Final Activity and Management Report Summary - AV IDENTITY IN ESCS (Specification of arterial venous identity in embryonic stem cells)
Targeted inactivation of genes involved in cardiovascular development in mice frequently leads to abnormalities in blood flow. We recently showed that blood flow played a crucial role in shaping vessel morphology; thus the presence of flow defects generally prohibited the precise assignment of the role of the mutated gene product in the vasculature, since primary defects due to the gene could not be separated from secondary defects due to the flow abnormalities. In this research we developed a method to distinguish between genetic defects caused by targeted inactivation of the neuropilin-1 (Nrp-1) receptor and hemodynamic or blood flow defects occurring when the Nrp-1 gene was deleted in embryos.
We showed that vessel remodeling defects occurred concomitantly with the onset of blood flow and caused death of embryos at 10.5 days of gestation. Using mouse embryo culture, we established that flow defects were already present as soon as the heart began to beat and continuous circulation was never established in the mutant mice. The geometry of yolk sac blood vessels was altered and remodelling into yolk sac arteries and veins did not occur. To separate flow induced deficiencies from those caused by the Nrp-1 mutation we arrested blood flow in cultured normal and mutant embryos and followed their vascular development. We found that loss of Nrp-1 function rather than flow induced the altered geometry of the vascular plexus. Endothelial cell migration, but not replication, was altered in Nrp-1 mutants. Gene expression analysis of endothelial cells isolated from freshly dissected wild type and mutants and after culture in no-flow conditions showed down-regulation of the arterial marker genes connexin-40 and ephrinB2 related to the loss of Nrp-1 function. This method allowed genetic defects caused by loss of function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.
We showed that vessel remodeling defects occurred concomitantly with the onset of blood flow and caused death of embryos at 10.5 days of gestation. Using mouse embryo culture, we established that flow defects were already present as soon as the heart began to beat and continuous circulation was never established in the mutant mice. The geometry of yolk sac blood vessels was altered and remodelling into yolk sac arteries and veins did not occur. To separate flow induced deficiencies from those caused by the Nrp-1 mutation we arrested blood flow in cultured normal and mutant embryos and followed their vascular development. We found that loss of Nrp-1 function rather than flow induced the altered geometry of the vascular plexus. Endothelial cell migration, but not replication, was altered in Nrp-1 mutants. Gene expression analysis of endothelial cells isolated from freshly dissected wild type and mutants and after culture in no-flow conditions showed down-regulation of the arterial marker genes connexin-40 and ephrinB2 related to the loss of Nrp-1 function. This method allowed genetic defects caused by loss of function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.