Abstract
Our understanding of how human embryos develop before gastrulation, including spatial self-organization and cell type ontogeny, remains limited by available two-dimensional technological platforms1,2 that do not recapitulate the in vivo conditions3,4,5. Here we report a three-dimensional (3D) blastocyst-culture system that enables human blastocyst development up to the primitive streak anlage stage. These 3D embryos mimic developmental landmarks and 3D architectures in vivo, including the embryonic disc, amnion, basement membrane, primary and primate unique secondary yolk sac, formation of anterior–posterior polarity and primitive streak anlage. Using single-cell transcriptome profiling, we delineate ontology and regulatory networks that underlie the segregation of epiblast, primitive endoderm and trophoblast. Compared with epiblasts, the amniotic epithelium shows unique and characteristic phenotypes. After implantation, specific pathways and transcription factors trigger the differentiation of cytotrophoblasts, extravillous cytotrophoblasts and syncytiotrophoblasts. Epiblasts undergo a transition to pluripotency upon implantation, and the transcriptome of these cells is maintained until the generation of the primitive streak anlage. These developmental processes are driven by different pluripotency factors. Together, findings from our 3D-culture approach help to determine the molecular and morphogenetic developmental landscape that occurs during human embryogenesis.
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Data availability
Source Data for Extended Data Figs. 1f, k–m, 4b, f, p, 5g, h, l, m, p, q, r, 9f, 10d–h are provided with the paper. scRNA-seq data have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE136447 (scRNA-seq data). The SC3-seq data of cynomolgus monkey embryos (for Extended Data Fig. 10) are GEO accession GSE74767 (ref. 39); the scRNA-seq data of human pre-implantation embryos (for Extended Data Fig. 3d, e) are with GEO accession GSE66507 (ref. 16) and GSE36552 (ref. 17) and ArrayExpress accession E-MTAB-3929 (ref. 18).
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Acknowledgements
This work was supported by the National Key Research and Development Program of China (2018YFA0108500), the Yunnan Key R&D Program (2018ZF007-02 and 2018FA008), the National Natural Science Foundation of China (31760268 and 81660266), Chinese Postdoctoral Science Foundation (2017M623317XB), Yunnan Provincial Innovation Team (2018HC003 and 2017HC009) and Yunnan Innovation Talents of Science and Technology (2013HA027). We thank Brandi J. Mattson for editing assistance.
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T.L. and W.J. initiated the project. T.L. designed the experiments, organized and supervised the entire project, and wrote the manuscript. T.L. supervised L.X. and Y.Y. to perform data analysis. L.X., Y.Y., Y.M., Y.L., Z.A., D.W., K.D., J. H. and Y.B. performed embryo culture, single-cell isolation and data collection. Y. Z., Y.Y., Z.Z., J.G., S.R. Z.S., X.D. and Y.N. analysed RNA-seq data.
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Extended data figures and tables
Extended Data Fig. 1 Establishing a 3D blastocyst-culture system.
This figure is related to Fig. 1. a–f, Modification of human embryo culture medium. To test human embryo development, human embryos at 5–6 d.p.f. were cultured on low attachment plate without any 3D extracellular matrix up to 14 d.p.f. By sequentially culturing in IVC1 (6–8 d.p.f.) and IVC2 (8–14 d.p.f.) media, 6.67% (2 out of 30 embryos) of embryos could survive until 14 d.p.f. a, Schematics of improving culture medium. Sodium lactate, sodium pyruvate and ROCK inhibitor (Y27632) were added to the IVC1 and IVC2 media, referred to as mIVC1 (6–8 d.p.f.) and mIVC2 (8–14 d.p.f.), respectively. Culture in mIVC1 and mIVC2 enabled 25% (4 out of 16 embryos) of human blastocysts to develop up to 14 d.p.f. (c). b, Representative developing embryos based on morphological observation. n = 16 independent embryos from three independent experiments. c, Representative embryos with abnormal development. Abnormal embryos displayed growth arrest or had obvious cell death or fragmentations. n = 13 independent embryos from three independent experiments. d, Representative staining of abnormal embryos with CK7, GATA6 and OCT4 at 14 d.p.f. In all six examined embryos, consistent data were obtained. e, Staining of developing embryos (2 out of 3 embryos) with CK7, GATA6 and OCT4 at 14 d.p.f. f, Quantification of developmental rates of human embryos cultured in control medium (IVC1 and IVC2) and modified medium (mIVC1 and mVIC2). n = 30 and 16 biologically