bauer Moritz Bauer orcid.org/0000-0002-5937-8169 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Conceptualization, Data curation, Software, Formal analysis, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Tom Mattimoe Tom Mattimoe orcid.org/0000-0002-7174-6729 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Data curation, Formal analysis, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Niccolò Arecco Niccolò Arecco orcid.org/0000-0003-1760-3898 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Data curation, Software, Formal analysis, ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Luca Cozzuto Luca Cozzuto orcid.org/0000-0003-3194-8892 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Software, Methodology Search for more papers by this author Patricia Lorden Patricia Lorden orcid.org/0000-0003-3400-2576 CNAG-CRG, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Contribution: ​Investigation Search for more papers by this author Norio Hamada Norio Hamada orcid.org/0000-0002-0906-8277 Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Contribution: Resources, Methodology Search for more papers by this author Yoshiaki Nosaka Yoshiaki Nosaka orcid.org/0000-0001-8395-1212 Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Contribution: Resources, Methodology Search for more papers by this author So I Nagaoka So I Nagaoka orcid.org/0000-0002-2377-4936 Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Contribution: Resources, Methodology Search for more papers by this author Pauline Audergon Pauline Audergon orcid.org/0000-0002-1624-7388 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Methodology Search for more papers by this author Antonio Tarruell Antonio Tarruell orcid.org/0000-0002-2664-9700 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Contribution: Methodology Search for more papers by this author Holger Heyn Holger Heyn orcid.org/0000-0002-3276-1889 CNAG-CRG, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Contribution: Resources, Supervision, Methodology Search for more papers by this author Katsuhiko Hayashi Katsuhiko Hayashi orcid.org/0000-0002-2479-2941 Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Contribution: Resources, Supervision, Methodology Search for more papers by this author Mitinori Saitou Mitinori Saitou orcid.org/0000-0002-2895-6798 Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan Contribution: Resources, Supervision, Methodology Search for more papers by this author Bernhard Payer Corresponding Author Bernhard Payer [email protected] orcid.org/0000-0002-4694-2082 Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain Universitat Pompeu Fabra (UPF), Barcelona, Spain Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Author Information Jacqueline Severino1,†, Moritz Bauer1,9,†, Tom Mattimoe1, Niccolò Arecco1, Luca Cozzuto1, Patricia Lorden2, Norio Hamada3, Yoshiaki Nosaka4,5,6, So I Nagaoka4,5,6, Pauline Audergon1, Antonio Tarruell1, Holger Heyn2,7, Katsuhiko Hayashi8, Mitinori Saitou4,5,6 and Bernhard Payer *,1,7 1Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, Barcelona, Spain 2CNAG-CRG, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology (BIST), Barcelona, Spain 3Department of Obstetrics and Gynecology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan 4Institute for the Advanced Study of Human Biology (ASHBi), Kyoto University, Kyoto, Japan 5Department of Anatomy and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan 6Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan 7Universitat Pompeu Fabra (UPF), Barcelona, Spain 8Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan 9Present address: Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands † These authors contributed equally to this work *Corresponding author. Tel: +34933160159; E-mail: [email protected] The EMBO Journal (2022)e109457https://doi.org/10.15252/embj.2021109457 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X-chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X-inactivation and reactivation dynamics using a tailor-made in vitro system of primordial germ cell-like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X-inactivation in PGCLCs in vitro and in germ cell-competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X-inactivation is followed by step-wise X-reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X-inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine-tuned X-chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis. Synopsis The kinetics and biological function of epigenetic