Letter | Published:

NANOG alone induces germ cells in primed epiblast in vitro by activation of enhancers

Nature volume 529, pages 403407 (21 January 2016) | Download Citation

Abstract

Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGCs) in mice1, where its precise role is yet unclear2,3,4. We investigated this in an in vitro model, in which naive pluripotent embryonic stem (ES) cells cultured in basic fibroblast growth factor (bFGF) and activin A develop as epiblast-like cells (EpiLCs) and gain competence for a PGC-like fate5. Consequently, bone morphogenetic protein 4 (BMP4), or ectopic expression of key germline transcription factors Prdm1, Prdm14 and Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ES cells6,7,8. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that after the dissolution of the naive ES-cell pluripotency network during establishment of EpiLCs9,10, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG-binding patterns between ES cells and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ES cells, they show contrasting roles in EpiLCs, as Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development.

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Primary accessions

Gene Expression Omnibus

Data deposits

Microarray data have been deposited in the Gene Expression Omnibus under accession number GSE71933.

References

  1. 1.

    , , , & Nanog expression in mouse germ cell development. Gene Expr. Patterns 5, 639–646 (2005)

  2. 2.

    et al. Nanog safeguards pluripotency and mediates germline development. Nature 450, 1230–1234 (2007)

  3. 3.

    et al. Conditional knockdown of Nanog induces apoptotic cell death in mouse migrating primordial germ cells. Development 136, 4011–4020 (2009)

  4. 4.

    , , , & Nanog-independent reprogramming to iPSCs with canonical factors. Stem Cell Reports 2, 119–126 (2014)

  5. 5.

    , , , & Reconstitution of the mouse germ cell specification pathway in culture by pluripotent stem cells. Cell 146, 519–532 (2011)

  6. 6.

    et al. Bmp4 is required for the generation of primordial germ cells in the mouse embryo. Genes Dev. 13, 424–436 (1999)

  7. 7.

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

  8. 8.

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

  9. 9.

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

  10. 10.

    et al. Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development. eLife 4, e09571 (2015)

  11. 11.

    , & Genetic and epigenetic regulators of pluripotency. Cell 128, 747–762 (2007)

  12. 12.

    , , , & Sequence-specific regulator Prdm14 safeguards mouse ESCs from entering extraembryonic endoderm fates. Nature Struct. Mol. Biol. 18, 120–127 (2011)

  13. 13.

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

  14. 14.

    et al. Prdm14 promotes germline fate and naive pluripotency by repressing FGF signalling and DNA methylation. EMBO Rep. 14, 629–637 (2013)

  15. 15.

    et al. Nanog co-regulated by Nodal/Smad2 and Oct4 is required for pluripotency in developing mouse epiblast. Dev. Biol. 392, 182–192 (2014)

  16. 16.

    , & Tcf7l1 prepares epiblast cells in the gastrulating mouse embryo for lineage specification. Development 140, 1665–1675 (2013)

  17. 17.

    et al. Extensive and orderly reprogramming of genome-wide chromatin modifications associated with specification and early development of germ cells in mice. Dev. Biol. 278, 440–458 (2005)

  18. 18.

    et al. Chromatin dynamics during epigenetic reprogramming in the mouse germ line. Nature 452, 877–881 (2008)

  19. 19.

    et al. Germline DNA demethylation dynamics and imprint erasure through 5-hydroxymethylcytosine. Science 339, 448–452 (2013)

  20. 20.

    , & Retinoic acid is a potent growth activator of mouse primordial germ cells in vitro. Dev. Biol. 168, 683–685 (1995)

  21. 21.

    et al. A role for Lin28 in primordial germ-cell development and germ-cell malignancy. Nature 460, 909–913 (2009)

  22. 22.

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

  23. 23.

    et al. The germ cell determinant Blimp1 is not required for derivation of pluripotent stem cells. Cell Stem Cell 11, 110–117 (2012)

  24. 24.

    et al. A mesodermal factor, T, specifies mouse germ cell fate by directly activating germline determinants. Dev. Cell 27, 516–529 (2013)

  25. 25.

    et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature 461, 614–620 (2009)

  26. 26.

    et al. Context-dependent wiring of Sox2 regulatory networks for self-renewal of embryonic and trophoblast stem cells. Mol. Cell 52, 380–392 (2013)

  27. 27.

    et al. Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nature Cell Biol. 9, 625–635 (2007)

  28. 28.

    et al. Essential role of Sox2 for the establishment and maintenance of the germ cell line. Stem Cells 31, 1408–1421 (2013)

  29. 29.

    et al. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl Acad. Sci. USA 107, 21931–21936 (2010)

  30. 30.

    et al. Quantitative dynamics of chromatin remodeling during germ cell specification from mouse embryonic stem cells. Cell Stem Cell 16, 517–532 (2015)

  31. 31.

