X-chromosome dosage compensation in female placental mammals is achieved by X-chromosome inactivation (XCI). Human pre-implantation embryos are an exception, in which dosage compensation occurs by X-chromosome dampening (XCD). Here, we examined whether XCD extends to human prenatal germ cells given their similarities to naive pluripotent cells. We found that female human primordial germ cells (hPGCs) display reduced X-linked gene expression before entering meiosis. Moreover, in hPGCs, both X chromosomes are active and express the long non-coding RNAs X active coating transcript (XACT) and X inactive specific transcript (XIST)—the master regulator of XCI—which are silenced after entry into meiosis. We find that XACT is a hPGC marker, describe XCD associated with XIST expression in hPGCs and suggest that XCD evolved in humans to regulate X-linked genes in pre-implantation embryos and PGCs. Furthermore, we found a unique mechanism of X-chromosome regulation in human primordial oocytes. Therefore, future studies of human germline development must consider the sexually dimorphic X-chromosome dosage compensation mechanisms in the prenatal germline.
Subscribe to Journal
Get full journal access for 1 year
only $17.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The scRNA-seq data of prenatal tissues reported in this paper are available under the following accession numbers: GSE143380 (female cell data) and GSE143356 (male cell data). scRNA-seq datasets are also available online for interactive exploration (http://germline.mcdb.ucla.edu). Previously published RNA-seq data of male and female hPGCs and somatic cells25,52,53 and single-cell RNA-seq data from female germ cells30 and from female FCGs40 that were reanalysed here are available under the following accession codes: GSE63392 (ref. 25), GSE60138 (ref. 52), GSE93126 (ref. 53), GSE79280 (ref. 30) and GSE86146 (ref. 40), respectively. Human conceptus tissue requests can be made to email@example.com. All other data supporting the findings of this study are available from the corresponding authors on reasonable request. Source data are provided with this paper.
Custom scripts used for aligning population RNA-seq, scRNA-seq, data processing and plotting are available on request.
Deng, X., Berletch, J. B., Nguyen, D. K. & Disteche, C. M. X chromosome regulation: diverse patterns in development, tissues and disease. Nat. Rev. Genet. 15, 367–378 (2014).
Payer, B. & Lee, J. T. X chromosome dosage compensation: how mammals keep the balance. Annu. Rev. Genet. 42, 733–772 (2008).
Wutz, A. Gene silencing in X-chromosome inactivation: advances in understanding facultative heterochromatin formation. Nat. Rev. Genet. 12, 542–553 (2011).
Gendrel, A.-V. & Heard, E. Fifty years of X-inactivation research. Development 138, 5049–5055 (2011).
Plath, K., Mlynarczyk-Evans, S., Nusinow, D. A. & Panning, B. Xist RNA and the mechanism of X chromosome inactivation. Annu. Rev. Genet. 36, 233–278 (2002).
Robert Finestra, T. & Gribnau, J. X chromosome inactivation: silencing, topology and reactivation. Curr. Opin. Cell Biol. 46, 54–61 (2017).
Brockdorff, N. Localized accumulation of Xist RNA in X chromosome inactivation. Open Biol. 9, 190213 (2020).
Sahakyan, A., Plath, K. & Rougeulle, C. Regulation of X-chromosome dosage compensation in human: mechanisms and model systems. Philos. Trans. R. Soc. Lond. B 372, 20160363 (2017).
Chuva de Sousa Lopes, S. M. et al. X chromosome activity in mouse XX primordial germ cells. PLoS Genet. 4, e30 (2008).
Sugimoto, M. & Abe, K. X chromosome reactivation initiates in nascent primordial germ cells in mice. PLoS Genet. 3, 1309–1317 (2007).
de Napoles, M., Nesterova, T. & Brockdorff, N. Early loss of Xist RNA expression and inactive X chromosome associated chromatin modification in developing primordial germ cells. PLoS ONE 2, e860 (2007).
Sangrithi, M. N. et al. Non-Canonical and sexually dimorphic X dosage compensation states in the mouse and human germline. Dev. Cell 40, 289–301 (2017).
Nesterova, T. B. et al. Characterization of the genomic Xist locus in rodents reveals conservation of overall gene structure and tandem repeats but rapid evolution of unique sequence. Genome Res. 11, 833–849 (2001).
