Fig. 3: Formation of DVE/AVE-like population in VE-like compartment.

a UMAP representation of VE-like cluster revealing two subclusters diverging in their transcriptional profile. b Comparison of EmVE-like cluster with published scRNA-Seq dataset for EmVE of natural murine embryos²³ revealing high AUCell-based enrichment score and mean expression levels of EmVE genes within EmVE-like cluster. c Schematic representation of DVE and AVE formation in murine embryos between E5.5 and E5.75. d Analysis of AVE marker expression in VE-like subclusters displays higher expression of AVE marker gens inside the EmVE-like cell cluster. e Comparison of published AVE-signatures²³ showing high AUCell-based enrichment score and high mean expression for cells of EmVE-like cluster, in contrast to a lower AUC score and mean expression levels in cells of ExVE-like cluster. f Expression of DVE/AVE markers EOMES and OTX2 was detected to colocalize in cells of the EmVE-compartment in RtL-embryoids, indicated by white arrows. OTX2, red; EOMES, green; DAPI, blue. Scale bar = 50 µm. g DVE/AVE marker LEFTY1 was found colocalizing with EOMES + cells, which was detected to be restricted to one side of the EmVE-like compartment in RtL-embryoids (white squares are magnified in h. Scale bar = 25 µm. h Magnification of the EmVE-like compartment as indicated by the white, dotted squares in g. LEFTY1, red; EOMES, green; DAPI, blue. Scale bar = 20 µm. White arrows indicate EOMES + /LEFTY1 + cells. i LEFTY1 + cells were detected in distal, transition or anterior position in RtL-embryoids. LEFTY1, red; GFP, green; DAPI, blue. Scale bar = 50 µm. j Quantification of LEFTY1 + cell location in RtL-embryoids at day 4 (4d) and day 5 (5d) into the protocol. The majority of RtL-embryoids displayed LEFTY1 + cells in either distal or transition positions, with few RtL-embryoids showing localization of LEFTY1 + in an anterior position, relative to the Epi-like compartments, both 4d (red bars) and 5d (blue bars) into the protocol. Total n = 375 for each, 4d and 5d conditions. Data are presented as mean values ± SD. Source data of j are provided as a Source Data file. Experiments were repeated independently at least three times with similar results (f–h).