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VASCULAR BIOLOGY
DLL1-mediated Notch activation regulates endothelial identity in mouse fetal
arteries
Inga Sörensen,1 Ralf H. Adams,2,3 and Achim Gossler1
1Institute
for Molecular Biology, Medizinische Hochschule Hannover, Hannover; 2Department of Tissue Morphogenesis, Max-Planck-Institute for Molecular
Biomedicine, Münster; and 3Faculty of Medicine, University of Münster, Münster, Germany
Notch signaling has been shown to regulate various aspects of vascular development. However, a specific role of the
ligand Delta-like 1 (DLL1) has not been
shown thus far. Here, we demonstrate
that during fetal development, DLL1 is an
essential Notch ligand in the vascular
endothelium of large arteries to activate
Notch1 and maintain arterial identity. DLL1
was detected in fetal arterial endothelial
cells beginning at embryonic day 13.5.
While DLL4-mediated activation has been
shown to suppress vascular endothelial
growth factor (VEGF) pathway components in growing capillary beds, DLL1Notch signaling was required for VEGF
receptor expression in fetal arteries. In
the absence of DLL1 function, VEGF receptor 2 (VEGFR2) and its coreceptor,
neuropilin-1 (NRP1), were down-regulated
in mutant arteries, which was followed by
up-regulation of chicken ovalbumin upstream promoter-transcription factor II
(COUP-TFII), a repressor of arterial differentiation and Nrp1 expression in veins.
Consistent with a positive modulation of
the VEGF pathway by DLL1, the Nrp1
promoter contains several recombinant
signal-binding protein 1 for J ␬ (RBPJ␬)–
binding sites and was responsive to Notch
activity in cell culture. Our results establish DLL1 as a critical endothelial Notch
ligand required for maintaining arterial
identity during mouse fetal development
and suggest context-dependent interrelations of the VEGFA and Notch signaling
pathways. (Blood. 2009;113:5680-5688)
Introduction
A functional cardiovascular system with precisely formed and
connected arteries and veins is established early during mammalian
embryogenesis and is essential for subsequent development and
survival. First, endothelial precursors, the angioblasts, differentiate
into endothelial cells and form a primitive vascular network in a
process called vasculogenesis. These first vessels are simple tubes
composed entirely of endothelial cells. Subsequently, the simple
vascular network is remodeled into a hierarchical network of veins,
arteries and capillaries in a process called angiogenesis.1 Angiogenesis is a precisely orchestrated multistep process that involves
localized cell proliferation and migration, the extension of endothelial sprouts and their conversion into patent tubules, as well as the
selective pruning of some vessel connections. During angiogenesis,
the division into morphologically, functionally, and molecularly
unique arteries and veins is established and supporting cell types,
such as smooth muscle cells and pericytes, are recruited to form
mature and fully functional vessels.2
Vascular endothelial growth factor (VEGF) signaling is essential for establishment of the embryonic vasculature as is indicated
by absence or severe malformations of vessels in embryos lacking
VEGF or its receptors (reviewed in Rossant and Howard3). Early
during angiogenesis, ie, before development of a functional
circulation, the distinction between arteries and veins is established
in a process termed arteriovenous differentiation. Accordingly,
certain markers and signaling molecules, such as the transmembrane protein ephrinB2 (EFNB2), are specifically expressed in the
endothelium of developing arteries. Conversely, the expression of
the receptor tyrosine kinase EPHB4, an interaction partner of
ephrinB2, and other gene products are confined to the venous
endothelium. Loss of either EphrinB2 or EPHB4 leads to very
similar defects in vascular remodeling, suggesting that they
mediate bidirectional signaling essential for angiogenesis and the
establishment of boundaries between venous and arterial domains.4-6
The evolutionarily conserved Notch signaling pathway plays a
central role in the specification of cell fates by mediating local cell
interactions in different tissues in diverse organisms.7-9 The Notch
gene of Drosophila, as well as its vertebrate homologues, encode
large cell surface receptors that interact with their ligands, namely
the products of the Delta and Serrate (in vertebrates called Jagged)
genes, in a cell-to-cell contact-dependent fashion. Notch, Delta,
and Serrate/Jagged encode transmembrane proteins with multiple
EGF-like repeats in their extracellular domains.10 Upon ligand
binding, the intracellular portion of Notch is proteolytically
cleaved, translocates to the nucleus, and forms a complex with the
transcriptional regulator recombinant signal-binding protein 1 for J
␬ (RBPjk), which will then activate transcription of target genes
such as Hes and Hey family bHLH transcriptional regulators.11-13
Four different Notch receptors (Notch 1-4) and 4 activating
ligands, Delta-like (Dll) 1, 4, Jagged1, and Jagged2, have been
identified in mice. Several receptors and ligands are expressed in
the developing vasculature. Notch1, Notch4, Delta1, Delta4, and
Jagged2 are specifically expressed in the arterial endothelium.
