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. Zi-Jiang Chen  Is a corresponding author
  19. Emre Seli  Is a corresponding author
  20. Maria J de los Santos
  21. Magdalena Zernicka Goetz
  1. Mammalian Embryo and Stem Cell Group, University of Cambridge, Department of Physiology, Development and Neuroscience, United Kingdom
  2. Blavatnik Institute, Harvard Medical School, Department of Genetics, United States
  3. MRC Laboratory of Molecular Biology. Francis Crick Avenue, Biomedical Campus., United Kingdom
  4. IVIRMA Valencia, IVI Foundation, Spain
  5. Center for Reproductive Medicine, Cheeloo College of Medicine, Shandong University, China
  6. Key laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, China
  7. Developmental Plasticity and Self-Organization Group, California Institute of Technology, Division of Biology and Biological Engineering, United States
  8. Yale School of Medicine, Department of Genetics, United States
  9. California Institute of Technology, Division of Biology and Biological Engineering,, United States
  10. USC Fertility, University of Southern California, Keck School of Medicine, United Kingdom
  11. IVIRMA New Jersey, United States
  12. CARE Fertility Group, John Webster House, 6 Lawrence Drive, Nottingham Business Park, United Kingdom
  13. School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, United Kingdom
  14. Yale School of Medicine, Department of Obstetrics, Gynecology, and Reproductive Sciences, United States
6 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Timing of compaction and polarization in human embryos.

(a) Scheme for human embryo culture. Supernumerary in vitro fertilized human embryos were warmed at day 3, and cultured for 2, 9, or 24 hr to examine the localization of polarization markers. (b) …

Figure 1—source data 1

Source data for Figure 1.

This file contains the source data used to make the graphs presented in Figure 1 and Figure 1—figure supplement 1. GraphPad Prism was utilized to visually represent the quantitative data.

https://cdn.elifesciences.org/articles/65068/elife-65068-fig1-data1-v3.xlsx
Figure 1—figure supplement 1
Morpho-kinetic analysis of human embryos cultured in vitro from day 3 to day 4.

(a-b) Histograms showing the number of total (a) and intact (b) cells at the time of embryo warming, N = 132 control embryos. (c–d) Histograms showing the number of total (c) and intact (d) cells …

Figure 2 with 2 supplements
PLC activity regulates cell polarization in the human embryo.

(a) Scheme of the PLCE1/PLCB1 siRNA injections. (b), Representative images of embryos injected with control siRNA or PLCE1/PLCB1 siRNA and cultured until embryonic day four to reveal the …

Figure 2—source data 1

Source data for Figure 2.

This file contains the source data used to make the graphs presented in Figure 2 and Figure 2—figure supplements 1 and 2. GraphPad Prism was utilized to visually represent the quantitative data.

https://cdn.elifesciences.org/articles/65068/elife-65068-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Morphokinetic analysis of the consequences of pharmacological inhibition of PLC.

(a) Scheme of the PLC inhibitor treatment. (b) Quantification of the total number of cells in embryos from panel d. Each dot represents one embryo. N = 42 embryos for media control group, N = 19 …

Figure 2—figure supplement 2
Morphokinetic analysis of PLC loss of function experiments.

(a) Expression profile for all PLC isoforms at different stages of human preimplantation development. Data retrieved from Yan et al., 2013. (b) The expression level of PLCE1 and PLCB1 in human …

Figure 3 with 1 supplement
GATA3 expression is initiated independently of cell polarization.

(a) Representative images of in vitro fertilized human embryos warmed at day 3 and cultured for 2, 9, or 24 hr (see scheme in Figure 1a ) to reveal the localization of F-actin, PARD6, and GATA3. …

Figure 3—source data 1

Source data for Figure 3 and Figure 3—figure supplement 1.

This file contains the source data used to make the graphs presented in Figure 3. GraphPad Prism was utilized to visually represent the quantitative data.

https://cdn.elifesciences.org/articles/65068/elife-65068-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
Trophectoderm specification and inner cell generation in human embryos.

(a) Number of cells showing nuclear GATA3 (defined as a nucleus to cytoplasm ratio of more than 1.5) in relation to their polarity status in human embryos at embryonic day 4. Numbers in each bar …

Generation of inner cells in the human embryo.

(a) Representative images of embryos that have inner cells with a low number of outer polarized cells. White arrowheads indicate the presence of inner cells. (b) Line chart showing the relation …

Figure 4—source data 1

Source data for Figure 4.

This file contains the source data used to make the graphs presented in Figure 4. GraphPad Prism was utilized to visually represent the quantitative data.

https://cdn.elifesciences.org/articles/65068/elife-65068-fig4-data1-v3.xlsx
Schematic summarizing the main findings of this study.

F-actin polarization precedes PAR complex polarization and it is triggered by PLC activation in mouse and human embryos. In human embryos, blastomeres initiate the expression of TE factors …

Author response image 1
Correlation analysis of the variables ‘duration of compaction’ and ‘female age’ in a validation dataset comprising 810 embryos.

Duration of compaction was defined as the time in hours between the start of compaction and morula formation. r: pearson correlation coefficient; p: p-value. No correlation was found between both …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Sequenced-based reagentsiRNA to PLCE1 and PLCB1QiagenHs_PLCB1_4, SI00115521; Hs_PLCB1_6, SI02781184Hs_PLCE1_1, SI00115521; negative control siRNA:1022076.A 20 μM concentration of siRNA solution was used for injection.
Biological sample (Human)Human embryosDonated supernumerary embryos generated from in vitro fertilization experiments
Antibody(Rabbit monoclonal), anti-PARD6Santa Cruzsc-67393(1:200)
Antibody(Goat polyclona)l, anti-GATA3R&D systemsAF2605(1:200)
Antibody(Mouse monoclonal), anti-aPKCSanta Cruzsc-17781(1:50)
Antibody(Mouse monoclonal), anti-YAP1Santa Cruzsc-101199(1:200)
Recombinant DNA reagentpRN3P- GAP-GFPZhu et al., 2017
Sequence-based reagentGAPDH-FThis paperqPCR primersGATCATCAGCAATGCCTCCT
Sequence-based reagentGAPDH-RThis paperqPCR primersTTCAGCTCAGGGATGACCTT
Sequence-based reagentPLCB1-FThis paperqPCR primersGGAAGCGGCAAAAAGAAGCTC
Sequence-based reagentPLCB1-RThis paperqPCR primersCGTCGTCGTCACTTTCCGT
Sequence-based reagentPLCE1-FThis paperqPCR primersTGCAGCCTCTCATCCAGTT
Sequence-based reagentPLCE1-RThis paperqPCR primersCCCTGCGGTAAATAGTCTGC
Commercial assay or kitSMART-Seq v4 Ultra Low Input RNA KitTakaraCat. No. 634,888
Commercial assay or kitAgencourt AMPure XP KitBeckman CoulterA63880
Chemical compound, drugU73122CaymanchemNo. 70,740
Chemical compound, drugDMSOSigma-AlrichD2650−5 × 10 ML
Software, algorithmPrism 8Graphpad

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