Lyve1-Cre mediated loss of MEKK3 impairs definitive hematopoiesis in the yolk sac despite preserved cardiac development. (A) Hematoxylin and eosin (H&E)-stained sections of E10.5 control and Lyve1-Cre;Map3k3^f/f (YS EC MEKK3^KO) hearts. Scale bars, 50 μm. Boxed regions are shown at higher magnification on the right; scale bars, 20 μm. N = 3 for each genotype. (B) E10.5 control and YS EC MEKK3^KO embryos surrounded by intact yolk sacs. Scale bars, 500 μm. Representative of n = 8 to 10 embryos of each genotype. (C) H&E-stained sections of E10.5 control and YS EC MEKK3^KO yolk sacs. Note the reduced number of round hematopoietic cells in the YS EC MEKK3^KO vascular lumen despite preserved vessel size. Representative of n = 8 to 10 embryos of each genotype at both timepoints. Scale bars, 20 μm. (D) Confocal images of E9.5 control and YS EC MEKK3^KO yolk sacs stained for CD31. Scale bars, 100 μm. Boxed regions are shown at higher magnification on the right. Scale bars, 50 μm. Representative of 5 embryos per genotype. (E) E10.5 control and YS EC MEKK3^KO yolk sac sections stained for CD31 and RUNX1. Scale bars, 20 μm. (F) The number of RUNX1⁺ cells per mm² of vascular area. (G) The number of RUNX1⁺ CD31⁺ cells per mm² of vascular area. (H) The ratio of round/flat RUNX1⁺ cells in E10.5 control and YS EC MEKK3^KO yolk sacs. Each data point in panels F-G represents the mean of >25 vessels in a single animal’s yolk sac. N = 5 E10.5 control and 4 E10.5 YS EC MEKK3^KO yolk sacs. Error bars represent ± standard deviation; significance determined by an unpaired, 2-tailed Student t test. *P < .05; **P < .01.