Notch3 was detected in the smooth muscle cells surrounding
arteries but not veins, whereas Jagged1 is expressed in both the
endothelial and smooth muscle cells surrounding arteries.14,15
Notch4 and Delta4 expression appear to be vessel-specific and
Submitted August 14, 2008; accepted January 4, 2009. Prepublished online as
Blood First Edition paper, January 14, 2009; DOI 10.1182/blood-2008-08-174508.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
An Inside Blood analysis of this article appears at the front of this issue.
The online version of this article contains a data supplement.
5680
© 2009 by The American Society of Hematology
BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
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BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
entirely restricted to the endothelium, including developing
capillaries.16,17
Studies in zebrafish and mice have demonstrated essential functions
of Notch signaling for vascular remodeling of the primitive vascular
plexus and establishment of arterial fate (reviewed in Rossant and
Howard,3 Alva and Iruela-Arispe,18 Shawber and Kitajewski,19 Adams
and Alitalo,20 and Siekmann et al21). Mouse embryos deficient for
Jagged1, Notch1, or Notch1/Notch4 die between embryonic day (E) 9.5
and 10.5 and display severe vascular malformations.22,23 In all these
mutants, initial establishment of the vascular network appears to be
largely normal, indicating essential roles in angiogenesis, but not in
vasculogenesis. During zebrafish development, loss of Notch or the
downstream Notch target gene gridlock (the ortholog of mouse Hey2),
leads to a loss of the arterial cell marker ephrinB2 (in zebrafish:
ephrinB2a) and an increase in the expression of the venous marker
EphB4 in the dorsal aorta.24,25 Similarly, in early mouse embryos
DLL4-mediated Notch activity is required in a dose-dependent manner
for arterial differentiation and ephrinB2 expression,26,27 indicating that
DLL4 is the critical endothelial Notch ligand directing normal arterial
patterning in early mammalian embryos. In zebrafish embryos, Notch
activity is regulated by VEGF-A suggesting that arterial specification
occurs via a VEGF-A/Notch signaling cascade resulting in the upregulation of the arterial endothelial cell marker ephrinB2.28
In adult heterozygous Dll1 mutants, ischemia-induced arteriogenesis was severely impaired,29 indicating that DLL1 regulates
postnatal arteriogenesis and suggesting that DLL1 might also play
a role in the vascular endothelium earlier in development. Here we
show that DLL1 is specifically expressed in fetal arterial endothelial cells beginning at E13.5, is a critical regulator of arterial
patterning, and, in contrast to the role of the Notch pathway in
growing capillaries, positively regulates VEGF signaling. Embryos
with reduced DLL1 levels or specific ablation of DLL1 in vascular
endothelium rapidly lost Notch1 activation after E13.5 although
DLL4 expression was maintained. Arterial endothelial and smooth
muscle markers, notably VEGF receptor 2 (VEGFR2) and its
coreceptor neuropilin-1 (NRP1), were down-regulated. These
changes in the mutant arteries preceded the ectopic expression of
venous markers such as chicken ovalbumin upstream promotertranscription factor II (COUP-TFII), which normally suppresses
Nrp1 expression and the arterial differentiation program in the
venous endothelium.30 Our results establish DLL1 as a critical
endothelial Notch ligand required for maintaining arterial identity
during fetal development and suggest context-dependent interrelations of the VEGF-A and Notch signaling pathways.
Methods
Immunohistochemistry
Immunostraining was performed on 10-␮m sections of MeOH/DMSO
(dimethyl sulfoxide)–fixed paraffin embedded embryos of the indicated
age. Mutant and wild type embryos were analyzed in parallel with the
indicated antibody under identical conditions. At least 3 embryos were
analyzed with each marker at a given developmental stage. Slides were
dehydrated, for antigen retrieval slides were boiled in 10 mM sodium citrate
(pH 6.0). Blocking was performed in 1% bovine serum albumin (BSA) and
sections were incubated with the primary antibody overnight. After
incubation with the appropriate horseradish peroxidase (HRP)–conjugated
secondary antibody, the signal was developed with a TSA-Cy3 System
(Tyramide Signal Amplification; Perkin Elmer, Waltham, MA). Vascular
smooth muscle cells were marked using ␣–smooth muscle actin antibodies
and cell nuclei with Draq5 (Biostatus Limited, Leicestershire, United
Kingdom). Antibodies used were: anti-Notch1 ICD Val-1744 (1:100; Cell
Dll1 FUNCTION IN FETAL ARTERIES
5681
Signaling Technology, Danvers, MA); anti-EphrinB2 (1 ␮g/mL), antiEphB4 (1␮g/mL), anti-Neuropilin1 (Nrp1, 5 ␮g/mL; all R&D Systems,
Minneapolis, MN); anti-VEGF (147; 2 ␮g/mL), anti-VEGFR2 (Flk1, C-20;
2 ␮g/mL), anti-VEGFR1 (Flt1, C-17; 2 ␮g/mL), anti-Smoothelin (c-20;
2 ␮g/mL), anti-Jagged1 (H-114; 2 ␮g/mL; all Santa Cruz Biotechnology,
Santa Cruz, CA); anti-DLL4 (15 ␮g/mL), anti-DLL1 (10 ␮g/mL; Abcam,
Cambridge, MA); anti-COUPTFII (0.01 ␮g/mL; Perseus Proteomics,
Tokyo, Japan); anti-eNOS (2 ␮g/mL; Affinity Bioreagents, Rockford, IL);
anti-Notch1 (bTan; 1/50; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA); anti–phospho-VEGFR2 (pFlk1-Tyr 951;
2 ␮g/mL; Santa Cruz Biotechnology); anti–phospho-VEGFR2 (pFlk1-Tyr
1175; 1/100; Cell Signaling Technologies); anti–␣-smooth muscle actinFITC (1/800; Sigma-Aldrich, St Louis, MO). Secondary antibodies used
were: anti–mouse IgG-HRP, anti–rabbit IgG-HRP, anti–rat IgG-HRP (all
1/200; GE Healthcare Europe, LittleChalfont, United Kingdom); anti–goat
IgG-HRP (1/100; Dianova, Hamburg, Germany).
