Loss of endothelial cell KLF2/4 function phenocopies endothelial MEKK3 loss in the yolk sac and AGM region. (A) Expression level (log₂ size-factor-normalized UMI counts) of Klf2 and Klf4 in Wnt^hi/lo AE, conflux AE, pre-HE, and HEC + IAHC cell populations in E10.0 EC MEKK3KO and control littermates. An unpaired, 1-sided Mann-Whitney U test was applied to test if the expression of Klf2 and Klf4 was significantly lower in EC MEKK3KO endothelial cells compared with control. HEC+IAHC cells were combined in the analysis due to the low cell numbers in these populations in the E10.0 EC MEKK3KO sample. ****P < .0001; *P < .05. ns, not significant. (B) Stereo microscopic images and ematoxylin and eosin (H&E)-stained sections of E9.5 and E10.5 control and EC KLF2^KO/KLF4^HET yolk sacs. Images on the left are of intact embryos surrounded by yolk sacs. Boxed regions are shown at higher magnification in the images to their right. Images to the right are H&E-stained sections. Images are representative of 8 to 10 embryos per genotype at both timepoints. Scale bars of stereo microscopic images, 500 μm; H&E-stained sections, 20 μm. (C) Number of RUNX1⁺ cells per mm² of vascular area in E9.5 and E10.5 control and EC KLF2^KO/KLF4^HET yolk sacs. (D) Number of RUNX1⁺ CD31⁺ cells per mm² of vascular area in E9.5 and E10.5 control and EC KLF2^KO/KLF4^HET yolk sacs. (E) Ratio of round/flat RUNX1⁺ cells in E9.5 and E10.5 control and EC KLF2^KO/KLF4^HET yolk sacs. Each data point in B-D represents the mean of >25 vessels measured per yolk sac. N = 5 E9.5 control, 13 E10.5 control, 5 E9.5 EC KLF2^KO/KLF4^HET, and 9 E10.5 EC KLF2^KO/KLF4^HET yolk sacs. Error bars represent ± SD; significance determined by an unpaired, 2-tailed Student t test. **P < .01; ****P < .0001. (F) Wholemount confocal images of aortas from E9.5 control and EC KLF2^KO/KLF4^HET embryos stained for CD31, RUNX1, and c-Kit. Images on right show higher magnification views of boxed regions on the left. Images are representative of 5 embryos per genotype. Scale bars, 100 μm and 20 μm in panels on the left and right, respectively. (G) Number of RUNX1⁺CD31⁺c-Kit^lo/− HECs per mm of dorsal aorta in E9.5 and E10.5 control and EC KLF2^KO/KLF4^HET embryos. N = 10 E9.5 control and EC KLF2^KO/KLF4^HET embryos, 11 E10.5 control, and 7 EC KLF2^KO/KLF4^HET embryos. (H) Number of RUNX1⁺CD31⁺c-Kit^hi IAHC cells per mm of dorsal aorta in E9.5 and E10.5 control and EC KLF2^KO/KLF4^HET embryos. N = 10 E9.5 control and EC KLF2^KO/KLF4^HET embryos, 11 E10.5 control, and 7 E10.5 EC KLF2^KO/KLF4^HET embryos. Error bars represent mean ± standard deviation; significance determined by an unpaired 2-tailed Student t test. ***P < .001; ****P < .0001.