independent embryos, respectively. Developmental rates of human embryos at 11 and 13 d.p.f. were based on the two following requirements by morphology: obvious expansion over culture and absence of obvious cell death mass and fragmented phenotypes. At 14 d.p.f., we determined the embryo development ratio by staining CK7 (TrB), GATA6 (PrE,) and OCT4 (EPI). g–q, Representative human embryo development after culture under four different 3D conditions over development. The limited number of embryos only enabled us to compare embryo development under four conditions. As the implantation time window is 8–10 d.p.f., we embedded embryos with Matrigel at 9 d.p.f. Embryo development was verified on basis of morphological observation and staining of specific markers for OCT4, GATA6 and CK7. g–j, Top, schematics of in vitro 3D culture of human blastocysts under different culture conditions. Middle, representative images of human embryos during development. Bottom, representative stained images of cultured human embryos under different 3D conditions at 14 d.p.f. g, Human embryos at 5–6 d.p.f. were cultured on low attachment plate without Matrigel (W/O Matr) up to 14 d.p.f. The outermost TrBs showed signs of apoptosis (blue arrowheads), as determined by morphological observations and CK7 staining, which suggests that the condition was unsuitable for TrB development and survival required for attachment. In total, 2 of 20 embryos (three independent experiments) survived up to 14 d.p.f. and displayed normal EPI, PrE and TrB development. h, Human embryos were embedded in 25% Matrigel at 9 d.p.f. for continuous culture up to 14 d.p.f. n = 25 embryos from three independent experiments. The invasion and outgrowth of TrBs were observed and embryos became flat, which suggests a higher concentration of Matrigel is advantageous to differentiation and development of TrBs, as confirmed by CK7 staining. i, Human embryos were embedded in 10% Matrigel on the new well, which was pre-coated with 100% Matrigel (30 μl) (Matr+10%Matr), at 9 d.p.f. for continuous culture up to 14 d.p.f. Although embryos displayed considerable expansion over culture, staining with lineage markers showed that EPIs in most embryos were lost over development. Only 1 of 33 embryos from three independent experiments grew to 14 d.p.f and was accompanied by EPI, PrE and TrB development. The negative outcome may indicate that high concentrations of Matrigel can inhibit EPI development. In h and i, red arrowheads indicate TrBs invading into Matrigel. j, Human embryos were embedded in 10% Matrigel at 9 d.p.f. for continuous culture up to 14 d.p.f. Compared with the 25% Matrigel and Matr+10% Matr conditions, human embryos in 10% Matrigel increased in size at the thickness (Z axis) and showed better 3D spatial structures. In total, 4 of 17 embryos from three independent experiments grew to 14 d.p.f. with normal development. k, Quantification of the mean diameter of human embryos cultured under different 3D conditions during development by analysis of 5–14 embryos from three independent experiments. Data are mean ± s.d. l, Quantification of human developmental embryos during culture in different 3D conditions. Data were based on morphological observations only. Human developing embryos met the two following requirements: obvious expansion over culture; absence of obvious cell death mass or fragmented phenotypes. m, Quantification of developing embryos in different 3D conditions. n = 20 (W/O Matr), 25 (25% Matr), 33 (Matr+10% Matr) and 17 (10% Matr) blastocysts. Developing embryos met the following requirements: obvious expansion over culture; absence of obvious cell death mass or fragmented phenotypes; and development of EPIs, PrEs and TrBs and formation of amnion identified by OCT4, GATA6 and CK7 staining. Although embryos in the 25% Matr and Matr+10% Matr culture conditions have normal morphologies, some embryos lacked OCT4+ EPIs or GATA6+ PrEs and gave rise to a high proportion of TrBs, which suggest that high concentrations of Matrigel could inhibit EPI development and promote TrB proliferation. n–q, Three-dimensional construction of human embryos cultured in 10% Matrigel (3 out of 3 embryos from three independent experiments). n, A representative 3D reconstruction of EPIs. Inset shows OCT4 staining of one section from the same embryo. o, A representative 3D reconstruction of SYS and amnion including an embryonic disc and an amniotic cavity (see Supplementary Video 1). p, Three-dimensional reconstruction of TrBs (see Supplementary Video 2). q, Three-dimensional magnification of TrBs close to the amnion side. Scale bars, 100 μm (phase-contrast) or 50 μm (staining).
