Letter

Pluripotent state transitions coordinate morphogenesis in mouse and human embryos

Received:
Accepted:
Published online:

Abstract

The foundations of mammalian development lie in a cluster of embryonic epiblast stem cells. In response to extracellular matrix signalling, these cells undergo epithelialization and create an apical surface in contact with a cavity1,2, a fundamental event for all subsequent development. Concomitantly, epiblast cells transit through distinct pluripotent states3,4, before lineage commitment at gastrulation. These pluripotent states have been characterized at the molecular level5, but their biological importance remains unclear. Here we show that exit from an unrestricted naive pluripotent state is required for epiblast epithelialization and generation of the pro-amniotic cavity in mouse embryos. Embryonic stem cells locked in the naive state are able to initiate polarization but fail to undergo lumenogenesis. Mechanistically, exit from naive pluripotency activates an Oct4-governed transcriptional program that results in expression of glycosylated sialomucin proteins and the vesicle tethering and fusion events of lumenogenesis. Similarly, exit of epiblasts from naive pluripotency in cultured human post-implantation embryos triggers amniotic cavity formation and developmental progression. Our results add tissue-level architecture as a new criterion for the characterization of different pluripotent states, and show the relevance of transitions between these states during development of the mammalian embryo.

  • Subscribe to Nature for full access:

    $199

    Subscribe

Additional access options:

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

Accessions

Primary accessions

ArrayExpress

References

  1. 1.

    & Self-organizing properties of mouse pluripotent cells initiate morphogenesis upon implantation. Cell 156, 1032–1044 (2014)

  2. 2.

    , & A description of 34 human ova within the first 17 days of development. Am. J. Anat. 98, 435–493 (1956)

  3. 3.

    et al. Hallmarks of pluripotency. Nature 525, 469–478 (2015)

  4. 4.

    & Mapping the route from naive pluripotency to lineage specification. Phil. Trans. R. Soc. Lond. B 369, 20130540 (2014)

  5. 5.

    , , & Dynamic stem cell states: naive to primed pluripotency in rodents and humans. Nat. Rev. Mol. Cell Biol. 17, 155–169 (2016)

  6. 6.

    , & Otx2 is an intrinsic determinant of the embryonic stem cell state and is required for transition to a stable epiblast stem cell condition. Development 140, 43–55 (2013)

  7. 7.

    & The role of podocalyxin in health and disease. J. Am. Soc. Nephrol. 20, 1669–1676 (2009)

  8. 8.

    et al. Lineage-specific profiling delineates the emergence and progression of naive pluripotency in mammalian embryogenesis. Dev. Cell 35, 366–382 (2015)

  9. 9.

    , , & In vitro culture of mouse blastocysts beyond the implantation stages. Nat. Protoc. 9, 2732–2739 (2014)

  10. 10.

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

  11. 11.

    et al. Self-organization of the human embryo in the absence of maternal tissues. Nat. Cell Biol. 18, 700–708 (2016)

  12. 12.

    et al. Lumen formation is an intrinsic property of isolated human pluripotent stem cells. Stem Cell Reports 5, 954–962 (2015)

  13. 13.

    et al. Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3. Cell 153, 335–347 (2013)

  14. 14.

    , , , & DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Nat. Genet. 39, 380–385 (2007)

  15. 15.

    et al. Self-renewal versus lineage commitment of embryonic stem cells: protein kinase C signaling shifts the balance. Stem Cells 29, 618–628 (2011)

  16. 16.

    , , , & Defining an essential transcription factor program for naïve pluripotency. Science 344, 1156–1160 (2014)

  17. 17.

    et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655 (2003)

  18. 18.

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

  19. 19.

    et al. Otx2 and Oct4 drive early enhancer activation during embryonic stem cell transition from naive pluripotency. Cell Rep. 7, 1968–1981 (2014)

  20. 20.

    et al. A molecular network for de novo generation of the apical surface and lumen. Nat. Cell Biol. 12, 1035–1045 (2010)

  21. 21.

    , , , & Rab11 regulates recycling through the pericentriolar recycling endosome. J. Cell Biol. 135, 913–924 (1996)

  22. 22.

    , , & Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells. J. Cell Biol. 168, 303–313 (2005)

  23. 23.

    et al. Anuria, omphalocele, and perinatal lethality in mice lacking the CD34-related protein podocalyxin. J. Exp. Med. 194, 13–28 (2001)

  24. 24.

    et al. Electrostatic cell-surface repulsion initiates lumen formation in developing blood vessels. Curr. Biol. 20, 2003–2009 (2010)

  25. 25.

    , & Polarized protein transport and lumen formation during epithelial tissue morphogenesis. Annu. Rev. Cell Dev. Biol. 31, 575–591 (2015)

  26. 26.

    et al. Cingulin and actin mediate midbody-dependent apical lumen formation during polarization of epithelial cells. Nat. Commun. 7, 12426 (2016)

  27. 27.

    et al. Defining the three cell lineages of the human blastocyst by single-cell RNA-seq. Development 142, 3151–3165 (2015)

  28. 28.

    et al. Self-organization of the in vitro attached human embryo. Nature 533, 251–254 (2016)

  29. 29.

    et al. Systematic identification of culture conditions for induction and maintenance of naive human pluripotency. Cell Stem Cell 15, 471–487 (2014)

  30. 30.

    et al. Comprehensive Cell surface protein profiling identifies specific markers of human naive and primed pluripotent states. Cell Stem Cell 20, 874–890 (2017)

  31. 31.

    et al. Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158, 1254–1269 (2014)

  32. 32.

    , , & Culture of human embryos through implantation stages in vitro. Protoc. Exch. (2016)

  33. 33.

    et al. In vivo imaging and differential localization of lipid-modified GFP-variant fusions in embryonic stem cells and mice. Genesis 44, 202–218 (2006)

  34. 34.

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

  35. 35.

    et al. Tracking the embryonic stem cell transition from ground state pluripotency. Development 144, 1221–1234 (2017)

  36. 36.

    et al. The BAF chromatin remodelling complex is an epigenetic regulator of lineage specification in the early mouse embryo. Development 143, 1271–1283 (2016)

  37. 37.

    , , & Three-dimensional culture models of normal and malignant breast epithelial cells. Nat. Methods 4, 359–365 (2007)

  38. 38.

    et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013)

  39. 39.

    , & NIH image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012)

  40. 40.

    et al. Cell-polarity dynamics controls the mechanism of lumen formation in epithelial morphogenesis. Curr. Biol. 18, 507–513 (2008)

  41. 41.

    et al. De novo lumen formation and elongation in the developing nephron: a central role for afadin in apical polarity. Development 140, 1774–1784 (2013)

  42. 42.

    et al. Full-length RNA-seq from single cells using Smart-seq2. Nat. Protoc. 9, 171–181 (2014)

  43. 43.

    , & HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015)

  44. 44.

    & Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010)

  45. 45.

    , & Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014)

Download references

Acknowledgements

We thank K. McNagny, J. Hanna and R. Jaenisch for reagents and discussions; F. Martin-Belmonte, D. Glover, C. Lynch, M. Serrano, A. Hupalowska, F. Antonica and M. Petruzzelli for feedback; J. N. Skepper for help with electron microscopy; W. Mansfield for help with embryo transfer. This work was supported by Wellcome Trust (098287/Z/12/Z) and ERC (669198) grants to M.Z.-G. Work in the laboratory of T.V. was supported by Wellcome Trust and KU Leuven (SymBioSys PFV/10/016). Work in the laboratory of J.C.M. was supported by EMBL and Cancer Research UK. M.N.S. was supported by Ramon Areces and EMBO postdoctoral fellowships; A.S. by a Wellcome Trust strategic award (105031/D/14/Z) and G.R. by a Newton fellowship.

Author information

Author notes

    • Antonio Scialdone
    • , Gaelle Recher
    • , Iain C. Macaulay
    •  & Christa Buecker

    Present addresses: Institute of Epigenetics and Stem Cells, Helmholtz Zentrum München, München 85764, Germany (A.S.); Bioimaging and Optofluidics group, IOGS, CNRS & University of Bordeaux, Rue Francois Mitterrand, 33400 Talence, France (G.R.); Earlham Institute, Norwich Research Park, Norwich NR4 7UG, UK (I.C.M.); Max F. Perutz Laboratories, Vienna Biocenter (VBC), Dr Bohr-Gasse 9, Vienna 1030, Austria (C.B.).

