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First published online 5 November 2008
doi: 10.1242/dev.029736
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Research Report |
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.
Author for correspondence (e-mail:
cjkuo{at}stanford.edu)
Accepted 7 October 2008
SUMMARY
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 Egfl7
and
miR-126 alleles revealed that
Egfl7/ mice were phenotypically
normal, whereas miR-126/ 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.
Key words: Angiogenesis, miRNA, miR-126 (Mirn126), Egfl7, p85β (Pik3r2)
INTRODUCTION
MicroRNAs (miRNAs) are essential regulators of physiology and
pathophysiology (Zhao and Srivastava,
2007
). The inadvertent dysregulation of intronic miRNAs has been
predicted to be a general complication in the design and interpretation of
mouse knockout studies (Osokine et al.,
2008). miR-126 (Mirn126 - Mouse Genome
Informatics) is an endothelial miRNA residing within intron 7 of
Egfl7, resulting in pan-vascular developmental coexpression of
miR-126 and Egfl7 and their abundant expression in cultured
endothelium (Fitch et al.,
2004; Kloosterman et al.,
2006; Kuehbacher et al.,
2007; Poliseno et al.,
2006). Egfl7 is an endothelial secreted extracellular
matrix protein, which, in zebrafish, regulates embryonic vascular tube
assembly (De Maziere et al.,
2008; Parker et al.,
2004). In vitro, various functions have been ascribed to
Egfl7, including the regulation of endothelial or vascular smooth
muscle migration and adhesion (Campagnolo
et al., 2005; Parker et al.,
2004; Soncin et al.,
2003). Two different mouse knockout alleles of Egfl7 have
been described: a gene-trap insertion into intron 2, and an IRES lacZ
knock-in replacing exons 5-7, both upstream of miR-126 in intron 7.
Both the Egfl7 gene-trap and lacZ knock-in are associated
with edema, angiogenic deficits and 
50% embryonic lethality
(Schmidt et al., 2007). Here,
we explored the functions of both Egfl7 and its embedded miRNA,
miR-126, using floxed alleles to selectively disrupt each gene
without reciprocal perturbation.
MATERIALS AND METHODS
Generation of Egfl7/ and miR-126/ mice
For targeting Egfl7, a loxP site (Pl452) and a neomycin selection
cassette plus a loxP site (Pl451) were cloned into an AflII site
5' of exon 5 and into an NheI site 3' of exon 7,
respectively. For targeting the 73-bp miR-126 precursor, Pl452 and
Pl451 were cloned into an NheI site 194 bp 5' of
miR-126 and an NsiI site 22 bp 3' of miR-126,
respectively (flanking 289 bp total) (for details, see Figs S1 and S2 in the
supplementary material). Delta () alleles were generated by crossing to
CMV- or HPRT-Cre mice. Mutant mice were analyzed in a mixed
129sV/C57Bl/6 genetic background. All mice were treated according to the
Stanford Institutional Animal Care and Use Committee and the Stanford
Administrative Panel on Laboratory Animal Care.
miRNA in situ hybridization
In situ hybridization was performed as described
(Obernosterer et al., 2007).
Mouse miR-126 locked nucleic acid (LNA) probes were from Exiqon.
Generation of rabbit anti-Egfl7 antibody and immunofluorescence staining
Rabbits were immunized against the bacterially expressed C-terminal 112
amino acids of murine Egfl7 fused to the C-terminus of maltose binding protein
(MBP). Antiserum was affinity purified against the C-terminal 112 amino acids
of Egfl7 fused to the C-terminus of glutathione-S-transferase (GST). PFA-fixed
frozen uterus sections were stained with 0.1 µg of affinity-purified rabbit
anti-Egfl7 antibody and imaged with a Zeiss Z1 Axioimager with Apotome.
Quantitative real-time PCR
miR-126 expression was analyzed using the Taqman MicroRNA Assay
(Applied Biosystems) utilizing looped RT primers to detect processed
miR-126, and expression was normalized to that of miR-16.
