Extended Data Fig. 9: Further characterization of embryoids at S8.

a, Representative bright field images of abnormally developed S8 embryoids. Scale bars, 500 μm. Similar images were obtained in at least 3 independent experiments. b, c, Immunofluorescent analysis showing the expression of SOX1 in S8 embryoids (b) and E8.5 embryos (c), highlighting formation of the neural tube. n = 2 S8 embryoids from 2 experiments, n = 2 E8.5 embryos from 2 experiments. Scale bars, 100 μm. d, e, Immunofluorescent analysis showing the expression of GATA6 and MHC-II in S8 embryoids (d) and E8.5 embryos (e), highlighting initiation of heart formation. n = 3 S8 embryoids from 2 experiments, n = 2 E8.5 embryos from 2 experiments. Scale bars, 100 μm. f, Dot plots showing the average levels and proportion of cells expressing cell type-specific marker genes in embryo-like structures at S8. Cell type-specific marker genes for different lineages in E8.5 mouse embryos were identified using published single-cell transcriptomic datasets (E-MTAB-6967). Expressions of these marker genes in different linages from E8.5 mouse embryos are shown as the control. VE, Visceral endoderm; SE, Surface ectoderm; SC, Spinal cord; SM, Somitic mesoderm; RN, Rostral neurectoderm; PhM, Pharyngeal mesoderm; PGC, Primordial germ cell; PE, Parietal endoderm; PxM, Paraxial mesoderm; NMP, Neuromesodermal progenitor; NC, Neural crest; Mes, Mesenchyme; IM, Intermediate mesoderm; HP, Haematoendothelial progenitors; FB, Forebrain; ExE_M, Extraembryonic mesoderm; ExE_E, Extraembryonic endoderm; Eryth, Erythroid; Endo, Endothelium; DE, Definitive endoderm; CdM, Caudal mesoderm; CMs, Cardiomyocytes; BP, Blood progenitors; Al, Allantois. g, Correlation of gene expression for each identified lineage between embryo-like structures at S8 and natural E8.5 embryos. Correlation coefficients are indicated. Abbreviations are as defined in (f).