Stimulation of definitive hematopoiesis by the inflammatory mediators LPS and IFN-γ requires endothelial MEKK3. (A) Schematic of experimental procedure for ex vivo stimulation of embryonic trunk explants with inflammatory mediators. (B) Whole mount staining of control and EC MEKK3^KO embryonic trunks for CD31 and RUNX1 after exposure to vehicle or LPS. Arrows denote RUNX1⁺ cells. Scale bars, 20 μm. (C) Average number of RUNX1⁺CD31⁺ cells per millimeter of dorsal aorta for each treatment cohort. Each data point represents the average number of RUNX1⁺CD31⁺ cells in a single embryo. N = 8 vehicle-treated and 17 LPS-treated control embryos; 9 vehicle-treated and 12 LPS-treated EC MEKK3^KO embryos. (D) Whole mount staining of control and EC MEKK3^KO embryo trunks for CD31 and RUNX1 after exposure to vehicle or IFN-γ. Arrows indicate examples of RUNX1⁺ cells. Scale bars, 20 μm. (E) Average number of RUNX1⁺CD31⁺ cells per millimeter of dorsal aorta per embryo. N = 7 vehicle- and 8 IFN-γ–treated control embryos; 6 vehicle- and 11 IFN-γ–treated EC MEKK3^KO embryos. Error bars represent ± standard deviation; significance determined by an unpaired, 2-tailed Student t test. ns, not significant. *P < .05; **P < .01; ***P < .001.