Egfl7 expression was determined using the SYBR Green Quantitect PCR Kit
(Qiagen) and normalized to that of Gapdh. Egfl7 primers:
5'-TGCGACGGACACAGAGCCTGCA-3' and
5'-CAAGTATCTCCCTGCCATCCCA-3'. Assays were performed in triplicate
and results from at least three independent experiments are presented.
Whole-mount retina staining
P5 eyes were dissected and fixed in 4% paraformaldehyde (PFA) in PBS
overnight at 4°C. Retinas were isolated, blocked in PBS containing 1% BSA
and 0.5% Triton X-100 overnight at 4°C, incubated overnight with 10 µg
of FITC-conjugated isolectin B4 (Vector Labs) in 500 µl of the same
solution, washed and then flat mounted.
Western blot analysis
Antibodies used were: rabbit anti-p85, rabbit anti-phospho-Akt (Akt1 -
Mouse Genome Informatics) (Ser 473), rabbit anti-phospho-Erk (Mapk1 - Mouse
Genome Informatics) (all from Cell Signaling), rabbit anti-Spred1, rabbit
anti-p85β, rabbit anti-
-actin (all from Abcam) and rat anti-HA
(Roche).
|
Scratch wound assay
HUVEC were serum starved overnight 24 hours after transfection of miRNA
inhibitors, and scraped with a sterile P200 tip to generate a cell-free zone.
Cells were stimulated with human VEGF165 (R&D Systems) (10
ng/ml) for 24 hours. Migration was quantified by counting the number of cells
per scratched area (n=6).
Corneal micropocket assay
The corneal micropocket assay was performed as described
(Kuo et al., 2001).
miR-126 target luciferase reporter assay
The 3'UTR of Pik3r2 and Spred1 were amplified and
cloned downstream of a Renilla luciferase reporter gene. The
miR-126 binding sites were mutated from 5'-ACGGTAC-3' to
5'-GTAACGA-3' and from 5'-GGTACG-3' to
5'-AAGCAT-3' in the 3'UTR of Pik3r2 and
Spred1, respectively. The Lin41 (Trim71 - Mouse
Genome Informatics) 3'UTR was used as a negative control. 293T cells in
24-well plates were transfected with 3.35 ng/well of firefly luciferase, 0.667
ng/well of Renilla 3'UTR construct, and either 0, 10 or 100
ng/well of miR-126 expression vector. Empty vector was added to
provide a total of 337 ng of DNA per transfection. Forty-eight hours after
transfection, the Renilla/firefly luciferase was measured using the
Dual Reporter Luciferase Kit (Promega).
Akt/Erk phosphorylation assay
Akt/Erk phosphorylation assays were performed as described
(Gerber et al., 1998).
Statistical analysis
P-values were determined using a two-tailed Student's
t-test assuming unequal variances.
RESULTS AND DISCUSSION
We explored the mouse Egfl7/miR-126 locus using selectively floxed
Egfl7 and miR-126
alleles to replace either a 289 bp segment of intron 7 containing
miR-126 or exons 5-7 of Egfl7 with a single loxP site,
without disruption of the reciprocal gene or miRNA
(Fig. 1A; see Figs S1 and S2 in
the supplementary material).
miR-126/, but not
Egfl7/, embryos exhibited loss of
miR-126 expression as assessed by in situ hybridization or
quantitative PCR (qPCR) using looped RT primers to detect processed
miR-126 (Fig. 1B,C).
Conversely, Egfl7/, but not
miR-126/, mice exhibited loss of
Egfl7 by qPCR and by immunofluorescence with an affinity-purified
rabbit anti-Egfl7 antiserum (Fig.
1C,D). Furthermore, sequencing of the Egfl7 ORF amplified
from miR-126/ cDNA revealed a lack
of occult Egfl7 splicing alterations resulting from the
miR-126 microdeletion (Fig.
1E). These studies indicated the successful generation of two
monospecific alleles for miR-126 and Egfl7,
respectively.
