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. 2025 Jan 2;16:142. doi: 10.1038/s41467-024-55597-w

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — a UMAP plots of the Inhibit dataset of genes enriched in the presumptive JZP cluster. Phlda2 is a well-known marker of the JZ. Yet SynT-I markers Slc16a1 and Hbegf are enriched in the same or adjacent cluster (arrow), as is Cxadr. Colour gradient shows normalised expression. b UMAP plots of locations of cells with top 10% expression of Phlda2, Slc16a1 and overlap between the two cell populations. c Immunostaining for SynT-I marker STRA6 (green) and junctional zone marker NCAM1 (red) reveals non-overlap between individual LP and JZP cells entering these lineages. Staining was carried out after 48 h of culture in Inhibit conditions. Image is representative of three independent experiments. d Diagram depicting the organization of emerging trophoblast cell types at sites of fetal vessel invagination into the chorionic ectoderm. JZP = junctional zone precursor, mbs = maternal blood space, sTGC =sinusoidal trophoblast giant cell, LP = labyrinth precursor. e Cxadr expression dynamics across the 48 h differentiation time course in Inhibit and Remove conditions. During these early stages of differentiation, Cxadr expression is up-regulated, in particular in Inhibit conditions that promote LP differentiation. Data are normalized to stem cell conditions and plotted as mean +/- SEM of three independent replicates. Statistical significance was calculated using 2-way ANOVA. **** p < 0.0001. Source data are provided as a Source Data file which contains exact P values. f Representative image of differentiated TSCs at 3 days (3D) of differentiation stained for CXADR (green) and cell membrane marker ZO1 (red). The encircled area highlights early-stage syncytializing cells that have lost CXADR while still retaining some ZO1. Image is representative of three independent experiments. g E10.5 placenta stained for CXADR (green) and MCT1 (red) showing the immediately adjacent localisation of CXADR-positive single cells to syncytial MCT1-positive cells. Images are representative of n = 4 placentas. The middle column of images depicts a higher magnification of the boxed area in the left column. The right-hand column of images depicts an area at the base of the placental labyrinth taken from a consecutive section of that shown on the left. Images are representative of three independent samples.