The epiblast (EPI) is the origin of all somatic and germ cells in mammals, and of pluripotent stem cells in vitro. To explore the ontogeny of human and primate pluripotency, here we perform comprehensive single-cell RNA sequencing for pre- and post-implantation EPI development in cynomolgus monkeys (Macaca fascicularis). We show that after specification in the blastocysts, EPI from cynomolgus monkeys (cyEPI) undergoes major transcriptome changes on implantation. Thereafter, while generating gastrulating cells, cyEPI stably maintains its transcriptome over a week, retains a unique set of pluripotency genes and acquires properties for ‘neuron differentiation’. Human and monkey pluripotent stem cells show the highest similarity to post-implantation late cyEPI, which, despite co-existing with gastrulating cells, bears characteristics of pre-gastrulating mouse EPI and epiblast-like cells in vitro. These findings not only reveal the divergence and coherence of EPI development, but also identify a developmental coordinate of the spectrum of pluripotency among key species, providing a basis for better regulation of human pluripotency in vitro.
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This work was supported in part by a Grant-in-Aid from MEXT and by JST-ERATO. We thank Y. Nagai, R. Kabata, N. Konishi, Y. Sakaguchi, M. Kasawaki, T. Sato, M. Kabata, J. Matsushita, and M. Matsutani for their technical assistance. We are grateful to the Center for Anatomical, Pathological and Forensic Medical Researches, Kyoto University, for histology, to H. Suemori for CMK6/9, to M. Ema for encouragement, and to the animal care staff at Research Center for Animal Life Science, Shiga University of Medical Science for assistance.
The authors declare no competing financial interests.
Reviewer Information Nature thanks T. Li, K. Niakan and H. Niwa for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Figure 1 Summary of monkey pre-implantation development.
a, A phylogenic tree of primates55. Cynomolgus, rhesus, and Japanese monkeys are members of macaques of Ceropithecoidae, which are classified as old-world monkeys. b, Summary of super-ovulation and oocyte collection for this study. c, Summary of monkey pre-implantation development. No insemination, embryos without cleavage; degenerated, degenerated embryos; arrested, embryos that failed to form a blastocoel; blastocyst formation, embryos with blastocoel formation by E8. d, Developmental progression of monkey pre-implantation embryos. Embryos with more than 16 cells without blastocoel cavities, those with blastocoel cavities, and those with cells outside the zona pellucida were classified as morula, blastocysts, and hatched, respectively. e, The cell numbers (counted by DAPI) of pre-implantation embryos from E6 to E9. The cells with degenerated nuclei were excluded. The colour coding is as indicated. f, Scatter plots of the cell numbers that were positive for each marker (y axis, the colour coding indicated) against the whole-cell numbers (x axis). Each plot indicates the numbers in one embryo. The orange and red bars indicate the range of embryonic days and developmental stages, respectively. g, Expression of CDX2/OCT4/GATA4 from E6 to E9 (embryos n = 7, 6, 12, 12, respectively). The numbers of cells positive for each marker are indicated. h, Expression of GATA6/OCT4 from E6 to E9 (embryos n = 4, 13, 11, 3, respectively). The numbers of cells positive for each marker are indicated. i, Expression of TFAP2C/OCT4/GATA6 at E8 (embryos n = 5). ICM is magnified (right). Arrowheads indicate hypoblast. j, Summary of the expression of key markers in monkey, human and mouse pre-implantation embryos. Blastocysts of the three species show grossly similar morphology, but notably, monkey hypoblast extends parietally to cover mural trophectoderm. OCT4 expression appears to be equal in EPI and hypoblast of mouse blastocysts56, whereas OCT4 is expressed at a higher level in EPI than in hypoblast and trophectoderm in human and monkey blastocysts13,42. NANOG and GATA4 exhibit a similar expression pattern among the three species56,57,58. CDX2 shows a similar expression in human and monkeys, but an earlier expression in morula in mice13,56. GATA6 exhibits the most variable expression pattern among the three species: it is expressed only in hypoblast in humans and mice57,59, but is uniformly expressed in hypoblast and trophectoderm in monkeys. Scale bars, 100 μm.