Surprisingly, Egfl7/ mice were
phenotypically normal and born at the expected Mendelian ratios despite
previous reports from gene-trap and conventional knockout alleles
(Schmidt et al., 2007)
(Fig. 2A). By contrast,
miR-126/ mice recapitulated
numerous previously described Egfl7 mutant phenotypes
(Schmidt et al., 2007)
including 50% embryonic lethality
(Fig. 2B), which appeared
obligately associated with the development of prominent subcutaneous embryonic
edema by E14.5 (Fig. 2C,D). At
E15.5, multifocal, progressive hemorrhage of varying severity from ruptured
blood vessels was observed in 20% of the
miR-126/ embryos, most prominently
in the jugular and subcutaneous regions
(Fig. 2E), with resultant
embryonic lethality becoming first apparent at E16.5. The embryonic edema was
phenocopied by
miR-126flox//;Tie2-Cre embryos,
consistent with a cell-autonomous mechanism in the endothelium
(Fig. 2F).
|
|
/ neonates,
which were obtained at 50% of the expected frequency
(Fig. 2B), exhibited delayed
postnatal retinal angiogenesis (Fig.
3A-C). This was particularly notable in terms of compromised
radial migration, a decreased area of retinal vascularization, and abnormally
thickened endothelial sprouts (Fig.
3A-C), as previously described in Egfl7 gene-trap and
knock-in mice (Schmidt et al.,
2007). miR-126/ mice
further displayed delayed developmental cranial angiogenesis
(Fig. 3D), again reminiscent of
previously described Egfl7 mutant phenotypes
(Schmidt et al., 2007).
Consistent with a more substantial role for miR-126 in the regulation
of adult angiogenic processes, surviving
miR-126/ mice demonstrated impaired
angiogenesis in a VEGF-dependent corneal micropocket assay
(Fig. 3E,F). None of the
aforementioned phenotypes was observed in
Egfl7/ or wild-type mice
(Fig. 3A-F), with the deficits
in retinal, head and corneal vasculature all supporting the in vivo regulation
of angiogenesis by miR-126.
The mechanisms of miR-126 regulation of angiogenesis were further
explored in cultured endothelial cells. Transfection of an RNA hairpin
inhibitor induced a greater than 95% depletion of mature miR-126 in
HUVEC (Fig. 4A). This was
accompanied by significant decreases in migration in scratch assays, as well
as impaired VEGF-dependent activation of the downstream kinase Akt
(Fig. 4B,C). The basis for this
impaired VEGF signaling in miR-126-deficient endothelium was examined
at the level of miRNA target genes. miR-126 directly repressed
expression of the Pik3r2-encoded p85β subunit of PI3 kinase
(PI3K) in co-transfection assays, whereas p85β protein was increased in
both primary miR-126/ endothelium and
miR-126 knockdown HUVEC (Fig.
4D-G). Either the knockdown of miR-126 or the
overexpression of the target p85β in HUVEC was sufficient to impair
VEGF-mediated activation of the PI3K downstream target Akt, paralleling
inhibition of insulin receptor tyrosine kinase signaling by p85 overexpression
(Barbour et al., 2005;
Brachmann et al., 2005;
Ueki et al., 2002)
(Fig. 4C,H). miR-126
knockdown additionally impaired VEGF activation of Erk
(Fig. 4C), further reiterating
compromised signal transduction in angiogenesis by miR-126 knockdown
in vitro. In this regard, the Erk pathway inhibitor Spred1
(Taniguchi et al., 2007) was
directly repressed by miR-126 co-transfection and was upregulated in
miR-126 knockdown HUVEC (see Fig. S3 in the supplementary
material).
|
/ embryos and was tightly correlated
with the lethality observed in 50% of embryos. This edema did not appear
secondary to intrinsic cardiac defects (data not shown). The incompletely
penetrant embryonic lethality and angiogenic delay of
miR-126/ mice contrast with the more
classical embryonic lethal angiogenic phenotypes
(Gale and Yancopoulos, 1999)
and appear consonant with the comparatively subtle action of miRNAs in
fine-tuning global gene expression profiles
(Kloosterman and Plasterk,
2006; Zhao and Srivastava,
2007).
