The embryonic heart and vessels are dynamic and form and remodel while functional. Much has been learned about the genetic mechanisms underlying the development of the cardiovascular system, but we are just beginning to understand how changes in heart and vessel structure are influenced by hemodynamic forces such as shear stress. Recent work has shown that vessel remodeling in the mouse yolk sac is secondarily effected when cardiac function is reduced or absent. These findings indicate that proper circulation is required for vessel remodeling, but have not defined whether the role of circulation is to provide mechanical cues, to deliver oxygen or to circulate signaling molecules. Here, we used time-lapse confocal microscopy to determine the role of fluid-derived forces in vessel remodeling in the developing murine yolk sac. Novel methods were used to characterize flows in normal embryos and in embryos with impaired contractility (Mlc2a -/-). We found abnormal plasma and erythroblast circulation in these embryos, which led us to hypothesize that the entry of erythroblasts into circulation is a key event in triggering vessel remodeling. We tested this by sequestering erythroblasts in the blood islands, thereby lowering the hematocrit and reducing shear stress, and found that vessel remodeling and the expression of eNOS (Nos3) depends on erythroblast flow. Further, we rescued remodeling defects and eNOS expression in low-hematocrit embryos by restoring the viscosity of the blood. These data show that hemodynamic force is necessary and sufficient to induce vessel remodeling in the mammalian yolk sac.

Rationale The density of native (pre-existing) collaterals varies widely and is a significant determinant of variation in severity of stroke, myocardial infarction and peripheral artery disease. However, little is known about mechanisms responsible for formation of the collateral circulation in healthy tissues. Objective We previously found that variation in VEGF expression causes differences in collateral density of newborn and adult mice. Herein, we sought to determine mechanisms of collaterogenesis in the embryo and the role of VEGF in this process. Methods and Results Pial collaterals begin forming between embryonic day (E) 13.5 and 14.5 as sprout-like extensions from arterioles of existing cerebral artery trees. Global VEGF-A overexpressing mice (Vegf hi/+) formed more—and Vegf lo/+ formed fewer—collaterals during embryogenesis, in association with differences in vascular patterning. Conditional global reduction of Vegf or Flk1 only during collaterogenesis significantly reduced collateral formation, but now without affecting vascular patterning, and the effects remained in adulthood. Endothelial-specific Vegf reduction had no effect on collaterogenesis. Endothelial-specific reduction of a disintegrin-and-metalloprotease-domain-10 (Adam10) and inhibition of γ-secretase increased collateral formation, consistent with their roles in VEGF-induced Notch1 activation and suppression of “pro-sprouting” signals. Endothelial-specific knockdown of Adam17 reduced collateral formation, consistent with its roles in endothelial cell migration and embryonic vascular stabilization, but not in activation of ligand-bound Notch1. These effects also remained in adulthood. Conclusions Formation of pial collaterals occurs during a narrow developmental window via a sprouting angiogenesis-like mechanism, requires paracrine VEGF-stimulation of Flk1-Notch signaling, and adult collateral number is dependent on embryonic collaterogenesis.

During developmental hematopoiesis, multilineage hematopoietic progenitors are thought to derive from a subset of vascular endothelium. Herein, we define the phenotype of such hemogenic endothelial cells and demonstrate, on a clonal level, that they exhibit multilineage hematopoietic potential. Furthermore, we have begun to define the molecular signals that regulate their development. We found that the formation of yolk sac hemogenic endothelium and its hematopoietic potential were significantly impaired in the absence of retinoic acid (RA) signaling, and could be restored in RA-deficient (Raldh2−/−) embryos by provision of exogenous RA in utero. Thus, we identify a novel, critical role for RA signaling in the development of hemogenic endothelium that contributes to definitive hematopoiesis.

Rationale Severity of tissue injury in occlusive disease is dependent on the extent (number and diameter) of collateral vessels, which varies widely among healthy mice and humans. However, the causative genetic elements are unknown. Recently, much of the variation among different mouse strains, including C57Bl/6J (B6, high extent) and BALB/cByJ (Bc, low), was linked to a QTL on chromosome 7 (Candq1). Objective We used congenic mapping to refine Candq1 and its candidate genes and create an “isogenic” strain-set with large differences in collateral extent to assess their and Candq1’s impact, alone, on ischemic injury. Methods and Results Six congenic strains possessing portions of Candq1 introgressed from B6 into Bc were generated and phenotyped. Candq1 was refined from 27 to 0.737 Mb with full retention of effect, ie, return/rescue of phenotypes from the poor values in Bc to nearly those of wildtype B6 in the B6/B6 congenic mice: 83% rescue of low pial collateral extent, and 4.5-fold increase in blood flow and 85% reduction of infarct volume after middle cerebral artery occlusion; 54% rescue of low skeletal muscle collaterals, and augmented recovery of perfusion (83%) and function after femoral artery ligation. Gene deletion and in-silico analysis further delineated the candidate genes. Conclusion We have significantly refined Candq1 (now designated Determinant of collateral extent-1, Dce1), demonstrated that genetic background-dependent variation in collaterals is a major factor underlying differences in ischemic tissue injury, and generated a congenic strain-set with wide, allele-dose-dependent variation in collateral extent for use in investigations of the collateral circulation.