Extended Data Fig. 2 Representative z-series of an 8- and a 10-d.p.f. embryo.
This figure is related to Fig. 1f, g. a, Series of confocal z-sections of the 8-d.p.f. embryo stained for OCT4 (green), GATA6 (grey) and CK7 (red). The PYS cavity (white arrows) was surrounded by few GATA6+ PrEs. Similar phenotypes were observed in 3 out of 4 embryos from three independent experiments. b, Series of confocal z-sections of the whole embryo stained for OCT4 (green), GATA6 (grey) and CK7 (red) showing that the PYS cavity in the 10-d.p.f. embryo becomes more distinct. Similar phenotypes were observed in 3 out of 3 embryos from three independent experiments. The yolk sac cavity (red arrows) was surrounded by GATA6+ PrEs. Scale bars, 50 μm.
Extended Data Fig. 3 Lineage delineation by transcriptome.
This figure is related to Fig. 2. a–c, Quality control of single-cell RNA-sequencing data. a, Sequence quality was evaluated by FastQC. b, Total reads, mapped reads and mapping ratios of 557 single cells. c, Saturation curve of sequencing. d, e, Integrated analysis of embryonic single-cell data from different source. We used the analytical strategies developed previously15 to analyse single-cell RNA-seq data (255 single cells) from 6–9-d.p.f. embryos in the study and single-cell RNA-seq data (216 single cells) from three previous reports16,17,18 (later blastocysts or 6–7-d.p.f. blastocysts). The three datasets have previously been analysed15. d, PCA based on 12 lineage markers (NANOG, SOX2, KLF17 and TDGF1 for EPI; PDGFRA, GATA6, GATA4 and SOX17 for PrE; GATA3, GATA2, KRT18 and TEAD3 for TrB) showed clear separation between EPI, TrB or PrE could be attained for nearly all samples including our single cells from 6–9-d.p.f. blastocysts, which indicates that lineage delamination occurs at 6 d.p.f. The result is consistent with previous findings15. e, t-SNE analyses using 4,333 viable genes across all samples. The samples from previous studies were defined into four types: intermediate cells, EPI, PrE and TrB15. The combined Seurat revealed that most of cells independent of cell resource mixed well. Although most of samples were clustered into EPI, PrE or TrB, similar to the results using 12 lineage genes (d), some cells from 6-d.p.f. embryos remained in an intermediate state with overlapping expression of POU5F1, GATA6, PDGFRA and GATA3. Compared with cells from 6-d.p.f. embryos, cells from 7–9-d.p.f. embryos displayed a clearer separation. These data showed that cell fates of 7–9-d.p.f. embryos became more fixed. f–j, Lineage delineation by transcriptome. Analysis of genes corresponding to EPI, PrE and TrB from 7–9-d.p.f. embryos to understand the regulators involved in the segregation process. f, Heat map of lineage-specific genes of EPI, TrB and PrE from 7–9-d.p.f. embryos (Supplementary Table 1). Their representative transcriptional factors and KEGG pathways are shown, respectively. GO terms and KEGG pathways showed EPI-specific genes associated with signalling pathways regulating stem-cell pluripotency including PI3K–AKT, p53, RAP1 and MAPK. PrE-expressing genes related to TGFβ, PPAR and Ras signalling pathways. TrB-specific genes contributed to Hippo, HIF, PPAR and thyroid receptor signalling pathways. Notably, the PI3K–AKT signalling pathway was enriched in EPIs, PrEs and TrBs. To explain the difference, we examined gene expressions of the PI3K–AKT signalling pathway components in three cell types and found that cell types specifically expressed different genes of PI3K–AKT pathway (Supplementary Table 1.4). g, WGCNA dendrogram indicating different gene modules in all single cell samples from 7–9-d.p.f embryos. Three major branches corresponded to PrE (brown module), TrB (blue module) and EPI (turquoise module). h–j, Hub-gene-network analysis of transcriptional factors specific for PrE (brown module), TrB (blue module) and EPIs (turquoise module). The size of dots represents hubness. h, Hub-gene network of the EPI-specific gene module. In addition to well-known transcription factors (NANOG, PRDM14, SOX2, OCT4 (also known as POU5F1), ZSCAN10 and KLF17), new candidate factors may associate with EPI differentiation, such as VENTX, BCL11A, PBX1 and ARGFX. i, Hub-gene network of the PrE-specific gene module. High