Slides were analyzed by confocal laser-scanning microscopy using a
TCS-SP2 AOBS with a PLAPO 63⫻/1.4 objective (Leica Microsystems,
Wetzlar, Germany). For image acquisition, Leica Confocal Software
version 2.5 (Leica Microsystems) was used. Pictures were processed and
assembled using Photoshop and Illustrator CS (Adobe Systems, San Jose, CA).
Western blot analysis
Outflow tracts of E15.5 embryos were dissected, flash-frozen in liquid
nitrogen, and minced in Ripa buffer. Equal amounts of protein from cell or
pooled tissue lysates were separated by SDS–polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes (Immobilon; Millipore,
Billerica, MA) and probed with primary and secondary antibodies. Except
for Neuropilin1 (anti-Neuropilin1, 1 ␮g/mL; Abcam), the same antibodies
as for immunohistochemistry were used. As a loading control, a mouse
anti-actin antibody (MP Biochemicals, Solon, OH) was used.
Cre induction and blocking of VEGF and Notch activity
To generate the endothelial-specific knockout, mice were injected on
3 consecutive days intraperitoneally with 100 ␮L Tamoxifen (Sigma-Aldrich) at
a concentration of 10 mg/mL in corn oil (Sigma-Aldrich). The monoclonal
neutralizing anti–mouse VEGF antibody (N-mVEGF) was purchased from
ReliaTech (Braunschweig, Germany), diluted in physiologic salt solution and
injected into the tail vein of pregnant females on 2 consecutive days. For
Notch inhibition experiments, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-Sphenylglycine t-butyl ester (DAPT; 50 mg/kg; Alexis Biochemicals, San Diego,
CA) was injected (10 ␮L in DMSO) into the tail vein. All animal studies were
approved by the institutional review board at the Institute for Molecular Biology
according to German rules and stipulations.
Nrp1 promoter constructs
Five kilobases of the upstream region of Neuropilin1 were amplified from a
BAC clone (RP23-320F22; ImaGenes, Berlin, Germany) using the Expand
High-Fidelity PCR System (Roche, Basel, Switzerland). Mutagenesis was
carried out using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) and confirmed by sequencing. Constructs were cloned
into the luciferase reporter construct pGL4.27 (luc2P/minP/Hygo; Promega, Madison, WI).
In situ hybridization
In situ hybridization was performed by standard procedures.
Mice
Dll1lacz 31, Dll1Dll1kineo,
previously.
32Dll1loxp 33,
and R-NICD34 mice were described
Cell lines
CHO cells stably expressing DLL1 or DLL4 were generated by transfection
of CHO cells with expression plasmids using Jetpei (BIOMOL Research
Laboratories, Plymouth Meeting, PA) according to the manufacturer’s
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5682
BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
SÖRENSEN et al
instructions followed by G418 selection. HeLa cells stably expressing
Notch1 were provided by A. Israel (Institut Pasteur, Paris, France).
Retina staining
Eyes were dissected at P6, P9, and P15, fixed in 4% paraformaldehyde
(PFA) for 1 hour at room temperature, washed twice with phosphatebuffered saline (PBS), and enucleated. Retinas were dissected, incubated
with biotinylated Griffonia simplicifolia lectin1 (Isolectin B4; Vector
Laboratories, Burlingame, CA; detected with streptavidin 405 Alexa),
anti-Desmin (Abcam; detected with donkey anti–rabbit 488 Alexa), and
Cy3-conjugated ␣–smooth muscle actin and flat mounted. For DLL1
staining, eyes were fixed in 4% PFA for 5⬘ and dissected retinas were
refixed in 100% MeOH. Mounted retinas were analyzed using the
microscope Leica DMI 6000B with a HC PL Fluotar 10⫻/0.3 objective
(Leica Microsystems). For image acquisition, LAS AF software (Leica
Microsystems) was used.