Affiliations

  1. Mammalian Embryo and Stem Cell Group, University of Cambridge, Department of Physiology, Development and Neuroscience, Downing Street, Cambridge CB2 3EG, UK.

    • Marta N. Shahbazi
    • , Natalia Skorupska
    • , Antonia Weberling
    • , Gaelle Recher
    • , Meng Zhu
    • , Agnieszka Jedrusik
    •  & Magdalena Zernicka-Goetz
  2. EMBL-European Bioinformatics Institute (EMBL-EBI), Wellcome Genome Campus, Cambridge CB10 1SD, UK.

    • Antonio Scialdone
    •  & John C. Marioni
  3. Faculty of Life Sciences and Medicine, King’s College London, Women’s Health Academic Centre, Assisted Conception Unit, Guy’s Hospital, Great Maze Pond, London SE1 9RT, UK.

    • Liani G. Devito
    • , Laila Noli
    • , Yakoub Khalaf
    •  & Dusko Ilic
  4. Wellcome Trust Sanger Institute, Wellcome Genome Campus, Cambridge CB10 1SA, UK.

    • Iain C. Macaulay
    • , Thierry Voet
    •  & John C. Marioni
  5. Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California 94305, USA.

    • Christa Buecker
  6. Laboratory of Reproductive Genomics, Department of Human Genetics, KU Leuven, Herestraat 49, Leuven 3000, Belgium.

    • Thierry Voet
  7. Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge CB2 0RE, UK.

    • John C. Marioni

Authors

  1. Search for Marta N. Shahbazi in:

  2. Search for Antonio Scialdone in:

  3. Search for Natalia Skorupska in:

  4. Search for Antonia Weberling in:

  5. Search for Gaelle Recher in:

  6. Search for Meng Zhu in:

  7. Search for Agnieszka Jedrusik in:

  8. Search for Liani G. Devito in:

  9. Search for Laila Noli in:

  10. Search for Iain C. Macaulay in:

  11. Search for Christa Buecker in:

  12. Search for Yakoub Khalaf in:

  13. Search for Dusko Ilic in:

  14. Search for Thierry Voet in:

  15. Search for John C. Marioni in:

  16. Search for Magdalena Zernicka-Goetz in:

Contributions

M.N.S. designed, performed and analysed most of the experiments. A.S. analysed the sequencing data. N.S. and A.W. performed experiments in Fig. 4g and Extended Data Figs 57. M.Z. and G.R. helped with embryo experiments and image analysis. A.J., L.G.D. and L.N. helped with human embryo cultures. I.C.M. prepared cDNA libraries. C.B. generated and analysed ChIP–seq data. D.I. and Y.K. supervised the human embryo experiments. T.V. supervised the cDNA library preparation. J.C.M. supervised the computational analyses of the sequencing data. M.Z.-G. supervised the study. M.N.S. and M.Z.-G. conceived the project and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Magdalena Zernicka-Goetz.

Reviewer Information Nature thanks J.-L. Maitre and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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

Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

  2. 2.

    Supplementary Table 2

    This file contains antibodies used in this study.

  3. 3.

    Supplementary Table 3

    This file contains a list of RT-PCR primers used in this study.

Excel files

  1. 1.

    Supplementary Table 1

    This file contains genes expressed in the mouse epiblast at peri-implantation stages.

Videos

  1. 1.

    ΔPE-Oct4-GFP mESCs cultured in 3D matrigel without 2i/LIF and imaged every 30 minutes.

    The arrow points to the cell shown in Extended Data Fig. 2e. The maximum projection is shown throughout the movie. Scale bars, 50 μm.

  2. 2.

    Rex1::GFPd2 mESCs cultured in 3D matrigel without 2i/LIF and imaged every 30 minutes.

    The arrow points to the cell shown in Extended Data Fig. 2e. The maximum projection is shown throughout the movie. Scale bars, 50 μm.

  3. 3.

    Nanog-YFP mTmG mESCs cultured in 3D matrigel without 2i/LIF and imaged every 30 minutes.

    The arrow points to the cell shown in Extended Data Fig. 2e. The maximum projection is shown throughout the movie. Scale bars, 50 μm.

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.