correlations of some transcription factors with PrE differentiation included GATA4, SOX17, GATA6 and HNF1B. j, Hub-gene network of the TrB-specific gene module. TrB-specific transcription factors, such as MYBL2, TFAP2A, DLX6 and GCM1. k, Comparison of lineage-specific total genes overlapping between a previous study18 and this study. In the previous study18, by analysing 5–7-d.p.f. embryos and combining the lineage-specific results, 439, 820 and 222 genes—which significantly maintained TrB-, EPI- and PrE-specific genes, respectively—were identified. Comparison analysis showed that although we identified more EPI-, PrE- and TrB-specific genes by our resource data, core lineage transcription factors (NANOG, POU5F1 and SOX2 for EPI; GATA6, SOX17 and GATA4 for PrE; GATA2 and GATA3 for TrB) are maintained cross different samples. The difference in gene expressions may be contributed by different development stages of embryos. Difference of gene expressions, including transcription factors, is summarized in Supplementary Table 1.6.
Extended Data Fig. 4 AME separation from EPI.
a, E-cadherin expression in amnion from a 12-d.p.f. embryo. Right panels show magnified squares (3 out of 3 embryos). White and red lines indicate nuclei and apical orientation, respectively. b, Quantification of expression of E-cadherin (AME = 33 cells, EPI = 44 cells) and OCT4 (AME = 22 cells, EPI = 44 cells) in columnar EPIs and squamous AME. Data are mean ± s.d from 3 embryos. ***P < 0.001, two-sided t-test. c, d, Representative staining of laminin at 12 and 14 d.p.f. (3 out of 3 embryos). Right panels show magnified squares from d. e, f, Representative staining and quantification of NANOG expression (AME = 33 cells, EPI = 30 cells, and SOX2 expression (AME = 34 cells, EPI = 30 cells) in 14-d.p.f. EPIs and AME (3 out of 3 embryos). Data are mean ± s.d. **P < 0.01, ***P < 0.001, two-tailed t-test. Right panels show magnified squares from e. g, h, Representative staining and quantification of laminin and E-cadherin in 6-d.p.f. embryos (3 out of 3 embryos). i, j, Representative staining and quantification of laminin and E-cadherin in 8-d.p.f. embryos (3 embryos). White long lines in h and j show positions used to plot intensity profiles (right). k, l, Representative staining of laminin and E-cadherin in 10-d.p.f. embryos (3 out of 3 embryos). Together, we conclude that AME separation from EPIs correlates with asymmetrical distributions of E-cadherin and laminin (a–l). m, Representative staining of EZRIN in 14-d.p.f amnion (2 out of 2 embryos). White arrows indicate EZRIN expression in apical surface in EPIs and AME. n, Representative staining of WGA in 14-d.p.f. embryos (2 out of 2 embryos). Red arrowheads indicate WGA expression in extra-embryonic cells. o, t-SNE analyses revealed three clusters of 12- and 14-d.p.f. embryos—AME, intermediate state cells and EPIs. p, Compared to EPIs, the violin plots show AME significantly downregulated pluripotency genes and upregulated genes that specifically expressed in the AME of 12–17-d.p.f monkey embryos or self-organized amnion from human pluripotent stem cells. All violins have the same maximum width; black dot denotes the mean. o, p, AME, n = 13 cells; intermediate state cells, n = 26 cells; EPI, 53 cells. q–t, Gene expression profiles of AME and EPI in the 12- and 14-d.p.f. embryos (Supplementary Table 2). AME, n = 12 cells (one single cell with high NANOG expression was not included); EPI, 53 cells. q, Volcano map of differentially expressed genes (DEGs) between AME and EPI in the 12- and 14-d.p.f. embryos. DEGs were defined with uncorrected P < 0.01 (two-sided likelihood ratio tests) and log2-transformed fold change > 1 or <−1, and median FPKM > 1 in one group. r, Heat map of DEGs between the AME and EPI. Right panel presents representative transcription factors. s, Compared to the EPIs, GO terms of upregulated genes in the AME. t, Compared to EPI, KEGG pathways of genes enriched in the AME. u, hCG was expressed in the AMEs, but not in the EPIs (2 out of 2 embryos). White arrows indicate the AMEs have squamous nuclear shape, expressed hCG, but downregulated the pluripotent gene, OCT4. Scale bars, 15 μm (a, l), 20 μm (c, e), 25 μm (d, g, h, k, m, n, u) or 50 μm (i).