Petropoulos, S. et al. Single-cell RNA-seq reveals lineage and X chromosome dynamics in human preimplantation embryos. Cell https://doi.org/10.1016/j.cell.2016.03.023 (2016).
Okamoto, I. et al. Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development. Nature 472, 370–374 (2011).
Moreira de Mello, J. C. et al. Random X inactivation and extensive mosaicism in human placenta revealed by analysis of allele-specific gene expression along the X chromosome. PLoS ONE 5, e10947 (2010).
Vallot, C. et al. XACT noncoding RNA competes with XIST in the control of X chromosome activity during human early development. Cell Stem Cell 20, 102–111 (2017).
Vallot, C. et al. XACT, a long noncoding transcript coating the active X chromosome in human pluripotent cells. Nat. Genet. 45, 239–241 (2013).
Patrat, C., Ouimette, J.-F. & Rougeulle, C. X chromosome inactivation in human development. Development 147, dev183095 (2020).
Moreira de Mello, J. C., Fernandes, G. R., Vibranovski, M. D. & Pereira, L. V. Early X chromosome inactivation during human preimplantation development revealed by single-cell RNA-sequencing. Sci. Rep. 7, 10794 (2017).
Sousa, E. J. et al. Exit from naive pluripotency induces a transient X chromosome Inactivation-like state in males. Cell Stem Cell 22, 919–928 (2018).
Sahakyan, A. et al. Human naive pluripotent stem cells model X chromosome dampening and X inactivation. Cell Stem Cell 20, 87–101 (2016).
Patel, S. et al. Human embryonic stem cells do not change their X inactivation status during differentiation. Cell Rep. 18, 54–67 (2016).
Tang, W. W. C. et al. A unique gene regulatory network resets the human germline epigenome for development. Cell 161, 1453–1467 (2015).
Gkountela, S. et al. DNA demethylation dynamics in the human prenatal germline. Cell 161, 1425–1436 (2015).
Guo, F. et al. The transcriptome and DNA methylome landscapes of human primordial germ cells. Cell 161, 1437–1452 (2015).
Anderson, R. A., Fulton, N., Cowan, G., Coutts, S. & Saunders, P. T. K. Conserved and divergent patterns of expression of DAZL, VASA and OCT4 in the germ cells of the human fetal ovary and testis. BMC Dev. Biol. 7, 136 (2007).
Gkountela, S. et al. The ontogeny of cKIT+ human primordial germ cells proves to be a resource for human germ line reprogramming, imprint erasure and in vitro differentiation. Nat. Cell Biol. 15, 113–122 (2012).
Perrett, R. M. et al. The early human germ cell lineage does not express SOX2 during in vivo development or upon in vitro culture. Biol. Reprod. 78, 852–858 (2008).
Vértesy, Á. et al. Parental haplotype-specific single-cell transcriptomics reveal incomplete epigenetic reprogramming in human female germ cells. Nat. Commun. 9, 1873 (2018).
Plath, K. et al. Role of histone H3 lysine 27 methylation in X inactivation. Science 300, 131–135 (2003).
Silva, J. et al. Establishment of histone H3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 polycomb group complexes. Dev. Cell 4, 481–495 (2003).
Chen, D. et al. The TFAP2C-regulated OCT4 naive enhancer is involved in human germline formation. Cell Rep. 25, 3591–3602 (2018).
Kobayashi, T. & Surani, M. A. On the origin of the human germline. Development 145, dev150433 (2018).
Sasaki, K. et al. Robust in vitro induction of human germ cell fate from pluripotent stem cells. Cell Stem Cell 17, 178–194 (2015).
Diaz Perez, S. V. et al. Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin. Hum. Mol. Genet. 21, 751–764 (2011).
Pandolfi, E. C. et al. Generation of three human induced pluripotent stem cell sublines (MZT04D, MZT04J, MZT04C) for reproductive science research. Stem Cell Res. 40, 101576 (2019).
Deglincerti, A. et al. Self-organization of the in vitro attached human embryo. Nature 533, 251–254 (2016).
Shahbazi, M. N. et al. Self-organization of the human embryo in the absence of maternal tissues. Nat. Cell Biol. 18, 700–708 (2016).
