Abstract
Development requires coordinated interactions between the epiblast, which generates the embryo proper; the trophectoderm, which generates the placenta; and the hypoblast, which forms both the anterior signalling centre and the yolk sac. These interactions remain poorly understood in human embryogenesis because mechanistic studies have only recently become possible. Here we examine signalling interactions post-implantation using human embryos and stem cell models of the epiblast and hypoblast. We find anterior hypoblast specification is NODAL dependent, as in the mouse. However, while BMP inhibits anterior signalling centre specification in the mouse, it is essential for its maintenance in human. We also find contrasting requirements for BMP in the naive pre-implantation epiblast of mouse and human embryos. Finally, we show that NOTCH signalling is important for human epiblast survival. Our findings of conserved and species-specific factors that drive these early stages of embryonic development highlight the strengths of comparative species studies.
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Main
Many human pregnancies fail during implantation1,2, yet this stage is difficult to study in vivo. Successful development beyond implantation stages requires the specification of the three major lineages: the epiblast, which gives rise to the embryo proper, and two extra-embryonic tissues: the trophectoderm, that generates the placenta, and the hypoblast, which forms the yolk sac. Interactions between extra-embryonic and epiblast tissues are essential to embryo implantation, survival and patterning, and coordinate to set the stage for gastrulation3,4.
Although some of the mechanisms underlying early embryo development differ between human and mouse3,4,5, several events, including the formation of the pro-amniotic cavity within the polarized epiblast, concomitant exit from naive pluripotency6,7,8, and specification of a subset of hypoblast (primitive endoderm in mouse)-derived visceral endoderm cells into the anterior visceral endoderm (AVE), or anterior hypoblast, are conserved across mammalian species. The anterior hypoblast signalling centre secretes antagonists of the BMP, WNT and NODAL pathways, which protect the adjacent epiblast from primitive streak formation9,10,11,12,13,14. In the mouse, the AVE first appears at the distal tip of the egg cylinder in response to synergistic NODAL induction between the visceral endoderm and epiblast15,16,17. Trophectoderm-derived extra-embryonic ectoderm-secreted Bmp4 couples morphogen gradients to morphogenesis by repressing AVE specification until the embryo reaches the appropriate size16,18,19. The presence of the analogous anterior hypoblast signalling centre has also been observed in non-human primate20,21 and in vitro cultured human embryos10. However, the role of morphogens in driving anterior hypoblast specification during human development and their conservation with the mouse is unknown.
In this Article, we sought to determine the signalling dynamics underpinning the pre-to-post implantation transition, focusing specifically on anterior hypoblast specification in human embryos. Functional testing in human embryos, as well as stem cell models of the hypoblast and epiblast, revealed crucial roles for NODAL, BMP and NOTCH during human peri-implantation development.
Results
Signalling dynamics during human embryo implantation
Recent advancements adapting culture conditions originally developed for the mouse to the in vitro culture of human embryos beyond implantation have allowed us and others to investigate this critical window of development for the first time22,23,24,25. We, and other groups, have utilized these approaches to generate large single-cell RNA sequencing (scRNA-seq) datasets of early primate development10,26,27,28,29.
Here we used existing scRNA-seq datasets to guide a cross-species study of the conserved and divergent signalling dynamics underpinning anterior hypoblast specification. To identify candidate signalling pathways, we first integrated existing scRNA-seq datasets spanning 5–14 days post-fertilization (days 5–14) in the human embryo, as well as equivalent stages in cynomolgus macaque (days 6–17) and mouse embryos (embryonic day (E)3.5–E7.5) (Fig. 1a–c and Extended Data Figs. 1a–c and 2a–c).
a, Diagram depicting human development from blastocyst through pre-gastrulation stages. The datasets integrated to generate our combined object are presented below. b, Uniform manifold approximation projection (UMAP) of datasets coloured by original publication. N = 9,862 single cells. c, UMAP of human datasets coloured by embryo age. d, UMAPs of human datasets coloured by a gradient of canonical marker gene expression for epiblast (EPI), hypoblast (HYPO), trophectoderm/trophoblast (TE/TrB) and terminal extravillous trophoblast (EVT) or syncytiotrophoblast (STB) lineages. e, UMAP coloured by assigned cell type. f, Heatmap of WikiPathways signalling pathway module score for each developmental stage of each individual cell type. Visualized value is calculated average scaled score from individual single-cell module scores. g,h, CellPhoneDB dotplots for blastocyst (g) and peri-implantation (h) stages depicting predicted activity of individual receptor–ligand pairs. Each ligand–receptor pair is colour matched to the expressing lineage on the x axis. N = 9,862 single cells. AP, anterior-posterior; dep, dependent; ind, independent; reg, regulation; comp, complex.
In our integrated, species-specific datasets, we could identify key clusters on the basis of conserved expression patterns, including POU5F1, SOX2 and NANOG in the epiblast clusters, GATA6 and SOX17 in the hypoblast/primitive and visceral endoderm clusters and GATA3, GATA2 and TFAP2C in the trophectoderm/trophoblast/extra-embryonic ectoderm clusters (Fig. 1d,e, Extended Data Figs. 1d and 2d, and Supplementary Tables 1–6). Two terminally differentiated CGA-positive trophoblast (a post-implantation derivative of the trophectoderm) lineages could also be detected in the human: SDC1-positive syncytiotrophoblast and CGB1-positive extravillous trophoblast10,30,31,32,33. In the macaque, a population of GATA6, COL6A1, VIM, POSTN, LUM-positive extra-embryonic mesenchyme was also detected, which was not observed in in vitro cultured human embryos21,34,35 (Extended Data Fig. 1d).
Next, we leveraged our integrated dataset to identify signalling dynamics during human in vitro development, focusing on stages when the anterior hypoblast is specified. Module scoring (using WikiPathways gene lists, Supplementary Table 7) for genes associated with key signalling pathways identified enrichment within and between different cell lineages (Fig. 1f and Extended Data Fig. 3a,b). Module scoring was also carried out in mouse and macaque datasets (Extended Data Figs. 1e and 2e).
Our module scoring closely aligned with patterns previously described in mammalian embryos, including enrichment of NODAL in the epiblast and hypoblast15,36,37, WNT pathway enrichment in the epiblast before gastrulation38,39, upregulation of FGFR (FGFR1, FGFR3 and FGFR4) modules across lineages at implantation10 and Hedgehog signalling module enrichment in the syncytiotrophoblast40 (Fig. 1f). The NODAL module was noticeably enriched in the blastocyst-stage epiblast in human and peri-implantation-stage epiblast in macaque, in contrast to mouse, where enrichment in genes associated with NODAL signalling occurred upon implantation (Fig. 1f and Extended Data Figs. 1e and 2e). Likewise, the mouse extra-embryonic ectoderm was enriched for the BMP module following implantation, but this pattern was predominantly absent in macaque and human trophoblast, where the BMP module is enriched in the hypoblast and extra-embryonic mesenchyme instead (Fig. 1f and Extended Data Figs. 1e, 2e and 3a,b). Overall, this analysis provided a global insight into the gene expression patterns associated with specific signalling pathways during implantation of the human embryo.
To dissect potential crosstalk between lineages of the peri-implantation human embryo, we employed the computational tool CellPhoneDB, which utilizes a curated heteromeric ligand–receptor repository to provide a tissue-to-tissue interaction map41 (Fig. 1g,h and Extended Data Fig. 3c,d). We found that epiblast-secreted NODAL was predicted to be received by all lineages of the blastocyst. In contrast, the predicted NODAL receptivity was enriched in the hypoblast upon implantation (Fig. 1g,h). In agreement with our global analysis, BMP signalling interactions were predicted to be present in the hypoblast, which expressed both BMP ligands and receptors. Notably, upon implantation, predicted epiblast-to-epiblast NOTCH signalling was specifically enriched.
Using our combined transcriptomic analysis as a guide, we next sought to identify signalling pathways important for human anterior hypoblast signalling centre specification. We selected candidate pathways on the basis of three criteria: (1) lineage-specific expression; (2) temporal expression changes during implantation; and (3) predicted role in anterior hypoblast formation and/or epiblast maturation based on other model organisms. This analysis led us to select two TGFb superfamily members: NODAL and BMP; and the NOTCH pathway.
NODAL signalling is essential for the anterior hypoblast
To quantify inferred NODAL activity, we examined the NODAL effector SMAD2.3. by calculating the nuclear, cytoplasmic and nuclear-to-cytoplasmic ratio (n/c) of total SMAD2.3 in individual cells (Extended Data Fig. 4a–c)42,43. To accurately capture early blastocyst development, we further classified blastocysts at day 5 or 6 as ‘segregating’ (inner cell mass co-expressing epiblast and hypoblast markers) or ‘segregated’ (inner cell mass with mutually exclusive epiblast and hypoblast protein expression) (Extended Data Fig. 4d,e)37,44,45.