Extended Data Fig. 5 Human embryos at 14 d.p.f. initiate anterior–posterior polarity and generation of PSA in 3D-culture conditions.
This figure relates to Fig. 3. a, OCT4, GATA6 and TBXT T staining of sections from a 14-d.p.f. embryo (2 out of 2 embryos), showing that T+ cells originated from the EPI compartment close to the AME compartment boundary at 14 d.p.f. b, Representative OCT4, OTX2 and SOX2 staining. Arrow indicates an OTX2+ cell. n = 4 of 5 embryos from three independent experiments displayed consistent data. c–e, LEFTY1 (c, d; n = 3 out of 4 embryos from two independent experiments) and OTX2 (e; n = 3 out of 5 embryos from two independent experiments) immunofluorescence was only detected on the side of 14-d.p.f. embryonic disc. Arrows indicate LEFTY1+ or OTX2+ cells. Right image in d is magnification of the square in the left image. The exclusive expression of NANOG and SOX2 was not observed in EPIs (e). f, Staining of OCT4, HESX1 and GATA6 in the 12-d.p.f. embryos (2 out of 2 embryos). g, The violin plots show dynamic expression of HESX1 during EPI development. All violin plots have the same maximum width, black dot denotes the mean. h, Correlation of HESX1 and T expression of 14-d.p.f. EPIs, as determined by scRNA-seq. Each plot represents a single cell. i, Volcano plots show DEGs in HESX1+T− (10 single cells) and HESX1−T+ (12 single cells) EPIs by scRNA-seq. DEGs were defined as those with uncorrected P < 0.01 (likelihood ratio test) and fold change of >2 or <−2, and median FPKM > 1 in one group. j, k, Staining of OCT4, FLK1 and T at 14 d.p.f. (2 out of 2 embryos from two independent experiments). Red arrows denote migrating T+ cells; white arrows denote FLK1+ extra-embryonic mesenchyme. l, The violin plots show expression dynamics of primitive streak genes over pluripotent-stem-cell development. All violins have the same maximum width, black dot denotes the mean. In total, 136 cells were included (Extended Data Fig. 9e): ICM, n = 49 cells; pre-EPI, n = 23 cells; post-EPI, n = 48 cells; PSA-EPI, n = 16 cells. *P < 0.05, ** P < 0.01, two-sided Wilcoxon rank-sum test. m–o, Absence of specific neural gene expression indicates 14-d.p.f embryos do not generate the initial nervous system, which meets the internationally recognized ethical limit for human embryo culture. m, Violin plots of dynamic expressions of neural-specific genes in EPIs over embryo culture. All violins have the same maximum width, black dot denote the mean. In total, 136 cells were included: ICM, n = 49 cells; pre-EPI, n = 23 cells; post-EPI, n = 48 cells; PSA-EPI, n = 16 cells. n, o, Representative staining of PAX6, OCT4, SOX1 and FOXA2 in human 14-d.p.f embryos (3/3 embryos). p–r, Development and cell proliferation of human embryos cultured in the Matr+10% Matr condition. Quantified data at each stage were based on five embryos from three independent experiments. Data are presented as mean ± s.d. p, Quantification of the dynamics of total cell number per embryo during culture. *P < 0.05, **P < 0.01, two-sided Student’s t-test. q, Dynamics of OCT4+ EPIs and GATA6+ PrEs per embryo over culture. r, Dynamics of CK7+ TrBs per embryo over culture. EPIs and PrEs maintained gradual proliferation at 8–10 d.p.f., after which their proliferation speeds accelerated. However, TrBs always maintained a rapid proliferation rate, which may be for establishing cell connections with the maternal environment. Scale bars, 25 μm.
Extended Data Fig. 6 Z-series of a 14-d.p.f. embryo with a PSA.
