Stimulation of definitive hematopoiesis by catecholamines requires endothelial MEKK3. (A) Experimental procedure for in vivo treatment of E9.5 embryos with isoproterenol and phenylephrine (ISO/PHE). (B) Heart rates of E9.5 control and EC MEKK3^KO embryos with and without exposure to maternal ISO/PHE measured by transabdominal ultrasound. (C) CD31⁺ cells in whole mount E9.5 control and EC MEKK3^KO yolk sacs ^+/− administration of ISO/PHE to pregnant dams. The CD31⁺ vascular pattern is outlined in yellow dotted lines. Images are representative of ≥5 embryos per genotype. Bars represent 50 μm. (D) Quantitative analysis of vessel diameters in the yolk sac of E9.5 control and EC MEKK3^KO embryos isolated from vehicle and ISO/PHE-treated dams. N = 4 control and 5 EC MEKK3^KO embryos from vehicle-treated dams; 6 control and 3 EC MEKK3^KO embryos from ISO/PHE-treated dams. (E) Dorsal aortas from E9.5 control and EC MEKK3^KO embryos isolated from pregnant dams treated with ISO/PHE, labeled with antibodies for CD31, RUNX1, and c-Kit. Bars represent 50 μm. (F) Number of RUNX1⁺CD31⁺c-Kit^lo/− HECs (left) and RUNX1⁺CD31⁺c-Kit^hi IAHC cells (right) per mm of dorsal aorta in E9.5 control and EC MEKK3^KO embryos ± ISO/PHE. N = 11 control and 12 EC MEKK3^KO embryos from vehicle-treated dams; 5 control and 6 EC MEKK3^KO embryos from ISO/PHE-treated dams. (G) Experimental procedure for stimulating EHT in E9.5 embryo trunks with ISO/PHE ex vivo. (H) Whole mount staining of E9.5 embryonic trunks for CD31 and RUNX1 following vehicle or ISO/PHE treatment ex vivo. Arrows denote sites of RUNX1⁺ cells. Bars represent 50 μm. (I) Number of RUNX1⁺CD31⁺ cells per mm of aorta in E9.5 control embryo trunks ± ISO/PHE. N = 6 vehicle- and 7 ISO/PHE-treated embryo trunk explants. Error bars represent ± SD; significance determined with unpaired two-tailed Student t test. ns, not significant. *P < .05; ***P < .001; ****P < .0001.