Brain staining
Brains were dissected at P6, P9, and P15, fixed overnight in 4% PFA,
washed in PBS, dehydrated to 100% MeOH, and incubated in 5% H2O2 in
Methanol for 5 hours to destroy endogenous peroxidase. Brains were
rehydrated, incubated with anti-Pecam (Pharmingen, San Diego, CA) and a
biotinylated secondary antibody (Vector Laboratories), incubated in avidinbiotin-peroxidase complex (ABC)–Reagent (Vector Laboratories) and the
signal was developed using DAB (diaminobenzidin; Vector Laboratories).
Images were taken with a Leica M420 macroscope with Leica Apozoom 1.6
(Leica Microsystems). A FUJIK digital camera HC-300Z and the software
Photograb-300Z (Fujifilm, Tokyo, Japan) were used to acquire images.
Skin staining
Heads of E17.5 embryos were fixed in 4% PFA overnight. The skin was
dissected and incubated for 4 hours in blocking solution (1%BSA, 0.5%
Tween-20 in PBS) incubated with the primary antibody (anti-Endomucin
[gift of D. Vestweber, Max-Planck-Institute for Biomedicine, Münster,
Germany]), anti–collagen typeIV [Chemicon, Billerica, MA]) O/N at 4°C,
washed, incubated with the secondary antibody (donkey anti–rat Alexa 488
and donkey anti–rabbit Alexa 543; Molecular Probes, Carlsbad, CA) and
flat mounted with Flouromount (Southern Biotech, Birmingham, AL).
Mounted skin preparations were analyzed with a Leica DM 5000B
microscope with a HC PL Fluo 10⫻/0.3 objective (Leica Microsystems).
Images were acquired with a Leica DFC 300FX camera and Leica FireCam
software (Leica Microsystems).
Ink injection
Ink (diluted 1:100 in PBS) was injected into the carotid artery of E17.5
embryos. Embryos were fixed in 4% PFA O/N, dehydrated to 100%
methanol, and cleared in benzyl alcohol/benzyl benzoate (2:1). For
microscope and image acquisition, see brain staining.
Morphometric and branchpoint analysis
Analysis of the vessel wall thickness and vessel lumen was carried out on
hematoxylin-eosin (HE)–stained paraffin sections of E15.5 and E18.5
embryos. Images were taken with a Leica DM 5000B microscope with a
HC PL Fluo 20⫻/0.5 objective (Leica Microsystems). Images were
acquired with a Leica DFC 300FX camera and Leica FireCam software
(Leica Microsystems). Wall thickness and lumen were measured using
ImageJ (National Institutes of Health, Bethesda, MD). Branchpoints were
counted on endomucin-stained skin preparations of E17.5 embryos using
Imaris (Bitplane, Zurich, Switzerland). Microscope and image acquisition
were the same as for skin stainings.
TA assay
CHO cells stably expressing DLL1 or DLL4 were transiently transfected
with Jag1 expression plasmids using Jetpei (Peqlab Biotechnologie,
Erlangen, Germany) following the manufacturer’s instructions. HeLa cells
Figure 1. DLL1 expression in fetal arteries. DLL1 (red) is highly expressed in the
endothelium (arrows in C⬘ and E⬘) and in the smooth muscle layer (arrowheads in C⬘
and E⬘) starting at embryonic day (E) 13.5 in arterial vessels, but not in veins (D). At
E12.5, there is no detectable expression in arteries or veins (A,B). This pattern
persists until E18.5 (E,F). A⬘ to F⬘ shows magnifications of panels A through F. Green
indicates antibody staining for ␣–smooth muscle actin.
stably expressing Notch1 were transiently transfected with a Jag1 or manic
fringe Mfng expression plasmid and a RBPJ␬ luciferase reporter construct.
Aliquots of 106 transfected HeLaN1 cells were cocultivated on 6-well plates
for 24 hours with 106 CHO cells, stably expressing the respective ligands.
Luciferase activity was measured using the Dual-Luciferase reporter Assay
System (Promega). Firefly luciferase activity was normalized to cotransfected
Renilla luciferase activity (pRL-TK; Promega).
