Article

A PAX5–OCT4–PRDM1 developmental switch specifies human primordial germ cells

Received:
Accepted:
Published:

Abstract

Dysregulation of genetic pathways during human germ cell development leads to infertility. Here, we analysed bona fide human primordial germ cells (hPGCs) to probe the developmental genetics of human germ cell specification and differentiation. We examined the distribution of OCT4 occupancy in hPGCs relative to human embryonic stem cells (hESCs). We demonstrated that development, from pluripotent stem cells to germ cells, is driven by switching partners with OCT4 from SOX2 to PAX5 and PRDM1. Gain- and loss-of-function studies revealed that PAX5 encodes a critical regulator of hPGC development. Moreover, an epistasis analysis indicated that PAX5 acts upstream of OCT4 and PRDM1. The PAX5–OCT4–PRDM1 proteins form a core transcriptional network that activates germline and represses somatic programmes during human germ cell differentiation. These findings illustrate the power of combined genome editing, cell differentiation and engraftment for probing human developmental genetics that have historically been difficult to study.

  • Subscribe to Nature Cell Biology for full access:

    $59

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

Additional information

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  1. 1.

    Ramathal, C. et al. DDX3Y gene rescue of a Y chromosome AZFa deletion restores germ cell formation and transcriptional programs. Sci. Rep. 5, 15041 (2015).

  2. 2.

    Dominguez, A., Chiang, H., Sukhwani, M., Orwig, K. & Reijo Pera, R. A. Human germ cell formation in xenotransplants of induced pluripotent stem cells carrying X chromosome aneuploidies. Sci. Rep. 4, 6432 (2014).

  3. 3.

    Ramathal, C. et al. Fate of iPSCs derived from azoospermic and fertile men following xenotransplantation to seminiferous tubules. Cell Rep. 7, 1284–1297 (2014).

  4. 4.

    Durruthy, J. D. et al. Fate of induced pluripotent stem cells following transplantation to murine seminiferous tubules. Hum. Mol. Genet. 23, 3071–3084 (2014).

  5. 5.

    Kee, K., Angeles, V., Flores, M., Nguyen, H. & Reijo Pera, R. A. Human DAZL, DAZ and BOULE genes modulate primordial germ cell and haploid gamete formation. Nature 462, 222–225 (2009).

  6. 6.

    Salto Mamsen, L., Lutterodt, M. C., Andersen, E. W., Byskov, A. G. & Andersen, C. Y. Germ cell numbers in human embryonic and fetal gonads during the first two trimesters of pregnancy: analysis of six published studies. Hum. Reprod. 26, 2140–2145 (2011).

  7. 7.

    Clark, A. T. et al. Spontaneous differentiation of germ cells from human embryonic stem cells in vitro. Hum. Mol. Genet. 13, 727–739 (2004).

  8. 8.

    Reijo Pera, R. A., Alagappan, R. K., Patrizio, P. & Page, D. C. Severe oligospermia resulting from deletions of the azoospermia factor gene on the Y chromosome. Lancet 347, 1290–1293 (1996).

  9. 9.

    Irie, N., Tang, W. W. & Azim Surani, M. Germ cell specification and pluripotency in mammals: a perspective from early embryogenesis. Reprod. Med. Biol. 13, 203–215 (2014).

  10. 10.

    Magnusdottir, E. et al. A tripartite transcription factor network regulates primordial germ cell specification in mice. Nat. Cell Biol. 15, 905–915 (2013).

  11. 11.

    Tang, W. W., Kobayashi, T., Irie, N., Dietmann, S. & Surani, M. A. Specification and epigenetic programming of the human germ line. Nat. Rev. Genet. 17, 585–600 (2016).

  12. 12.

    Saitou, M., Barton, S. C. & Surani, M. A. A molecular programme for the specification of germ cell fate in mice. Nature 418, 293–300 (2002).

  13. 13.

    Saitou, M. & Yamaji, M. Primordial germ cells in mice. Cold Spring Harb. Perspect. Biol. 4, a008375 (2012).

  14. 14.

    Nakaki, F. et al. Induction of mouse germ-cell fate by transcription factors in vitro. Nature 501, 222–226 (2013).