Li, L. et al. Single-cell RNA-seq analysis maps development of human germline cells and gonadal niche interactions. Cell Stem Cell 858–873 (2017); https://doi.org/10.1016/j.stem.2017.03.007
Wolf, F. A. et al. PAGA: graph abstraction reconciles clustering with trajectory inference through a topology preserving map of single cells. Genome Biol. 20, 59 (2019).
Anderson, E. L. et al. Stra8 and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesis and oogenesis in mice. Proc. Natl Acad. Sci. USA 105, 14976–14980 (2008).
Törmälä, R. M. et al. Zona pellucida components are present in human fetal ovary before follicle formation. Mol. Cell. Endocrinol. 289, 10–15 (2008).
Zhang, Y. et al. Transcriptome landscape of human folliculogenesis reveals oocyte and granulosa cell interactions. Mol. Cell 72, 1021–1034 (2018).
Casanova, M. et al. A primate-specific retroviral enhancer wires the XACT lncRNA into the core pluripotency network in humans. Nat. Commun. 10, 5652 (2019).
Larsson, A. J. M., Coucoravas, C., Sandberg, R. & Reinius, B. X-chromosome upregulation is driven by increased burst frequency. Nat. Struct. Mol. Biol. 26, 963–969 (2019).
Deng, X. et al. Evidence for compensatory upregulation of expressed X-linked genes in mammals, Caenorhabditis elegans and Drosophila melanogaster. Nat. Genet. 43, 1179–1185 (2011).
Yamashiro, C. et al. Generation of human oogonia from induced pluripotent stem cells in vitro. Science 362, 356–360 (2018).
Folsom, L. J. & Fuqua, J. S. Reproductive issues in women with Turner syndrome. Endocrinol. Metab. Clin. North Am. 44, 723–737 (2015).
Franik, S. et al. Klinefelter syndrome and fertility—impact of X-chromosomal inheritance on spermatogenesis. Andrologia 50, e13004 (2018).
Reynaud, K. et al. Number of ovarian follicles in human fetuses with the 45,X karyotype. Fertil. Steril. 81, 1112–1119 (2004).
Irie, N. et al. SOX17 is a critical specifier of human primordial germ cell fate. Cell 160, 253–268 (2015).
Chen, D. et al. Germline competency of human embryonic stem cells depends on eomesodermin. Biol. Reprod. 97, 850–861 (2017).
Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 19, 15 (2018).
Blondel, V. D., Guillaume, J.-L., Lambiotte, R. & Lefebvre, E. Fast unfolding of communities in large networks. J. Stat. Mech. Theory Exp. 2008, P10008 (2008).
Solovei, I. Fluorescence in situ hybridization (FISH) on tissue cryosections. Methods Mol. Biol. 659, 71–82 (2010).
Ramírez, F. et al. High-resolution TADs reveal DNA sequences underlying genome organization in flies. Nat. Commun. 9, 189 (2018).
We thank J. Scholes, F. Codrea and J. Calimlim for support with FACS; J. Tang for banking and culturing of the UCLA hESC lines; staff at the microscopy cores at the UCLA Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research Center (BSCRC) for help with imaging; staff at the Technology Center for Genomics and Bioinformatics at the UCLA Johnson Comprehensive Cancer Center (JCCC) and the Next Generation Sequencing core at BSCRC for help with genomics approaches; and staff at the Translational Pathology Core Laboratory for help with histology. T.C. was supported by a Boehringer Ingelheim PhD Fellowship. This work is supported by funds from the NIH to A.C. (R01HD079546). K.P. was supported by the BSCRC at UCLA, the David Geffen School of Medicine at UCLA, and the UCLA JCCC, the NIH (R01HD098387, P01GM099134) and a Faculty Scholar grant from the Howard Hughes Medical Institute. All human pre-implantation embryo and human embryo attachment culture studies were performed using funds from the UCLA Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research Innovation Award. No NIH funds were used for research with human pre-implantation embryos. Human fetal tissue research is supported by a grant to I. Glass at the University of Washington Birth Defects laboratory (5R24HD000836-53).
The authors declare no competing interests.