Results
Dll1 is critical for Notch1 activation in arterial endothelial cells
Before E13.5, expression of DLL1 was not detected in blood
vessels. At E13.5, DLL1 expression in vascular endothelial cells
was detected in arteries, but not in capillaries or veins, and
persisted in arterial vascular endothelium throughout subsequent
development (Figure 1, and Figure S4, available on the Blood
website; see the Supplemental Materials link at the top of the online
article), suggesting a function in fetal arteries well after early
Notch-regulated angiogenic processes. To analyze the function of
Dll1 in the fetal vascular endothelium, we made use of a
hypomorphic Dll1 allele (Dll1Dll1kineo) that significantly reduces
Dll1 transcription32 when combined with a Dll1lacZ null allele.31
Heteroallelic mice (Dll1lacZ/Dll1kineo) carrying these alleles survive
until birth and have, among other malformations, severe skeletal
muscle defects. Muscle defects in early heteroallelic embryos were
indistinguishable from muscle differentiation defects in Dll1 null
mutants,32 suggesting that in Dll1lacZ/Dll1kineo embryos, Dll1 activity
is essentially abolished. To detect a potential requirement of DLL1
for Notch activity in fetal blood vessels, we analyzed the aorta and
other large vessels of the trunk at the level of the outflow tract
(Figure 2A). We first analyzed Notch1 activation using an antibody
(Val1744) specifically recognizing the activated form of Notch1,
NICD. At E13.5, activated Notch1 (NICD) was detected in wild
type and mutant arteries (Figure 2B a, a⬘, b, b⬘). In contrast, no
NICD was detected in mutant vascular endothelium at E15.5 and
E18.5 (Figure 2B d, d⬘, f, f ⬘), although Notch1 protein was present
at all analyzed stages (Figure 2B g-l, g⬘-l⬘). Consistent with
absence of NICD, Hey2 transcription was down-regulated in
mutant vessels (Figure S2A n, n⬘). To address whether loss of
Notch1 activation affects expression of other receptors or Notch
pathway components, we analyzed their expression by in situ
hybridization. Notch2, 3, and 4, and Dll4 expression was apparently unaffected, whereas Jag1 and Mfng appeared up-regulated in
mutant vessels (Figure S2A c-j, l, p). Because DLL4 is critical for
Notch activity in early embryonic vessels,26,27 we analyzed whether
DLL4 protein expression is maintained in Dll1 mutant embryos.
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BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
Figure 2. Notch1 activation is reduced in Dll1 mutants. (A) Histologic section of an
E15.5 embryo indicating the level along the anterior-posterior body axis used for the
analysis of the fetal vessel phenotype. (B) Antibody staining for cleaved Notch (a-f),
full-length Notch1 (g-l), and DLL4 (m-r). Green: ␣–smooth muscle actin, red indicates
antigen. Notch activation is abolished in Dll1 mutant embryos at E15.5 and E18.5
(c-f), whereas Notch1 and DLL4 are still expressed at these embryonic stages (g-r).
At E13.5, mutant vessels show expression patterns of the analyzed markers similar
to wild type (a,b,g,h,m,n). Although DLL4 is normally expressed it does not seem to
be able to activate Notch1. a⬘ to r⬘ show magnification of the boxed areas indicated in
subpanels a through r.
DLL4 protein was clearly detected in mutant vessels by antibody
staining (Figure 2B n, n⬘, p, p⬘, r, r⬘), indicating that DLL4 is not
sufficient to efficiently activate Notch1 in this context, although we
cannot exclude that DLL4 might activate (an)other Notch receptor(s) expressed in fetal arteries. Collectively, these results indicate
that DLL1 is a critical activator of Notch1 in fetal arterial
endothelium, and suggest nonredundant functions of DLL1 and
DLL4 in this context.
Normal levels of Notch ligands in vascular endothelial cells are
crucial for embryonic angiogenesis26,27 and ischemia-induced arteriogenesis.29 Thus, loss of DLL1 might simply reduce the concentration of ligands below a threshold required for sufficient Notch
activity. Therefore, we tested whether DLL1 can enhance the
ability of DLL4 to activate Notch in a coculture assay in vitro.
However, coexpression of DLL1 and DLL4 did not improve Notch
activation in vitro (Figure S2B) arguing against a synergistic role of
the 2 ligands. Likewise, coexpression of JAG1 weakly improved
Notch activation by DLL1, but not DLL4, in vitro (data not shown).
Because fringe glycosyltransferases can modify Notch receptors
and thereby enhance or attenuate the responsiveness of Notch to
different ligands,35 and Mfng expression appeared up-regulated in
mutant endothelium, we tested whether MFNG differently influences the ability of DLL1 and DLL4 to activate Notch in vitro.
Expression of MFNG in Notch1-expressing HeLa cells (HeLa-N1)
enhanced Notch activity approximately 8-fold when these cells
Dll1 FUNCTION IN FETAL ARTERIES
5683
Figure 3. Arterial maker expression is severely reduced in Dll1 mutant
embryos. At E13.5, mutant embryos still show normal expression of arterial markers
comparable to wild-type embryos (A, B, G, H, M, N, S, T). At E15.5 and E18.5, the
arterial markers Neuropilin1 (C-F), VEGFR2 (I-L), EphrinB2 (O-R), and Smoothelin
(U-X) are severely reduced in arterial vessels. The venous marker COUP-TFII, which
is normally expressed in perivascular cells surrounding arteries, is ectopically
up-regulated in arterial endothelial cells (Y-ZD) at E15.5 (arrowheads in ZB⬘) and
E18.5 (arrowheads in ZD⬘). Green indicates ␣–smooth muscle actin; red indicates
antigen. A⬘ to ZD⬘ show magnifications of the boxed areas indicated in A-ZD.
were cocultured with either DLL1 or DLL4 expressing CHO cells
(Figure S2B) compared with HeLa-N1 cells without MFNG. When
MFNG was coexpressed with DLL1 or DLL4 in CHO cells, Notch
activation in HeLa-N1 cells was unaffected. Thus, up-regulation of
Mfng expression has no inhibitory effect on DLL4 activity but
might rather indicate the induction of a compensatory (albeit
insufficient) mechanism for the restoration of Notch signaling in
Dll1 mutant fetal arteries.