reprogramming during mammalian germ cell development remain poorly understood. Here, single-cell analysis of X-chromosome activation changes during primordial germ cell-like cell (PGCLC) differentiation uncovers hallmarks of female germ cell development. Time-resolved tracing of X-chromosome dynamics during PGCLC specification from embryonic stem cells shows heterogeneous and moderate X-inactivation compared to somatic cell lineages. Germ cell-competent epiblast cells show heterogeneous and incomplete X-inactivation in vivo. RNA expression profiling highlights uncoupling of X-inactivation and PGCLC fate acquisition. Subsequent X-chromosome reactivation occurs step-wise through prophase I of meiosis. PGCLCs with deficient X-inactivation or premature X-reactivation are abnormal and have reduced meiotic potential. Introduction The germ cell lineage is unique in its critical function to transmit genetic and epigenetic information from one generation to the next. In mice, primordial germ cells (PGCs), the precursors of eggs and sperm, are specified during early postimplantation development from somatic precursors in the proximal epiblast by inductive signals (Lawson et al, 1999; Ohinata et al, 2005, 2009). Thereafter, PGCs migrate and enter the future gonads where they receive sex-specific somatic signals, which determine the germ cell sex and promote differentiation towards a spermatogenic or oogenic fate (Miyauchi et al, 2017; Spiller et al, 2017). While in males, germ cells enter mitotic arrest and differentiate into prospermatogonia, in females, germ cells instead progress into meiosis and oogenesis. A hallmark feature of early germ cell development is the extensive epigenetic reprogramming (Kurimoto & Saitou, 2019), characterized by global changes in histone marks (Seki et al, 2005; Hajkova et al, 2008), DNA demethylation and erasure of genomic imprints (Hajkova et al, 2002; Seisenberger et al, 2012; Shirane et al, 2016). This establishes an epigenetic naive state (Ohta et al, 2017), which is required in order for PGCs to progress towards gonadal germ cell fate (Hill et al, 2018) and to control their timing to enter female meiosis (Yokobayashi et al, 2013). Ultimately, this erasure of parental information allows the reestablishment of new paternal and maternal marks during spermatogenesis and oogenesis, respectively, which are critical for the competence of egg and sperm to facilitate embryonic development in the next generation (Reik & Surani, 2015; Ohta et al, 2017). In addition to these global changes, another important epigenetic reprogramming event takes place in the female germline; the reversal of silencing of the inactive X chromosome by X-chromosome reactivation. While X-chromosome inactivation (Lyon, 1961; Payer & Lee, 2008; Galupa & Heard, 2018) is the process by which female mammals (XX) achieve X-linked gene dosage parity with males (XY), X-reactivation takes place specifically in pluripotent epiblast cells of the mouse blastocyst (Mak et al, 2004; Borensztein et al, 2017) and in PGCs during their migration and upon their entry into the gonads (Sugimoto & Abe, 2007; Chuva de Sousa Lopes et al, 2008). Therefore, while X-inactivation is associated with pluripotency exit and the differentiated state (Schulz et al, 2014), X-reactivation is a key feature of naive pluripotency and germ cell development (Pasque et al, 2014; Payer, 2016; Janiszewski et al, 2019; Panda et al, 2020; Bauer et al, 2021; Talon et al, 2021). X-reactivation in mouse PGCs is a multistep process, which initiates during PGC migration with downregulation of Xist, the long non-coding master regulator RNA of X-inactivation and concomitant loss of the associated histone H3K27me3 mark from the inactive X (Sugimoto & Abe, 2007; Chuva de Sousa Lopes et al, 2008). This process is regulated by repression of the Xist gene by the germ cell transcription factor PRDM14 (Payer et al, 2013; Mallol et al, 2019) and potentially by other members of the pluripotency network such as NANOG or OCT4 (Navarro et al, 2008), which are all expressed during PGC development. Subsequently, X-linked genes become progressively reactivated during migration, with the process being completed after PGCs have reached the gonads, and following the initiation of oogenesis and meiosis (Sugimoto & Abe, 2007; Sangrithi et al, 2017). X-linked gene reactivation is thereby thought to be enhanced by a female-specific signal from gonadal somatic cells (Chuva de Sousa Lopes et al, 2008). Although the molecular nature of the X-reactivation-promoting signal is currently