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Initiation of a conserved trophectoderm program in human, cow and mouse embryos

Nature volume 587, pages 443–447 (2020)Cite this article

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Abstract

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac1. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human2,3,4,5 and cow6. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical–basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.

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Fig. 1: Transcriptional and protein expression differences between cells in human morula embryos.
Fig. 2: Apical expression of aPKC and AMOT in outer cells in human morula embryos, in which SOX2 expression is retained.
Fig. 3: aPKC activity is required for YAP1 and GATA3 expression in mouse, cow and human morula embryos.

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Data availability

The datasets analysed in this study have previously been published, and are available at the Gene Expression Omnibus (GEO) in repositories GSE36552 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE36552) and GSE66507 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE66507); at EMBL-EBI ArrayExpress accession number E-MTAB-3929 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3929/); and at EMBL-EBI European Nucleotide Archive (ENA) accession code PRJNA494280 (https://www.ebi.ac.uk/ena/browser/view/PRJNA494280). Source data are provided with this paper.

Code availability

The data processing and analysis pipelines are publicly available at https://github.com/galanisl/TE_differentiation.

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Acknowledgements

We thank the donors whose contributions have enabled this research; P. Patel, A. Srikantharajah, M. Summers, A. Handyside, K. Ahuja, S. Lavery, A. Rattos and M. Jansa Perez for the coordination and donation of embryos to our research project; members of the laboratories of K.K.N., J. M. A. Turner and R. Lovell-Badge, as well as M. Marass, T. Rayon Alonso, B. Thompson and N. Goehring for discussion, advice and feedback on the manuscript; the laboratories of B. Thompson, N. Goehring and J. Briscoe for sharing reagents and advice; the Advanced Light Microscopy and Biological Research Facilities (Francis Crick Institute); and A. Brodie and K. Bacon for assisting with cow embryo culture. The AMOT antibody (Amot-C no. 10061-1) used in this paper for mouse embryos was provided by the laboratory of H. Sasaki. CRT0276121 was provided by Cancer Research Technology. Work in the laboratory of L.D. was funded by a donation from MSD to ‘Fondation de l’Université de Nantes’. Work in the laboratory of H.V.d.V. was funded by the Fonds Wetenschappelijk Onderzoek Flanders (FWOAL722) and the Wetenschappelijk Fonds Willy Gepts (WFWG, UZ Brussel, G142). Work in the laboratory of A.A.F.-N. was supported by Comparative Biomedical Sciences Departmental fund from the Royal Veterinary College. Work in the laboratory of K.K.N. was supported by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001120), the UK Medical Research Council (FC001120) and the Wellcome Trust (FC001120), and by the Rosa Beddington Fund.

Author information

Author notes

  1. Norah M. E. Fogarty

    Present address: Centre for Stem Cells and Regenerative Medicine, King’s College London, London, UK

  2. Kathy K. Niakan

    Present address: The Centre for Trophoblast Research, Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge, UK

Authors and Affiliations

  1. Human Embryo and Stem Cell Laboratory, The Francis Crick Institute, London, UK

    Claudia Gerri, Afshan McCarthy, Gregorio Alanis-Lobato, Norah M. E. Fogarty & Kathy K. Niakan

  2. Department of Reproduction and Immunology, Vrije Universiteit Brussel, Brussels, Belgium

    Andrej Demtschenko & Hilde Van de Velde

  3. Université de Nantes, CHU Nantes, Inserm, Centre de Recherche en Transplantation et Immunologie, UMR 1064, ITUN, Nantes, France

