Fig 2: 3D-geec recapitulates in utero development from E4.5 to E6.0 by 48 h(A) Comparison of 3D-geec embryos from day 0 (D0) to 2 (D2; bottom) with embryos developed in utero from E4.5 to E6.0 (top). Immunofluorescence images of a representative embryo (see Figure S3 for additional stages) stained for Sox2+ or Oct3/4+ EPI, Gata4+ VE, and asymmetrically localized Lefty1+ AVE (yellow arrowheads).(B and C) Scatterplots showing the length against diameter (B), and cell numbers of PrE/VE covering EPI against EPI (C) for 3D-geec and in utero embryos in log scale.(D) The diameter-to-length ratio for embryos shown in (B).(E) The total (EPI and PrE/VE) cell numbers for embryos shown in (C).(F) Scaled timeline of 3D-geec development based on the total cell numbers (E). 3D-geec embryos correspond to E5.20 and E6.04 in utero embryos after 24 and 48 h in culture, respectively. n = 21 (E4.5), 28 (E4.75), 20 (E5.0), 20 (E5.25), 21 (E5.5), 21 (E5.75), 22 (E6.0), 20 (D1), and 26 (D2).p values calculated using t test (D) and Kruskal-Wallis ANOVA followed by multiple Mann-Whitney U test (E). Scale bars, 50 μm. See also Figures S2 and S3.

Fig 3: Formation of DVE/AVE-like population in VE-like compartment.a UMAP representation of VE-like cluster revealing two subclusters diverging in their transcriptional profile. b Comparison of EmVE-like cluster with published scRNA-Seq dataset for EmVE of natural murine embryos23 revealing high AUCell-based enrichment score and mean expression levels of EmVE genes within EmVE-like cluster. c Schematic representation of DVE and AVE formation in murine embryos between E5.5 and E5.75. d Analysis of AVE marker expression in VE-like subclusters displays higher expression of AVE marker gens inside the EmVE-like cell cluster. e Comparison of published AVE-signatures23 showing high AUCell-based enrichment score and high mean expression for cells of EmVE-like cluster, in contrast to a lower AUC score and mean expression levels in cells of ExVE-like cluster. f Expression of DVE/AVE markers EOMES and OTX2 was detected to colocalize in cells of the EmVE-compartment in RtL-embryoids, indicated by white arrows. OTX2, red; EOMES, green; DAPI, blue. Scale bar = 50 µm. g DVE/AVE marker LEFTY1 was found colocalizing with EOMES + cells, which was detected to be restricted to one side of the EmVE-like compartment in RtL-embryoids (white squares are magnified in h. Scale bar = 25 µm. h Magnification of the EmVE-like compartment as indicated by the white, dotted squares in g. LEFTY1, red; EOMES, green; DAPI, blue. Scale bar = 20 µm. White arrows indicate EOMES + /LEFTY1 + cells. i LEFTY1 + cells were detected in distal, transition or anterior position in RtL-embryoids. LEFTY1, red; GFP, green; DAPI, blue. Scale bar = 50 µm. j Quantification of LEFTY1 + cell location in RtL-embryoids at day 4 (4d) and day 5 (5d) into the protocol. The majority of RtL-embryoids displayed LEFTY1 + cells in either distal or transition positions, with few RtL-embryoids showing localization of LEFTY1 + in an anterior position, relative to the Epi-like compartments, both 4d (red bars) and 5d (blue bars) into the protocol. Total n = 375 for each, 4d and 5d conditions. Data are presented as mean values ± SD. Source data of j are provided as a Source Data file. Experiments were repeated independently at least three times with similar results (f–h).

Fig 4: Characterisation of the putative anterior hypoblast in human embryos at 9 d.p.f.a UMAP of hypoblast cells subdivided into sub-clusters 0-2. Most enriched genes expressed in each sub-cluster are reported in Supplementary Data 8. b Sub-cluster 0 expresses CER1. c, d Immunofluorescence at 9 d.p.f.: CER1 is expressed asymmetrically in a subset of hypoblast cells (GATA6+). N (experimental replicates): 3. N(embryos): 19 e, Quantification CER1+ cells (n = 19 embryos, mean in red ± SEM). f Quantification hypoblast cells (GATA6+) in contact with basal epiblast (n = 19 embryos, mean in red ±SEM). g Percentage of CER1+ cells versus total hypoblast cells per embryo. Boxes represent the 25th/75th percentiles, red line the median, cross the mean, whiskers the min/max, dots individual embryos (n = 19); N(experimental replicates): 3. Source data provided as a Source Data file. h Quantification of angular distribution of CER1+ cells along the hypoblast hemisphere (0° to 180°). N(embryos) n = 28, combined from Supplementary Figure 7f (all embryos included). CER1+ cells show a significant localisation bias towards one side of the hypoblast in 10/28 embryos (Supplementary Figure 7f). i, Correlation analysis of CER1 with WNT, BMP and NODAL antagonists. Co-expression corrected by Benjamini–Hochberg: significant correlations between CER1 and LEFTY1 (p = 4.21E−13), LEFTY2 (p = 2.75E−10), HHEX (p = 1.88E−07), NOG (p = 1.39E−08), DKK4 (p = 4.41E−07), DKK1 (p = 1.65E−05), SFRP1 (p = 0.0047). Correlation with NCLN, CHRD and SOSTDC1 are ns. j Immunofluorescence of LEFTY1 at 9 d.p.f.. N(experimental replicates): 3. N(embryos): 7. k Quantification of LEFTY1+ cells (n = 7 embryos, mean in red ± SEM). l Quantification of angular distribution of LEFTY1 + cells along the hypoblast hemisphere. N(embryos) n = 7, combined from Supplementary Fig. 10a. LEFTY1+ cells showed a statistically significant localisation bias in 2/7 embryos (Supplementary Figure 10a). m Immunofluorescence at 9 d.p.f. of nuclear pSMAD1.5 in a subset of OCT4+ cells (arrows) distant from CER1 domain, where pSMAD1.5 is not detected. N(experimental replicates): 8. N(embryos) stained for pSMAD1.5 = 29; 9/29 embryos (31%) show pSMAD1.5 in OCT4+ cells; 8/9 embryos (89%) display localisation of pSMAD1.5 distant to CER1. Scale bars: 50 μm (Fig. 3c,j, Fig. 3d,m (left)), 25 μm (Fig. 3d,m (right)). Source data are provided as a Source Data file.

Fig 5: Quantitative evaluation of 3D-geec outcome based on AVE asymmetry at D2(A) Schematic to calculate the AVE asymmetry index. A polar plot shows the distribution of VE and AVE cells in the embryo, using the distance and angle from the distal tip. The AVE asymmetry index is calculated based on the position of AVE centroid in this polar plot.(B) Representative 3D projections of E5.25, E6.0, and D2 embryos immunostained for Gata4 (VE) and Lefty1 or Cerl1 (AVE), and their respective polar plots.(C) AVE asymmetry index of E5.25, E6.0, and D2 embryos. AVE position of in utero embryos was first qualitatively classified as asymmetric (blue), symmetric (red), or borderline (gray). No embryos at E5.25 and E6.0 that were classified as symmetric have an AVE asymmetry index larger than 0.15. Thus, we used this value to evaluate 3D-geec D2 embryos for AVE asymmetry. n = 15 (E5.25), 13 (E6.0), and 18 (D2).(D) Summary of 3D-geec efficiency based on quantitative quality control and evaluation.Scale bars, 50 μm. See also Figure S2.