Yolk sac Flk1⁺c-Kit⁺CD45⁻ SP cells are endothelial cells with hematopoietic potential. (A) Whole mount immunohistochemical colocalization of (i) Flk1 (Alexa-488; green) and (ii) c-Kit (Alexa-594; red) on hemogenic endothelial cells of the E9.5 murine yolk sac arterial capillary bed. (Aiii,iv) Color-merged confocal z-stack projection of E9.5 yolk sac whole mount immunostain. White arrowheads indicate Flk1⁺c-Kit⁺ hemogenic endothelial cells localized to the vitelline vein. (B) Clonal analysis of Flk1⁺c-Kit⁺CD45⁻ SP hemogenic endothelial cells cultured on fibronectin with MethoCult GF M3434. (Bi) Day 1 culture, single Flk1⁺c-Kit⁺CD45⁻ SP cell stained for 4 hours at 37°C with 10 μg/mL DiI AcLDL. (Bii) Day 3 culture showing adherent cobblestone morphology of proliferating Flk1⁺c-Kit⁺CD45⁻ SP hemogenic endothelial cell colony. (Biii) Day 7 culture showing emergence of multipotent HPC from clonal Flk1⁺c-Kit⁺CD45⁻ SP hemogenic endothelial cell culture. White arrowheads indicate representative cells displaying characteristic endothelial cell morphology; black arrowheads indicate hematopoietic progenitor morphology. (C) Multilineage hematopoietic potential of Flk1⁺c-Kit⁺CD45⁻ SP hemogenic endothelial cell clones cultured on fibronectin in MethoCult GF M3434, (Ci) BFU-E erythroid colony, (Cii) CFU-GM granulocyte-monocyte colony, (Ciii) CFU-GEMM multipotent hematopoietic progenitor colony. (D) Immunohistochemical colocalization of c-Kit (Alexa-488; green) and RARα (Alexa-594; red) on hemogenic endothelial cells of the E9.5 murine yolk sac. White arrowheads indicate c-Kit⁺RARα⁺ endothelial and hematopoietic cells. Scale bars in all panels represent 100 μm (20× magnification).