We found that the trophectoderm, epiblast and hypoblast all expressed NODAL receptors at the blastocyst stage in human (Extended Data Fig. 4f–h). In segregating blastocysts, the n/c SMAD2.3 was significantly higher in trophectoderm cells than inner cell mass cells, though raw nuclear and cytoplasmic signals were lower, similar to the E3.5 early mouse blastocyst (Fig. 2a,b and Extended Data Fig. 5a–c). Concomitant with lineage segregation, GATA6-positive hypoblast cells exhibited increased n/c SMAD2.3 compared with epiblast cells (Fig. 2c). The hypoblast (human) and visceral endoderm (mouse) maintained these higher levels of n/c SMAD2.3 compared with the epiblast at implantation stages (Fig. 2d–f and Extended Data Fig. 5a,d–f), consistent with our module scoring and CellPhoneDB predictions from the scRNA-seq datasets. The n/c SMAD2.3 decreased over time in both human and mouse epiblasts (Fig. 2g and Extended Data Fig. 5a,g). However, levels within the inner cell mass in human embryos appeared higher and subsequently showed a larger decrease following lineage segregation. In contrast, high n/c SMAD2.3 was maintained in the human hypoblast upon implantation but increased in the visceral endoderm upon implantation in mouse (Fig. 2h and Extended Data Fig. 5a,h). The mouse extra-embryonic ectoderm showed a decrease in n/c Smad2.3 upon implantation (Extended Data Fig. 5i). Strikingly, on day 9 in the human embryo, n/c SMAD2.3 was higher in hypoblast cells in direct contact with the overlying epiblast (Fig. 2i,j), supporting predictions by CellPhoneDB that the epiblast is the primary NODAL source.
a, Immunofluorescence images of segregating (N = 3 embryos) and segregated human blastocysts (N = 3 blastocysts) stained for OCT4/E-CADHERIN, GATA6, total SMAD2.3 and DAPI. b,c, Quantification of nuclear and cytoplasmic SMAD2.3 fluorescence and their ratio in inner cell mass (ICM, n = 34 cells) and trophectoderm (TE, n = 31 cells) in segregating blastocysts and between epiblast (EPI, n = 26 cells), hypoblast (HYPO, n = 31 cells) and TE (n = 13 cells) of segregated blastocysts. d, Immunofluorescence images of day 7 (N = 17 embryos) and day 9 (N = 13 embryos) human embryos following in vitro implantation stained for OCT4/PODLX, GATA6, SMAD2.3 and DAPI. e,f, Quantification of nuclear and cytoplasmic SMAD2.3 fluorescence and their ratio in EPI and HYPO at day 7 (EPI n = 299 cells, HYPO n = 322 cells) and day 9 (EPI n = 297 cells, HYPO n = 214 cells). g,h, Plot of n/c SMAD2.3 over time in EPI (g; n = 11 day 5, 26 day 6, 299 day 7 and 197 day 9 cells) and HYPO (h; n = 10 day 5, 31 day 6, 322 day 7 and 214 day 9 cells). i, Quantification of n/c SMAD2.3 in HYPO cells at day 7 and day 9 depending on contact with overlying EPI. j, High-power images of SMAD2.3 in day 9 HYPO cells either contacting or not contacting the EPI. Regions shown here are outlined with a dashed box in d. For violin plots, central dotted line denotes median and dotted lines mark the 25th and 75th quartiles. Error bars denote standard error (g and h). Statistical tests: two-sided unpaired t-test (b (nuclear (nuc) and cytoplasmic (cyto))), two-sided Mann–Whitney (b (n/c), e and f), one-way analysis of variance with Tukey–Kramer post-hoc (c (nuc)), Kruskal–Wallis with Dunn’s post-hoc (c (cyto and n/c) and g–i); ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Unmarked pairwise comparisons are not significant (NS). Exact P values presented in Supplementary Table 8. Scale bars: 25 µm (a), 100 µm (b and e) and 20 µm (k).
To investigate the function of NODAL signalling across peri-implantation stages, we used Activin-A or the ALK4/5/7 inhibitor A83-01 to modulate activation of this pathway at two stages of human embryogenesis: in the human blastocyst from day 5 to day 7, when the inner cell mass segregates and the anterior hypoblast marker CER1 is first expressed; and from day 7 to day 9, which encompasses in vitro implantation, exit from naive pluripotency and regionalization of the hypoblast for anterior–posterior axis specification10. As amnion and extra-embryonic mesenchyme do not form robustly during in vitro culture of human embryos10,24,25,26,27,46, we limited our experimentation to time frames preceding their expected differentiation in vivo to mitigate signalling defects associated with the lack of these tissues. We validated Activin-A-mediated activation and A83-01-mediated inhibition of NODAL signalling in human embryonic stem (ES) cells (Extended Data Fig. 6a,b).
Activin-A or A83-01 treatment did not affect total cell numbers of OCT4-positive epiblast or GATA6-positive hypoblast (Fig. 3a–j and Extended Data Fig. 6c,d). In contrast, treating human embryos from days 5–7 or days 7–9 with A83-01 decreased the number of CER1-positive hypoblast cells (Fig. 3f,j and Extended Data Fig. 6c,d). NODAL is essential for the formation of the AVE at equivalent peri-implantation stages in the mouse15,16,47. In stage-matched in vitro cultured mouse embryos, A83-01 treatment between E3.5 + 48 h did not affect epiblast or hypoblast cell numbers (Extended Data Fig. 6e–g). A83-01 treatment between E5.0 + 36 h decreased epiblast size and AVE formation, as observed previously16 (Extended Data Fig. 6h–k).
a, Schematic of experimental design for human embryo treatment from day 5 to day 7 to modulate NODAL signalling. b, Immunofluorescence images of embryos cultured from day 5 to day 7 in control (N = 44 embryos), 2 µM A83-01 (N = 12 embryos) or 25 ng ml−1 Activin-A (N = 14 embryos) conditions. c–e, Quantification of the number of epiblast (c), hypoblast (d) and CER1-positive hypoblast cells (e) at day 7. f, Schematic of experimental design for human embryo treatment antagonists from day 7 to day 9 to modulate NODAL signalling. g, Immunofluorescence images of embryos cultured from day 7 to day 9 in control (N = 39 embryos), 2 µM A83-01 (N = 12 embryos) or 25 ng ml−1 Activin-A (N = 15 embryos) conditions. h–j, Quantification of the number of epiblast (h), hypoblast (i) and CER1-positive hypoblast cells (j) at day 9. k, Schematic and immunofluorescence images of YSLC differentiation ± 2 µM A83-01 for 48 h during YSLC specification or maturation. l, Quantification of GATA6 fluorescence levels relative to DAPI and normalized to control. D2–D4: control n = 4,085; A83-01 n = 4,255. D4–D6: control n = 4,139; A83-01 n = 4,180. N = 2 experiments. m, Percentage of GATA6-positive cells that are CER1 positive. D2–D4: control n = 2,502; A83-01 n = 2,347. D4–D6: control n = 2,500; A83-01 n = 2,013. N = 2 experiments. n, Schematic and immunofluorescence images of pSMAD1.5.9 expression in human (h)ES cell-derived spheroids cultured in mTeSR+ medium (mTeSR+ n = 1,060 cells) or mTeSR+ medium conditioned on either control YSLC (YSLC CM n = 1,836 cells) or on YSLC differentiated with 48 h of A83-01 treatment (YSLC + A83 CM n = 1,654 cells). N = 3 experiments. o, Quantification of normalized pSMAD1.5.9 fluorescence from n. For box plots, box encompasses 25th and 75th quartiles with median marked by central line. Minimum and maximum and denoted by whiskers. Plus symbol marks the mean. Error bars denote standard error (m–o). Statistical tests: two-sided Mann–Whitney test (c–e and h–j); two-sided unpaired t-test (l and m); one-way analysis of variance with Tukey–Kramer post-hoc (o). ****P < 0.0001, **P < 0.01, *P < 0.05. Unmarked pairwise comparisons are not significant (NS). Exact P values presented in Supplementary Table 8. Scale bars: 100 µm (b and g) and 50 µm (k and n). Embryos in b and g were re-stained for GATA6, and re-orientated second-stain images are placed on a grey background for transparency and to match the orientation of the merged image.
To further examine the role of NODAL in the anterior hypoblast, we generated yolk sac-like cells (YSLCs) from human ES cells, which express CER1 and resemble extra-embryonic endoderm48 (Fig. 3k). Treatment with A83-01 decreased both GATA6 and CER1 expression (Fig. 3l,m), as expected given the use of exogenous Activin-A to differentiate YSLCs. We have previously shown that YSLCs secrete signalling antagonists and that YSLC conditioned medium protects three-dimensional (3D) epiblast-like human ES cell spheroids from BMP-induced differentiation48. Conditioned medium generated from YSLCs treated with A83-01 exhibited impaired capacity to protect human ES cell spheroids, demonstrated by increased pSMAD1.5.9 expression (Fig. 3n,o). These data demonstrate that specification and maintenance of the anterior hypoblast are NODAL dependent.
A83-01 is commonly used to differentiate naïve human ES cells towards trophectoderm49,50,51. Therefore, we also assessed the response of the trophectoderm to NODAL perturbation from day 5 to day 7 post-fertilization. A83-01 treatment did not increase trophectoderm cell number. However, Activin-A treatment decreased the number of GATA3-positive trophectoderm cells (Extended Data Fig. 6l–p). Further analysis demonstrated that this reduction occurs specifically in the GATA3/GATA6-double positive population, which marks pre-implantation trophectoderm surrounding the blastocoel cavity52 (Extended Data Fig. 6o,p). Further, Activin-A treated embryos at day 7 showed higher rates of attachment and implantation-like morphology compared with A83-01 treated embryos (Extended Data Fig. 6q). The transition towards predominantly GATA3-positive/GATA6-negative trophoblast cells was reminiscent of the transition between day 7 and day 9 in control conditions (Extended Data Fig. 6r,s). Thus, our results show that NODAL signalling positively regulates trophectoderm maturation, in agreement with recent reports that epiblast-secreted signals drive local maturation of the trophectoderm51.
BMP signalling is active in the human naive epiblast
Next, we characterized the activity of BMP signalling by examining the expression of its nuclear effector pSMAD1.5.9 (refs. 43,53) (Extended Data Fig. 7a–c). In segregating blastocysts, the trophectoderm was enriched for pSMAD1.5.9 (Fig. 4a,b and Extended Data Fig. 7d) despite low predicted activity/module scoring of BMP. However, both BMPR1A and BMPR2 are expressed in pre-implantation trophectoderm and downregulated at implantation (Extended Data Fig. 3b). In contrast, there was no difference in pSMAD1.5.9 intensity between inner cell mass and trophectoderm lineages of stage-matched mouse embryos (Extended Data Fig. 7e,f). In segregated blastocysts, pSMAD1.5.9 was enriched in the hypoblast, and expression in the trophectoderm was maintained (Fig. 4c and Extended Data Fig. 7d). Comparatively, despite a global increase in pSMAD1.5.9 levels, we observed no lineage-specific enrichment in E4.5 late mouse blastocysts (Extended Data Fig. 7g). pSMAD1.5.9 was higher in the hypoblast than epiblast at day 7 in human embryos (Fig. 4d,e and Extended Data Fig. 7d), consistent with the module scoring, but levels were similar in the hypoblast and epiblast in human embryos at day 9 (Fig. 4f,g and Extended Data Fig. 7d). In the mouse, following implantation, only the visceral endoderm sustained high levels of BMP activity (Extended Data Fig. 7j–l).