This figure is related to Fig. 3. Series of confocal z-sections of the embryo stained for T (green), OCT4 (red) and N-cadherin (grey), showing formation of PSA . The thickness of every section was 12 μm. Numbers on the left indicate the number of sections. Red and white arrows indicate T+ and N-cadherin+ cells, respectively. Some T+ cells (red arrows) in the 13# section located in the amnion epithelium, consistent with the distribution of some T+ cells in the monkey amnion epithelium23. T+ cells (red arrows) in the 16# section disrupted the N-cadherin-forming basement-membrane barriers between epiblast and hypoblast and focally migrated from the embryonic disc to generate the PSA. Similar data were observed in 3 out of 3 embryos from two experiments. Scale bars, 50 μm.
Extended Data Fig. 7 Z-series of a 14-d.p.f. embryo with a cell emigration region.
This figure is related to Fig. 3. Series of confocal z-sections of the embryo stained for GATA6 (green), OCT4 (red) and T (grey). The thickness of every section was 12 μm. Numbers on the left indicate the number of sections. Red and white arrows denote T+GATA6+OCT4− cells and T+GATA6+OCT4+ cells, respectively. In the 7# and 8# sections, some migrating T+ EPIs invaded the space near the visceral endoderm and co-expressed GATA6 (white arrows). Similar data were observed in 4 out of 5 embryos from three experiments. Scale bars, 50 μm.
Extended Data Fig. 8 Development of TrB lineage.
a–i, Representative confocal staining of human embryo section. a, Staining of CK7 and F-actin. n = 5 independent embryos from three independent experiments. b, c, Cells near the EPI–PrE bilayer had a single nucleus that expressed TEAD4 and E-cadherin. E-cadherin displayed symmetrical distributions in the embryos (3 out of 3 embryos for each staining from two experiments). d, e, Multinucleated cells (d1, e1 and e2) and cells with a single nucleus (d2) (3 out of 3 embryos each staining from two experiments). Dashed lines in d and e outline the region of interest in cells. f, g, Representative staining of CK7 and HLA-G (3 out of 3 embryos) in 12-d.p.f. (f) and 14-d.p.f. (g) human embryo. h, i, Representative staining of TEAD4 and HLA-G (h) or TEAD4 and hCG (i) in 14-d.p.f human embryo (3 out of 3 embryos). In f, the inset is magnification of the square. In g–i, right panel is magnification of the square. In i, the inset is magnification of the region indicated by a red arrow. j–l, t-SNE plot of 352 TrBs. Cells (dots) coloured according to the original inferred lineage identity (k) and embryonic stage (l). m, Lineage segregation path constructed by Monocle based on developmental time (left) and cell types defined with selected markers (right). n, Heat map of polypeptide hormone genes expressed in the six types of trophoblasts during culture. o, Heat map indicates expression of genes specific for each cell type. Representative genes and key GO enrichments shown. GO terms and KEGG pathways of genes specific for different subtypes of TrBs (multiple test corrected P < 0.01, hypergeometric tests) in the six types of TrBs from pre-implantation stage embryos to 14-d.p.f embryos are shown in Supplementary Table 4. Pre-CTB-expressing genes related to cell metabolism, biosynthesis and cell differentiation, were in accord with the characteristics of trophoblast stem cells. High expressions of NF-κB, as well as canonical and non-canonical Wnt signalling pathway genes, indicate potential functions on CTB development. STB-specific genes indicated hormone secretion, whereas early-STB-expressing genes associated with cell differentiation and migration, dependent on several signalling pathways. EVT-specific genes contributed to regulating the immune system and angiogenesis. Scale bars, 50 μm (a–c, e–i) or 20 μm (b).
Extended Data Fig. 9 Epiblast development during embryo culture in the 3D condition.