Consistent with its vascular expression pattern, these miR-126
phenotypes occur cell-autonomously in endothelium as judged from the
compartment-specific deletion phenotypes of
miR-126flox//;Tie2-Cre embryos.
Mechanistically, this cell-autonomous action allows miR-126
deficiency to derepress and overexpress the p85β regulatory subunit of
PI3K and Spred1, which represent negative regulators of PI3K and MAP kinase
signaling, respectively (see Fig. S4 in the supplementary material). Although
p85β and Spred1 dysregulation clearly appears contributory to the
miR-126 phenotype, the promiscuous action of miRNA suggests the
likely action of numerous additional target genes.
During the preparation of this manuscript, miR-126 deletion
phenotypes in mouse and knockdown in zebrafish were described with impaired
angiogenesis and vascular integrity via dysregulation of Spred1 and p85β
(Fish et al., 2008;
Wang et al., 2008). These
phenotypes are both reinforced by similar findings in the current report and
are extended by our analysis of endothelial-specific deletion in
miR-126flox//;Tie2-Cre embryos.
Furthermore, an added significant feature of the current study is the
unexpected lack of abnormalities in
Egfl7/ mice and the widespread
phenocopying by miR-126/ mice of vascular
deficits of previously described Egfl7 alleles, consisting of a
gene-trap in intron 2 and a lacZ insertion into exons 5-7, both
upstream of intron 7 that contains miR-126
(Schmidt et al., 2007). These
data indicate that miR-126 might well regulate the collective
migration of endothelium as has been proposed for Egfl7
(Schmidt et al., 2007). These
miR-126/ mice should facilitate
additional exploration of miR-126 function in settings of adult
angiogenesis, as well as of divergent miR-126 roles such as in
metastasis suppression (Tavazoie et al.,
2008). Conversely, the
Egfl7/ mice will allow selective in
vivo analysis of Egfl7 without the confounding influence of miR-126.
Our data by no means exclude novel and essential Egfl7-specific
functions, either alone or in conjunction with the paralog Egfl8, or
as described in zebrafish knockdown, mouse overexpression and in vitro studies
(Campagnolo et al., 2005;
Lelievre et al., 2008;
Soncin et al., 2003;
Xu et al., 2008).
The inadvertent disruption of miRNA expression by conventional deletion and
gene-trap knockout approaches in mice was recently predicted in a
bioinformatics analysis by McManus and colleagues
(Osokine et al., 2008). Our
results comparing miR-126/ and
Egfl7/ mice provide the most
extensive documentation of this complication to date, which might be more
widespread than anticipated; this possibility was not formally examined in the
mouse miR-126 deletion, as a parallel Egfl7 knockout was not
engineered (Wang et al.,
2008). From these studies, evaluation of intronic miRNA should be
a general consideration in the design and interpretation of mouse knockout
studies (Osokine et al.,
2008), and complications thereof might be avoided by utilizing
minimally disruptive strategies such as floxed alleles covering small genomic
regions. Finally, the regulation of angiogenesis by a mammalian miRNA suggests
novel methods for the therapeutic modulation of vascularization, for instance
during cancer or macular degeneration.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/24/3989/DC1
ACKNOWLEDGMENTS
We are indebted to Gerald Crabtree, Andrew Fire, Cecile Chartier and Cullen Taniguchi for helpful discussions. Hprt-Cre mice and recombineering plasmids were kind gifts of Liqun Luo and Neal Copeland, respectively. This work was supported by grants from the NIH (1 R01 CA95654-01, 1 R01 NS052830-01 and 1 R01 HL074267-01) and the Brain Tumor Society to C.J.K.
Footnotes
* These authors contributed equally to this work 
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2=0.741), whereas
miR-126
