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First published online 5 November 2008
doi: 10.1242/dev.029736

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

Frank Kuhnert^1,*, Michael R. Mancuso^1,*, Jessica Hampton¹, Kryn Stankunas², Tomoichiro Asano³, Chang-Zheng Chen⁴ and Calvin J. Kuo^1,

¹ Division of Hematology, Department of Medicine, Stanford University School of Medicine, CCSR 1155, 269 Campus Drive, Stanford, CA 94305, USA.
² Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, CCSR 1155, 269 Campus Drive, Stanford, CA 94305, USA.
³ Department of Medical Science, Graduate School of Medicine, University of Hiroshima, 1-2-3 Kasumi, Minami-ku, Hiroshima City, Hiroshima 734-8553, Japan.
⁴ Baxter Laboratory and Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305, USA.

View larger version (59K): [in this window] [in a new window]   Fig. 1. Generation and validation of Egfl7 and miR-126 deletion alleles. (A) Egfl7 and miR-126 delta () alleles were generated by flanking exons 5-7 of Egfl7 or a 289 bp segment of intron 7 containing miR-126 with loxP sites, respectively, followed by in vivo deletion using Cre recombinase. Green arrowheads, remnant loxP sites after Cre deletion. Blue arrows, PCR primers used in E. Red line, Egfl7 epitope used for polyclonal antibody generation. (B) In situ hybridization for processed miR-126 (dark purple staining) demonstrates vascular expression in the trunk region of wild-type (wt) E14.5 mouse embryos (top panels) that is absent in miR-126/ embryos (bottom panels). Arrows in higher magnification images (taken from the boxed regions) highlight vascular miR-126 expression in the neural tube and carotid artery in wild-type embryos, and arrowheads the absence thereof in miR-126/ embryos. CA, carotid artery; JV, jugular vein; NT, neural tube; OE, esophagus; TR, trachea; VA, vertebral artery. (C) Quantitative PCR (n=6) confirmed the absence of Egfl7 mRNA in Egfl7/ embryos and the absence of mature (processed) miR-126 in miR-126/ embryos. A looped RT primer specifically detecting the mature miR-126 processed end was utilized. Notably, Egfl7/ embryos exhibited normal miR-126 processing and miR-126/ embryos exhibited normal levels of Egfl7 mRNA, indicating that microdeletion did not disrupt physiological expression of the adjacent gene/miRNA in either case. *P<0.001 versus wild type. (D) Immunofluorescence staining of uterus from a pregnant mouse with affinity-purified rabbit anti-Egfl7 antibody demonstrating loss of Egfl7 protein in adult Egfl7/, but not adult miR-126/, mice. (E) RT-PCR of full-length Egfl7 coding sequence from miR-126/ embryos indicates that microdeletion of miR-126 does not induce occult splicing of Egfl7 mRNA. A doublet is present in Egfl7/ embryos representing out-of-frame splicing from exon 4 to exon 8 or 9.

View larger version (80K): [in this window] [in a new window]   Fig. 2. miR-126/, but not Egfl7/, mice exhibit incompletely penetrant embryonic lethality, edema and vascular leakage. (A,B) Breeding tables from heterozygous intercrosses show that Egfl7/ mice are born at normal Mendelian ratios (2=0.741), whereas miR-126/ embryos exhibit 50% embryonic/perinatal lethality (2<0.001). Numbers in parenthesis indicate embryos found with edema. Red numbers indicate deviation from Mendelian ratios. (C) Wild-type (wt) and Egfl7/ embryos were phenotypically indistinguishable, whereas 50% of the miR-126/ embryos displayed subcutaneous edema (*) at E14.5. (D) Hematoxylin and Eosin staining reveals the severity of the edema (*) in E14.5 embryos. (E) Varying degrees of subcutaneous hemorrhage are detected in 20% of E15.5 miR-126/ embryos; a severely affected embryo is depicted. Histological analysis shows red blood cells extravasating from a representative ruptured, leaky vessel (arrow). (F) Tie2-Cre-mediated endothelial deletion of miR-126 phenocopies the miR-126/ edema (*) phenotype.

View larger version (83K): [in this window] [in a new window]   Fig. 3. Angiogenesis phenotypes in miR-126/ embryos. (A) Isolectin B4 staining of P5 postnatal retinas. Retinal vascularization was normal in Egfl7/ mice but was severely delayed in miR-126/ mice as indicated by arrows. The dashed line indicates the edge of the optic cup. wt, wild type. (B) Quantitation of retinal vascularization demonstrates a 40% reduction of retinal vascular coverage in miR-126/ mice. (C) High-magnification images of retinal vascular sprouts. Note the marked thickening (arrows) of vascular sprouts in miR-126/ retinas as compared with wild-type or Egfl7/ mice. (D) CD31 (Pecam1) whole-mount staining of E12.5 heads. Note the delayed vascularization and reduced complexity (arrow) of the cranial vasculature in miR-126/ embryos. (E) Adult animals of the indicated genotypes (n=7) received corneal implants of slow-release hydron pellets containing VEGF. Neovascularization was quantified after 6 days by slit lamp examination. Arrows highlight the length of vessel growth. (F) Adult miR-126/ mice exhibited 50% impairment of corneal vascularization relative to Egfl7/ or wild-type mice.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Regulation of p85β expression by miR-126. (A) Quantitative PCR analysis confirms the almost complete absence of miR-126 expression in HUVEC transfected with a miR-126 hairpin inhibitor, as opposed to a scrambled control (scr). (B) Impaired migration of HUVEC transfected with the miR-126 hairpin inhibitor, versus scr, in the in vitro scratch wound assay (*P<0.05 versus scrambled inhibitor-transfected). (C) Impaired VEGF-dependent Akt and Erk phosphorylation in HUVEC transfected with the miR-126 hairpin inhibitor, versus scr. (D) Target site alignment for miR-126 in the 3'UTR of Pik3r2, which encodes p85β. (E) p85β (Pik3r2) is a direct target of miR-126 as shown by dose-dependent repression by miR-126 of luciferase expression from the wild-type p85β 3'UTR, but not the control Lin41 3'UTR, reporters in 293T cells. Mutation of the miR-126 binding site in the p85β 3'UTR (p85β mut) abrogates repression by miR-126, identifying p85β as a direct target. *P<0.05 versus no miR-126 expression vector and P<0.05 versus p85β mut and Lin41 3'UTR reporter construct, for a given dose of miR-126 expression vector (0, 10 and 100 ng). NS, not significant. (F) p85 is upregulated in primary brain endothelial cells isolated from miR-126/, but not Egfl7/, mice as assessed by western blot with anti-pan p85 antibody; anti-actin antibody provided a loading control. (G) Upregulation of p85β expression in HUVEC transfected with a hairpin inhibitor targeting miR-126, versus scr. (H) Adenoviral expression of p85β in HUVEC is sufficient to inhibit VEGF-induced Akt phosphorylation.

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