Human embryo polarization requires PLC signaling to mediate trophectoderm specification

  1. Meng Zhu
  2. Marta Shahbazi
  3. Angel Martin
  4. Chuanxin Zhang
  5. Berna Sozen
  6. Mate Borsos
  7. Rachel S Mandelbaum
  8. Richard J Paulson
  9. Matteo A Mole
  10. Marga Esbert
  11. Shiny Titus
  12. Richard T Scott
  13. Alison Campbell
  14. Simon Fishel
  15. Viviana Gradinaru
  16. Han Zhao
  17. Keliang Wu
  18. Zijiang Chen  Is a corresponding author
  19. Emre Seli  Is a corresponding author
  20. Maria J. de los Santos  Is a corresponding author
  21. Magdalena Zernicka-Goetz  Is a corresponding author
  1. Blavatnik Institute, Harvard Medical School, United States
  2. MRC Laboratory of Molecular Biology, United Kingdom
  3. IVIRMA Valencia, Spain
  4. Shandong University, China
  5. Yale School of Medicine, United States
  6. California Institute of Technology, United States
  7. USC Fertility, University of Southern California, United States
  8. University of Cambridge, United Kingdom
  9. IVIRMA New Jersey, United States
  10. CARE Fertility, United Kingdom
  11. Shanghai Jiao Tong University, China

Abstract

Apico-basal polarization of cells within the embryo is critical for the segregation of distinct lineages during mammalian development. Polarized cells become the trophectoderm (TE), which forms the placenta, and apolar cells become the inner cell mass (ICM), the founding population of the fetus. The cellular and molecular mechanisms leading to polarization of the human embryo and its timing during embryogenesis have remained unknown. Here, we show that human embryo polarization occurs in two steps: it begins with the apical enrichment of F-actin and is followed by the apical accumulation of the PAR complex. This two-step polarization process leads to the formation of an apical domain at the 8-16 cell stage. Using RNA interference, we show that apical domain formation requires Phospholipase C (PLC) signaling, specifically the enzymes PLCB1 and PLCE1, from the 8-cell stage onwards. Finally, we show that although expression of the critical TE differentiation marker GATA3 can be initiated independently of embryo polarization, downregulation of PLCB1 and PLCE1 decreases GATA3 expression through a reduction in the number of polarized cells. Therefore, apical domain formation reinforces a TE fate. The results we present here demonstrate how polarization is triggered to regulate the first lineage segregation in human embryos.

Data availability

Source data files used for generating each figure has been uploaded as supplementary material.

Article and author information

Author details

  1. Meng Zhu

    Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6157-8840
  2. Marta Shahbazi

    MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1599-5747
  3. Angel Martin

    IVIRMA Valencia, IVIRMA Valencia, Valencia, Spain
    Competing interests
    The authors declare that no competing interests exist.
  4. Chuanxin Zhang

    National Research Center for Assisted Reproductive Technology and Reproductive Genetics, Shandong University, Jinan, China
    Competing interests
    The authors declare that no competing interests exist.
  5. Berna Sozen

    Department of Obstetrics, Gynaecology, and Reproductive Sciences, Yale School of Medicine, New Haven, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Mate Borsos

    California Institute of Technology, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Rachel S Mandelbaum

    Keck School of Medicine, USC Fertility, University of Southern California, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Richard J Paulson

    Keck School of Medicine, USC Fertility, University of Southern California, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Matteo A Mole

    Physiology, Development and Neuroscience, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  10. Marga Esbert

    IVIRMA, IVIRMA New Jersey, New Jersey, United States
    Competing interests
    The authors declare that no competing interests exist.
  11. Shiny Titus

    IVIRMA, IVIRMA New Jersey, New Jersey, United States
    Competing interests
    The authors declare that no competing interests exist.
  12. Richard T Scott

    IVIRMA, IVIRMA New Jersey, New Jersey, United States
    Competing interests
    The authors declare that no competing interests exist.
  13. Alison Campbell

    CARE Fertility,, CARE Fertility, Nottingham, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  14. Simon Fishel