Peer review information: Peer reviewer reports are available.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Female hPGCs from week 4 pf ovaries have lost the H3K27me3 nuclear accumulation.
a, Distribution of single nucleotide polymorphisms (SNPs) from maternally inherited (x-axis) and paternally inherited (y-axis) alleles in gene expression data of female hPGCs. Each dot represents sum of all detected SNPs per cell for genes on chromosome 1 (Chr1), X-linked genes subject to XCI and escapees of XCI, respectively, based on published scRNA-seq data30. b, Representative immunofluorescence staining of OCT4 (magenta), H3K27me3 (green), DAZL (grey) and DAPI (blue) on female hPGCs at week 4 pf prior to gonad formation, when hPGCs are migrating through the aorta-gonad-mesonephros (AGM) (1 sample was analyzed). Insets show a rare OCT4+/DAZL+ cell with no nuclear accumulation of H3K27me3 (inset 1) and an OCT4+/DAZL- cell with H3K27me3 accumulation (inset 2) along the genital ridge of the AGM. Scale bar upper panel 50 microns, lower panel 30 microns. c, Percentage of OCT4+/DAZL+ and OCT4+/DAZL- cells with an Xi-like nuclear accumulation of H3K27me3 from the experiment shown in (c); (n=58 cells from 1 AGM). d, Quantification of the proportion of DAZL+ female hPGCs at weeks 4, 7 and 12 pf with an Xi-like nuclear accumulation of H3K27me3 (n=50-100 cells per sample in 2 replicates). e, Representative immunofluorescence staining of a fetal ovary at week 12 pf with DAZL (magenta), H3K27me3 (green), OCT4 (grey) and DAPI (blue). Inset shows a DAZL+/OCT4 negative female germ cell that is negative for H3K27me3 (1 pair of ovaries were analyzed), scale bar 50 microns. Statistical source data are provided in Source Data Extended Data Fig. 1. Source data
a, Boxplots presenting the X/A ratio, calculated from the sum of X-linked linked gene expression and the sum of autosomal gene expression, of individual female (red) or male (cyan) germ cells (left panel) and surrounding somatic cells (right panel) obtained from gonads harvested from indicated developmental timepoints (week). b, As in (a), except that the X/A ratio was determined from the mean expression levels of X-linked and autosomal genes per cell. c, Boxplot showing the distribution of the sum of all autosomal (left) and X-linked (right) gene expression, respectively, in individual female and male germ cells across for developmental time points shown in (a). d, As in (a), except that the X/A ratios in female and male FGCs across developmental time from a published study are shown40. e, X/A ratio per single cell in female and male gonadal somatic cells from all developmental ages accompanying the data shown in (d). Wilcoxon statistical testing between age matched samples, NS- Not Significant, * p<0.05, ** p<0.01, *** p<0.001. a-c: n = 49528 cells analyzed across 10 independent experiments and d-e: 1016 cells analyzed from published dataset40 in total.
a, Boxplots of the X/A ratios of female germ cells along the developmental trajectory and in female gonadal somatic cells, as described in Fig. 5, except that the top 5% highest expressed genes were excluded from the analysis. b, As in (a), except that the bottom 5% of expressed genes were excluded from the analysis. c, Boxplots of the X/A ratios in female germ cells and female gonadal cells as described in Fig. 5, except that the X/A ratios were calculated from the mean value of X-linked and autosomal gene expression per cell. d, Sum of all autosomal gene expression normalized counts in female germ cells organized by clusters along the developmental trajectory. e, Sum of all X-linked gene expression in female germ cells organized by clusters along the developmental trajectory. X-linked gene expression increases in clusters 7-9 coincident with entrance into meiosis and repression of the naïve-like pluripotency program. f, Germline trajectory analysis of previously published scRNA-seq data from female FCGs40. The hPGCs state with pluripotency program expression is captured with clusters 0-4, and meiotic entry in cluster 5-7. g, Boxplots of the X/A ratios for female germ cells and female gonadal somatic cells for the data set shown in (f). From cluster 5 onwards, X/A ratios in differentiating female germ cells are higher than gonadal somatic cells. Wilcoxon statistical testing for (a), (b), (c), (g). NS- Not Significant, * p<0.05, ** p<0.01, *** p<0.001. Source data