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

  32. 32.

    et al. Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev. Growth Differ. 41, 675–684 (1999)

  33. 33.

    et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008)

  34. 34.

    et al. Fiji: an open-source platform for biological-image analysis. Nature Methods 9, 676–682 (2012)

  35. 35.

    et al. In vivo epigenomic profiling of germ cells reveals germ cell molecular signatures. Dev. Cell 24, 324–333 (2013)

  36. 36.

    et al. Epiblast stem cell-based system reveals reprogramming synergy of germline factors. Cell Stem Cell 10, 425–439 (2012)

  37. 37.

    , , & Manipulating the Mouse Embryo. A Laboratory Manual 3rd edn, 453–506 (2003)

  38. 38.

    et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013)

  39. 39.

    et al. The transcriptional and epigenomic foundations of ground state pluripotency. Cell 149, 590–604 (2012)

Download references

Acknowledgements

We thank H. Leitch for ES cell lines, C. Lee for help with animal husbandry, H. Niwa for vectors and conditional Sox2-knockout ES cells, N. Miller, R. Walker and A. Riddell for FACS sorting and J. Bauer for analysis of microarray data. K.M. was supported by the Japan Society for the Promotion of Science (JSPS) Institutional Program for Young Researchers Overseas Visits. U.G. was supported by a Marie Skłodowska-Curie and a Newton Trust/Leverhulme Trust Early Career fellowship. J.J.Z. was a recipient of a Wellcome Trust PhD Studentship (RG44593). T.K. was supported by a JSPS Fellowship for research abroad. This research was supported by Gurdon Institute core grants from the Wellcome Trust (092096) and Cancer Research UK (C6946/A14492), and a grant from the Wellcome Trust to M.A.S. (WT096738).

Author information

Author notes

    • Kazuhiro Murakami
    •  & Ufuk Günesdogan

    These authors contributed equally to this work.

Affiliations

  1. Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK

    • Kazuhiro Murakami
    • , Ufuk Günesdogan
    • , Jan J. Zylicz
    • , Walfred W. C. Tang
    • , Roopsha Sengupta
    • , Toshihiro Kobayashi
    • , Shinseog Kim
    • , Richard Butler
    •  & M. Azim Surani
  2. Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK

    • Kazuhiro Murakami
    • , Ufuk Günesdogan
    • , Jan J. Zylicz
    • , Walfred W. C. Tang
    • , Roopsha Sengupta
    • , Toshihiro Kobayashi
    • , Shinseog Kim
    •  & M. Azim Surani
  3. Wellcome Trust Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK

    • Kazuhiro Murakami
    • , Ufuk Günesdogan
    • , Jan J. Zylicz
    • , Walfred W. C. Tang
    • , Roopsha Sengupta
    • , Toshihiro Kobayashi
    • , Shinseog Kim
    • , Sabine Dietmann
    •  & M. Azim Surani
  4. Laboratory for Pluripotent Cell Studies, Center for Developmental Biology, RIKEN, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan

    • Kazuhiro Murakami
  5. Laboratory for Molecular and Cellular Biology, Faculty of Advanced Life Science, Hokkaido University, Kita21 Nishi11, Kita-ku, Sapporo, Hokkaido 001-0021, Japan

    • Kazuhiro Murakami

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Contributions

K.M. and U.G. designed and performed experiments, and wrote the paper; W.W.C.T. designed and carried out the luciferase assays; NANOG ChIP experiments were carried out by J.J.Z., while R.S. performed immunofluorescence analysis; T.K. and S.K. designed and carried out the chimaera experiments; S.D. performed bioinformatic analysis; R.B. developed the ‘Object Scan’ plugin; M.A.S. supervised the project, designed experiments and wrote the paper. All authors discussed the results and contributed to the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to M. Azim Surani.

Extended data

Supplementary information

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  1. 1.

    Supplementary Figure 1

    This file contains uncropped scans of Western blot gels.

Excel files

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    Supplementary Table 1

    This table contains oligonucleotide sequences.

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DOI

https://doi.org/10.1038/nature16480

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