    Alexandre Bruneau, Sophie Loubersac & Laurent David

  4. Service de Biologie de la Reproduction, CHU Nantes, Université de Nantes, Nantes, France

    Sophie Loubersac

  5. Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK

    Daniel Hampshire & Ali A. Fouladi-Nashta

  6. Bourn Hall Clinic, Cambridge, UK

    Kay Elder, Phil Snell & Leila Christie

  7. Université de Nantes, CHU Nantes, Inserm, CNRS, SFR Santé, Inserm UMS 016, CNRS UMS 3556, Nantes, France

    Laurent David

  8. Center for Reproductive Medicine, UZ-Brussel, Brussels, Belgium

    Hilde Van de Velde

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Contributions

C.G. and K.K.N. conceived the study; K.K.N. supervised the project; C.G., K.K.N. and A.M. designed the experiments; C.G., K.K.N., A.M. and N.M.E.F. performed experiments; G.A.-L. performed the bioinformatic analysis of scRNA-seq and ATAC-seq datasets; C.G., K.K.N., A.M. and G.A.-L. analysed data; S.L. managed human embryos donated to research in Nantes; C.G. and A.D. performed experiments on human embryos in Brussels; A.B. and S.L. performed experiments on human embryos in Nantes; K.E., P.S. and L.C. coordinated the donation of embryos to the research project in London; L.D. supervised experiments on human embryos in Nantes; H.V.d.V. supervised experiments on human embryos in Brussels; A.A.F.-N. and D.H. provided cow ovaries; A.A.F.-N. provided techniques for cow embryo generation, helped with the conceptualization and design of experiments on cow embryos, and hosted C.G. in his laboratory; C.G. and K.K.N. wrote the manuscript with help from all of the authors.

Corresponding author

Correspondence to Kathy K. Niakan.

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Extended data figures and tables

Extended Data Fig. 1 Morphokinetic analysis to benchmark key morphological events in mouse, cow and human pre-implantation development.

a, Representative images of mouse, cow and human embryos at each developmental stage analysed. Yellow arrows point to blastomere flattening. Yellow outline denotes the compacted morula. Yellow arrowheads mark microlumens. Yellow asterisks show single dominant cavity. Yellow bars show zona pellucida (ZP) thinning. dpf, days post-fertilization. Scale bars, 100 μm. b, c, Morphokinetic analysis of mouse, cow and human pre-implantation development, showing relative time in hours (b) and in percentage (c) (from 8-cell stage to the end of cavitation). n = 15 embryos for mouse and cow, and n = 16 for human. d, Morphokinetic analysis for each of the mouse, cow and human pre-implantation embryos used in the analysis, showing relative time in percentage (from the 8-cell stage up to the end of cavitation). n = 15 embryos for mouse and cow, and n = 16 for human. e, Quantification of the number of individual cells that divided between 8-cell stage and start of compaction in mouse, cow and human embryos. n = 15 embryos for mouse and human, and n = 14 for cow. Data are presented as mean ± s.d. Two-tailed Mann–Whitney U test, ****P < 0.0001, ns, not significant. f, Quantification of the total cell number in mouse (32-cell stage), cow and human morula embryos. n = 25 for mouse, n = 20 for cow and n = 34 for human. Data are presented as mean ± s.d. Two-tailed Mann–Whitney U test, ****P < 0.0001. g, Quantification of the number of inner and outer cells in mouse, cow and human morula embryos. n = 25 for mouse, n = 20 for cow and n = 34 for human. Data are presented as mean ± s.d. Two-tailed Student’s t-test, ****P < 0.0001, ns, not significant. h, Quantification of the number of ICM and TE cells in mouse, cow and human blastocysts. n = 14 for mouse, n = 10 for cow and n = 12 for human. Data are presented as mean ± s.d. Two-tailed Student’s t-test, ****P < 0.0001, ns, not significant. More details about statistics and reproducibility are provided in Methods.

Source data

Extended Data Fig. 2 Protein expression of TE-associated markers in mouse, cow and human morula embryos.

a, b, Immunofluorescence analysis of F-actin, TEAD4 and Hoechst-33342 nuclear staining in human morula embryo. Yellow arrowheads point to a cell without TEAD4 expression. c, Percentage of TEAD4-positive and -negative cells in human morula embryos (n = 112 cells from 5 embryos). d, Quantification of TEAD4 normalized fluorescence intensity in human morula embryos (n = 63 cells from 5 embryos). Data are presented as mean ± s.d. Two-tailed Student’s t-test, ns, not significant. e, Immunofluorescence analysis of GATA3, E-cadherin, YAP1 and DAPI nuclear staining in mouse morula embryos. f, g, Quantification of YAP1 (f) and GATA3 (g) normalized fluorescence intensity in mouse embryos shown in e (n = 182 cells for YAP1 from 13 embryos and n = 139 cells for GATA3 from 9 embryos). Data are presented as mean ± s.d. Two-tailed Mann–Whitney U test, ****P < 0.0001. h, Immunofluorescence analysis using various secondary antibodies and DAPI nuclear staining (blue) in mouse morula embryos (n = 3). i, Time-course immunofluorescence analysis of GATA3, β-catenin, YAP1 and DAPI nuclear staining in cow embryos at different developmental stages: 8-cell (n = 5), late compaction (n = 5), morula (n = 10) and expanded blastocyst (n = 5). j, k, Quantification of YAP1 (j) and GATA3 (k) normalized fluorescence intensity in cow morula embryos shown in i (n = 97 cells from 10 embryos). Data are presented as mean ± s.d. Two-tailed Mann–Whitney U test, ****P < 0.0001.l, Immunofluorescence analysis of GATA3, GATA2 and DAPI nuclear staining in a human morula embryo (n = 3). m, Immunofluorescence analysis of GATA3, OCT4, GATA2 and DAPI nuclear staining in a human expanded blastocyst embryo (n = 3). Yellow arrowheads point to outer cells expressing GATA3, and cyan arrow points to an inner cell devoid of GATA3 expression. Scale bars, 25 μm (mouse embryos), 50 μm (cow and human embryos). More details about statistics and reproducibility are provided in Methods.