Dll1 signals maintain arterial gene expression
To address the consequences of abolished Dll1 mediated Notch
activity in endothelial cells, we analyzed the expression of NRP1,
VEGFR2, and ephrinB2 proteins specifically expressed in endothelial cells of arteries, and of smoothelin, which is restricted to
arterial vessel walls. At E13.5, when NICD was still detected in
mutant vessels (Figure 2B b, b⬘), all markers were expressed in
apparently normal patterns (Figure 3B, B⬘,H, H⬘, N, N⬘, T, T⬘, Z,
Z⬘). However, at E15.5 and E18.5, expression of all markers was
severely down-regulated or abolished (Figure 3D, D⬘, F, F⬘, J, J⬘, L,
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5684
SÖRENSEN et al
BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
L⬘, P, P ⬘, R, R ⬘,V, V ⬘, W, W⬘). COUP-TFII, a transcription factor
that is normally exclusively expressed in venous endothelial cells
and perivascular cells surrounding arteries and veins, and is
essential for vein identity,30 was ectopically activated in the
endothelium of arteries at low levels at E15.5 and was readily
detected at E18.5 (arrowheads in Figure 3ZB, ZB⬘, ZD, ZD⬘). Loss
of NICD and artery-specific gene expression was also found in
large vessels in the head and abdomen (Figure S1). We conclude
that DLL1 mediated Notch activity is required to maintain arteryspecific gene expression in fetal arteries throughout the body.
Dll1 function is required in endothelial cells
As the analyzed allele combination affects DLL1 function systemically, some effects might not be specific for DLL1 function in the
vascular endothelium. To analyze whether the observed changes
can be attributed specifically to DLL1 function in endothelial cells,
we deleted DLL1 in endothelial cells using a floxed Dll1 allele33
and a transgenic mouse line, in which a tamoxifen-inducible Cre
(CreERT2) was inserted into a large PAC clone of the Cadh5
(VE-Cadherin) gene resulting in endothelium-specific expression
(R.H.A., unpublished data, August 5, 2006). Fetuses lacking DLL1
in the arterial endothelium showed a down-regulation of markers
similar to heteroallelic mutant embryos (Figure 4B b, b⬘, f, f⬘, j, j ⬘,
n, n⬘, r, r⬘; Figure 4C). Arguing further for a specific role of
DLL1-Notch signaling in the endothelium, expression of NICD
(a constitutively active form of Notch) using a mouse line carrying
a Cre-inducible NICD cDNA inserted into the ROSA26 locus34
restored arterial gene expression in Dll1 mutants (Figure 4B c, c⬘,
g, g⬘, k, k⬘, o, o⬘, s, s⬘; Figure 4C). Conversely, administration of the
␥-secretase inhibitor DAPT, an inhibitor of Notch signaling, led to
the down-regulation of arterial markers as in Dll1 mutants (Figure
4B d, d⬘, h, h⬘, l, l⬘, p, p⬘, t, t⬘; Figure 4C), confirming that DLL1 is
indeed the pivotal Notch ligand controlling arterial identity during
fetal development.