a, Immunofluorescence images of segregating (N = 4 blastocysts) and segregated human blastocysts (N = 9 blastocysts) stained for SOX2/F-Actin, GATA6, phosphorylated (p)SMAD1.5.9 and DAPI. b,c, Quantification of the normalized fluorescence of pSMAD1.5.9 in inner cell mass (ICM, n = 59) versus trophectoderm (TE, n = 80 cells) in segregating blastocysts and between epiblast (EPI, n = 77 cells), hypoblast (HYPO, n = 142 cells) and TE (n = 258 cells) of segregated blastocysts. d, Immunofluorescence images of day 7 (N = 9 embryos) and day 9 (N = 4 embryos) human embryos following in vitro implantation stained for SOX2/PODLX, GATA6, pSMAD1.5.9 and DAPI. e,f, Quantification of normalized fluorescence of pSMAD1.5.9 in EPI versus HYPO at day 7 (EPI n = 162 cells, HYPO n = 236 cells) and day 9 (EPI n = 29 cells, HYPO n = 87 cells). g,h, Plot of normalized pSMAD1.5.9 fluorescence over time in EPI (g; n = 44 day 5, 68 day 6, 162 day 7 and 29 day 9 cells) and HYPO (h; n = 20 day 5, 90 day 6, 236 day 7 and 87 day 9 cells). i, Quantification of normalized pSMAD1.5.9 fluorescence in HYPO cells at day 7 and day 9 depending on contact with EPI. j, High-power images of pSMAD1.5.9 in day 9 HYPO cells either contacting or not contacting the EPI. Regions shown here are outlined with a dashed box in d. For violin plots, central dotted line denotes median and dotted lines mark the 25th and 75th quartiles. Error bars denote standard error (g and h). Statistical tests: two-sided Mann–Whitney test (b, e and f); Kruskal–Wallis with Dunn’s post-hoc (c and g–i). ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Exact P values presented in Supplementary Table 8. Scale bars: 25 µm (a), 100 µm (b and e) and 20 µm (k).
pSMAD1.5.9 expression decreased over time in the human epiblast. Similarly, the human hypoblast exhibited a decrease in pSMAD1.5.9 upon implantation (Fig. 4h,i). Interestingly, at day 9, pSMAD1.5.9 expression was higher in hypoblast cells in contact with the epiblast than in those distal to the epiblast, despite no observable increase in epiblast expression of BMP ligands at this stage, implicating an additional regulator of pSMAD1.5.9 in the hypoblast (Fig. 4j).
To interrogate the role of BMP signalling in the embryo, we first examined the expression of BMP ligands. BMP2/BMP4/BMP6 expression was enriched in the human hypoblast (Extended Data Fig. 8a and Supplementary Table 4). In macaque, BMP2/BMP4 expression could also be observed in the extra-embryonic mesenchyme, while in mouse Bmp4/Bmp8b were enriched in the extra-embryonic ectoderm and Bmp2 was expressed in the visceral endoderm (Supplementary Tables 5 and 6). Addition of BMP2, BMP4 or BMP6 efficiently increased phosphorylation of SMAD1.5.9 in human ES cells (Extended Data Fig. 8b,c). We utilized BMP6 for subsequent experiments because it (1) showed specific upregulation upon implantation, (2) did not induce TBXT/BRACHYURY-positive primitive streak-like cell differentiation in vitro, and (3) was hypoblast specific in macaque. Additionally, we treated human embryos with the BMP receptor inhibitor LDN193189 (LDN; see Methods for details) to antagonize BMP signalling.
BMP inhibition with LDN from day 5 to day 7 decreased the number of epiblast cells (Fig. 5a–d and Extended Data Fig. 8d). In contrast, LDN treatment from day 7 to day 9 did not affect epiblast cell numbers, but conversely, the addition of BMP6 resulted in a reduction (Fig. 5e–h and Extended Data Fig. 8e). In contrast, mouse epiblast size decreased with LDN treatment in post- but not pre-implantation, as reported previously54 (Extended Data Fig. 8f–l). Together, these results suggest a switch in the requirement of BMP during pre- and post-implantation as well as a divergence between the mouse and human patterns.
a, Immunofluorescence images of embryos cultured from day 5 to day 7 in control (N = 44 embryos), 200 nM LDN (N = 8 embryos) or 50 ng ml−1 BMP6 (N = 8 embryos) conditions. b–d, Quantification of the number of epiblast (b), hypoblast (c) and CER1-positive hypoblast cells (d) at day 7. e, Immunofluorescence images of embryos cultured from day 7 to day 9 in control (N = 39 embryos), 200 nM LDN (N = 6 embryos) or 50 ng ml−1 BMP6 (N = 6 embryos) conditions. f–h, Quantification of the number of epiblast (f), hypoblast (g) and CER1-positive hypoblast cells (h) at day 9. i, Pearson regression correlation coefficient ID2 expression with naive pluripotency markers. j,k, Immunofluorescence and quantification of pSMAD1.5.9 in PXGL (naive: 3,057 cells) and mTeSR (primed: 5,761 cells) human (h)ES cells and in mouse (m)ES cells in 2iLif conditions (naive; 297 cells) or basal N2B27 (naive exit; 789 cells). N = 2 experiments. l, Immunofluorescence images of differentiated YSLCs ± 200 nM LDN for 48 h during specification or maturation of YSLCs. m, Quantification of GATA6 fluorescence levels in relative to DAPI and normalized to control. D2–D4: control n = 4,085; LDN n = 4,777. D4–D6: control n = 4,139; LDN n = 4,461. N = 2 experiments. n, Percentage of GATA6-positive cells that are CER1 positive. D2–D4: control n = 2,502; LDN n = 2,796. D4–D6: control n = 2,500; LDN n = 2,425. N = 2 experiments. o,p, Immunofluorescence images and quantification of pSMAD1.5.9 expression in hES cell-derived spheroids cultured in mTeSR+ medium (mTeSR+ n = 1,060 cells) or mTeSR+ medium conditioned on either control YSLC (YSLC CM n = 1,836 cells) or on YSLC differentiated with 48 h of LDN treatment (YSLC + LDN CM n = 1427 cells). N = 3 experiments. For box plots, box encompasses 25th and 75th quartiles with median marked by central line. Minimum and maximum and denoted by whiskers. Plus symbol marks the mean. Error bars denote standard error (k, m–n and p). Statistical tests: two-sided Mann–Whitney test (b–d and f–h); coefficients were tested (cor.test; two-tailed) and corrected for multiple hypothesis testing with the Benjamini–Hochberg method (i); two-sided unpaired t-test (k, m and n); one way analysis of variance with Tukey–Kramer post-hoc (p). ****P < 0.0001, ***P < 0.001, *P < 0.05. Unmarked pairwise comparisons are not significant (NS). Exact P values presented in Supplementary Table 8. Scale bars: 100 µm (a and e) and 50 µm (j, l and o).
To validate the role of BMP in the human epiblast, we utilized human and mouse ES cells across the pluripotency spectrum (that is naive or primed). We found that LDN treatment increased apoptosis in naive human ES cells but conversely decreased apoptosis in primed human ES cells (Extended Data Fig. 8m). These data suggest that pluripotency states may underscore the difference in pre- and post-implantation BMP dependence in the embryo. Examination of the correlation of BMP response genes ID1–3 (ref. 55) with naive pluripotency markers in the pre-implantation epiblast showed positive co-expression in human, but not mouse (Fig. 5i and Extended Data Fig. 8n). Similarly, pSMAD1.5.9 expression was enriched in naive compared with primed human ES cells, but this pattern was reversed in mouse ES cells (Fig. 5j,k). This difference in BMP activity appears functionally important, as LDN treatment decreased the human-specific naive marker AP2γ in human ES cells, while in mouse ES cells it decreased the mouse-specific primed marker Otx2 (Extended Data Fig. 8o,p). Overall, these data support a role for BMP in human pre-implantation epiblast and suggest that stage-dependent differences in BMP dependence between mouse and human are related to divergences in the pluripotency regulatory network during exit from the naive state.
BMP signalling is required for human anterior hypoblast
Strikingly, BMP inhibition by LDN treatment of human embryos from day 7 to day 9 ablated the anterior hypoblast, despite no change in total hypoblast cell numbers (Fig. 5e–h), revealing an essential role for BMP signalling in anterior hypoblast maintenance following implantation. In mouse, LDN treatment conversely increased the formation of the AVE (Extended Data Fig. 8i,j), in line with an inhibitory role of Bmp4. Further, we observed that LDN treatment during differentiation of YSLC from ES cells resulted in a decrease in GATA6 at all points in differentiation (Fig. 5l,m and Extended Data Fig. 8q). The addition of LDN to YSLC also reduced the proportion of CER1-positive cells but only at the endpoint of differentiation (Fig. 5n). This phenotype recapitulated the requirement for BMP in anterior hypoblast maintenance but not specification in the human embryo. Further, conditioned medium from YSLCs differentiated with LDN treatment during this later window showed reduced capacity to protect epiblast-like human ES cell spheroids, indicating functional impairment in the generation of secreted signalling antagonists (Fig. 5o,p). Taken together, our results indicate that the role of BMP signalling in anterior specification diverges between the mouse and human.