  15. 15.

    Ohinata, Y. et al. Blimp1 is a critical determinant of the germ cell lineage in mice. Nature 436, 207–213 (2005).

  16. 16.

    Weber, S. et al. Critical function of AP-2 gamma/TCFAP2C in mouse embryonic germ cell maintenance. Biol. Reprod. 82, 214–223 (2010).

  17. 17.

    Yamaji, M. et al. Critical function of Prdm14 for the establishment of the germ cell lineage in mice. Nat. Genet. 40, 1016–1022 (2008).

  18. 18.

    Zhou, Q. et al. Complete meiosis from embryonic stem cell-derived germ cells in vitro. Cell Stem Cell 18, 330–340 (2016).

  19. 19.

    Irie, N. et al. SOX17 is a critical specifier of human primordial germ cell fate. Cell 160, 253–268 (2015).

  20. 20.

    Tang, W. W. et al. A unique gene regulatory network resets the human germline epigenome for development. Cell 161, 1453–1467 (2015).

  21. 21.

    Li, L. et al. Single-cell RNA-seq analysis maps development of human germline cells and gonadal niche interactions. Cell Stem Cell 20, 858–873 (2017).

  22. 22.

    Kehler, J. et al. Oct4 is required for primordial germ cell survival. EMBO Rep. 5, 1078–1083 (2004).

  23. 23.

    Niwa, H., Miyazaki, J. & Smith, A. G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376 (2000).

  24. 24.

    Scholer, H. R., Dressler, G. R., Balling, R., Rohdewohld, H. & Gruss, P. Oct-4: a germline-specific transcription factor mapping to the mouse t-complex. EMBO J. 9, 2185–2195 (1990).

  25. 25.

    O’Neill, L. P., VerMilyea, M. D. & Turner, B. M. Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations. Nat. Genet. 38, 835–841 (2006).

  26. 26.

    Cotney, J. L. & Noonan, J. P. Chromatin immunoprecipitation with fixed animal tissues and preparation for high-throughput sequencing. Cold Spring Harb. Protoc. 2015, 191–199 (2015).

  27. 27.

    Nishimoto, M., Fukushima, A., Okuda, A. & Muramatsu, M. The gene for the embryonic stem cell coactivator UTF1 carries a regulatory element which selectively interacts with a complex composed of Oct-3/4 and Sox-2. Mol. Cell. Biol. 19, 5453–5465 (1999).

  28. 28.

    Boyer, L. A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).

  29. 29.

    Buecker, C. et al. Reorganization of enhancer patterns in transition from naive to primed pluripotency. Cell Stem Cell 14, 838–853 (2014).

  30. 30.

    Pardo, M. et al. An expanded Oct4 interaction network: implications for stem cell biology, development, and disease. Cell Stem Cell 6, 382–395 (2010).

  31. 31.

    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).

  32. 32.

    Vincent, S. D. et al. The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse. Development 132, 1315–1325 (2005).

  33. 33.

    Medvedovic, J., Ebert, A., Tagoh, H. & Busslinger, M. Pax5: a master regulator of B cell development and leukemogenesis. Adv. Immunol. 111, 179–206 (2011).

  34. 34.

    Kee, K. & Reijo Pera, R. A. Human germ cell lineage differentiation from embryonic stem cells. Cold Spring Harb. Protoc. https://doi.org/10.1101/pdb.prot5048 (2008).

  35. 35.

    Zhang, P., Xia, N. & Reijo Pera, R. A. Directed dopaminergic neuron differentiation from human pluripotent stem cells. J. Vis. Exp. https://doi.org/10.3791/51737 (2014).

  36. 36.

    Perrier, A. L. et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl Acad. Sci. USA 101, 12543–12548 (2004).

  37. 37.

    Yeom, Y. I. et al. Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development 122, 881–894 (1996).

  38. 38.

    Sasaki, K. et al. Robust in vitro induction of human germ cell fate from pluripotent stem cells. Cell Stem Cell 17, 178–194 (2015).

  39. 39.

    Wang, Z., Oron, E., Nelson, B., Razis, S. & Ivanova, N. Distinct lineage specification roles for NANOG, OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell 10, 440–454 (2012).

