Durrans Anna, Stuhlmann Heidi
Department of Cell and Developmental Biology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10065, USA.
J Angiogenes Res. 2010 Feb 19;2:4. doi: 10.1186/2040-2384-2-4.
Epidermal growth factor-like domain 7, Egfl7, is a largely endothelial restricted gene which is thought to have a role during the differentiation of embryonic stem cells (ESCs) along the endothelial lineage. While it has been shown that Egfl7 knock-down in zebrafish impairs endothelial cord formation, the role of the gene in mammals has been unresolved. Interpretation of mouse knockout studies has been complicated by the fact that deletion of miR-126, an intronic microRNA located within Egfl7, results in vascular defects. Here we use an siRNA knock-down approach to target specific regions of Egfl7 without affecting miR-126 expression. Egfl7 was knocked down in mouse ESCs and the effect on vascular development was assessed using the in vitro embryoid body (EB) model after either 7 or 14 days of differentiation. Knock-down of Egfl7 resulted in the formation of abnormal sheet-like CD31+ structures that were abundant within EBs after 7 days of differentiation. Only up to 60% of these sheets co-expressed basement membrane and endothelial cell junction markers. Similar CD31+ sheets were also seen as outgrowths from 7 day EBs into collagen gels. A partial remodelling occurred by 14 days of differentiation when fewer CD31+ sheets were seen both within EBs, and as outgrowths from EBs. Formation of these sheets was due, at least in part, to increased proliferation specifically of CD31+ cells. Cell death within EBs was unaffected by Egfl7 knock-down. In conclusion, our work shows that knock-down of Egfl7 causes defects in early vascular cord formation, and results in the development of CD31+ sheet-like structures. This suggests that Egfl7 is vital for the formation of endothelial cell cords, and that the gene has an important role during both vasculogenesis and angiogenesis in mammalian cells.
表皮生长因子样结构域7（Egfl7）是一个主要在内皮细胞中表达的基因，被认为在胚胎干细胞（ESC）沿内皮细胞谱系分化过程中发挥作用。虽然已经表明，斑马鱼中Egfl7基因敲低会损害内皮索形成，但该基因在哺乳动物中的作用尚未明确。由于位于Egfl7基因内的内含子微小RNA miR-126缺失会导致血管缺陷，因此对小鼠基因敲除研究的解读变得复杂。在这里，我们使用小干扰RNA（siRNA）敲低方法靶向Egfl7的特定区域，而不影响miR-126的表达。在小鼠胚胎干细胞中敲低Egfl7，并在分化7天或14天后使用体外胚状体（EB）模型评估其对血管发育的影响。敲低Egfl7导致形成异常的片状CD31+结构，在分化7天后，这些结构在胚状体内大量存在。这些片状结构中只有高达60%同时表达基底膜和内皮细胞连接标记物。类似的CD31+片状结构也可见于7天胚状体向胶原凝胶的生长物中。分化14天时发生了部分重塑，此时在胚状体内以及胚状体生长物中可见的CD31+片状结构减少。这些片状结构的形成至少部分是由于CD31+细胞的增殖增加。胚状体内的细胞死亡不受Egfl7基因敲低的影响。总之，我们的研究表明，敲低Egfl7会导致早期血管索形成缺陷，并导致CD31+片状结构的发育。这表明Egfl7对于内皮细胞索的形成至关重要，并且该基因在哺乳动物细胞的血管发生和血管生成过程中都发挥着重要作用。