NOTCH/γ-secretase signalling is important for human epiblast
Next, we investigated the putative role of NOTCH signalling as predicted in our CellPhoneDB analysis (Fig. 1h). We found that NOTCH ligands DLL3 and JAG1 and receptor NOTCH1 were expressed in the human epiblast, and the NOTCH effector RBPJ was increased upon embryo development through implantation in vitro (Extended Data Fig. 3b). To address the role of NOTCH signalling in the human embryo, we inhibited γ-secretase, which cleaves NOTCH receptors to generate the NOTCH intra-cellular domain. We found that γ-secretase inhibition (with DAPT, Compound-E or MK-0752) from day 5–7 embryos had minimal effect on total SOX2-positive epiblast or GATA6-positive hypoblast numbers. Similarly, epiblast and hypoblast were unaffected in E3.5 + 48 h mouse embryos treated with DAPT (Fig. 6a–d and Extended Data Fig. 9a–d). In contrast, DAPT addition between days 7 and 9 significantly decreased both epiblast and hypoblast total cell numbers. Compound-E and MK-0752 treatment also decreased the number of epiblast cells, albeit to a lesser extent (Fig. 6e–h and Extended Data Fig. 9b). Only γ-secretase inhibition using DAPT in human embryos showed a decrease in hypoblast cell numbers at day 9, indicating this loss may be related to the more drastic effect on the epiblast in this condition, rather than a direct effect on the hypoblast (Fig. 6e,g and Extended Data Fig. 9b). Mouse embryos treated with DAPT from E5.0 for an additional 36 h exhibited reduced epiblast length and aberrantly displayed epiblast cells within the pro-amniotic cavity, but no difference in total epiblast area (Extended Data Fig. 9f–j). Interestingly, 48-h γ-secretase inhibition (DAPT) of naive or primed human ES cells in adherent monolayer conditions (Matrigel-coated dishes) did not change the proportion of cleaved caspase-3-positive apoptotic cells relative to controls (Fig. 6i,j). In contrast, DAPT treatment of human ES cell-derived spheroids, which more closely resemble the epiblast architecture7,56, increased apoptosis, replicating the loss of the epiblast in human embryos post-implantation (Fig. 6i–k).
a, Immunofluorescence images of embryos cultured from day 5 to day 7 in control conditions (N = 44 embryos), 20 µM DAPT (N = 8 embryos), 10 µM Compound-E (N = 14 embryos) or 20 µM MK-0752 (N = 9 embryos). b–d, Quantification of the number of epiblast (b), hypoblast (d) and CER1-positive hypoblast cells (d) at day 7. e, Immunofluorescence images of embryos cultured from day 7 to day 9 in control conditions (N = 39 embryos), DAPT (N = 9 embryos), Compound-E (N = 12 embryos) or MK-0752 (N = 17 embryos). f–h, Quantification of the number of epiblast (f), total hypoblast (g) and CER1-positive hypoblast cells (h) at day 9. i–k, Immunofluorescence images and quantification of 2D naive PXGL and primed human (h)ES cells treated for 48 h in control conditions (PXGL n = 2,336, primed n = 7,796) or 20 µM DAPT (PXGL n = 2,386, primed n = 6,136; N = 2 experiments) and 3D spheroids derived from primed hES cells cultured in control conditions (n = 364) or DAPT (n = 643; N = 2 experiments). Cells were stained for SOX2 and cleaved caspase 3 (Ccasp3) with spheroids additionally stained for PODLX. l, Immunofluorescence images of the differentiation of YSLCs that were treated with DAPT for 48 h during YSLC specification or maturation. m, Percentage of GATA6-positive cells that are CER1 positive. D2–D4: control n = 2,502; DAPT n = 2,690. D4–D6: control n = 2,500; DAPT n = 2,701; N = 2 experiments. n, Immunofluorescence images of pSMAD1.5.9 expression in hES cell-derived spheroids cultured in mTeSR+ medium (mTeSR+ n = 1,060 cells) or mTeSR+ medium conditioned on either control YSLC (YSLC CM n = 1,836 cells) or on YSLC differentiated with 48 h of DAPT treatment (YSLC + DAPT CM n = 501 cells). N = 3 experiments. o, Quantification of normalized pSMAD1.5.9 fluorescence from n. For box plots, box encompasses 25th and 75th quartiles with median marked by central line. Minimum and maximum and denoted by whiskers. Plus symbol marks the mean. Error bars denote standard error (j, k and m–o). Statistics: two-sided Mann–Whitney test (b–d and f–h); two-sided unpaired t-test (j and m); two-way analysis of variance (ANOVA) with Šidák’s multiple comparison’s test (k); one way ANOVA with Tukey–Kramer post-hoc (o). P < 0.06 is noted. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Unmarked comparisons to control are not significant (NS). Exact P values presented in Supplementary Table 8. Scale bars: 100 µm (a and e) and 50 µm (i, l and n).
DAPT-treated mouse embryos also exhibited a loss of the AVE (Extended Data Fig. 9f–h). Similarly, all three γ-secretase inhibitors used to treat human embryos caused a reduction in CER1-positive anterior hypoblast cells, though their effects varied in severity and stage (Fig. 6e,h and Extended Data Fig. 9a). To investigate if NOTCH/γ-secretase inhibition within the epiblast has an indirect impact on the hypoblast, we utilized our directed YSLC differentiation approach48 in combination with γ-secretase inhibition. DAPT treatment did not prevent GATA6 expression. However, both initiation and maintenance of CER1 expression were reduced (Fig. 6l,m), suggesting a cell-autonomous role for NOTCH/γ-secretase in anterior hypoblast. Additionally, conditioned media from YSLCs differentiated in the presence of DAPT showed reduced functionality in the protection of epiblast-like spheroids (Fig. 6n,o). Together, these data support a species-conserved role of NOTCH/γ-secretase-mediated signalling in anterior specification, as well as in post-implantation epiblast survival in the human embryo.
Discussion
Our analysis of signalling dynamics during peri-implantation embryo development and anterior hypoblast specification revealed conserved and divergent roles of NODAL, BMP and NOTCH during early development across species. Taken together, our results demonstrate a crucial role of NODAL, BMP and NOTCH in human anterior hypoblast formation and suggest implantation as a switch point in BMP, NODAL and NOTCH signalling activity, particularly in the transition of the epiblast and maturation of the hypoblast (Extended Data Fig. 10).
The formation of the extra-embryonic anterior signalling centre is conserved in mouse and human, while the morphology and timing differ10. We demonstrate that, as in mouse, NODAL signalling is required in human for both the initial specification of the CER1-positive hypoblast and maintenance of CER1-positive cells. In contrast to the mouse, however, we find that BMP signalling is necessary for the maintenance of the anterior hypoblast after implantation. Importantly, by day 9, both BMP and NODAL show enriched activity in hypoblast cells directly underlying the epiblast. As both NODAL and BMP appear necessary for the maintenance of the anterior hypoblast, which underlies the epiblast, this pattern of enrichment supports their role. Further, the divergence in signalling activity between cells underlying the epiblast (visceral endoderm-like) and those lining the putative yolk sac cavity in post-implantation stages suggests the existence of spatially organized hypoblast-derived subpopulations with distinct signalling environments. NOTCH signalling also appears important in anteriorization as we observed a decrease in CER1-positive cells after γ-secretase inhibition in human and a loss of AVE formation in mouse embryos. This may be partly due to downstream loss of NODAL as has been reported in mouse57. The distinct roles of NODAL, BMP and NOTCH signalling we report here in human embryos could be recapitulated in YSLCs in vitro48.
Concomitant with anterior hypoblast formation, the mammalian epiblast transitions from a naive state of pluripotency to a primed state10,49,58, and these distinct stages can be captured in ES cell-derived human and mouse embryo models59,60. However, the conditions used to capture these distinct pluripotency states differ between mouse and human, indicative of evolutionary divergence in the pluripotency network. Here we find that the pre-implantation human epiblast exhibits high levels of both NODAL and BMP activity compared with the post-implantation epiblast. In line with this observation, BMP inhibition before, but not after, implantation decreases the number of human epiblast cells. These results contrast with analogous mouse treatments, where BMP or NODAL inhibition decreased epiblast size post-implantation. In vitro models of human and mouse naive and primed pluripotency also recapitulated these stage-based divergences in BMP activity between species. In addition, γ-secretase/NOTCH inhibition demonstrated distinct effects on the pre- or post-implantation human embryo, having minimal effects on epiblast cell number before implantation and decreasing this population when treated immediately following implantation. In mouse, γ-secretase/NOTCH inhibition altered epiblast tissue integrity with cells present in the pro-amniotic cavity, but did not appear to affect survival. NOTCH is active in the mouse epiblast from implantation to gastrulation61; however, its role is not well understood. Here we show its role in the survival of the human epiblast during its transition from a more disorganized pre-implantation state to the epithelialized rosette. The γ-secretase complex is known to mediate cleavage of several substrates in addition to NOTCH, including E-Cadherin, ERBB4, LRP6 and IGF1R62. Further work specifically targeting the NOTCH pathway will be essential for confirming its role in post-implantation development.
While we focus largely on the inner cell mass-derived lineages, our scRNA-seq analysis may also be used to identify key modulators of trophoblast development including terminal differentiation of the primate-specific syncytiotrophoblast and extravillous trophoblast. We show that NODAL signalling from the epiblast may drive maturation of the human trophectoderm to implantation-competent trophoblast. Previous work has shown that isolated inner cell masses in MEK and NODAL inhibitors generated trophectoderm cell outgrowths robustly and that these conditions can be used to differentiate trophectoderm cells from naïve human ES cells49. However, the addition of A83-01 from day 5 to day 7 does not appear to drive differentiation of the pre-implantation epiblast to trophectoderm. This suggests that either the combinatorial inhibition of both FGF-MAPK and NODAL could be crucial for trophectoderm differentiation, or that the differentiation of inner cell mass outgrowths and naive ES cells towards trophectoderm follows distinct pathways compared with trophectoderm specification in the morula. BMP signalling has also been associated with human trophoblast, and yet its role, tissue expression pattern and potential evolutionary divergence remain controversial46,49,63,64,65,66. BMP6 treatment alone may not be indicative of other BMP ligands' effects. Of note, the extra-embryonic mesenchyme and amnion in primates are likely to be sources of BMP ligands in addition to the hypoblast20,21,34,35,67; however, these populations do not differentiate robustly during in vitro culture of human embryos in current systems10,24,25,26,27,46. Further experiments will be required to fully investigate the pleiotropic role of BMP during human implantation.
In summary, we have sought to uncover roles of multiple signalling pathways operating during human implantation, which should be useful in efforts to improve stem cell-derived human embryo-like models while further contributing to our foundational knowledge of this crucial developmental period.