  40. 40.

    Kee, K., Gonsalves, J. M., Clark, A. T. & Reijo Pera, R. A. Bone morphogenetic proteins induce germ cell differentiation from human embryonic stem cells. Stem Cells Dev. 15, 831–837 (2006).

  41. 41.

    Hermann, B. P. et al. Spermatogonial stem cell transplantation into rhesus testes regenerates spermatogenesis producing functional sperm. Cell Stem Cell 11, 715–726 (2012).

  42. 42.

    Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

  43. 43.

    Trapnell, C. et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

  44. 44.

    Sandelin, A. et al. Arrays of ultraconserved non-coding regions span the loci of key developmental genes in vertebrate genomes. BMC Genomics 5, 99 (2004).

  45. 45.

    Wingender, E., Dietze, P., Karas, H. & Knuppel, R. TRANSFAC: a database on transcription factors and their DNA binding sites. Nucleic Acids Res. 24, 238–241 (1996).

  46. 46.

    McLean, C. Y. et al. GREAT improves functional interpretation of cis-regulatory regions. Nat. Biotechnol. 28, 495–501 (2010).

Download references

Acknowledgements

This work was supported by P50 HD 068158 to R.A.R.P. (Project I).

Author information

Affiliations

  1. Department of Cell Biology and Neurosciences, Montana State University, Bozeman, MT, USA

    • Fang Fang
    • , Benjamin Angulo
    • , Ninuo Xia
    • , Jun Cui
    •  & Renee A. Reijo Pera
  2. Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA

    • Fang Fang
    • , Benjamin Angulo
    • , Ninuo Xia
    • , Jun Cui
    •  & Renee A. Reijo Pera
  3. Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, School of Medicine, Magee Women’s Research Institute, Pittsburgh, PA, USA

    • Meena Sukhwani
    •  & Kyle E. Orwig
  4. Genomic Medicine Division, Hematology Branch, NHLBI/NIH, Rockville, MD, USA

    • Zhengyuan Wang
  5. Department of Microbiology and Immunology, Montana State University, Bozeman, MT, USA

    • Charles C. Carey
    • , Aurélien Mazurie
    • , Royce Wilkinson
    •  & Blake Wiedenheft
  6. Wellcome Trust Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK

    • Naoko Irie
    •  & M. Azim Surani

Authors

  1. Search for Fang Fang in:

  2. Search for Benjamin Angulo in:

  3. Search for Ninuo Xia in:

  4. Search for Meena Sukhwani in:

  5. Search for Zhengyuan Wang in:

  6. Search for Charles C. Carey in:

  7. Search for Aurélien Mazurie in:

  8. Search for Jun Cui in:

  9. Search for Royce Wilkinson in:

  10. Search for Blake Wiedenheft in:

  11. Search for Naoko Irie in:

  12. Search for M. Azim Surani in:

  13. Search for Kyle E. Orwig in:

  14. Search for Renee A. Reijo Pera in:

Contributions

The study was conceived and designed by F.F. and R.A.R.P. F.F. performed most experiments (including ChIP–seq, immunohistochemistry, RNA-seq, protein pull-down assays, luciferase reporter assays, gene expression profiling) and analysed the data. N.X. performed bioinformatics analyses for ChIP–seq and luciferase reporter assays, flow cytometry and gene expression analysis for the xenotransplantation experiments. B.A. generated the PAX5-knockout hESC lines and performed part of the immunohistochemistry in xenotransplantation samples. Z.W. performed the initial bioinformatics analysis for ChIP–seq data. M.S. and K.E.O. conducted the xenotransplantation. C.C.C and A.M. performed RNA-seq analysis. J.C. constructed the H1-DDX4 reporter. R.W. and B.W. designed and constructed the PAX5-knockout plasmids. M.A.S. and N.I. provided the PRDM1-knockout hESC line and protocol. The manuscript was written by F.F. and R.A.R.P. with input from the other authors.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Fang Fang.

Integrated supplementary information

  1. Supplementary Figure 1 Staining of OCT4-positive cells in human fetal testis and comparison between mixed ChIP-Seq and Standard ChIP-Seq.