Phenotypic consequences of lost artery-specific gene
expression
To address which consequences the loss of artery-specific gene
expression has for the structure of these vessels and the vasculature, we first examined Dll1lacZ/Dll1kineo and endothelium-specific
Dll1 mutant fetuses histologically, but detected no obvious structural defects (data not shown). Morphometric analyses did not
show significant differences in the thickness of the arterial walls,
but revealed a reduction of the lumens of the aorta and other large
arteries in both mutants (Figure S3A). Whole mount staining for
Collagen type IV and endomucin in skin preparations showed no
evidence for shunts or other gross abnormalities, but revealed a
statistically significant (P ⬍ .01) increase in capillary branch
points (Figure 4D). Ink injections into the carotid artery of E17.5
mutant embryos also did not show major abnormalities, although
main vessels in the head appeared disorganized in some mutant
embryos (Figure S3B). To analyze potential defects in the vasculature further, we turned to postnatal angiogenesis. Because pups
with deletion of DLL1 from the vascular endothelium during
embryogenesis were not viable, we induced recombination postnatally on P1, P2, and P3, and analyzed the vasculature in the retina
and brain of mutants on P6, P9, and P15, up to which point we did
not observe lethality in mutant pups. Staining of retinas for
Isolectin B4, Desmin, and ␣–smooth muscle actin revealed no
obvious differences in the capillary network or in pericyte and
smooth muscle cell coverage between wild-type and mutant pups
Figure 4. The endothelial-specific KO of Dll1 resembles the hypomorphic
arterial phenotype and can be rescued by constitutive overexpression of
activated Notch. (A) Regimen of Tamoxifen or DAPT application. Tamoxifen or the
␥-secretase inhibitor DAPT was injected into pregnant mice on 3 consecutive days
(E11.5-E13.5). Embryos were analyzed at E15.5. (B) Microphotographs of vessels
stained with antibodies. Antibody staining for Dll1 shows that is successfully removed
from the endothelium in tamoxifen-treated Dll1loxp; VE-Cadherin-Cre-ERT embryos
(a,b). Notch activation is disturbed in these embryos (e, f) and the expression of the
arterial markers Neuropilin1 (i,j), VEGFR2 (n,m), and EphrinB2 (q,r) is downregulated. Concomitant endothelial-specific overexpression of constitutively active
Notch (g) restored the expression of arterial markers (k,o,s). DLL1 expression is not
affected by constitutive Notch activity (c). Administration of the ␥-secretase inhibitor
DAPT blocked Notch activation (h) and reduced arterial makers (l,p,t) similar to loss
of DLL1-mediated Notch activation. DLL1 expression is not affected (d). Green
indicates ␣–smooth muscle actin; red indicates antigen. a⬘ to t⬘ show magnifications
of the boxed areas indicated in a through t. (C) Western blot analysis with protein
lysates of the heart outflow tract confirmed the results obtained by immunohistochemistry. Actin was used as a loading control. (D) Increased capillary branching in the skin
of E17.5 Dll1 hypomorphic embryos indicated by antibody staining for collagen typeIV
(red) and endomucin (green). Data are mean values of 5 embryos per genotype. Bars
indicate SD.
(Figure S3C a-c, f-h). In addition, PECAM staining of mutant
brains did not show severe abnormalities in the superficial vessel
network (Figure S3C d-e, i-j). However, we cannot exclude that
more severe defects develop over time in adult or aging mice.
DLL1 regulates VEGF responsiveness in arterial endothelial
cells
Loss of DLL1 led to down-regulation of NRP1, which is required
for efficient VEGFR2 function. COUP-TFII represses Nrp1 in
veins,30 and COUP-TFII itself is repressed by the Notch target
HEY2 in endothelial progenitor cells and differentiating ES cells.36
This suggested that loss of DLL1-mediated Notch activity in
arteries results in up-regulation of COUP-TFII, which in turn could
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BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
Figure 5. NRP1 expression depends on Notch activity. (A) Down-regulation of
Neuropilin1 at E14.5 precedes up-regulation of COUP-TFII. At E14, Notch activation
(a), and NRP1 and COUP-TFII expression (b,c) appeared normal in mutant embryos.
At E14.5, Notch activity (d) and expression of Neuropilin1 (NRP1) was reduced (e),
although COUP-TFII was not yet up-regulated in the arterial endothelium of E14.5
embryos (f). Green indicates ␣–smooth muscle actin; red indicates antigen. Nuclei
are counterstained with Draq5 (blue). (B) Arrangement of RBPJ␬ and COUP-TFII
binding sites in 5 kb of the Nrp1 promoter region. (C) The Neuropilin1 promoter
responds to Notch1 activation in vitro. Coculture of CHO cells stably expressing Dll1
with HeLa-N1 cells activated an RBP-luc reporter (set as 100%) and less strongly the
Nrp1-luc reporter. (D) Effective activation of the Nrp1 promoter fragment by Notch
requires all RBPJ␬ binding sites. M1-M12345 refers to mutations in the binding sites
indicated in panel B. Data are the results of 3 independent experiments. Bars indicate SD.
cause down-regulation of NRP1. However, no NRP1 protein was
detected in E15.5 mutant arteries although COUP-TFII was barely
expressed, arguing for a potential alternative mechanism of Nrp1
down-regulation. To clarify whether activation of COUP-TFII
underlies repression of Nrp1 in Dll1 mutants, we analyzed the
expression of NICD, NRP1 and COUP-TFII on serial sections of
mutant arteries at E14.0 and 14.5. At E14.0, NICD and NRP1 were
detected in arteries, and COUP-TFII was present in vascular
smooth muscle and perivascular cells, but not in endothelial cells
(Figure 5A a-c). At E14.5 NICD and NRP1 were no longer detected
(Figure 5A d-f) but also COUP-TFII was still absent from arterial
endothelial cells (arrowheads). This suggested that the initial
down-regulation of Nrp1 is not due to repression by COUP-TFII,
but independently caused by loss of activation by Notch1, which is
Dll1 FUNCTION IN FETAL ARTERIES
5685
consistent with the down-regulation of Nrp1 in Notch1 mutant
embryos.37 In support of a direct link between Notch and Nrp1
transcription, the promoter region of Nrp1 contains, in addition to
COUP-TFII binding sites, also 5 RBPj␬ sites (Figure 5B). To test
whether these sites are functional, we fused the Nrp1 promoter to a
luciferase reporter gene and measured its activation by Notch in
comparison to an established (RbpJ)6-luciferase reporter gene38 in
HeLa cells stably expressing Notch1 after coculture with DLL1
expressing CHO cells. The Nrp1 reporter was activated to levels of
approximately 40% of the efficient (RbpJ)6-luciferase reporter
(Figure 5C) indicating that the Nrp1 promoter responds to Notch
activation. Multiple RBPj␬ sites are also present in the Nrp1
promoters of the rat, human, canine and zebrafish Nrp1 genes,
although positions and spacing differ (data not shown), suggesting
that Nrp1 regulation by Notch is conserved. The lower activation of
the Nrp1 promoter construct compared with (RbpJ)6-luciferase
might be due to the presence of COUP-TFII in HeLa cells,39 which
could reduce expression from the promoter by competing with
activation by NICD bound to RBPj␬. Activation of the Nrp1
promoter was severely reduced or abolished by mutations in
individual or all RBPj␬ sites (Figure 5D), indicating specificity.
