Loss of MEKK3 in endothelial cells impairs definitive hematopoiesis in the dorsal aorta. (A) Whole-mount of E10 control and EC MEKK3^KO dorsal aortas stained for CD31, RUNX1, and c-Kit. Regions boxed are shown at higher magnification in images to their right. Scale bars, 100 μm (larger images) 20 μm (higher magnification views). Images are representative of 5 embryos of each genotype. (B) The number of RUNX1⁺CD31⁺c-Kit^hi IAHC cells per millimeter in the region of the dorsal aorta caudal to the vitelline artery in E9.5 and E10.5 control and EC MEKK3^KO embryos determined from confocal microscopic images. N = 12 E9.5 control, 7 E9.5 EC MEKK3^KO, 13 E10.5 control, and 5 E10.5 EC MEKK3^KO embryos. Error bars represent ± standard deviation (SD), and significance was determined by an unpaired, 2-tailed Student t test. *P < .05; ***P < .001. (C) Contour flow plots of RUNX1⁺c-Kit⁺ IAHC cells within the Ter119⁻CD31⁺CD61^low/−CD144⁺CD44⁺ population of cells from the AGM regions of E10.0 EC MEKK3^KO and control embryos. (See supplemental Figure 4A for gating strategy.) (D) Summary of data from panel C. Error bars represent mean ± SD; N = pooled embryos from 4 different experiments; ****P < .0001. (E) Representative histogram of RUNX1 MFI in IAHC cells from pooled embryos of each genotype. IAHC cells are gated as shown in supplemental Figure 4A. (F) RUNX1 MFI in IAHCs relative to control embryos, summarized from data in panel E. Data are from pooled embryos from litters harvested on 4 separate days. Error bars represent mean ± SD. **P < .01. (G) Number of RUNX1⁺CD31⁺c-Kit^low/− HECs per millimeter of the dorsal aorta in E9.5 and E10.5 control and EC MEKK3^KO embryos counted from confocal microscopic images. The number of embryos analyzed is the same as in panel B. (H) Representative contour flow plots of PROCR⁺ RUNX1⁺ HECs in the CD44⁺ subpopulation of CD45⁻Ter119⁻CD61^low/−CD31⁺CD144⁺ cells from the AGM regions of E10.0 EC MEKK3^KO and control embryos. (See supplemental Figure 4B for gating strategy.) (I) Summary of data from panel H. Data are from pooled embryos from litters harvested on 4 separate days. Error bars represent mean ± SD. ***P < .001. (J) Limiting dilution assays on OP9 stromal cells and OP9 cells expressing the Notch ligand DLL1 to determine the number of LMPs per AGM region in E10.5 control and EC MEKK3^KO embryos. Number of progenitors scored based on the number of wells containing CD45⁺ cells (left). Number of B, T, and myeloid progenitors (right). B cells were scored after 12 days of culture as CD45⁺CD19⁺B220^mid/lo, myeloid as Gr1⁺Mac1⁺ or Gr1⁺Mac1⁻, and T cells were CD90⁺ CD25⁺. Error bars represent mean ± SD. N = 3 experiments. (See supplemental Figure 4F-G for gating strategy.)