Fig. 4: Runx1+ HE emerge from Meis1-expressing pre-HE in vivo.

A Representative flow cytometry scatterplot gated for relevant subsets within the viable CD31⁺CD45⁻ population. B, C mRNA expression of selected EHT-related genes in FACS-isolated GFP⁻ and GFP⁺ EC from E10.5 AGM of (B) Meis1-GFP embryos (n = 5 pools of 4 embryos each from the same litter) and (C) Runx1+23GFP embryos (n = 7 pools of 3 embryos each). Quantification was performed by ddPCR and normalized to Gapdh. Bar-plots are showing the mean ± SEM. Indicated p-values were calculated using a two-sided t-test comparing two groups. D UMAP projections of the aEC, pre-HE, and EHT clusters showing the expression of selected genes in the CITE-seq data. E Immunostaining of the dorsal aorta from E10.5 Meis1-GFP embryos (gray: CD31, green: GFP, blue: Runx1, V: ventral, D: dorsal). Arrows point to Runx1+ cells and asterix (*) denotes adjacent Meis1-GFP⁺Runx1⁻ EC. The dotted line delineates the endothelial layer from the subaortic mesenchyme in upper panels. Scale bar = 20 µm. Staining was reproduced in 2 independent experiments. F Ex vivo differentiation assay on an OP9 stromal layer to assess hematopoietic colony formation from FACS-isolated GFP⁺ and GFP⁻ EC. Representative brightfield images (above, scale bar = 50 µm) of hematopoietic colonies formed and confirmation of CD45 expression by immunofluorescence staining (below, scale bar = 25 µm). The table on the right shows the number of colony-positive replicates at the endpoint for the various initial cell inputs tested. The frequency of HE cells was assessed using extreme limiting dilution assay (ELDA) calculation. Source data are provided as a Source Data file.