Extended Data Figure 2 Monkey early post-implantation development.
a, Ultrasound diagnosis of the recipient uterus for the implantation of transplanted embryos at E14 and E16. Dashed circles indicate the uterus and arrowheads indicate the chorionic cavity. Scale bars, 10 mm. b, Implantation (white arrowheads) and pseudo-implantation (black) sites on the recipient endometrium. The image at right is a higher magnification of the area boxed on the left. The implantation site was identified by maternal blood in the trophoblastic lacunae. The pseudo-implantation site was a reacted endometrium to the implantation on the overlying endometrium. Scale bar, 2 mm. c, Scheme of monkey early post-implantation development. AM, amnion; CS, connective stalk; CT, cytotrophoblast; EXCM, exocoelomic membrane; EXMC, extra-embryonic mesenchyme; Gast, gastrulating cells; SYS, secondary yolk sac; VE, visceral endoderm; YE, yolk-sac endoderm; TE, trophectoderm. d, Lower magnification images of Fig. 2b showing whole implantation sites at E14 (left) and E16 (right). PYS/CC, primary yolk sac/chorionic cavity. Scale bars, 500 μm (left) and 1.0 mm (right). e, Expression of OCT4/NANOG/GATA4 and OCT4/T/GATA6 in post-implantation embryos at E16 (embryos n = 2). Gastrulating cells positive for T and OCT4 migrated along visceral endoderm. Some cells (yellow arrowhead) showed ingression into visceral endoderm (white arrowhead). Scale bars, 100 μm. f, Expression of OCT4/GATA4 (left) and OCT4/GATA6 (right) in embryos at E14 (embryos n = 2). Arrowheads indicate extra-embryonic mesenchyme. Scale bars, 100 μm.
Extended Data Figure 3 Expression of key markers in single-cell cDNAs generated from monkey pre- and post-implantation embryos.
a, Summary of the SC3-seq samples. The numbers of embryos analysed (all of the embryos exhibited normal morphology), of synthesized cDNAs with appropriate quality, and of the cells analysed by SC3-seq are listed. b, c, qPCR analysis of the expression of key markers in single-cell cDNAs generated from pre-implanation (E6, E7, E8, E9) (b) and post-implantation (E13, E14, E16, E17) (c) embryos. The ∆Ct values from the average Ct values of GAPDH and PPIA are shown as heat maps and are used for clustering. The identities of the embryos and the samples used for the SC3-seq analyses (annotations are based on Fig. 3a) are indicated. The colour coding is as indicated.
Extended Data Figure 4 Comparison of the performance of the SC3-seq with that of other RNA-seq and single-cell RNA-seq methods.
a, Distributions of the expression levels of all annotated genes by the SC3-seq (diluted total RNA19, single cells; in this study) (left) and other methods24,49 (right) represented by violin plots. Medians are shown by white circles. For the SC3-seq, the transcript-level (log2(RPM+1)) distributions of 100 ng, 100 pg, and 10 pg of total RNA or single cells from 2i+L mESCs (cultured in N2B27 medium supplemented with a cytokine leukaemia inhibitory factor (LIF) and two kinase inhibitors (PD0325901 and CHIR99021))32 as starting materials are shown. For refs 24 and 49, the transcript levels are shown as log2(FPKM+0.1). Transcripts from 20 cells of 2i+L mESCs are amplified in ref. 24, and in ref. 49, 500 ng of polyA RNA from serum/LIF mESC/iPSCs are used for a standard RNA-seq procedure. b, Distributions of the expression levels of genes expressed at significant levels (log2(RPM+1) > 4 in at least one 100 ng RNA sample) among the same sample set in a. c, Comparisons of the distributions of the expression levels among corresponding samples in different data sets22,23,24. The geneset used in the first row consisted of all annotated genes. In the second, third, and fourth rows, the genesets used were genes expressed at significant levels as in b in at least one cell in the monkey pre-EPI group, in marmoset ICM samples, and in human EPI samples22 respectively. d, Scatter-plot analysis of the correlation between the expression level and the transcript length detected by the SC3-seq (2iLESC_MS68T82)19, (GSM1119616)49, and (ERR637931)24. Note that the method in ref. 24 tends to yield lower estimations of the levels of longer transcripts compared to the SC3-seq and standard RNA-seq.
Extended Data Figure 5 Characterization of extra-embryonic mesenchyme.
