Abstract
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.
Highlights
Epidermal growth factor-like domain 7, Egfl[7 ], was identified in a screen for genes with restricted expression during in vitro differentiation and mouse embryogenesis [ 1]
We looked at the effect of Egfl[7] knock-down on sprouting angiogenesis using embryoid body (EB) in a type I collagen gel
Quantitative PCR showed that Egfl[7] knock-down did not affect levels of the microRNAs miR-126-3p (3 prime end) or miR-126-5p (5 prime end) (Figure 1d ), which are generated as a stem loop encoded by intron 7 within the Egfl[7] gene (Figure 1a )
Summary
Epidermal growth factor-like domain 7, Egfl[7 ], was identified in a screen for genes with restricted expression during in vitro differentiation and mouse embryogenesis [ 1]. EGFL7 is expressed in undifferentiated mouse embryonic stem cells (ESCs), during early embryogenesis at sites of blood island formation and vasculogenesis, and in adults during pathological and physiological angiogenesis [ 1, 2] In this study we have used an siRNA knock-down approach, enabling us to target regions of the Egfl[7] gene other than intron 7 This has allowed us to investigate the role of Egfl[7] during vascular development, without affecting miR-126 expression. We show that Egfl[7] knock-down results in the formation of abnormal endothelial sheet-like structures, which form during the initial stages of in vitro vascular development. Our results suggest a role for Egfl[7] in EC organization, and indicate that the gene is necessary for normal vascular growth during both vasculogenesis and angiogenesis in mammalian cells
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