Methods
Ethics statement
Human embryo work was regulated by the Human Fertility and Embryology Authority under licence R0193. Approval was obtained from the Human Biology Research Ethics Committee at the University of Cambridge (reference HBREC.2021.26). All work is compliant with the 2021 International Society for Stem Cell Research (ISSCR) guidelines. Patients undergoing IVF at CARE Fertility, Bourn Hall Fertility Clinic, Herts & Essex Fertility Clinic, and King’s Fertility were given the option of continued storage, disposal or donation of embryos to research (including research project specific information) or training at the end of their treatment. Patients were offered counselling, received no financial benefit and could withdraw their participation at any time until the embryo had been used for research. Research consent for donated embryos was obtained from both gamete providers. Embryos were not cultured beyond day 14 post-fertilization or the appearance of the primitive streak. Human stem cell work was approved by the UK Stem Cell Bank Steering Committee (under approval SCSC21-38) and adheres to the regulations of the UK Code of Practice for the Use of Human Stem Cell Lines. Mice were kept in an animal house in individually ventilated housing on 12:12 h light–dark cycle with ad libitum access to food and water. Ambient temperature was maintained at 21–22 °C and humidity at 50%. Experiments with mice are regulated by the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 and carried out following ethical review by the University of Cambridge Animal Welfare and Ethical Review Body. Experiments were approved by the Home Office under licences 70/8864 and PP3370287. CD1 wild-type males aged 6–45 weeks and CD1 wild-type females aged 6–18 weeks were used for this study. Animals were inspected daily, and those showing health concerns were culled by cervical dislocation.
Sequencing analysis and code availability
Raw fastq files from human datasets26,27,36,45, cynomolgus monkey datasets28,35 and mouse datasets68,69,70,71 were obtained from public repositories with wget. All human datasets were aligned to the GRCh38 reference using kb-python’s kb ref function to generate a reference. For cynomolgus monkey, National Center for Biotechnology Information (NCBI) genome build 5.0 transcriptome fasta files were adjusted to Ensembl style and used in kb ref to generate a custom index. For the mouse, GRCm39 reference was used with kb ref to generate a custom index. All datasets were re-aligned using either kb-python or kallisto72,73, after data handling as below. Human datasets: 10x v2 data from Molè et al. were processed as previously described10. For Zhou et al.27, read1 files were trimmed using cutadapt74 for the reported adapter sequence. Trimmed reads were then aligned using the kb-python kb count function with custom specifications (-x 1,0,8:1,8,16:0,0,0) and the custom barcode whitelist available. Each pair of fastqs was processed individually into barcode–gene matrices and concatenated. For Xiang et al.26, a batch file was generated with cell ID, read1 and read2 for each fastq pair listed. Kallisto’s pseudo –quant command was then used to generate a cell ID–gene matrix. For Blakely et al.36, reads were aligned using kallisto pseudo –quant. For Petropoulos et al.45, single-end reads were processed with kallisto pseudo –quant with a pre-made batch file as above with 43 base pair read length specified. Cynomolgus datasets: for Ma et al.28, read1 fastqs were trimmed using cutadapt for TSO and polyA tail as described in the original publication. Next, kb python’s kb count function was used with custom specifications (–x 1,0,8:1,8,16:0,0,0). For Yang et al.35, reads were aligned using kb python’s kb count command with 10xv3 technology specified. For Nakamura et al.21, available count tables were used given the use of SOLiD sequencer limiting re-alignment program options. Mouse datasets: for Mohammed et al.70, kallisto pseudo –quant with a generated batch file was used to generate a cell ID–gene matrix. For Deng et al.69 and Cheng et al.68, single-end reads were aligned with kallisto pseudo –quant. Finally, for Pijuan-Sala et al.71, each sample set of 33 fastq files was aligned with kb count, with 10xv1 technology specified. The resulting set of barcode–gene matrices was then concatenated for downstream analysis.
Following re-alignment, any datasets not generated using unique molecular identifier counts were normalized using quminorm75. First, matrices were converted to transcripts per kilobase million (TPM), and then the TPM matrix ran through quminorm with a shape parameter up to a maximum of 2 that did not create not available/applicable (NA) values in the matrix. Then, each individual dataset was made into a Seurat object76. Each individual dataset was then merged into a species-specific Seurat object, with SCT batch correction applied across datasets. Clusters were identified on the basis of canonical marker expression. To perform module scoring, gene lists were obtained from rWikiPathways77. For the monkey and mouse, gene symbols were converted to human homologues using bioMart78. Seurat’s AddModuleScore function was used with WikiPathway gene lists of interests as input. For CellPhoneDB analysis41, human data were split on the basis of stage, and subset matrix and metadata for cell type were output as txt files. CellPhoneDB was then run with respective files and –counts-data set to gene_name. Data visualization was performed using Seurat’s DimPlot, FeaturePlot and VlnPlot functions, Scillus’ (https://scillus.netlify.app) Plot_Measure function, pheatmap and CellPhoneDB’s dotplot function.
The scripts used for analyses are available at ref. 79.
Thawing and in vitro culture of human embryos
Human embryos were thawed and cultured as described previously10,24. Briefly, cryopreserved human blastocysts (day 5 or 6) were thawed using the Kitazato thaw kit (VT8202-2, Hunter Scientific) according to the manufacturer’s instructions. The day before thawing, TS solution was placed at 37 °C overnight. The next day, IVF straws were submerged in 1 ml pre-warmed TS for 1 min. Embryos were then transferred to DS for 3 min, WS1 for 5 min and WS2 for 1 min. These steps were performed in reproplates (REPROPLATE, Hunter Scientific) using a STRIPPER micropipette (Origio). Embryos were incubated at 37 °C and 5% CO2 in normoxia and in pre-equilibrated human IVC1 supplemented with 50 ng ml−1 insulin growth factor-1 (IGF1) (78078, STEMCELL Technologies) under mineral oil for 1–4 h to allow for recovery. Following thaw, blastocysts were briefly treated with acidic Tyrode’s solution (T1788, Sigma) to remove the zona pellucida and placed in pre-equilibrated human IVC1 in eight-well µ-slide tissue culture plates (80826, Ibidi) in approximately 400 µl volume per embryo per well. Half medium changes were done every 24 h. For small-molecule experiments, human IVC1 was supplemented with either 2 µM A83-01 (72022, STEMCELL Technologies)80,81, 25 ng ml−1 Activin-A (Qk001, QKINE)82,83,84, 200 nM LDN (S2618, SelleckChem)85,86, 50 ng ml−1 BMP6 (SRP3017, Sigma Aldrich)85,86, 20 µM DAPT (72082, STEMCELL Technologies)87,88,89,90, 10 µM Compound-E (ab142164, Abcam)91,92,93, 20 µM MK-0752 (S2660, Selleck Chemicals)94,95,96 or dimethyl sulfoxide (DMSO) for 48 h. In all cases, these concentrations fall within a range of those used for either vertebrate embryos or complex human ES cell-derived models of the embryo. Within these ranges, a low-to-intermediate concentration was selected to avoid non-specific cytotoxic effects while still considering the higher concentration needed for embryo permeation compared with minimal 2D cell culture to achieve inhibitor action. Further, all small molecules and proteins were tested on human ES cells to validate the efficacy and test for cytotoxicity. For analysis, embryos were fixed in 4% paraformaldehyde for 20 min at room temperature for downstream analysis.
Recovery of mouse embryos and in vitro culture
Pregnant, time-staged mice were culled by cervical dislocation, and uteri were dissected and placed in M2 medium (pre-warmed if embryos were for in vitro culture, ice cold if for fixing). E3.5 blastocysts were flushed out of uteri of pregnant females and either fixed for immunofluorescence analysis or transferred to acidic Tyrode’s solution for zona pellucida removal. Embryos were cultured for 48 h in CMRL (11530037, Thermo Fisher Scientific) supplemented with 1× B27 (17504001, Thermo Fisher Scientific), 1× N2 (made in-house), 1× penicillin–streptomycin (15140122, Thermo Fisher Scientific), 1× GlutaMAX (35050-038, Thermo Fisher Scientific), 1× sodium pyruvate (11360039, Thermo Fisher Scientific), 1× essential amino acids (11130-036, Thermo Fisher Scientific), 1× non-essential amino acids (11140-035, Thermo Fisher Scientific) and 1.8 mM glucose (G8644, Sigma) supplemented with 20% foetal bovine serum5,28. Embryos were incubated with 25 ng ml−1 Activin-A, 200 nM LDN, 50 ng ml−1 BMP6, 20 µM DAPT or DMSO for 48 h. For E4.5, E5.5 and E5.75 collections, embryos were dissected directly from the uteri and fixed for analysis. For E5.0 collection, embryos were dissected from the uteri, and Reichert’s membrane was removed before culturing or 36 h with relevant small molecules as described above.
Human ES cell culture
Shef6 human ES cells (R-05-031, UK Stem Cell Bank) were routinely cultured on 1.6% v/v Matrigel (354230, Corning) in mTeSR1 medium (85850, STEMCELL Technologies) at 37 °C and 5% CO2. Cells were passaged every 3–5 days with TrypLE Express Enzyme (12604-021, Thermo Fisher Scientific). The ROCK inhibitor Y-27632 (72304, STEMCELL Technologies) was added for 24 h after passaging. Cells were routinely tested for mycoplasma contamination by polymerase chain reaction. To convert primed human ES cells to RSeT or PXGL naive conditions, cells were passaged onto mitomycin-C inactivated CF-1 MEFs (3 × 103 cells cm−2; GSC-6101G, Amsbio) in human ES cell medium containing Dulbecco’s modified Eagle medium (DMEM)/F12 supplemented with 20% Knockout Serum Replacement (10828010, Thermo Fisher Scientific), 100 µM β-mercaptoethanol (31350-010, Thermo Fisher Scientific), 1× GlutaMAX (35050061, Thermo Fisher Scientific), 1× non-essential amino acids, 1× penicillin–streptomycin and 10 ng ml−1 FGF2 (University of Cambridge, Department of Biochemistry) and 10 µM ROCK inhibitor Y-27632 (72304, STEMCELL Technologies). For RSeT conversion, cells were switched to RSeT medium (05978, STEMCELL Technologies). Cells were maintained in RSeT and passaged as above every 4–6 days. For PXGL conversion, previously described protocols were used97. Briefly, cells were cultured in hypoxia and medium was switched to chemically Resetting Media 1 (cRM-1), which consists of N2B27 supplemented with 1 µM PD0325901 (University of Cambridge, Stem Cell Institute), 10 ng ml−1 human recombinant LIF (300-05, PeproTech) and 1 mM valproic acid. N2B27 contains 1:1 DMEM/F12 and Neurobasal A (10888-0222, Thermo Fisher Scientific) supplemented with 0.5× B27 (10889-038, Thermo Fisher Scientific) and 0.5× N2 (made in-house), 100 µM β-mercaptoethanol, 1× GlutaMAX and 1× penicillin–streptomycin. cRM-1 was changed every 48 h for 4 days. Subsequently, medium was changed to PXGL–N2B27 supplemented with 1 µM PD0325901, 10 ng ml−1 human recombinant LIF, 2 µM Gö6983 (2285, Tocris) and 2 µM XAV939 (X3004, Merck). PXGL cells were passaged every 4–6 days using TrypLE (12604013, Thermo Fisher Scientific) for 3 min, and 10 µM ROCK inhibitor Y-27632 and 1 µl cm−2 Geltrex (A1413201, Thermo Fisher Scientific) were added at passage for 24 h.
