Fig. 3: AVE differentiation in BELAs.

a UMAP representation of single-cell transcriptomes (same as in Fig. 2b), colored according to Louvain clustering. b Heatmap showing the fraction and total number of cells from each sample in the four clusters from a. The small number of cells from VE cysts in clusters 1 and 2 likely originate from cells that were refractory to PrE differentiation (see above, and Raina et al.⁹), c Heatmap showing the 30 most upregulated genes between the cells of cluster 3 and cluster 4 in (a), ordered by log2-fold change. Single-cell expression is shown as the Pearson residual of the normalized counts. d Zoom-in in UMAP from (a) showing expression of Cer1, Sfrp1, and Lefty1 in VE cells. e UMAP of single-cell transcriptomes from BELA-VE cells (Cluster 3 and Cluster 4 in a, b), integrated with scRNAseq data from mouse embryos at E5.5 and E6.25 from¹⁵. f Same UMAP as in e, colored according to cell type annotation from¹⁵ after integration and label transfer. g Heatmap showing the fraction of BELA-VE cells assigned to particular cell types and developmental time points from the embryo. h Immunostaining for the AVE marker OTX2 (blue) and the VE marker GATA6 (magenta). Arrows highlight co-expression. i Immunostaining for the AVE marker OTX2 (blue) and the basement membrane marker LAM (magenta). h, i one representative out of a total of at least 13 structures from n = 2 independent experiments shown. j In situ HCR staining for the AVE markers Otx2 (blue) and Cer1 (orange), and the VE marker Gata6 (magenta). One representative out of a total of at least 60 structures from n = 3 independent experiments shown. k, l Orthogonal views (k) and 3D volume rendering (l) of a BELA stained for the Epi marker POU5F1 (green) and the AVE reporter Cer1:H2B-Venus (yellow) imaged with light sheet microscopy. One representative out of a total of 28 structures from n = 3 independent experiments shown. m Mean frequency of AVE marker gene expression in BELAs on different days after re-seeding. Otx2 expression was scored as AVE marker only if it could clearly be assigned to the outer layer of BELAs. n = 2 independent experiments, for number of BELAs analyzed at each timepoint see Source Data file. Error bars indicate SD. Scale bars: 25 µm.