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. 2022 Aug 25;610(7930):143–153. doi: 10.1038/s41586-022-05246-3

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 12 — a,b. Partially dissected natural embryos (a) cultured from E6.5 to E8.5 and day 8 ETiX embryoids (b) highlighting their development within extraembryonic membranes. Legend: HF: headfolds, H: heart, T: tailbud and All: allantois. Scale bars, 100 μm c. Subclustered UMAP of extraembryonic endoderm in the tiny sci-RNA-seq dataset and contribution of each individual timepoint to the subclustered UMAP of the extraembryonic endoderm. d,e. Expression of selected parietal endoderm genes in the subclustered UMAP of extraembryonic endoderm shown separately for natural embryos (d) and ETiX embryoids (e). f,g. Expression of selected extraembryonic visceral endoderm genes in the subclustered UMAP of extraembryonic endoderm shown separately for natural embryos (f) and ETiX embryoids (g). h. Expression of selected markers of endothelium Pecam1, Cd34, Icam1, Tek, Vegfa and Cdh5 in natural embryos and ETiX embryoids in tiny sci-RNA-seq dataset. i. Eomes, Cdx2, Sox2, Sox21 and Bmp4 identified a trophoblast progenitor population in natural embryos in the tiny sci-RNA-seq dataset. j. Expression of selected markers of the ectoplacental cone lineage. High levels of Hand1 (left side arrow) show the developmental progression of the ECP lineage towards spongiotrophoblast cells and trophoblast giant cells. Co-expression of Ascl2 and Chsy1 indicates committed ECP cells, Tpbpa identifies mature spongiotrophoblasts and expression of Hand1 and prolactin genes (Fig. 5i) denotes the trophoblast giant cells. k. Expression of selected markers of chorion progenitors (right side of arrow in Hand1 UMAP), chorion and differentiated chorion derivatives in natural embryos. Wnt7b indicates chorion progenitors, Tfrc indicates the chorion cluster, Epha4 identifies cells of the syncytiotrophoblast layer I and Gcm1 identifies the syncytiotrophoblast layer II. l–o Expression of marker genes presented in (i-k) in the ETiX embryoids UMAP

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