Role for hematopoietic stem cell cytokines and their receptors during quail developmental vascularization events. (A) Quail CAM tissue was isolated at day 6 of development, fixed, and double stained for the quail EC-specific marker, QH1, versus c-Kit, IL-3Rα, and CXCR4. Arrows indicate vessel wall borders; arrowheads indicate circulating blood cells. (B) Western blot analysis of embryonic 6-day quail vitelline vessels reveals the presence of hematopoietic cytokine receptors in the vasculature. (C) The chemical inhibitors AMD3100, imatinib, and ISCK03 were injected into quail eggs at day 3 of development (100nM) individually or in combination as well as blocking antibodies to SCF and IL-3 (20 μg/mL) versus controls. Embryos were allowed to develop until day 6. Embryo images reveal cranial and abdominal hemorrhage phenotypes (arrows) in embryos treated with hematopoietic cytokine or receptor antagonists, but not in controls. (D) Histologic analysis of developing quail tissue reveals marked hemorrhage in treated embryos. (E) Data are presented showing the frequency and location of hemorrhage visualized in treated versus control embryos. In addition, survival data as well as tissue mass are indicated (n ≥ 7; P ≤ .01). (F) Quail CAM tissue was collected from control versus imatinib/AMD3100 or α-IL-3/SCF–treated embryos at day 6 of development and immunostained for the quail EC-specific marker, QH1. The number of vessel branch points is quantified (top), as well as the area of nonvascularized tissue space (bottom; n ≥ 10; P ≤ .01). *Significance from control. (G) Left panels are representative sections from control versus α-IL-3/SCF–treated embryos at 6 days of development, where reduced sprouting is observed in forebrain parenchyma. Bar equals 100 μm. Representative QH1 stained images from 6-day quail CAM are shown from the indicated conditions showing marked vascular remodeling defects in the imatinib/AMD3100 or α-IL-3/SCF–treated embryos compared with control. Bar equals 100 μm.