For small-molecule experiments, primed or PXGL human ES cells were plated into ibiTreat dishes at normal passage densities. Forty-eight hours after passage, medium was changed to N2B27 supplemented with 25 ng ml−1 Activin-A, 2 µM A83-01, 50 ng ml−1 BMP6, 200 nM LDN or 20 µM DAPT. Plates were then fixed for 20 min in 4% paraformaldehyde for downstream analysis. For 3D culture of primed human ES cells, 30,000 cells were resuspended in 200 µl of ice-cold Geltrex and the resulting mix was plated into a single well of an 8 µ-well ibiTreat dish. Geltrex was polymerized by placement at 37 °C for 10 min. Two-hundred microlitres of mTeSR1 with ROCK inhibitor Y-27632 was added after polymerization. Twenty-four hours later, the medium was changed to N2B27 (±10 µM DAPT). Medium was refreshed 24 h later, and the plate was fixed in 4% paraformaldehyde for 30 min after a total of 48 h in experimental conditions. Conditioned medium experiments were performed as described previously48. Briefly, 80 µl of ice-cold Geltrex was added to an 8 µ-well ibiTreat dish to create a 100% Geltrex bed. This was polymerized at 37 °C for 4 min. A total of 1 × 103 cells cm−2 primed human ES cells were then added onto this bed in DMEM/F12 and allowed to settle for 15 min. After this, medium was carefully switched to conditioned medium (described below) with 5% Geltrex (v/v) and 10 µM ROCK inhibitor Y-27632. Conditioned medium with 5% Geltrex was refreshed daily for the next 2 days, and the resulting spheroids were fixed after a total of 72 h.
Human YSLC culture
YSLC differentiation was carried out as published48. Briefly, Shef6 human ES cells cultured in RSeT medium for at least 2 weeks were plated onto ibiTreat dishes at 1 × 103 cells cm−2 in RSeT medium with 10 µM Y-27632. Medium was changed the next day to ACL differentiation medium consisting of N2B27 supplemented with 5% v/v Knockout Serum Replacement, 100 ng ml−1 Activin-A, 3 µM CHIR99021 (University of Cambridge Stem Cell Institute) and 10 ng ml−1 human recombinant LIF. Medium was refreshed every 48 h, and 2 µM A83-01, 200 nM LDN or 20 µM DAPT was added to ACL medium for 48 h from either day 2 to day 4, followed by fixation, or day 4 to day 6 followed by fixation. For conditioned medium experiments, at day 6 cells were washed three times with phosphate-buffered saline and then mTeSR Plus medium (100-0276; STEMCELL Technologies) was added for 24 h. Medium was collected from YSLCs and passed through a 0.45-µm filter (16555, Sartorious), and stored for up to 1 week at 4 °C.
Mouse ES cell culture
CD1 mouse ES cells (generous gift from Prof. Jennifer Nichols (Stem Cell Institute, University of Cambridge, UK)) were routinely cultured on gelatin-coated (G7765, Sigma Aldrich) dishes in N2B27 supplemented with 1 µm PD0325901, 3 µm CHIR99021 and 10 ng ml−1 mouse Lif (University of Cambridge, Stem Cell Institute). Medium was changed every 48 h, and cells were passaged every 3–5 days using trypsin–ethylenediaminetetraacetic acid (25300062; Life Technologies). For experiments, cells were passaged as normal into ibiTreat dishes. The following day, medium was switched to either N2B27 + 2iLif, N2B27, or N2B27 + 200 nM LDN. Medium was refreshed after 24 h, and cells were fixed after 48 h.
Immunostaining
Embryos were fixed in 4% paraformaldehyde, permeabilized in 0.1 M glycine with 0.3% Triton X-100 and placed in blocking buffer containing 1% bovine serum albumin and 10% foetal bovine serum. Primary antibodies were diluted in blocking buffer and added overnight at 4 °C. Fluorescently tagged secondary antibodies were added for 2 h at room temperature. Primary antibodies used in this study are as follows: mouse monoclonal anti OCT3/4 (sc5279, Santa Cruz; 1:200 dilution), rat monoclonal anti SOX2 (14-19811-82, Thermo Fisher Scientific; 1:500 dilution), goat polyclonal anti NANOG (AF1997 R&D Systems; 1:500 dilution), rabbit monoclonal anti GATA6 (5851, Cell Signaling Technology; 1:2,000 dilution), goat polyclonal anti GATA6 (AF1700, R&D Systems; 1:200 dilution), mouse anti monoclonal Cdx2 (MU392-UC, Biogenex; 1:200 dilution), goat polyclonal anti CER1 (AF1075, R&D Systems; 1:250 dilution), rat monoclonal anti Cerebus1 (MAB1986, R&D Systems; 1:200 dilution), rabbit monoclonal anti Phospho-Smad1(Ser463/465)/Smad5(Ser463/465)/Smad9(Ser465/467) (13820T, Cell Signaling Technology; 1:200 dilution), rabbit monoclonal anti Smad2.3 (8685T, Cell Signaling Technology; 1:200 dilution), rabbit monoclonal anti-cleaved caspase 3 (9664, Cell Signaling Technology; 1:200 dilution), mouse monoclonal anti Podocalyxin (MAB1658, R&D Systems; 1:500 dilution), goat polyclonal anti Brachyury (AF2085, R&D Systems; 1:500 dilution), rat monoclonal anti GATA4 (14-9980-82, Thermo Fisher Scientific; 1:500 dilution), goat polyclonal anti AP2-gamma (AF5059, R&D Systems; 1:500 dilution), goat polyclonal anti Otx2 (AF1979, R&D Systems; 1:1,000 dilution) and Alexa Flour 594 Phalloidin (A12381, Thermo Fisher Scientific; 1:500 dilution).
Quantifications
Immunofluorescence images were captured on a Leica SP8 confocal and processed and analysed using Fiji (http://fiji.sc). Epiblast, hypoblast and CER1-positive cell numbers were manually counted using the multi-point cell counter plugin. Quantification of trophectoderm was performed using Imaris software (version 9.1.2) using the spots tool with manual curation. To quantify n/c SMAD2.3 in human and mouse embryos, the central three planes of individual cells were used to generate a three-plane z-stack. Individual 4′,6-diamidino-2-phenylindole (DAPI)-positive nuclei were used to generate a nuclear mask using the ‘Analyze Particles’ function on either the DAPI or lineage-associated transcription factor channel. The adjacent cytoplasmic area was drawn individually for each nucleus and the mean fluorescence of each region was measured, and the ratio computed. When embryos were stained with E-Cadherin, the membrane was delineated to allow for cytoplasmic region of interest determination. When embryos were stained with podocalyxin, the cytoplasmic region of interest was drawn to ensure delineation of a region captures suitable intra-cellular variation allowing for valid normalization. Measurements were computed on raw SMAD2.3 signal. To quantify pSMAD1.5.9 nuclear intensity, a nuclear mask generated on a central three-plane z-stack for each nucleus, and mean fluorescence values were measured. Within each three-plane z-stack, a background fluorescence taken adjacent to or within a cavity of the embryo was used for background normalization (\(\frac{\text{mean nuclear intensity}}{1+\text{mean background intensity}}\)). Background was normalized to (that is, to provide a comparable signal-to-noise ratio) rather than subtracted to account for the variability in laser penetration between experiments and z-planes. In stem cell experiments, nuclear masks were generated, mean fluorescence was measured, and all values were normalized to a control (DMSO) value of 1. To calculate the percentage of cleaved caspase-3-positive or CER1-positive cells, individual cells were manually counted using the cell counter plugin and presented as a percentage of all DAPI-negative or GATA6-positive cells. For 3D spheroid classification, the total number of structures was counted manually using the Cell Counter plugin, and each was assigned to a class of spheroid. For conditioned medium 3D spheroid quantifications, the central three planes of individual spheroids were used to generate a nuclear mask on the DAPI channel, and the mean nuclear pSMAD1.5.9 signal was quantified along with the signal of an acellular region for background normalization. To generate figures, images were processed by generating z-stacks of approximately five to ten planes to allow for visualization of embryo topology with cells on disparate planes followed by consistent adjustment of brightness and contrast.
