A single-cell resolution atlas of E10.5 AGM. (A) Schematic representation of FACS populations based on Runx1b^RFP, Gfi1^tomato/Gfi1b^GFP, and Gfi1^GFP/Gfi1b^GFP reporter mouse models. Cells isolated from Runx1b^RFP reporter include FACS-MES^Runx1b-het (CD41⁻CD45⁻TER119⁻CDH5⁻KIT⁻Runx1b:RFP⁺), FACS-ENDO^Runx1b-het (CD41⁻CD45⁻TER119⁻CDH5⁺KIT⁻Runx1b:RFP^-), and FACS-HE^Runx1b-het (CD41⁻CD45⁻TER119⁻CDH5⁺KIT⁻Runx1b:RFP⁺). Cells isolated from Runx1b^KO include FACS-MES^Runx1b-KO (CD41⁻CD45⁻TER119⁻CDH5⁻KIT⁻Runx1b:RFP⁺), FACS-ENDO^Runx1b-KO (CD41⁻CD45⁻TER119⁻CDH5⁺KIT⁻Runx1b:RFP⁻), and FACS-HE^Runx1b-KO (CD41⁻CD45⁻TER119⁻CDH5⁺KIT⁻Runx1b:RFP⁺). Cells isolated from Gfi1^tomato/Gfi1b^GFP are as described in Figure 1A. FACS-HE^Gfi1/1b-KO (CD41⁻CD45⁻TER119⁻CDH5⁺KIT⁻Gfi1:GFP⁺) was isolated from Gfi1^GFP/Gfi1b^GFP reporter. (B) Uniform Manifold Approximation and Projection (UMAP) plots showing all cells (left), heterozygote cells only (middle), and KO cells only (right). Cells are colored by FACS populations. (C) Two-dimensional UMAP with cells colored by in silico clustering. (D) Expression of venous endothelial (Nrp2), arterial endothelial (Gja5, Vwf), hematopoietic (Gfi1b), and mesenchymal (Bmp4, Dlk1) genes in the UMAP plot. (E) Violin plots combined with box and whisker plots showing the normalized expression (logcounts) of indicated genes in each cluster. The color coding is identical to panel C. Medians are shown as a solid black line; boxes indicate the upper (75%) and lower (25%) percentile; whiskers indicate ±1.5 times the interquartile range (IQR). Outliers (values > or <1.5 IQR) are shown as dots. AE, arterial endothelial; EHT, endothelial-to-hematopoietic transition; ENDO, endothelial; HE, hemogenic endothelium; IAHC, intra-aortic hematopoietic clusters; UC, unclassified; VE, venous endothelial.