Source data

Extended Data Fig. 3 WWTR1, KRT18 and GRHL2 expression in human morula embryos.

a, Immunofluorescence analysis of YAP1, F-actin, WWTR1 and DAPI nuclear staining in a human morula embryo (n = 3). b, Immunofluorescence analysis of YAP1, Nanog, WWTR1 and DAPI nuclear staining in a human expanded blastocyst embryo (n = 3). Yellow arrowheads point to outer and TE cells co-expressing YAP1 and WWTR1. c, Immunofluorescence analysis of GATA3, KRT18 and Hoechst-33342 nuclear staining in a human expanded blastocyst embryo (n = 3). d, Genome browser view of the ATAC-seq signal at the GRHL2 locus. High-confidence peaks (FDR-corrected P < 0.001; MACS2 dynamic Poisson model) were used to identify transcription factor (TF) motifs. Representative binding motifs associated with the footprints are highlighted. The average expression of GRHL2 in GATA3-high- and GATA3-low-expressing cells at the morula is shown and the TPM units are indicated. e, Immunofluorescence analysis of GRHL2, F-actin, GATA3 and DAPI nuclear staining in human morula embryos (n = 3). Yellow arrowheads point to outer cells co-expressing GRHL2 and GATA3, and cyan arrows point to inner cells expressing GRHL2 only. f, Immunofluorescence analysis of GRHL2, OCT4, GATA3 and DAPI nuclear staining in human expanded and hatching blastocyst embryo (n = 3 each stage). Scale bars, 50 μm. More details about statistics and reproducibility are provided in Methods.

Extended Data Fig. 4 Correlation analysis of GATA3 expression in human morula cells.

a–d, Scatter plots showing positive correlation of GATA3 expression profile with GRHL2, CLDN4, RAB20 (a), PTGES, TFEB, PLAC8 (b), ATP6V1B1, FXYD4, SLC7A2 (c), and VGLL4 (d) expression profiles in human morula cells. n = 197 cells. r, Pearson correlation coefficient. Values are displayed as log-transformed size-factor-normalized counts. The black line corresponds to a linear regression model fitted to the data with 95% confidence bands. e, Scatter plots of selected genes implicated in embryonic stem cell pluripotency and/or enriched in precursors of the epiblast and ICM in human morula cells that were identified as negatively correlated with GATA3 expression. n = 197 cells. r, Pearson correlation coefficient. Values are displayed as log-transformed size-factor-normalized counts. The black line corresponds to a linear regression model fitted to the data with 95% confidence bands. f, Box plots of selected genes showing positive or negative correlation with GATA3 expression in human morula cells. n = 197 cells. Data are shown as TPM + 1. Boxes correspond to the first and third quartiles, horizontal lines to the median, whiskers extend to 1.5× the interquartile range and dots are outliers. More details about statistics and reproducibility are provided in Methods.

