Fig. 3: 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.