unknown, the timing of X-linked gene reactivation around meiotic entry and the dependency of both processes on a female somatic signal suggest a potential mechanistic link. Until now it has not been formally tested, if, and to which degree, the X-inactivation status might impact the meiotic and oogenic potential of germ cells. Furthermore, previous studies on the X-inactivation and -reactivation dynamics during mouse germ cell development have been limited to few individual genes (Sugimoto & Abe, 2007) or have not been allelically resolved and therefore been unable to discriminate between transcripts expressed from either one or two X chromosomes (Sangrithi et al, 2017). Therefore, a comprehensive analysis of X-inactivation and -reactivation kinetics and its functional relation to germ cell developmental progression is necessary to gain mechanistic insight. Based on in vitro germ cell differentiation from mouse embryonic stem cells (ESCs) (Hayashi et al, 2011, 2012; Nakaki et al, 2013), we developed an X-chromosome reporter system (XRep) to study the kinetics of X-inactivation and -reactivation during germ cell development. We thereby provide a high-resolution allelic analysis of X-chromosome dynamics and discovered that germ cells with high meiotic and oogenic competence are characterized by a moderate degree of X-inactivation and gradual X-reactivation kinetics. In contrast, germ cells that failed to undergo X-inactivation or which reactivated the X chromosome too rapidly displayed abnormal gene expression and differentiation characteristics. Thus, we found first evidence that a controlled sequence of X-inactivation followed by X-reactivation to be a characteristic hallmark of normal female germ cells. This suggests that both dosage control and epigenetic reprogramming of the X chromosome may be critical indicators for female germ cells' developmental potential to progress towards meiosis and oogenesis. Results XRep, a tailor-made system for tracing X-chromosome dynamics during in vitro germ cell development In order to achieve a better understanding of the X-chromosome dynamics during mouse germ cell development, we created a tailor-made in vitro model system called XRep (Fig 1A). XRep combines the following features. First, it is based on a hybrid female embryonic stem cell (ESC) line containing one Mus musculus (Xmus) and one Mus castaneus (Xcas) X chromosome (Lee & Lu, 1999; Ogawa et al, 2008), allowing allele-specific determination of gene expression. Moreover, this line was shown to be karyotypically highly stable (Lee & Lu, 1999; Bauer et al, 2021), therefore preventing X-loss, a crucial prerequisite for X-inactivation and -reactivation studies. Additionally, the cell line contains a Tsix truncation (TST) on Xmus, forcing non-random X-inactivation of the Xmus upon cell differentiation (Luikenhuis et al, 2001; Ogawa et al, 2008). This enabled us to study the X-inactivation and -reactivation dynamics specifically of the Xmus chromosome, while the Xcas would remain constitutively active. Second, primordial germ cell-like cells (PGCLCs) can be obtained highly efficiently from XRep cells by doxycycline-inducible overexpression of the germ cell fate specifier transcription factors BLIMP1 (also known as PRDM1), PRDM14 and TFAP2C (also known as AP2γ) (Nakaki et al, 2013), therefore bypassing the need for addition of cytokines. Last, the X-chromosome status of XRep cells can be traced by dual X-linked reporter genes placed in the Hprt locus (Wu et al, 2014), a GFP reporter on Xmus (XGFP) and a tdTomato reporter on Xcas (XTomato). This allows us to isolate distinct populations of cells, harbouring either two active X chromosomes (XGFP+/XTomato+) or one inactive and one active X (XGFP−/XTomato+), using fluorescence-activated cell sorting (FACS). This gives us a unique advantage over in vivo studies, as it enables us to test the importance of X-inactivation and -reactivation for germ cell development by isolating and further culturing cells of different X-inactivation states. Taken together, this tailor-made system allows us to assess X-chromosome dynamics and its importance for female mouse germ cell development in vitro. Figure 1. 