Fig. 7: Basement membrane heterogeneity and the AP axis specification in human embryos.

a Schematics of human blastocyst to pre-gastrulation embryogenesis, illustrating the cross-sections of the embryo from pre-implantation blastocyst, peri-implantation (peri), early post-implantation (early post), and mid post-implantation (mid post) focusing on 3 major cell types: epiblast (EPI), hypoblast (HYPO), and trophectoderm/trophoblast (TE/TrB). b Heatmap of scaled average expression (Z-scores) from scRNAseq analyses of in vitro cultured human embryos across pre- to post-implantation embryogenesis showing differential gene expressions of selected genes of basement membrane, MMP, and anterior hypoblast markers in 3 major cell types. c Diagram illustrating the tissue architecture of the embryo in (d). d Representative Airyscan2 super-resolution image showing a fixed in vitro cultured 7dpf human embryo stained for POU5F1(EPI), GATA6 (HYPO), LAMININ (basement membrane), and CER1 (anterior hypoblast). Basement membrane between EPI and HYPO is becoming visible with tiny perforations (green arrowheads). Two CER1 expressing GATA6⁺ cells are visible (yellow arrowheads). Scale bars: 20μm and 10 μm in the inset. e Quantification of EPI, HYPO, and CER1⁺ cell numbers in in vitro cultured Day 7 human embryos. Only GATA6⁺ and CER1⁺ cells are counted as anterior HYPO. n = 10 embryos. f Diagram illustrating embryo architecture in (g) showing a forming primary yolk sac surrounded by GATA6⁺ cells and an amniotic cavity surrounded by the POU5F1⁺ cells. g Cross-section of a fixed in vitro cultured 9dpf human embryo stained with POU5F1 (EPI), GATA6 (HYPO), LAMININ (basement membrane), and CER1 (anterior HYPO). The basement membrane between EPI and HYPO is more visible than in a 7dpf embryo. Four CER1⁺ and GATA6⁺ cells are located at the lower end of the EPI and the left end of the basement membrane. h Quantification of EPI, HYPO, and CER1⁺ cell numbers in the in vitro cultured 9dpf human embryos. n = 14 embryos. i Maximum Z projection of an Airyscan2 super-resolution image, showing uneven basement membrane perforations in a 9dpf human embryo along the EPI-HYPO interface. Scale bars: 20 μm. j Maximum Z projection of the 9dpf embryo in (g), showing a subset of GATA6⁺ cells are CER1⁺ and is located at the bottom-left region. Scale bars: 20μm. k Zoomed-in image of (j), comparing basement membrane perforations in CER1⁺ and CER1^- region. More basement membrane perforations are present in the CER1⁺ region than in the adjacent CER^- region. Scale bars: 5 μm. l Quantification of the basement membrane perforation percentage in CER1⁺ and CER1^- region of 9dpf embryos. n = 3 embryos.