    CARE Fertility, CARE Fertility, Nottingham, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  15. Viviana Gradinaru

    Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5868-348X
  16. Han Zhao

    Cheeloo College of Medicine, Shandong University, Jinan, China
    Competing interests
    The authors declare that no competing interests exist.
  17. Keliang Wu

    Cheeloo College of Medicine, Shandong University, Jinan, China
    Competing interests
    The authors declare that no competing interests exist.
  18. Zijiang Chen

    Shanghai Key Laboratory for Assisted Reproduction and Reproductive Genetics, Renji Hospital, Shanghai Jiao Tong University, Shanghai, China
    For correspondence
    Chenzijiang@vip.163.com
    Competing interests
    The authors declare that no competing interests exist.
  19. Emre Seli

    Department of Obstetrics, Gynaecology, and Reproductive Sciences, Yale School of Medicine, New Heaven, United States
    For correspondence
    Emre.Seli@ivirma.com
    Competing interests
    The authors declare that no competing interests exist.
  20. Maria J. de los Santos

    IVIRMA Valencia, IVIRMA Valencia, Valencia, Spain
    For correspondence
    MariaJose.DelosSantos@ivirma.com
    Competing interests
    The authors declare that no competing interests exist.
  21. Magdalena Zernicka-Goetz

    Department of Physiology, Neuroscience and Development, University of Cambridge, Cambridgeshire, United Kingdom
    For correspondence
    mz205@cam.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7004-2471

Funding

Wellcome Trust (207415/Z/17/Z)

  • Magdalena Zernicka-Goetz

Open Philanthropy Project

  • Magdalena Zernicka-Goetz

Curci and Weston Heavens Foundations

  • Magdalena Zernicka-Goetz

Leverhulme Trust (RPG-2018-085)

  • Meng Zhu

European molecular biology organisation

  • Marta Shahbazi

Medical Research Council (MC_UP_1201/24)

  • Marta Shahbazi

National Key Research and Development Program of China (2018YFC1004000)

  • Zijiang Chen

Shandong Provincial Key Research and Development Program (2018YFJH0504)