Extended Data Fig. 5 SOX2 is an inner-cell-specific marker in mouse, but it is broadly expressed in cow and human morula embryos.

a, Immunofluorescence analysis of SOX2, E-cadherin, GATA3 and DAPI nuclear staining in mouse morula embryos. b, Quantification of SOX2 normalized fluorescence intensity in mouse embryos shown in a (n = 93 cells from 7 embryos). Data are presented as mean ± s.d. Two-tailed Mann–Whitney U test, ****P < 0.0001. Yellow arrows mark outer cells expressing only GATA3. c, Time-course immunofluorescence analysis of SOX2, β-catenin, GATA3 and DAPI nuclear staining in cow embryos at 8-cell (n = 5), late compaction (n = 5), morula (n = 10) and expanded blastocyst (n = 5) stages. d, Immunofluorescence analysis of SOX2, β-catenin, GATA3 and DAPI nuclear staining in human 8-cell (n = 5) and late-compaction (n = 5) stage embryos. e, Quantification of SOX2 normalized fluorescence intensity in cow morula embryos shown in c (n = 136 cells from 10 embryos). Data are presented as mean ± s.d. Two-tailed Student’s t-test, ns, not significant. f, Quantification of SOX2 normalized fluorescence intensity in human morula embryos shown in Fig. 2b (n = 68 cells from 6 embryos). Data are presented as mean ± s.d. Two-tailed Student’s t-test, ns, not significant. g, Immunofluorescence analysis of SOX2, F-actin, GATA3 and DAPI nuclear staining in human morula and expanded blastocyst embryo (n = 3 each stage). The SOX2 antibody used in g in this figure is MAB2018 (R & D), whereas the one used in Fig. 2 is 14-9811-82 (Ebioscience); both show a consistent signal for SOX2. Yellow arrowheads point to outer cells co-expressing GATA3 and SOX2, and cyan arrow points to an inner cell showing SOX2 expression only. Scale bars, 25 μm (mouse embryos), 50 μm (cow and human embryos). More details about statistics and reproducibility are provided in Methods.

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Extended Data Fig. 6 Apical PAR complex and AMOT expression in mouse, cow and human morula embryos.

a, Immunofluorescence analysis of aPKC, E-cadherin, AMOT and DAPI nuclear staining in mouse morula embryos (n = 10). b, Fluorescence intensity profile of aPKC and AMOT shown along the yellow arrows in the mouse morula embryo shown in a. a.u., arbitrary units. c, Immunofluorescence analysis of aPKC, E-cadherin and DAPI nuclear staining in mouse morula embryo (n = 10). d, Immunofluorescence analysis of aPKC, β-catenin, AMOT and DAPI nuclear staining in cow morula embryos (n = 10). e, Fluorescence intensity profile of aPKC and AMOT shown along the yellow arrows in the cow morula embryo shown in d. a.u., arbitrary units. f, Immunofluorescence analysis of aPKC and DAPI nuclear staining in cow morula embryo (n = 3). g, Immunofluorescence analysis of aPKC, E-cadherin and DAPI nuclear staining in human morula embryo (n = 3). The aPKC antibody used in c, f, g is LC-C354069 (LSBio), whereas the aPKC antibody used in Fig. 2c and in a, d, h, i is sc-17781 (Santa Cruz). Both antibodies show consistent and strong aPKC apical expression. h, Immunofluorescence analysis of PARD6B, F-actin, aPKC and DAPI nuclear staining in human morula embryo (n = 3). i, Immunofluorescence analysis of PARD6B, Nanog, aPKC and DAPI nuclear staining in human expanded blastocyst embryo (n = 3). Scale bars, 25 μm (mouse embryos), 50 μm (cow and human embryos). More details about statistics and reproducibility are provided in Methods.

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Extended Data Fig. 7 aPKC inhibitor dose–response experiments in mouse, cow and human embryos.

a, Schematic of aPKC inhibitor treatment in mouse embryos. b, Immunofluorescence analysis of GATA3, E-cadherin, YAP1 and DAPI nuclear staining in mouse morula embryos treated with different concentrations of aPKC inhibitor (n = reported in Supplementary Table 6 for each condition). c, Schematic of aPKC inhibitor treatment in cow embryos. d, Immunofluorescence analysis of β-catenin, YAP1 and DAPI nuclear staining in cow morula embryos treated with different concentrations of aPKC inhibitor (n = reported in Supplementary Table 7 for each condition). e, Schematic of aPKC inhibitor treatment in human embryos. f, Immunofluorescence analysis of GATA3, β-catenin, YAP1 and DAPI nuclear staining in human morula embryos treated with different concentrations of aPKC inhibitor (n = reported in Supplementary Table 8 for each condition). Scale bars, 25 μm (mouse embryos), 50 μm (cow and human embryos). More details about statistics and reproducibility are provided in Methods.