a, PCA of all cells by all expressed genes (390 cells, 18,353 genes). The colour coding is as indicated. b, Heat map of correlation coefficients among cells during monkey development. The values were calculated using the averaged expression levels of 6,167 DEGs in each cell type (the genes exhibiting more than fourfold changes among the groups (FDR < 0.01), and the mean of the expression level of at least one group was >(log2(RPM+1) = 4). c, PCA of cells from pre-implantation embryos by all expressed genes among these groups (193 cells, 15,187 genes). d, Scatter-plot comparison of the expression of key genes with that of POU5F1 in post-implantation cells. e, Expression of COL6A1/FOXA1 in embryos at E14 and E16 (embryos n = 2, 2, respectively). Scale bars, 100 μm. f, Venn diagram showing the overlap among the genes expressed at higher levels in EXMC (>4-fold) compared to postE-EPI, Gast1, or post-implantation parietal trophectoderm at E13 and E14. g, Heat map of the expression of the 228 genes identified, as in f, among monkey pre- and post-implantation cell types. h, Enrichment of Gene Ontology terms in the 228 genes identified, as in f. i, Venn diagram showing the overlap between the genes expressed at higher levels in hypoblast (>4-fold) compared to pre-EPI or preL-TE. j, Heat map of the expression of the 134 genes identified, as in i, among monkey pre- and post-implantation cell types. k, Pearson’s correlation coefficients shown by violin plots between all possible pairs of single-cell cDNAs of all cells, postE-EPI, Gast1, and extra-embryonic mesenchyme at E13 and E14. The numbers of cell analysed are indicated on the right. l, Schemes for the differentiation of primitive endoderm into visceral or parietal endoderm (VE or PE) in mice (top) or for the differentiation of hypoblast into VE/YE or extra-embryonic mesenchyme in cynomolgus monkeys (bottom).
Extended Data Figure 6 DEGs during the cyEPI development.
a, Expression of selected genes during EPI development (black bars, median values). b, DEGs during the cyEPI development. Orange and blue bars indicate the numbers of up- and downregulated genes, respectively, in the pair-wise comparisons indicated. c, Enrichment of Gene Ontology terms and representative genes (all genes are shown in Supplementary Table 2) in DEGs in the pair-wise comparisons indicated. d, Scatter-plot comparison of the averaged gene-expression levels between ICM and pre-EPI (top), pre-EPI and postE-EPI (middle), and postE-EPI and postL-EPI (bottom). Orange, upregulated; blue, downregulated (>4-fold difference (flanking diagonal lines), mean log2(RPM+1) > 4 in one cell type, FDR <0.01). Key genes are annotated and the numbers of DEGs are indicated. The correlation coefficient is indicated above the scatter plot. e, Expression of SOX11/OCT4 in embryos at E14 (top) and E16 (bottom) (embryos n = 2, 2, respectively). Scale bars, 100 μm.
Extended Data Figure 7 SC3-seq analyses of cells in mouse pre- and post-implantation embryos.
a, Summary of the SC3-seq samples of mouse embryonic cells. The numbers of synthesized cDNAs of appropriate quality and of the cells analysed by SC3-seq are listed. b, qPCR analysis of the expression of key markers in single-cell cDNAs from mouse E5.5 (pre-gastrulation) and E6.5 (early/mid-streak stage) embryos. The ∆Ct values from the average Ct values of Gapdh and Arbp are shown as heat maps and are used for clustering. For E5.5 embryos, cells were picked from EPI and visceral endoderm. For E6.5 embryos, cells were picked from proximal EPI and visceral endoderm. The samples used for the SC3-seq analyses (the annotations are based on c) are indicated. c, UHC of cells from mouse E4.5 (EPI, primitive endoderm, mural trophectoderm (mTE), and polar trophectoderm (pTE)19), E5.5, and E6.5 embryos by all expressed genes (log2(RPM+1) > 4 in at least one sample among 86 cells, 13,761 genes), and heat map of the levels of selected marker genes. Colour bars under the dendrogram indicate the cell types. Orange and green bars in the E6.5 EPI cluster indicate E6.5EPI-Tlo (green) and E6.5EPI-Thi (orange) cells, respectively. See e for details. d, PCA of cells by all expressed genes among the indicated cells (67 cells, 13,761 genes). e, Scatter-plot comparison of the expression of key genes for pluripotency or primitive streak formation against that of T in E6.5 EPI. Cells were classified by the levels of T (orange, E6.5EPI-Thi; green, E6.5EPI-Tlo). f, Heat map of the levels of genes with correlation or anti-correlation with T in E6.5 EPI. Genes were selected as follows: the levels > log2(RPM+1) = 6 in at least one cell, correlation coefficient with T > 0.6 (102 genes) or <−0.6 (99 genes). Enrichment of Gene Ontology terms and representative genes are indicated. g, DEGs during the mEPI development. Top, Orange and blue bars indicate the numbers of up- and downregulated genes, respectively, in the pair-wise comparisons indicated. Bottom, Enrichment of Gene Ontology terms and representative genes in DEGs in the pair-wise comparisons indicated.
