Separating genetic and hemodynamic defects in neuropilin 1 knockout embryos
Separating genetic and hemodynamic defects in neuropilin 1 knockout embryos
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DOI：
10.1242/dev.014902
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发表时间：
2008-07-15
期刊：
影响因子：
4.6
通讯作者：
Eichmann, Anne
中科院分区：
文献类型：
--
作者：
Jones, Elizabeth A. V.;Yuan, Li;Eichmann, Anne
靶向灭活参与鼠心血管发育的基因经常导致血流异常。由于血液流体动力学在形成血管形态中起着至关重要的作用，因此流动缺陷的存在通常阻止了突变基因产物在脉管系统中的作用的精确分配。在这项研究中，我们展示了如何区分由神经纤毛蛋白1（Nrp 1）受体的靶向失活引起的遗传缺陷和纯合子敲除胚胎中发生的血液动力学缺陷。我们对C57 BL/6背景下的Nrp 1无效等位基因的分析表明，血管重塑缺陷伴随着血流的发生而发生，并导致E10.5纯合突变体的死亡。使用小鼠胚胎培养，我们确定，血流动力学缺陷已经存在于E8.5和连续循环是从来没有建立在纯合子突变体。卵黄囊血管的几何形状发生改变，并且不发生重塑为卵黄囊动脉和静脉。为了将血流诱导的缺陷与Nrp 1突变引起的缺陷分开，我们在培养的野生型和突变型胚胎中阻止了血流，并跟踪了它们的血管发育。我们发现，Nrp 1功能的损失，而不是流量引起的毛细血管丛的几何形状改变。Nrp 1突变体改变了内皮细胞的迁移，但没有复制。从新鲜解剖的野生型和突变体中分离的内皮细胞的基因表达分析，并在无流量条件下培养后显示下调的动脉标记基因连接蛋白40和肝配蛋白B2相关的Nrp 1功能的丧失。该方法允许即使在存在血液动力学缺陷的情况下也分离由对心血管发育重要的基因的功能丧失引起的遗传缺陷。
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.