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. Download figure Download PowerPoint We first set out to assess competence for PGCLC differentiation of our XRep cell line. We slightly adapted published protocols (Hayashi & Saitou, 2013; Nakaki et al, 2013), by differentiating ESCs into epiblast-like cells (EpiLCs) for 4 days, as differentiation for 2 days, as demonstrated in said previous studies, did not yield PGCLCs with our XRep cells likely due to their specific genetic background (Fig EV1A). Furthermore, we extended the induction time of PGCLC generation from 4 to 5 days to ensure sufficient time to undergo X-inactivation (Fig 1B). We quantified PGCLC induction efficiency by FACS analysis, using SSEA1 and CD61 double-positive staining to mark successfully induced PGCLCs (Fig 1C). At PGCLC day 5, we found ~ 60% of the cell population to be double-positive for SSEA1/CD61, indicating a very high PGCLC induction efficiency when compared to the cytokine-based protocol (Hayashi & Saitou, 2013) and in line with previous observations on transcription factor-based PGCLC induction (Nakaki et al, 2013). To further assess the quality of our PGCLCs, we stained cryosections of PGCLC bodies at day 5 of induction for SOX2 and TFAP2C, both germ-line expressed transcription factors. We observed that > 50% of cells were double-positive for SOX2 and TFAP2C (Fig 1D), confirming PGCLC cell identity. We next wanted to assess X-inactivation kinetics using our XGFP and XTomato reporters. As expected, XTomato stayed active throughout the differentiation (Fig 1E and F). In contrast, we observed downregulation of the XGFP reporter at day 2 of PGCLC differentiation, with the XGFP− population gradually increasing until day 5 (Fig 1E and F). Nevertheless, even at day 5, up to 40% of PGCLCs remained XGFP+ in our system (Fig EV1B). Despite this, the large majority of EpiLCs showed H3K27me3 foci (Fig EV1D–F), which suggests that both XGFP− and XGFP+ PGCLCs originated from EpiLCs that had initiated X-inactivation. We do, however, note that EpiLCs still retained XGFP protein staining (Figs 1E and F, and EV1D and F), while XGFP transcripts were being downregulated (Fig EV1C), indicating that protein stability of GFP gives a delayed read-out of X-inactivation kinetics. Nevertheless, at the PGCLC stage, the XGFP signal faithfully reflected the X-inactivation state, as XGFP was only detected in cells without the X-inactivation-specific H3K27me3 spot (Fig EV1D–F). Click here to expand this figure. Figure EV1. 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. Download figure Download PowerPoint In summary, using our tailor-made XRep cell line, we could show that X-inactivation initiates early during PGCLC differentiation. Additionally, our system enables the isolation of distinct PGCLC populations, either having undergone X-inactivation or harbouring two active X chromosomes, suggesting that PGCLC specification can occur in the absence of X-inactivation as well. XGFP+ and XGFP− PGCLCs define distinct subpopulations Having identified two distinct PGCLC populations, we set out to characterize the transcriptional changes taking place during differentiation. We induced EpiLCs from ESCs for 4 days and subsequently induced PGCLCs for 5 days, at which stage we isolated XGFP+ and XGFP− PGCLCs by FACS (Fig EV2A). With these samples, we performed allele-specific RNA-sequencing on two biological replicates (different clones) with two technical replicates each. Principal component analysis (PCA) of the expression profiles showed a high coherence between replicates, with ESCs, EpiLCs and PGCLCs occupying distinct clusters (Fig 2A). Moreover, we observed that XGFP+ and XGFP− PGCLCs clustered separately, indicating distinct expression profiles of the two populations. To exclude the possibility that the distinct clustering of PGCLC populations was influenced by the different X-status of the two, we repeated the PCA while eliminating X-chromosome-linked genes from the analysis. We observed a highly similar clustering of samples with minimal changes in component variances (Fig EV2B). In order to assess whether transcriptional differences in XGFP+ and XGFP− PGCLCs could be explained by differences in developmental timing, we took advantage of published datasets of female in vivo PGCs from E9.5, E10.5, E11.5 and E12.5 embryos (Nagaoka et al, 2020) and compared expression profiles to our in vitro derived PGCLCs (Fig 2B). PCA revealed a trajectory where PC1 defined the developmental timing of in vivo and in vitro samples, whereas PC2 did not greatly contribute to the separation of our in vitro PGCLCs (PC loadings in Dataset EV4). We found that both PGCLC populations clust" @default.
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• W4281381837 title "Controlled X‐chromosome dynamics defines meiotic potential of female mouse <i>in vitro</i> germ cells" @default.
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