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Female human primordial germ cells display X-chromosome dosage compensation despite the absence of X-inactivation

Nature Cell Biology volume 22, pages 1436–1446 (2020)Cite this article

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Abstract

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.

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Fig. 1: Male and female hPGCs express the lncRNA XACT and female hPGCs carry two active X chromosomes in vivo.
Fig. 2: The lncRNA XACT is restricted to male and female hPGCLCs and is not expressed in somatic cells in vitro.
Fig. 3: XACT is predominantly expressed in NANOG+ pre- and post-implantation epiblast cells.
Fig. 4: Female germ cells undergo dynamic and ordered transcriptional changes between 7-16 weeks p.f.
Fig. 5: XIST expression is associated with XCD in female hPGCs.
Fig. 6: XIST is repressed as hPGCs enter meiosis.

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Data availability

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 bdrl@u.washington.edu. 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.

Code availability

Custom scripts used for aligning population RNA-seq, scRNA-seq, data processing and plotting are available on request.

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Acknowledgements

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).

Author information

Authors and Affiliations

  1. Department of Biological Chemistry, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA

    Tsotne Chitiashvili, Iris Dror, Rohan Chaudhari & Kathrin Plath

  2. Molecular Cell and Developmental Biology Department, University of California Los Angeles, Los Angeles, CA, USA

    Tsotne Chitiashvili, Fei-Man Hsu, Rohan Chaudhari, Erica Pandolfi, Di Chen & Amander Clark

  3. Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA, USA

    Tsotne Chitiashvili, Kathrin Plath & Amander Clark

  4. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California Los Angeles, Los Angeles, CA, USA

    Rachel Kim, Kathrin Plath & Amander Clark

  5. Department of Women’s Health, Research Institute for Women’s Health, Eberhard Karls University Tübingen, Tübingen, Germany

    Simone Liebscher & Katja Schenke-Layland

  6. NMI Natural and Medical Sciences Institute, University Tübingen, Reutlingen, Germany

    Katja Schenke-Layland

  7. Department of Medicine/Cardiology, Cardiovascular Research Laboratories, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA

    Katja Schenke-Layland

  8. Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, CA, USA

    Kathrin Plath & Amander Clark

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Contributions

T.C., K.P. and A.C. designed the experiments. T.C. and R.C. conducted immuno-RNA FISH experiments on tissues and in vitro PSC-derived cells. T.C., R.K. and E.P. generated female hiPSC lines from fibroblasts and conducted human blastocyst experiments. D.C. contributed to hPGCLC differentiation experiments. F.-M.H. created an interactive web-site to explore our scRNA-seq datasets. S.L. and K.S.-L. provided the human fetal tissues. T.C. performed scRNA-seq experiments, and the resulting data were analysed by T.C and I.D.; T.C analysed bulk RNA-seq data. T.C., K.P. and A.C. interpreted all data and wrote the manuscript.

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Correspondence to Kathrin Plath or Amander Clark.

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Extended data

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

Extended Data Fig. 2 The X/A ratio is higher in female germ cells than in male germ cells.

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.

Extended Data Fig. 3 Female hPGCs dampen X linked gene expression before entering meiosis.

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

Extended Data Fig. 4 Male hPGCs do not change X/A ratio upon sex specific differentiation.

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

Extended Data Fig. 5 XIST expression correlates with the X/A ratio.

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

Extended Data Fig. 6 XACT marks male and female hPGCLCs in vitro.

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 information

Supplementary Information

Supplementary Fig. 1. Gating strategy for hPGCLC sorts. Includes figure and brief description how gates were established to sort hPGCLCs.

Reporting Summary

Peer Review Information

Supplementary Table 1

First tab: sequencing summary of the scRNA-seq experiments. Second tab: a list of all the antibodies used in this study.

Source data

Source Data Fig. 1

Statistical source data for Fig. 1.

Source Data Fig. 2

Statistical source data for Fig. 2.

Source Data Fig. 3

Statistical source data for Fig. 3.

Source Data Fig. 4

Statistical source data for Fig. 4.

Source Data Fig. 5

Statistical source data for Fig. 5.

Source Data Fig. 6

Statistical source data for Fig. 6.

Source Data Extended Data Fig. 1

Statistical source data for Extended Data Fig. 1.

Source Data Extended Data Fig. 3

Statistical source data for Extended Data Fig. 3.

Source Data Extended Data Fig. 4

Statistical source data for Extended Data Fig. 4.

Source Data Extended Data Fig. 5

Statistical source data for Extended Data Fig. 5.

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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

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