Extended Data Fig. 8 Characterization of the effects of aPKC inhibition in mouse, cow and human embryos.

a, Immunofluorescence analysis of SOX2, E-cadherin, YAP1 and DAPI nuclear staining in control and aPKC-inhibitor-treated mouse morula embryos. Yellow arrowheads point to outer cells expressing SOX2 in mouse morula embryos. b, c, Quantification of SOX2 (b) and YAP1 (c) normalized fluorescence intensity in outer cells of mouse embryos shown in a (n = 155 cells from 19 embryos). Data are presented as mean ± s.d. Two-tailed Mann–Whitney U test for SOX2, two-tailed Student’s t-test for YAP1, ****P < 0.0001. d, Schematic of aPKC inhibitor treatment in mouse embryos. e, Morphokinetic analysis of control and aPKC-inhibitor-treated mouse embryos showing relative time in percentage (from 2-cell to morula stage) (n = 88). f, Schematic of aPKC inhibitor treatment in mouse embryos. g, Quantification of percentage of mouse embryos either developing to form an expanded blastocyst or arrested morula in control (n = 30) and aPKC inhibitor (n = 30) conditions. h, Schematic of aPKC inhibitor treatment in cow embryos. i, Quantification of percentage of cow embryos either developing to form an expanded blastocyst or arrested morula in control (n = 10) and aPKC inhibitor (n = 10) conditions. j, Schematic of aPKC inhibitor treatment in human embryos. k, Quantification of percentage of human embryos either developing to form an expanded blastocyst or arrested morula in control (n = 12) and aPKC inhibitor (n = 12) conditions. l, Immunofluorescence analysis of SOX2, β-catenin, YAP1 and DAPI nuclear staining in control and aPKC-inhibitor-treated cow morula embryos. m, n, Quantification of SOX2 (m) and YAP1 (n) normalized fluorescence intensity in outer cells of cow embryos shown in l (n = 218 cells from 15 embryos). Data are presented as mean ± s.d. Two-tailed Student’s t-test for SOX2, ns, not significant. Two-tailed Mann–Whitney U test for YAP1, ****P < 0.0001. o, Immunofluorescence analysis of SOX2, F-actin, YAP1 and DAPI nuclear staining in control and aPKC-inhibitor-treated human morula embryos. p, q, Quantification of SOX2 (p) and YAP1 (q) normalized fluorescence intensity in outer cells of human embryos shown in o (n = 140 cells from 10 embryos). Data are presented as mean ± s.d. Two-tailed Student’s t-test for SOX2, ns, not significant. Two-tailed Mann–Whitney U test for YAP1, ****P < 0.0001. Scale bars, 25 μm (mouse embryos), 50 μm (cow and human embryos). More details about statistics and reproducibility are provided in Methods.

Source data

Extended Data Fig. 9 Trim-Away optimization in mouse embryos.

a, Immunofluorescence analysis of TEAD4, E-cadherin, YAP1 and DAPI nuclear staining in control and aPKC-inhibitor-treated mouse morula embryos. b, c, Quantification of TEAD4 (b) and YAP1 (c) normalized fluorescence intensity in outer cells of mouse embryos shown in a (n = 101 cells from 10 embryos). Data are presented as mean ± s.d. Two-tailed Student’s t-test for TEAD4, ns, not significant. Two-tailed Mann–Whitney U test for YAP1, ****P < 0.0001. d, Immunofluorescence analysis of TEAD4, F-actin, YAP1 and DAPI nuclear staining in control and aPKC-inhibitor-treated human morula embryos. e, f, Quantification of TEAD4 (e) and YAP1 (f) normalized fluorescence intensity in outer cells of human embryos shown in d (n = 85 cells from 6 embryos). Data are presented as mean ± s.d. Two-tailed Student’s t-test for TEAD4, ns, not significant. Two-tailed Mann–Whitney U test for YAP1, ****P < 0.0001. g, Schematic of the Trim-Away approach. h, Schematic of the Trim-Away experiment in mouse. i, Schematic of the two anti-aPKC primary antibodies used in the Trim-Away experiment. j, Immunofluorescence analysis of anti-mouse IgG secondary antibody to detect the electroporated mouse anti-aPKC primary antibody, YAP1, GATA3 and DAPI nuclear staining in mouse morula embryos in control and Trim-Away conditions at different concentrations (n = reported in Supplementary Table 9 for each condition). Scale bars, 25 μm (mouse embryos), 50 μm (human embryos). More details about statistics and reproducibility are provided in Methods.