a, Male germ cells from the scRNA-seq data shown in Fig. 4a–c were projected along the developmental trajectory, and five clusters (0-4) were identified (n= 282 cells pulled from 5 samples). The pluripotency program is repressed in cluster 4, coincident with increased expression of prospermatogonia genes and exit from the cell cycle. b, Expression of marker genes along the developmental trajectory of male germ cells defined in (a). c, Box plots showing that X/A ratios in male germ cells along the developmental trajectory and in surrounding male somatic cells. d, As in (c), except that the top 5% highest expressed genes were excluded from the analysis. e, As in (c), except that bottom 5% of genes were excluded. f, As in (c), except that the top and bottom 5% of expressed genes were excluded. In total n = 24740 cells analyzed across 5 independent experiments in c-f. g, Sum of all autosomal gene expression per cell in male germ cells along the developmental trajectory, showing no dramatic differences across the clusters. h, As in (g), except for X-linked gene expression, showing no dramatic differences across the clusters. i, Germline trajectory analysis for male FGCs40, identified 7 clusters (0-6). Marker gene expression is given for these clusters. j, Box plots of the X/A ratios in male FCGs along the developmental trajectory defined in (i), showing an increase in cluster 2 relative to cluster 1. In total, n = 779 cells analyzed from published dataset40 in j, Wilcoxon statistical testing used for (c-f) and (j). NS- Not Significant, * p<0.05, ** p<0.01, *** p<0.001. Source data
a, Boxplot depicting the expression of XIST in female germ cells organized by clusters along the developmental trajectory defined for the published FCG data set40 in Extended Data Fig. 3f,g. XIST expression is significantly reduced from cluster 5 onwards. b, XIST expression in mature oocytes and granulosa cells from scRNA-seq data of adult ovary44. c, Scatter plot of average XIST expression (y-axis) and average X/A ratio (x-axis) for female germ cells clusters 6-9 (from Fig. 4d), capturing the entrance into meiosis. d, X/A ratios in female hPGCs and meiotic germ cells clustered based on expression of lncRNA XIST, ZP3+ primordial oocytes are clustered separately. e, Boxplots depicting XIST expression in male germ cells ordered along the developmental trajectory defined in Extended Data Fig. 4a–h, indicating that XIST transcripts are rarely detected in male germ cells. f, As in (e), except for male FGCs40 from Extended Data Fig. 4i,j. g, Average XACT cloud size in week 8 and 14 pf hPGCs with different patterns of XIST expression, error bars show standard deviation of the cloud sizes (76 cells analyzed in total from 2 independent experiments). Wilcoxon statistical testing used for (a), (d), (f), (g). NS- Not Significant, * p<0.05, ** p<0.01, *** p<0.001. Number of cells analyzed across 5 independent experiments: a. n = 1016 cells40, b. n = 148 cells44, d. n = 1938 cells, f, n = 779 cells40. Source data
Summary of the hPSC differentiation figure shown in Fig. 2. Due to XCI erosion, XACT is expressed from the Xa and the eroded X-chromosome in primed, female human pluripotent stem cells. The Xe state is transmitted into differentiated cells and upon hPGCLC differentiation. Moreover, XACT is maintained in hPGCLCs, whereas somatic cells silence XACT. Consequently, female hPSC-derived PGCLCs carry two XACT clouds and male hPSC-derived PGCLCs one.
Supplementary Fig. 1. Gating strategy for hPGCLC sorts. Includes figure and brief description how gates were established to sort hPGCLCs.
First tab: sequencing summary of the scRNA-seq experiments. Second tab: a list of all the antibodies used in this study.
Statistical source data for Fig. 1.
Statistical source data for Fig. 2.
Statistical source data for Fig. 3.
Statistical source data for Fig. 4.
Statistical source data for Fig. 5.
Statistical source data for Fig. 6.
Statistical source data for Extended Data Fig. 1.
Statistical source data for Extended Data Fig. 3.
Statistical source data for Extended Data Fig. 4.
Statistical source data for Extended Data Fig. 5.
About this article
Cite this article
Chitiashvili, T., Dror, I., Kim, R. et al. Female human primordial germ cells display X-chromosome dosage compensation despite the absence of X-inactivation. Nat Cell Biol 22, 1436–1446 (2020). https://doi.org/10.1038/s41556-020-00607-4
Nature Cell Biology (2020)