Extended Data Figure 8 Comparison of monkey and mEPI development.
a, PCA of cells during monkey (circles, colour-coded as in Fig. 3a) and mouse (squares) EPI development. Orthologues among humans, cynomolgus monkeys and mice were annotated (15,220 genes), and 13,473 genes expressed among these cells (monkey, 213 cells; mouse, 44 cells) were used for PCA. b, Heat map of the expression of 784 genes that contributed highly to the PC1 axis (>2 s.d. of PC1: cyEPI or mEPI genes (PCA as in a)). The genes are ordered by UHC, and representative cyEPI or mEPI genes and their key Gene Ontology enrichments are shown. c, Heat map of the levels of monkey and mouse common EPI genes (473 genes) (defined as: radius of PC2 and 3 > 3 s.d. and of PC1: −2 s.d. < PC1 < 2 s.d.) during monkey and mEPI development. d, Heat map of correlation coefficients among cells during cyEPI and mEPI development. The values were calculated using the averaged expression level of monkey and mouse common EPI genes (473 genes (c, Supplementary Table 2)). e, Signalling pathways enriched in genes upregulated during the pre-EPI to postE-EPI transition (top, 258 genes) or during the E4.5–E5.5 mEPI transition (bottom, 455 genes) by the KEGG pathway analysis. f, Heat map of the expression changes of key genes in the NOTCH pathway and NODAL/Nodal during upon implantation. g, A proposed pathway operating in monkey post-EPI, which acquires a property for ‘neuron differentiation’. NICD, NOTCH intracellular domain.
Extended Data Figure 9 Correlations between hPSCs and cyPSCs and cells during cyEPI development.
a, Morphology of cyESCs (CMK6) cultured with (left) or without (right) feeders. Scale bars, 200 μm. b, qPCR analysis of the expression of key markers in single-cell cDNAs generated from cyESCs (CMK6 and CMK9) cultured with or without feeders. The ∆Ct values from the average Ct value of GAPDH and PPIA are shown as heat maps. c, Summary of the SC3-seq samples of cyESCs and hiPSCs19. The numbers of synthesized cDNAs of appropriate quality and of the cells analysed by SC3-seq are listed. d, UHC with all expressed genes (421 cells, 18,527 genes). Note that one cyESC is clustered with postL-EPI. e, Enrichment of Gene Ontology terms in genes upregulated in cyESC against pre-EPI (520 genes). f, UHC with expression of genes for ‘positive (left, 351 genes of Gene Ontology: 0008284) or negative (right, 391 genes of Gene Ontology: 0008284) regulation of cell proliferation’. g, Heat map of the expression of cyEPI ontogenic genes among the indicated cells, including those reported by others22,23,24,33,34,35,36,37,38. The genes in common for all platforms were used (628/776 genes). N, ‘naive’; C, conventional. h, Heat map of the correlation coefficients among cells as in g. Correlation coefficients were calculated using the averaged expression levels of genes in g.
Extended Data Figure 10 Correlations among mPSCs and cells during cyEPI and mEPI development.
a, Morphology of mESCs with 2i+L and day 2 epiblast-like cells (EpiLC) induced from the mESCs32. Scale bars, 200 μm. b, Summary of the SC3-seq samples of mESCs and epiblast-like cells. The numbers of synthesized cDNAs of appropriate quality and of the cells analysed by SC3-seq are listed. c, qPCR analysis of the expression of key markers in single-cell cDNAs generated from 2i+L mESCs and epiblast-like cells. The ∆Ct values from the Ct values of Arbp are shown as heat maps and are used for clustering. d, UHC of cells from E4.5, E5.5, and E6.5 embryos, 2i+L mESCs, and epiblast-like cells with all expressed genes (108 cells, 14,628 genes). Colour bars under the dendrogram indicate the cell types. e, PCA of mEPI, primitive endoderm/visceral endoderm, 2i+L mESCs, and epiblast-like cells by all expressed genes among these cells (89 cells, 14,341 genes). f, Scatter-plot comparisons of the averaged gene-expression levels between E4.5 EPI and 2i+L mESCs (left), and between E5.5 mEPI and epiblast-like cells (right). Key genes are annotated and the numbers of DEGs are indicated. The correlation coefficient is indicated above the scatter plots. g, Heat map of the expression of monkey and mouse common EPI genes (473 genes, as in Extended Data Fig. 8b) in cells during cyEPI and mEPI development and in cyPSCs and mPSCs. h, Heat map of the correlation coefficients among cells as in g. Correlation coefficients were calculated using the averaged expression levels of genes in g.
Supplementary Table 1
This table contains a list of antibodies, primers, SC3-seq samples, mapping information, and published data used in the study. (XLSX 87 kb)
Supplementary Table 2
This table contains a list of genes used in the study. (XLSX 3787 kb)
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Nakamura, T., Okamoto, I., Sasaki, K. et al. A developmental coordinate of pluripotency among mice, monkeys and humans. Nature 537, 57–62 (2016). https://doi.org/10.1038/nature19096
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