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Extended Data Fig. 10 Characterization of the effects of aPKC Trim-Away in mouse embryos.

a, Immunofluorescence analysis of E-cadherin, mCherry and DAPI nuclear staining in mouse morula embryos in control and Trim-Away conditions (n = 10). b, Immunofluorescence analysis of anti-mouse IgG secondary antibody to detect the electroporated mouse anti-aPKC primary antibody, E-cadherin, rabbit anti-aPKC primary antibody to detect the aPKC protein and DAPI nuclear staining in mouse morula embryos in control and Trim-Away conditions. c, Quantification of aPKC cortical intensity (based on rabbit anti-aPKC signal) in outer cells at the morula stage of mouse embryos shown in b (n = 270 cells from 30 embryos). Data are presented as mean ± s.d. Two-tailed Student’s t-test, ****P < 0.0001. d, Immunofluorescence analysis of GATA3, YAP1, anti-mouse IgG secondary antibody to detect the electroporated mouse anti-aPKC primary antibody and DAPI nuclear staining in mouse morula embryos in control and Trim-Away conditions. Yellow arrowheads point to decreased YAP1 and GATA3 expression. e, f, Quantification of YAP1 (e) and GATA3 (f) normalized fluorescence intensity in outer cells of mouse embryos shown in d (n = 281 cells for YAP1 from 32 embryos, n = 263 cells for GATA3 from 31 embryos). Data are presented as mean ± s.d. Two-tailed Student’s t-test, ***P = 0.00078 for GATA3 distribution, ****P < 0.0001 for YAP1 distribution. g, Immunofluorescence analysis of SOX2, rabbit anti-aPKC primary antibody to detect the aPKC protein, anti-mouse IgG secondary antibody to detect the electroporated mouse anti-aPKC primary antibody and DAPI nuclear staining in mouse morula embryos in control and Trim-Away conditions. Yellow arrows point to outer cells expressing SOX2 in a mouse morula embryo. n = 2 independent experiments. h, Quantification of SOX2 normalized fluorescence intensity in outer cells of mouse embryos shown in g (n = 120 cells 18 embryos). Data are presented as mean ± s.d. Two-tailed Student’s t-test, *P = 0.026. i, Immunofluorescence analysis of TEAD4, anti-mouse IgG secondary antibody to detect the electroporated mouse anti-aPKC primary antibody and DAPI nuclear staining in mouse morula embryos in control and Trim-Away conditions. n = 2 independent experiments. j, Quantification of TEAD4 normalized fluorescence intensity in outer cells of mouse embryos shown in i (n = 90 cells from 10 embryos). Data are presented as mean ± s.d. Two-tailed Student’s t-test, ns, not significant. Scale bars, 25 μm. More details about statistics and reproducibility are provided in Methods.

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Extended Data Fig. 11 Trim-Away experiments in cow and human embryos.

a, b, Schematic of the Trim-Away experiments. c, Immunofluorescence analysis of anti-mouse IgG secondary antibody to detect the electroporated mouse anti-aPKC primary antibody, rabbit anti-aPKC primary antibody to detect the aPKC protein, and DAPI nuclear staining in human morula embryos in control and Trim-Away conditions. d, Quantification of aPKC cortical intensity (based on rabbit anti-aPKC signal) in outer cells of human embryos shown in c (n = 54 cells from 6 embryos). Data are presented as mean ± s.d. Two-tailed Student’s t-test, **P = 0.006. e, Immunofluorescence analysis of anti-mouse IgG secondary antibody to detect the electroporated mouse anti-aPKC primary antibody, mCherry, and DAPI nuclear staining in cow morula embryos in control and Trim-Away conditions at a pulse length of 5 or 7.5 ms (n = reported in Supplementary Table 10 for each condition). f, Immunofluorescence analysis of GATA3, YAP1, anti-mouse IgG secondary antibody to detect the electroporated mouse anti-aPKC primary antibody and DAPI nuclear staining in cow morula embryos in control and Trim-Away conditions. g, h, Quantification of YAP1 (g) and GATA3 (h) normalized fluorescence intensity in outer cells of cow embryos shown in f (n = 154 cells for YAP1 from 16 embryos, and n = 207 cells for GATA3 from 20 embryos). Data are presented as mean ± s.d. Two-tailed Student’s t-test, ns, not significant. Scale bars, 50 μm. i, Proposed model for human early lineage specification. EPI, epiblast; PrE, primitive endoderm; E-CAD, E-cadherin; β-CAT, β-catenin. More details about statistics and reproducibility are provided in Methods.