Statistics and reproducibility
No statistical method was used to pre-determine sample sizes. Sample sizes are similar to previous publications7,10,24. For characterization of normal development, embryos lacking any of the three lineages were excluded. Multiple SMAD fluorescence intensities were taken per lineage per embryo. All embryos were included in functional experiments and each cell type count is taken from individual embryos. All stem cell experiments were performed independently at least twice. Investigators were not blinded to group allocation during the experiment or analysis, as blinding would not have been possible due to medium preparation and changing requirements. Group allocation was not performed randomly; rather, based on visual assessment of embryos, investigators attempted to ensure balanced distributions of blastocysts/implanting embryos assessed as expanded with nice inner cell masses versus embryos that appeared delayed or with visible cell death across experimental groups. Statistical tests, except the Bayesian distribution model, were performed in Prism 9 (GraphPad), and where relevant, two-sided tests were used. Normality was tested with a Shapiro–Wilk test. Bayesian distribution modelling, which is suited to the small sample sizes used in human embryo studies, was used as a supplemental tool to assess how each small-molecule treatment affected the distribution of cell number. To do this, the brms R package was used98,99, with the assumption of a Poisson distribution and the Control counts set to inform the priors and be used as reference. Brms’ default Markov chain Monte Carlo settings were used. Coefficient ± credible intervals were either below −0.33, denoting a decrease in the distribution compared to control, or above 0.33, indicating an increase in the distribution compared to control. Credible intervals that bridged this range indicate no significant difference. All coefficients and credible intervals in addition to Mann–Whitney test P values are presented in Supplementary Table 8. For data presentation, box plots encompass the 25th to 75th percentile in the box, with the median marked by the central line, and the mean marked by a cross. The minimum and maximum are marked by the whiskers. For violin plots, the dashed line marks the median value and dotted lines mark the 25th and 75th percentiles. For summary plots (for example, Fig. 2h,i), the mean ± standard error of the mean is plotted.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
All raw data used here are previously published and publicly available. For aligning sequencing data, GRCh38 (https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.26/), Genome assembly Macaca_fascicularis_5.0 (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000364345.1/) and GRCm39 (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000001635.27/) were used. For human data: Molè et al.10, ArrayExpress E-MTAB-8060; Xiang et al.26, Gene Expression Omnibus GSE136447; Zhou et al.27, Gene Expression Omnibus GSE109555; Petropoulos et al.45, ArrayExpress E-MTAB-3929; Blakely et al.36, Gene Expression Omnibus GSE66507. For cynomolgus monkey data: Yang et al.35, Gene Expression Omnibus GSE148683; Ma et al.28, Gene Expression Omnibus GSE130114; Nakamura et al.21, Gene Expression Omnibus GSE74767. For mouse data: Pijuan-Sala et al.71, ArrayExpress E-MTAB-6967; Mohammed et al.70, Gene Expression Omnibus GSE100597; Cheng et al.68, Gene Expression Omnibus GSE109071; Deng et al.69, Gene Expression Omnibus GSE45719. Scripts used for analysis are available at ref. 79. The integrated Seurat objects for each species are available on Zenodo100 (https://doi.org/10.5281/zenodo.7689580). All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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Acknowledgements
The authors thank patients at CARE, Herts & Essex, Bourn Hall Fertility and King’s Fertility Clinics for their generous donations, as well as the embryologists and members of each clinic for facilitating donations. We thank G. Serapio-García for advice on Bayesian statistical analysis and the bioinformaticians and data scientists who made their code, packages and vignettes available. This work is supported by Wellcome Trust (207415/Z/17/Z) and Open Atlas and NOMIS awards to M.Z.-G. B.A.T.W. was supported by the Gates Cambridge Trust. C.W.G. was supported by a Leverhulme Trust Early Career Fellowship. L.K.I.-S. was supported by the Rosetrees Trust.
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B.A.T.W., A.W., C.W.G. and L.K.I.-S. thawed and cultured human embryos for research. B.A.T.W. performed single-cell sequencing analyses. Z.B., A.B., A.C., P.C., C.D., P.E., S.F., S.G.V., M.L., R.O., C.P., N.C., L.R., A.M., L.W., L.C., K.E. and P.S. interfaced with patients, prepared informed consent documentation and prepared embryos for transfer from clinical to research setting. A.W. dissected and cultured mouse embryos. B.A.T.W. and C.W.G. cultured and differentiated human and mouse stem cells. B.A.T.W. and C.W.G. performed quantitative image analyses. B.A.T.W., C.W.G., A.W. and M.Z.-G. wrote the manuscript and conceived the study. B.A.T.W., A.W. and C.W.G. contributed equally.
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Nature Cell Biology thanks Alta Charo, Guoping Fan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Generation of combined cynomolgus monkey dataset.
a, Uniform Manifold Approximation Projection (UMAP) of cynomolgus monkey scRNA-seq datasets colored by original publication. N = 10221 single cells. b, UMAP of monkey datasets colored by age of embryo. c, UMAP of monkey datasets colored by assigned cell type. d, UMAPs of monkey datasets colored by a gradient indicating gene expression for canonical markers of epiblast (EPI), hypoblast (HYPO), extraembryonic mesenchyme (EXMC), primitive streak, and trophectoderm/trophoblast (TE/TrB). Extraembryonic mesenchyme markers are also plotted on human dataset. e, Heatmap of average WikiPathways signaling pathway module score split by cell type and stage. Visualized value is calculated average scaled score from individual single-cell module scores. Gene names were converted from mfac to hgnc gene symbols for module scoring.
Extended Data Fig. 2 Generation of combined mouse dataset.
a, Uniform Manifold Approximation Projection (UMAP) of mouse scRNA-seq datasets colored by original publication. N = 16395 single cells. b, UMAP of mouse datasets colored by age. c, UMAP of mouse datasets colored by assigned cell type. d, UMAPs of mouse datasets colored by a gradient indicating gene expression for canonical markers of epiblast (EPI), primitive endoderm/visceral endoderm (PrE/VE), primitive streak, and trophectoderm/extraembryonic ectoderm (TE/ExE). e, Heatmap of average WikiPathways signaling pathway module score split by cell type and stage. Visualized value is calculated average scaled score from individual single-cell module scores. Gene names were converted from mmus to hgnc gene symbols for module scoring.
Extended Data Fig. 3 Signaling dynamics during human implantation.
a, Average scaled WikiPathways module scores for combined human dataset between lineages. b, Expression of WNT, FGF, BMP and NODAL associated genes plotted by stage during human, macaque, and mouse pre-gastrulation development. c-d, CellphoneDB dotplots for early post-implantation (c) and late post-implantation (d) stages depicting the predicted activity of individual receptor-ligand pairs. Each ligand-receptor pair is color matched to the expressing lineage on the x-axis.
Extended Data Fig. 4 Total SMAD2.3 quantification methodology.
a, Examples from a segregated blastocyst presented in Fig. 2a of Epiblast (EPI), Hypoblast (HYPO) and Trophectoderm (TE) cells with nuclear (solid line) and cytoplasmic (dotted line) regions of interest (ROI) outlined. The measured nuclear (Avg. Nuc) and cytoplasmic (Avg. Cyto) and calculated nuclear/cytoplasmic ratio (N/C) of total SMAD2.3 intensity for individual cells is shown. b-c, Examples from a segregating blastocyst (b) and day 7 embryo (c) of cells with nuclear and cytoplasmic ROIs outlined. Measured nuclear, cytoplasmic, and calculated nuclear-to-cytoplasmic ratios of total SMAD2.3 intensities are shown for each cell. Note that here the raw, unprocessed SMAD2.3 signal is shown for the central 3-plane z-stacks used directly for quantification. N = 14 experiments. d-e, Examples of segregating (d) and segregated (e) human blastocysts. Inner cell mass cells co-expressing epiblast and hypoblast markers are marked with grey asterisks. N = 3 experiments. f-h, Violin plots depicting expression of NODAL receptors ACVR1B, ACVR2A, and ACVR2B across stages and lineages in human single cell RNA sequencing data. N = 9862 single cells. Scale bars: 100 µm.
Extended Data Fig. 5 Characterization of NODAL signaling across mouse implantation.
a, Mean ± S.E.M. nuclear-to-cytoplasmic ratio of total SMAD2.3 within each lineage of segregating and segregated blastocysts, day 7, and day 9 embryos, separated by individual embryo. Note consistency of trends, particularly of TE versus ICM enrichment in segregating blastocysts, and of hypoblast versus epiblast. b, Immunofluorescence images of mouse embryos at E3.5 (N = 10 embryos), E4.5 (N = 6 embryos), E5.5 (N = 6 embryos) and E5.75 (N = 6 embryos) stained for total Smad2.3, DAPI, and either Sox2 or Gata6. c-f, Quantification of nuclear and cytoplasmic Smad2.3. fluorescence and their ratio. g-i, Quantification of the nuclear/cytoplasmic ratio of Smad2.3 within the epiblast (g; n = 63 E3.5, 50 E4.5, 95 E5.5, 95 E5.75 cells), primitive endoderm/visceral endoderm (PrE/VE; n = 69 E3.5, 42 E4.5, 105 E5.5, 129 E5.75 cells) (h) or trophectoderm/extraembryonic ectoderm (TE/ExE) (i; n = 62 E3.5, 60 E4.5, 89 E5.5, 69 E5.75 cells) over time. For Violin plots c-f, central dotted line denotes median and dotted lines mark the 25th and 75th quartiles. Statistical tests: (c) two-sided unpaired T-test; (d-f) Kruskal Wallis with Dunn’s post-hoc; ****p < 0.0001, ***p < 0.001, *p < 0.05. Unmarked pairwise comparisons are not significant (ns). Exact p-values presented in Supplemental Table 8. Scale bars: 100 µm.
Extended Data Fig. 6 NODAL modulation in human ESC, mouse embryos, and effect of NODAL modulation on human trophectoderm.
a-b, Immunofluorescence images and quantification of primed hESCs cultured with either DMSO (n = 2588), 50 ng/ml Activin-A (n = 2465), or Activin-A with 2 µM A83-01 (n = 2463) for 48 hours. c-d, Bayesian coefficients ± credible interval for counts presented in Fig. 3c–e (c) and Fig. 3h–j. (d). e, Immunofluorescence images of mouse embryos recovered at E3.5 and cultured in vitro for 48 h in control (N = 19), 25 ng/ml Activin-A (N = 13 embryos) or 2 µM A83-01 (N = 13 embryos) conditions. f-g, Quantification of the number of epiblast (f) and primitive endoderm/visceral endoderm (g) from e. h, Immunofluorescence images of mouse embryos recovered at E5.0 and cultured in vitro for 36 h in control (N = 8), Activin-A (N = 8), or A83-01 (N = 6) conditions. i, Quantification of the proportion of embryos that successfully made an egg cylinder and the proportion of egg cylinders with a Cer1-positive DVE/AVE. j, Quantification of epiblast length. k, Quantification of epiblast area. l, Immunofluorescence images of embryos cultured from day 5 to 7 in control (N = 8 embryos), 2 µM A83-01 (N = 6 embryos), or 25 ng/ml Activin-A (N = 6 embryos) conditions. m-o, Quantification of the number of total GATA3-positive (GATA3 + ) (m), GATA3 + /GATA6-negative (-) (n), and GATA3 + /GATA6+ trophectoderm/trophoblast cells (o) at day 7. p, Bayesian coefficients ± credible interval for counts presented in m-o. (N = 8 control, 8 Activin-A, and 6 A83-01 treated embryos). q, Quantification of the percentage of embryos cultured from day 5 to 7 in control conditions (N = 16), A83-01 (N = 12), or Activin-A (N = 13) that underwent in vitro implantation. r, Immunofluorescence images of a day 9 embryo cultured in control conditions (N = 4 embryos). s, Quantification of the number of total GATA3 + , GATA3 + /GATA6-, and GATA3 + /GATA6+ cells at day 9. For box plots, the box encompasses the 25th-75th quartiles with the median marked by the central line. The minimum and maximum are denoted by the whiskers. For violin plots, the mean is marked by the red line. Statistical tests: (b, j-k) One-way ANOVA with Tukey-Kramer post-hoc; (f-g, m-o) two-sided Mann-Whitney test; (q) Fisher’s Exact Test. ****p < 0.0001. **p < 0.01, *p < 0.05. Unmarked pairwise comparisons are not significant (ns). Exact p-values presented in Supplemental Table 8. Scale bars: (a) 50 µm, (e, h, l, r) 100 µm.
