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First published online 11 June 2008
doi: 10.1242/dev.014902
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1 INSERM U833, F-75005, Paris, France.
2 Collège de France, 11 Place Marcelin Berthelot, 75005 Paris,
France.
3 School of Life Science, Xiamen University 361005 Xiamen, Fujian, China.
4 Department of Tumor Biology and Angiogenesis, Genentech, 1 DNA Way, South San
Francisco, CA 94080, USA.
* Author for correspondence (e-mail: liz.jones{at}mcgill.ca)
Accepted 15 May 2008
Targeted inactivation of genes involved in murine cardiovascular development frequently leads to abnormalities in blood flow. As blood fluid dynamics play a crucial role in shaping vessel morphology, the presence of flow defects generally prohibits the precise assignment of the role of the mutated gene product in the vasculature. In this study, we show how to distinguish between genetic defects caused by targeted inactivation of the neuropilin 1 (Nrp1) receptor and hemodynamic defects occurring in homozygous knockout embryos. Our analysis of a Nrp1 null allele bred onto a C57BL/6 background shows that vessel remodeling defects occur concomitantly with the onset of blood flow and cause death of homozygous mutants at E10.5. Using mouse embryo culture, we establish that hemodynamic defects are already present at E8.5 and continuous circulation is never established in homozygous mutants. The geometry of yolk sac blood vessels is altered and remodeling into yolk sac arteries and veins does not occur. To separate flow-induced deficiencies from those caused by the Nrp1 mutation, we arrested blood flow in cultured wild-type and mutant embryos and followed their vascular development. We find that loss of Nrp1 function rather than flow induces the altered geometry of the capillary plexus. Endothelial cell migration, but not replication, is altered in Nrp1 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 ephrin B2 related to the loss of Nrp1 function. This method allows genetic defects caused by loss-of-function of a gene important for cardiovascular development to be isolated even in the presence of hemodynamic defects.
Key words: Neuropilin 1, VEGF, Hemodynamics, Endothelial cell migration, Arterial-venous differentiation
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