  • Zijiang Chen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Human subjects: Ethical approvalThis study was performed in five different avenues: Clinical Embryology Laboratory at IVIRMAValencia (Spain), University of Cambridge (United Kingdom), National Research Center forAssisted Reproductive Technology and Reproductive Genetics (China), California Institute ofTechnology (United States), and Foundation for Embryonic Competence New Jersey (UnitedStates). All the work complies with The International Society for Stem Cell Research (ISSCR)guidelines.Experiments performed in Spain: The work in the Clinical Embryology Laboratory at IVIRMAValencia was approved by the National Commission of Human Reproduction (CNRHA), theGeneral direction of research, innovation, technology and quality and the ethics committee ofClinical Research IVI Valencia, which complies with Spanish law on assisted reproductivetechnologies (14/2006). A total of 260 excess and cryopreserved donated day 3 humanembryos from 95 IVF patients were used. Embryos were not created for research. Couplesdonating their embryos were informed of the objectives of the project, the nature of theexperiments, and the conditions of the research. No financial inducements were offered fordonation. The average age of women was 28.12 {plus minus} 4. The fixed embryos were analysed at theUniversity of Cambridge. This work was evaluated by the Human Biology Research EthicsCommittee of the University of Cambridge (reference HBREC.2017.27).Experiments performed in the United Kingdom: The work performed at the University ofCambridge was in accordance with the Human Fertility and Embryology Authority (HFEA)regulations (license reference R0193). Ethical approval was obtained from the "HumanBiology Research Ethics Committee" of the University of Cambridge (referenceHBREC.2017.21). Informed consent was obtained from all patients from CARE Fertility Groupand Herts & Essex fertility clinics, who donated excess and cryopreserved embryos used inthis study after completing their IVF treatment. Embryos were not created for research. Priorto giving consent, patients were informed about the specific objectives of the project, and theconditions that apply within the license, offered counselling and did not receive any financialinducements. In this study, we used 15 donated embryos at the 2 pronuclei stage (day 1 d.p.f.),which were warmed and cultured in the Cambridge Laboratory according to the aboveregulations.Experiments performed in China: The work performed in the National Research Center forAssisted Reproductive Technology and Reproductive Genetics was conducted under theregulations of the Human Biomedical Research Ethics Guidelines (regulated by NationalHealth Commission of the People's Republic of China on 1 December 2016) and the HumanEmbryonic Stem Cell Research Ethics Guidelines (regulated by China National Center forBiotechnology Development on 24 December 2003). These regulations and guidelines allowexcess 3PN human gametes, and/or human embryos created or genetically manipulated invitro and those cultured for no more than 14 days, to be used for scientific researches. Theaim and protocols involved in this study were reviewed and approved by the InstitutionalReview Board (IRB) of Reproductive Medicine, Shandong University. The protocols includethe use of 3PN embryos and the siRNA injections. All embryos were donated after informedconsent was obtained. The embryos were excess and fresh 3PN zygotes generated from invitro fertilisation, with the donor females age ranging from 22 to 40 years old. These embryoswere used for microinjection experiments. Embryos were not created for research. Patientswere informed about the objectives, experimental approach and potential outcomes of theresearch prior to the donation. They were offered counselling to aid them in their decision.Experiments performed in California (United States): The work at California Institute ofTechnology was approved by the California Institute of Technology Committee for theProtection of Human Subjects (IRB number 19-0948). Human embryos at the zygote stagewere obtained from the University of Southern California (USC) through the pre-existing USCIRB-approved Biospecimen Repository for Reproductive Research (HS-15-00859) afterappropriate approval was obtained unanimously from the Biorepository Ethics Committee. AtUSC Fertility, excess cryopreserved embryos were donated after completion of IVF; embryoswere not created for research purposes. Patients were informed of the general conditions ofthe donation, objectives, and methodology of human embryo research. They were offeredcounselling and alternatives, including discarding embryos and continued cryopreservation.Patients were informed that they would not benefit directly from donation of embryos toresearch. A total of 41 donated human embryos at the zygote 2 pronuclei stage (day 1 postfertilisation.) from 4 IVF patients were used. The average age of women was 44. The embryoswere warmed at USC Fertility per usual IVF procedure and then transferred to the CaliforniaInstitute of Technology for the remainder of the protocol.Experiments performed in New Jersey (United States): the research was approved by theWestern IRB (Clinical IRB 20031397). Embryos used in this study were excess fresh embryosto the IVF treatment of IVI-RMA patients. Informed consent was obtained from all the couplesthat donated their embryos. They were informed of the general objective, methodology andpotential outcomes of the research. Patients did not receive any financial compensation forthe donation. Embryos were not created for research.

Reviewing Editor

  1. Joshua M Brickman, University of Copenhagen, Denmark

Publication history

  1. Received: November 20, 2020
  2. Accepted: September 25, 2021
  3. Accepted Manuscript published: September 27, 2021 (version 1)
  4. Version of Record published: October 13, 2021 (version 2)
  5. Version of Record updated: October 19, 2021 (version 3)

Copyright

© 2021, Zhu et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Meng Zhu
  2. Marta Shahbazi
  3. Angel Martin
  4. Chuanxin Zhang
  5. Berna Sozen
  6. Mate Borsos
  7. Rachel S Mandelbaum
  8. Richard J Paulson
  9. Matteo A Mole
  10. Marga Esbert
  11. Shiny Titus
  12. Richard T Scott
  13. Alison Campbell
  14. Simon Fishel
  15. Viviana Gradinaru
  16. Han Zhao
  17. Keliang Wu
  18. Zijiang Chen
  19. Emre Seli
  20. Maria J. de los Santos
  21. Magdalena Zernicka-Goetz
(2021)
Human embryo polarization requires PLC signaling to mediate trophectoderm specification
eLife 10:e65068.
https://doi.org/10.7554/eLife.65068

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