    (a) Cross-section of human fetal testis (22 weeks) with immunostaining of OCT4. Scale bar represents 50 µm. (b) Immunostaining of OCT4 and cKIT in human fetal testis. Arrows indicate co-staining cells. Scale bar represents 50 µm. (c) Immunostaining of OCT4 and DDX4 proteins in human fetal testis. Arrows indicate cells that only express OCT4. Scale bar represents 50 µm. Immunostaining experiments in (a-c) were independently repeated a minimum of three times with similar results. (d) Schematic strategy for comparing mixed ChIP with conventional ChIP. (e) ChIP-qPCR for detection of peaks at OCT4 locus. ChIP-qPCR were independently repeated a minimum of three times with similar results. (f) Scatterplot comparing OCT4 ChIP-seq data generated in pure ESCs and 1% ESCs mixed with fibroblast cells. Correlation was computed using whole genome data within 10kb of transcription start site (TSS) of RefSeq genes. Sample size n=2 and Pearson’s correlation coefficient was used for the correlation analysis. (g) Venn diagram showing overlapping genes bound by OCT4 generated by ChIP-seq data in pure ESCs and 1% ESCs mixed with fibroblast cells. (h) Venn diagram showing overlapping genes bound by OCT4 generated by ChIP-seq data derived from two biological replicates.

  2. Supplementary Figure 2 PAX5 and PRDM1 expression and binding in hPGCs.

    (a) Cross-section of human fetal testis (22 weeks) with immunostaining for OCT4, PAX5, PRDM1, SOX2 and IgG control. Scale bars represent 50 µm. Immunostaining experiments were independently repeated a minimum of three times with similar results. (b) Expression level of transcription factors in hESCs and human fetal testis. Data are represented as mean ± SD of n=3 independent replicates. (c) Heatmap visualization of PAX5 and PRDM1 ChIP-seq data, depicting all binding events centered on the peak region within a 5kb window around the peak. (d) GST-pull down assay to assess protein interactions between PRDM1 and PAX5. Western blot images are representative of three independent experiments with similar results. Unprocessed scans of western blot analysis are available in Supplementary Fig. 8. Source data for b are in Supplementary Table 2.

  3. Supplementary Figure 3 Overexpression of PAX5 and PRDM1 enhances germ cell potential of hESCs during in vitro differentiation.

    (a) RT-qPCR analysis of expression level of PAX5 in H1 ESCs, PAX5 KO and cells overexpressing PAX5. Data are represented as mean ± SD of three replicates. (b) Bright field view of cells PAX5 OE and PRDM1 OE cells. Scale bars represent 50 µm. Experiments were independently repeated a minimum of three times with similar results. (c) Immunostaining of OCT4, PAX5 and PRDM1 in PAX5 and PRDM1 overexpression hESCs. OE represents overexpression. Scale bars represent 25 µm. Immunostaining experiments were independently repeated a minimum of three times with similar results. (d) RT-qPCR analysis of control, PAX5 OE and PRDM1 OE H1 hESCs before and after BMPs-induced differentiation. Data are represented as mean ± SD of n=3 independent replicates. (e) Genome browser representation of ChIP-seq tracks for PAX5 at the SYCP3 and SYCP1 loci. ChIP-seq were independently repeated twice with similar results. (f) RT-qPCR analysis of control and PAX5&PRDM1 double OE H1 hESCs after BMPs-induced differentiation. Data are represented as mean ± SD of n=3 independent replicates. Source data for a, d and f are in Supplementary Table 2.

  4. Supplementary Figure 4 Overexpression of PAX5 and PRDM1 enhances germ cell differentiation of hESCs in vivo in xenotransplantation.

    (a) Immunostaining of GFP and DAPI in untransplanted mouse testis. Scale bars represent 100 µm. Immunostaining experiments were independently repeated a minimum of three times with similar results. (b) Immunostaining analysis of testis xenografts derived from PAX5&PRDM1 double OE H1 hESCs. All images are merged from DDX4 (red), GFP (green) and DAPI-stained nuclei. Scale bars represent 50 µm. Immunostaining experiments were independently repeated a minimum of three times with similar results. (c) Percentage of tubules positive for GFP+/ DDX4+ cells was calculated across multiple cross-sections (relative to total number of tubules). Data are represented as mean ± SD of n=3 independent replicates. (d) For each positive tubule, the ratio of GFP+/DDX4+ cells per tubule was determined. Data are represented as mean ± SD of n=3 independent replicates. Source data for c and d are in Supplementary Table 2.