This figure relates to Fig. 4. a–d, Dynamic expressions of pluripotent genes over human embryo development in the 3D condition. a, The dynamic expressions of OCT4, TFCP2L1 and KLF4 during human embryo development (3 out of 3 embryos). b, The dynamic expressions of OCT4, SOX17 and KLF17 during human embryo development (3 out of 3 embryos). Loss of TFCP2L1, KLF4 and KLF17 at 10 d.p.f. (implantation stage) indicate the pluripotent state transition of epiblasts. c, Expression of NANOG and PRDM14 at 10-d.p.f. human embryos (2 out of 2 embryos). d, Dynamic expressions of OCT4, CD24 (a primed pluripotency gene) and KLF17 during human embryo development (2 out of 2 embryos). e, t-SNE of pluripotent stem cells. To exclude cells from the AME and intermediate state in the epiblast cluster, we excluded the NANOG-negative cells and maintained 136 cells with high expression of NANOG. f, The violin plots show the dynamics of naive, primed and common pluripotency genes in pre-gastrulation embryos. All violins have the same maximum width, black dot denotes the mean. AME and intermediate state cells were excluded in the synthesis. *P < 0.05, **P < 0.01, ***P < 0.001, two-sided Wilcoxon rank-sum test. g–j, PluriNetWork analysis of EPIs from ICM (g), pre-EPI (h), post-EPI (i) and PSA-EPI (j) stages revealed that key pluripotency regulators dominated the networks. k, DEGs during EPI development. GO terms and representative genes in DEGs in the pairwise comparisons are indicated. l–n, Scatter-plot comparison of the gene-expression levels between ICM and pre-EPI (l), pre-EPI and post-EPI (m), and post-EPI and PSA-EPI (n). Key genes are annotated (Supplementary Table 6). Red denotes upregulated; green denotes downregulated; >twofold difference, uncorrected P < 0.01 (likelihood ratio test) and median FPKM > 1 in one group. Scale bars, 50 μm (a, b), 100 μm (c) or 25 μm (d).
Extended Data Fig. 10 Comparison of monkey and human EPI development.
a, PCA of the EPI lineage as determined by the expressed genes among all groups of EPIs during development in human (hu) (circles) and cynomolgus monkey (cy) (squares). In total, 12,475 out of 16,487 annotated gene expressed among human and monkey cells (human, 222 cells; monkey 213 cells) were used. b, Heat map of 966 genes that highly contributed to PC1 (>2 s.d. of PC1). c, Heat map of 1,152 genes with significant scores for PC2 and PC3 loading (radius of PC2 and PC3 > 3 s.d.) during monkey and human EPI development. None of the 1,151 genes overlapped with 996 genes with significant scores for PC1 loading. d, The violin plots of pluripotency genes over EPI pluripotency transition in monkey embryos. Monkey scRNA-seq data were from a published database39. e, The violin plots of pluripotency genes during the EPI pluripotency transition in human embryos. We observed stable expressions of STAT3 and TBX3 with a trend of gradually increasing of UTF1, NR0B1, LIFR and SOX15 during human EPI pluripotent state transition. f, The violin plots showed dynamics of BMP signalling pathway gene expression in EPI pluripotency transition of human and monkey embryos. Monkey scRNA-seq data were obtained from a published database8. g, The violin plots showed dynamics of FGF signalling pathway gene expression in EPI pluripotency transition of human and monkey embryos. h, The violin plots showed dynamics of Notch signalling pathway gene expression in EPI pluripotency transition of human embryos. In d–h, all violins have the same maximum width, black dot denotes the mean. In e–h, AME and intermediate state cells were excluded in the synthesis and only included 136 single cells. Monkey scRNA-seq data were obtained from a published database8. See also Supplementary Table 7.
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Supplementary Information
This file contains full legends for Supplementary Tables 1-8.
41586_2019_1875_MOESM4_ESM.xlsx
Supplemental Table 2 | Differentially expressed genes between amnion epithelium and epiblast in d.p.f 12 and 14 embryos.
Supplemental Video 1 | 3D-reconstruction of amnion and secondary yolk sac in d.p.f 14 embryo
The embryo was viewed using two-photon microscopy and 3D-reconstruction of 100 μm z-axis. 3 of 3 embryos from three independent experiments displayed similar results. Staining: Green, GATA6; Red, OCT4; Blue, DAPI.
Supplemental Video 2 | 3D reconstruction of TrBs with 200 μm z-axis
TrBs exhibited spatial asymmetric development predominantly located in the amnion side. Gray, CK7 staining. 3 of 3 embryos from three independent experiments displayed similar results.
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Xiang, L., Yin, Y., Zheng, Y. et al. A developmental landscape of 3D-cultured human pre-gastrulation embryos. Nature 577, 537–542 (2020). https://doi.org/10.1038/s41586-019-1875-y
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DOI: https://doi.org/10.1038/s41586-019-1875-y
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