Extended Data Fig. 7 Comparative characterization of BMP signaling across mouse implantation.
a, Examples from a segregated blastocyst presented in Fig. 4a of Epiblast (EPI), Hypoblast (HYPO) and Trophectoderm (TE) cells with nuclear region of interest (ROI) outlined. The measured nuclear (Avg. Nuc) and background and calculated normalized (Nuc/1+Background) pSMAD1.5.9 intensity for individual cells is shown. b-c, Examples from a segregating blastocyst (b) and day 7 embryo (c) of cells with nuclear and background ROIs outlined. Measured nuclear and background (bg) and calculated normalized pSMAD1.5.9 intensities are shown for each cell. Note that here the raw, unprocessed pSMAD1.5.9 signal is shown for the central 3-plane z-stacks used directly for quantification. Cells that are central to the same 3-plane z-stack use the same background value for normalization. d, Mean ± S.E.M. normalized pSMAD1.5.9 intensity within each lineage of segregating (N = 9) and segregated blastocysts (N = 9), day 7 (N = 6), and day 9 embryos, separated by individual embryo. Note consistency of trends, particularly of TE versus ICM enrichment in segregating blastocysts, and of hypoblast versus epiblast in segregated blastocysts and at day 7. e, Immunofluorescence images of mouse embryos at E3.5 (N = 9 embryos), E4.5 (N = 4 embryos), E5.5 (N = 6 embryos) and E5.75 (N = 5 embryos) stained for phosphorylated (p)Smad1.5.9, DAPI and either Cdx2 or Gata6. f-i, Quantification of normalized pSmad1.5.9 fluorescence between lineages at the different stages of mouse development depicted in e. j-l, Quantification of normalized pSmad1.5.9 in epiblast (EPI) (j; n = 69 E3.5, 22 E4.5, 82 E5.5, 93 E5.75 cells), primitive endoderm/visceral endoderm (PrE/VE) (k; n = 69 E3.5, 26 E4.5, 102 E5.5, and 121 E5.75 cells) (j) or trophectoderm/extraembryonic ectoderm (TE/ExE) (l; n = 60 E3.5, 28 E4.5, 62 E5.5, and 69 E5.75 cells) across stages. For Violin plots e-h, central dotted line denotes median and dotted lines mark the 25th and 75th quartiles. Statistical tests: (f) two-sided Mann Whitney, (g-i) Kruskal Wallis with Dunn’s post-hoc; ****p < 0.0001, ***p < 0.001, *p < 0.05. Unmarked pairwise comparisons are not significant (ns). Exact p-values presented in Supplemental Table 8. Scale bars: 100 µm.
Extended Data Fig. 8 Validation of BMP modulation, comparative functional modulation in mouse, and divergent roles for BMP in naïve pluripotency.
a, Violin plots of BMP2/4/6 expression. N = 9862 single cells. b-c, Immunofluorescence images and quantification of primed hESCs cultured in control conditions (n = 2825), 25 ng/ml BMP2 (n = 2184), 25 ng/ml BMP4 (n = 1681), 50 ng/ml BMP6 (n = 889), or 50 ng/ml BMP6 + 200 nM LDN193189 (n = 1406). d-e, Bayesian coefficients ± credible interval for counts presented in Fig. 5b-d (d) and Fig. 5f-h (e). f, Immunofluorescence images of mouse embryos recovered at E3.5 and cultured in vitro for 48 h in control (N = 19), 50 ng/ml BMP6 (N = 11), or 200 nM LDN193189 (N = 13) conditions. g-h, Quantification of the number epiblast (g) and primitive endoderm/visceral endoderm (h) from f. i, Immunofluorescence images of mouse embryos recovered at E5.0 and cultured in vitro for 36 h in control (N = 8), BMP6 (N = 10) or LDN193189 (N = 8) conditions. j, Quantification of the proportion of embryos that successfully made an egg cylinder and have a Cer1-positive DVE/AVE. k-l, Quantification of the epiblast length (k) and area (l). m, Immunofluorescence images and quantification of hESCs treated for 48 hours with control conditions (PXGL n = 2336, primed n = 7796) or 200 nM LDN193189 (PXGL n = 2398, primed n = 6500, N = 2 experiments). n, Pearson regression correlation coefficients for ID1 and ID3 expression with naïve pluripotency markers. o, Immunofluorescence and quantification of AP2γ in hESCs (LDN: n = 1695 untreated cells; n = 1426 LDN treated cells; N = 2 experiments). p, Immunofluorescence and quantification of Otx2 in mESCs cultured in either 2iLif naïve conditions (n = 1570 cells), N2B27 (n = 3602 cells), or N2B27 + LDN (n = 1064 cells). N = 3 experiments. q, Immunofluorescence images and quantification of pSMAD1.5.9 in YSLCs (control: n = 1150; LDN: n = 827; N = 2 experiments). For box plots, the box encompasses the 25th-75th quartiles with the median marked by the central line. The minimum and maximum are denoted by the whiskers. For violin plots, the mean is marked by the red line. Statistical tests: (c, k-l, p) One-way ANOVA with Tukey-Kramer post-hoc; (e-f) two-sided Mann-Whitney test, (m, o, q) two-sided unpaired T-test; (o) Coefficients were tested (cor.test; two-tailed) and corrected for multiple hypothesis testing with the Benjamini-Hochberg method; ****p < 0.0001. ***p < 0.001. **p < 0.01. *p < 0.05. Unmarked pairwise comparisons are not significant (ns). Exact p-values presented in Supplemental Table 8. Scale bars: (b, m, o-q) 50 µm, (f, i) 100 µm.
Extended Data Fig. 9 Comparative functional Notch interrogation of mouse.
a-b, Bayesian coefficients ± credible interval for counts presented in Fig. 6b–d (a) and Fig. 6f–h (b). c, Immunofluorescence images of representative mouse embryo recovered at E3.5 and cultured in vitro for 48 hours in either control conditions (N = 19 embryos) or with 20 µM DAPT added to the media (N = 12 embryos). d-e, Quantification of number of epiblast (d) and primitive endoderm/visceral endoderm (e) from c. f, Immunofluorescence images of a representative mouse embryo recovered at E5.0 and cultured in vitro for 36 h in either control conditions (N = 8 embryos) with 20 µM DAPT added to the media (N = 8 embryos). g, Quantification of the proportion of embryos that successfully made an egg cylinder and of the proportion of egg cylinders that had a Cer1-positive DVE or AVE. h, Quantification of epiblast length in µm at the central plane. i, Quantification of epiblast area in µm2 at the central plane. For box plots, the box encompasses the 25th-75th quartiles with the median marked by the central line. The minimum and maximum are denoted by the whiskers. Statistical tests: (d-e) Two-sided Mann-Whitney test; (h-i) two-sided unpaired T-test; **p < 0.01. Exact p-values presented in Supplemental Table 8. Scale bars = 100 µm.
Extended Data Fig. 10 Proposed model of peri-implantation signaling activity and anterior specification.
a, Proposed model of BMP, NODAL and NOTCH signaling activity in the Hypoblast (top) and Epiblast (bottom) during human peri-implantation. Note implantation as a switch point in the activity of pathways for the epiblast, and a point of divergence of subpopulations within the hypoblast. The role of NOTCH in the hypoblast remains less clear, indicated by a gradient. b, Summary of signaling perturbation effects on human anterior hypoblast (top) and mouse distal/anterior visceral endoderm (DVE/AVE; bottom) specification. Schematics of human embryos correspond to day 5, day 7, and day 9 and mouse embryo schematics correspond to E3.5, E5.0, E5.75.
Supplementary information
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Captions for Supplementary Tables 1–8.
Supplementary Table
Supplementary Table 1. Human differential gene expression analysis. Tables denoting differentially expressed genes identified by receiver-operator characteristic (ROC) analysis either between epiblast versus hypoblast versus trophectoderm/trophoblast, between trophoblast lineages, and across stages within each lineage for integrated human embryo scRNA-seq data. Supplementary Table 2. Cynomolgus monkey differential gene expression analysis. Tables denoting differentially expressed genes identified by ROC analysis either between epiblast versus hypoblast versus trophectoderm/trophoblast versus extra-embryonic mesenchyme and across stages within each lineage for integrated cynomolgus monkey embryo scRNA-seq data. Supplementary Table 3. Mouse differential gene expression analysis. Tables denoting differentially expressed genes identified by ROC analysis either between epiblast versus visceral endoderm versus extra-embryonic ectoderm and across stages within each lineage for integrated mouse embryo scRNA-seq data. Supplementary Table 4. Human Embryo average expression. Average expression following Seurat’s SCTransform normalization for each stage across lineages in combined human scRNA-seq data. Supplementary Table 5. Cynomolgus monkey embryo average expression. Average expression following Seurat’s SCTransform normalization for each stage across lineages in combined cynomolgus monkey scRNA-seq data. Supplementary Table 6. Mouse embryo average expression. Average expression following Seurat’s SCTransform normalization for each stage across lineages in combined mouse scRNA-seq data. Supplementary Table 7. List of WikiPathways gene modules used. List and annotation of gene lists used for module scoring. Supplementary Table 8. Statistical analysis results. Coefficients and credible intervals for Bayesian analysis and exact P values for all statistical tests presented.
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Weatherbee, B.A.T., Weberling, A., Gantner, C.W. et al. Distinct pathways drive anterior hypoblast specification in the implanting human embryo. Nat Cell Biol 26, 353–365 (2024). https://doi.org/10.1038/s41556-024-01367-1
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DOI: https://doi.org/10.1038/s41556-024-01367-1
