FIGURE 1.

Deletion of the 3.9-kb upstream promoter fragment of the Etv2 gene results in lethality and a lack of hematoendothelial lineages. A, schematic of the gRNA and PCR screening primer designs for deleting the Etv2 3.9-kb upstream promoter. Fwd, forward; Rev, reverse. B, PCR genotyping of Mut ES cells demonstrates the detection of only the deleted band. C, gene expression of Etv2 during EB differentiation of WT, Mut, and Null ES cells. D and G, representative FACS profiles of dissociated D6 EBs for expression of hematopoietic and endothelial markers, respectively. E and F, quantitative analysis of the FACS data in D. CD41 single- and CD41/CD45 double-positive cells are completely absent in the Mut as well as the Etv2 Null ES cells. H, quantitative analysis of the FACS data in G. CD31/Flk1 double-positive cells are absent in the Mut. I, histological analyses of the WT and Mut embryo, demonstrating an absence of the endocardial/endothelial lineage (End) and vasculature (dorsal aorta (DA)) in the Mut embryo compared with a WT littermate. h, heart). J, immunohistochemical analysis using endomucin antibody (α-endomucin) in WT and Mut embryos, further demonstrating an absence of the endocardial lineage (arrowhead) and the vasculature (arrows) in the Mut embryo. K, immunohistochemical analysis using anti-endomucin in the WT and Mut yolk sacs showing the presence of endothelium and blood cells (arrow), whereas they are not detectable in the Mut yolk sac. K' and K” are enlarged regions of those indicated by rectangles. L, gene expression of Etv2, CD31, and CD41 in E8.5 WT and Mut embryos. Scale bars = 100 μm (I and J) and 50 μm (K).