  5. Supplementary Figure 5 Construction of PAX5 knockout hESC line with CRISPR.

    (a) Targeting strategy of PAX5 knockout in hESC with the designated guide RNA (gRNA) and the resulting deleted sequences. (b) Sequences of wide type PAX5 and PAX5 KO line that show homologous recombination and deletions are shown. Grey box indicates CRISPR recognition site and black bars indicate deleted sequences. (c) Immunofluorescence of PAX5 on wild-type (WT) and PAX5 KO after BMPs-induced differentiation. Scale bars represent 50 µm. Immunostaining experiments were independently repeated a minimum of three times with similar results. (d) Western blot of PAX5 and ACTIN on wild type (WT) and PAX5 knockout (KO) cells. Western blot images are representative of three independent experiments. Unprocessed scans of Western blot analysis are available in Supplementary Fig. 8.

  6. Supplementary Figure 6 Gene expression of in vitro derived hPGCs and sorting of hPGCs derived from mouse seminiferous tubules.

    (a) PAX5 expression level from previously published RNA-seq data19,31,38. Data are represented as mean of three technical replicates. (c) Expression level of germ cell genes from RNA-seq data19,38. Data are represented as mean of three technical replicates. (d) RT-qPCR analysis of OCT4 expression in control and PAX5 KO differentiated cells in vitro. “protocol” refers to the in vitro human germ cell differentiation protocols developed in the specific paper19,38. Data are represented as mean ± SD of n=3 independent replicates. P-value was calculated by two-tailed Student’s t-test and "ns" means not significant. Source data for a-c are in Supplementary Table 2.

  7. Supplementary Figure 7 Identification and mutation of PAX5 binding motifs in OCT4 and PRDM1 enhancer.

    (a, e) scanning of OCT4 (a) and PRDM1 (e) enhancer regions to look for key regulatory elements (RE). OCT4 or PRDM1 enhancer region is shown on the top. Red bars represent putative RE. Luciferase activity is shown on the bottom. Red box indicates key RE that has the strongest enhancer activity. Activity is presented relative to the full-length enhancer construct and minimal promoter construct (MP). MP: minimal promoter; RE: regulatory element; Full-length: Full-length enhancer. Data are represented as mean ± SD of n=3 independent replicates. (b, f) Sequence of OCT4 RE4 (b) or PRDM1 RE1 (f). Grey boxes indicate putative binding site for PAX5. PBS: putative binding site. (c, g) The effects of deleting the specific PBS regions in RE4 for OCT4 enhancer (c) and in RE1 for PRDM1 enhancer (g) on luciferase reporter activity. Activity is presented relative to the wild-type construct (RE4 or RE1) and minimal promoter construct (MP). Red box indicates PBS whose deletion abolished the induction of luciferase activity by PAX5 OE. Data are represented as mean ± SD of n=3 independent replicates. (d, h) The effects of mutating PBS2 for OCT4 enhancer (d) or PBS2 for PRDM1 enhancer (h) on luciferase reporter activity. Sequence of wide-type PBS and mutated PBS is shown on the top. Luciferase activity is presented relative to the wild-type construct (RE4 or RE1) and minimal promoter construct (MP). Data are represented as mean ± SD of n=3 independent replicates. Source data for a, c, d, e, g and h are in Supplementary Table 2.

  8. Supplementary Figure 8 Unprocessed gel blots.

    Of note, for some immunoblotting assays membranes were cut into several pieces to incubate with different antibodies, and therefore the raw images of these membranes are small in size.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–8 and Supplementary Table legends

  2. Reporting Summary

  3. Supplementary Table 1

    Primer sequences used by category for qRT-PCR analysis

  4. Supplementary Table 2

    Statistics source data for Figure 1–7 and Supplementary Figures 1–7