Source data

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Table 1. Morphokinetic analysis in mouse, cow and human embryos. Statistical significance of the morphokinetic analysis, displaying P values and the statistical test used for each stage considered.

Supplementary Table

Supplementary Table 2. List of genes expressed with GATA3 in human morula cells. List of genes co-expressed in GATA3-high human TE cells at the blastocyst stage, which are also detectable in human morula cells. The expression profile of each gene in morula cells was compared with GATA3 by means of Pearson and Spearman correlation coefficients.

Supplementary Table

Supplementary Table 3. List of selected genes positively correlating with GATA3 expression in human morula cells. List of selected genes showing positive correlation with GATA3 expression in human morula cells. The corresponding Pearson and Spearman correlation coefficients are shown, along with significantly associated gene ontology (GO) and REACTOME (R-HSA) terms.

Supplementary Table

Supplementary Table 4. List of genes highly expressed in human morula cells with low GATA3 expression. List of genes highly expressed in the 10% of the human morula cells with the lowest levels of GATA3 expression. The expression profile of each gene in morula cells was compared with GATA3 by means of Pearson and Spearman correlation coefficients.

Supplementary Table

Supplementary Table 5. List of selected genes negatively correlating with GATA3 expression in human morula cells. List of selected genes negatively correlated with GATA3 expression in human morula cells. The corresponding Pearson and Spearman correlation coefficients are shown.

Supplementary Table

Supplementary Table 6. aPKC inhibitor concentrations tested in mouse embryos. List of concentrations of the aPKC inhibitor tested in mouse embryos, treating from the 4-cell to morula stage. We report the total number of embryos treated, the number of embryos arrested during the treatment and number of embryos that reached the morula stage. Schematic representation of the treatment is shown in Extended Data Figure 7a.

Supplementary Table

Supplementary Table 7. aPKC inhibitor concentrations tested in cow embryos. List of concentrations of the aPKC inhibitor tested in cow embryos, treating from pre-compaction to the morula stage. We report the total number of embryos treated, the number of embryos arrested during the treatment and number of embryos that reached the morula stage. Schematic representation of the treatment is shown in Extended Data Figure 7c.

Supplementary Table

Supplementary Table 8. aPKC inhibitor concentrations tested in human embryos. List of concentrations of the aPKC inhibitor tested in human embryos, treating from pre-compaction to the morula stage. We report the total number of embryos treated, the number of embryos arrested during the treatment and number of embryos that reached the morula stage. Schematic representation of the treatment is shown in Extended Data Figure 7e.

Supplementary Table

Supplementary Table 9. Trim-Away concentrations tested in mouse embryos. List of concentrations of mCherry-TRIM21 mRNA and anti-mouse aPKC primary antibody used for the Trim-Away aPKC depletion experiments in mouse embryos from the 4-cell to morula stage. We report the total number of embryos tested, the number of embryos arrested during the experiment and number of embryos that reached the morula stage. Schematic representation of the treatment is shown in Extended Data Figure 9h.

Supplementary Table

Supplementary Table 10. Trim-Away concentrations tested in cow embryos. List of concentrations of mCherry-TRIM21 mRNA and anti-mouse aPKC primary antibody used for the Trim-Away aPKC depletion experiments in cow embryos from the 4-cell to morula stage. We report the total number of embryos tested, the number of embryos arrested during the experiment and number of embryos that reached the morula stage. Schematic representation of the treatment is shown in Extended Data Figure 11b.

Supplementary Table

Supplementary Table 11. Antibodies used in this study. List of antibodies and concentrations used in this study.

Video 1

Mouse pre-implantation development. Time-lapse Embryoscope+ video of a mouse pronuclear stage zygote developing to the blastocyst stage.

Video 2

Cow pre-implantation development. Time-lapse Embryoscope video of a cow 2-cell stage embryo developing to the blastocyst stage.

Video 3

Human pre-implantation development. Time-lapse Embryoscope+ video of a human pronuclear stage zygote developing to the blastocyst stage.

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Gerri, C., McCarthy, A., Alanis-Lobato, G. et al. Initiation of a conserved trophectoderm program in human, cow and mouse embryos. Nature 587, 443–447 (2020). https://doi.org/10.1038/s41586-020-2759-x

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