Attribution of vascular phenotypes of the murine Egfl7 locus to the microRNA miR-126

Intronic microRNAs have been proposed to complicate the design and interpretation of mouse knockout studies. The endothelial-expressed Egfl7/miR-126 locus contains miR-126 within Egfl7 intron 7, and angiogenesis deficits have been previously ascribed to Egfl7 gene-trap and lacZ knock-in mice. Surprisingly, selectively floxed Egfl7Δ and miR-126Δ alleles revealed that Egfl7Δ/Δ mice were phenotypically normal, whereas miR-126Δ/Δ mice bearing a 289-nt microdeletion recapitulated previously described Egfl7 embryonic and postnatal retinal vascular phenotypes. Regulation of angiogenesis by miR-126 was confirmed by endothelial-specific deletion and in the adult cornea micropocket assay. Furthermore, miR-126 deletion inhibited VEGF-dependent Akt and Erk signaling by derepression of the p85β subunit of PI3 kinase and of Spred1, respectively. These studies demonstrate the regulation of angiogenesis by an endothelial miRNA, attribute previously described Egfl7 vascular phenotypes to miR-126, and document inadvertent miRNA dysregulation as a complication of mouse knockout strategies.

[1]  L. Campagnolo,et al.  Egfl7, a novel epidermal growth factor‐domain gene expressed in endothelial cells , 2004, Developmental dynamics : an official publication of the American Association of Anatomists.

[2]  W. Gerald,et al.  Endogenous human microRNAs that suppress breast cancer metastasis , 2008, Nature.

[3]  J. Klumperman,et al.  Egfl7 knockdown causes defects in the extension and junctional arrangements of endothelial cells during zebrafish vasculogenesis , 2008, Developmental dynamics : an official publication of the American Association of Anatomists.

[4]  G. Yancopoulos,et al.  Growth factors acting via endothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development. , 1999, Genes & development.

[5]  Michael T. McManus,et al.  Unintentional miRNA Ablation Is a Risk Factor in Gene Knockout Studies: A Short Report , 2008, PLoS genetics.

[6]  J. Fallon,et al.  EGFL7 is a chemoattractant for endothelial cells and is up-regulated in angiogenesis and arterial injury. , 2005, The American journal of pathology.

[7]  I. Kasman,et al.  EGFL7 regulates the collective migration of endothelial cells by restricting their spatial distribution , 2007, Development.

[8]  Ru-Fang Yeh,et al.  miR-126 regulates angiogenic signaling and vascular integrity. , 2008, Developmental cell.

[9]  F. Soncin,et al.  VE‐statin, an endothelial repressor of smooth muscle cell migration , 2003, The EMBO journal.

[10]  Yong Zhao,et al.  A developmental view of microRNA function. , 2007, Trends in biochemical sciences.

[11]  W. Truog,et al.  Epidermal growth factor-like domain 7 protects endothelial cells from hyperoxia-induced cell death. , 2008, American journal of physiology. Lung cellular and molecular physiology.

[12]  C. Kahn,et al.  Increased P85α Is a Potent Negative Regulator of Skeletal Muscle Insulin Signaling and Induces in Vivo Insulin Resistance Associated with Growth Hormone Excess* , 2005, Journal of Biological Chemistry.

[13]  F. Soncin,et al.  VE‐statin/egfl7 regulates vascular elastogenesis by interacting with lysyl oxidases , 2008, The EMBO journal.

[14]  Laura Mariani,et al.  MicroRNAs modulate the angiogenic properties of HUVECs. , 2006, Blood.

[15]  R. D'Amato,et al.  Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[16]  Mattias Alenius,et al.  Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections , 2007, Nature Protocols.

[17]  Wigard P Kloosterman,et al.  In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes , 2005, Nature Methods.

[18]  Didier Y. R. Stainier,et al.  The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation , 2004, Nature.

[19]  John McAnally,et al.  The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis. , 2008, Developmental cell.

[20]  R. Plasterk,et al.  The diverse functions of microRNAs in animal development and disease. , 2006, Developmental cell.

[21]  Y. Maehara,et al.  Spreds Are Essential for Embryonic Lymphangiogenesis by Regulating Vascular Endothelial Growth Factor Receptor 3 Signaling , 2007, Molecular and Cellular Biology.

[22]  J. Engelman,et al.  Phosphoinositide 3-Kinase Catalytic Subunit Deletion and Regulatory Subunit Deletion Have Opposite Effects on Insulin Sensitivity in Mice , 2005, Molecular and Cellular Biology.

[23]  Kohjiro Ueki,et al.  Molecular Balance between the Regulatory and Catalytic Subunits of Phosphoinositide 3-Kinase Regulates Cell Signaling and Survival , 2002, Molecular and Cellular Biology.

[24]  Stefanie Dimmeler,et al.  Role of Dicer and Drosha for Endothelial MicroRNA Expression and Angiogenesis , 2007, Circulation research.