The drastic effect of mutations already in individual RBPj␬ sites, in
particular sites 1, 2, and 3 might be explained by the increased ratio
of COUP-TFII and RBPj␬ binding sites (and the balance between
repressor and activator).
These results suggested that Notch activity directly regulates
Nrp1 expression and thus efficient reception of VEGF-A signals in
fetal arteries. To address whether VEGF-A activity affects Notch
expression or activity in fetal arteries, we blocked VEGF-A by
repeated injection of a neutralizing anti–mouse VEGF-A antibody
into pregnant females. This treatment did not affect expression of
VEGFR2 (Figure 6B b, b⬘; Figure 6C), but prevented its activation
as indicated by the absence of tyrosine phosphorylated (activated)
VEGFR2, and by down-regulation of eNOS, which is positively
regulated by VEGF-A40 (Figure 6B d, d⬘, f, f⬘; Figure 6C).
Importantly, neither expression of DLL1 (Figure 6B j, j⬘; Figure
6C) or DLL4 (Figure 6B l, l⬘; Figure 6C), nor activation of Notch1
(Figure 6B n, n⬘; Figure 6C) and ephrinB2 expression (Figure 6B h,
h⬘; Figure 6C) was down-regulated. EphrinB2 expression in the
absence of activated VEGFR2 also suggests a direct role for Notch
signaling in regulating the expression of this arterial marker, as has
recently been shown for the ventricular chamber.41
Collectively, these findings suggest that in mouse fetal arteries
Notch signaling maintains arterial identity independent from the
VEGF-A pathway.
Discussion
A role for Notch signaling in vascular development is firmly
established. However, a specific role of the Notch ligand DLL1 in
vessel development has been elusive. Here, we show that DLL1 is
pivotal for activation of Notch in fetal arteries to maintain
artery-specific gene expression and responsiveness to VEGFR2/
NRP1-mediated VEGF signals.
DLL1 is a critical ligand for Notch activation in fetal arteries
Dll1 null mutant embryos show prominent hemorrhaging around
E10.31 However, Dll1 expression in the vasculature was not
detected before E13.5 (Figure S4), suggesting that the early
hemorrhagic phenotype in Dll1 mutant embryos arises secondarily
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5686
SÖRENSEN et al
Figure 6. Blocking of VEGF signaling does not impair Notch signaling.
(A) Regimen of antibody application. The neutralizing antibody against mouse VEGF
(N-mVEGF) was injected into the tail vein on 2 consecutive days (E13.5 and E14.5),
and embryos were analyzed at E15.5. (B) Analysis of VEGF-A and Notch activity.
Staining for phosphorylated VEGFR2 and eNos as a downstream target of VEGFR2
confirms that VEGF-A signaling is effectively inhibited (a,b,e,f). VEGFR2 itself is still
normally expressed (c,d). Notch activation (m,n), the expression of Notch ligands Dll1
and Dll4 (i,l) and of EphrinB2 (g,h) is not changed in N-mVEGF–treated embryos.
Green indicates ␣–smooth muscle actin; red indicates antigen. a⬘ to n⬘ show
magnifications. (C) Western blot analysis of markers analyzed in panel B. Actin was
used as a loading control.
to the defects affecting tissue architecture and patterning, and does
not reflect a specific requirement for DLL1 function in endothelial
cells of early blood vessels, whereas DLL4 and JAG1 are essential
for the early specification of the arterial fate, the regulation of tip
cell behavior, and vascular remodeling.22,27,42,43 After E13.5, activa-
BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
tion of Notch1, which is the essential Notch receptor in the vascular
endothelium regulating embryonic and postnatal angiogenesis,23,44,45 was virtually abolished in arteries lacking DLL1.
Surprisingly, NICD was not generated at detectable levels despite
the presence of both DLL4 and JAG1 in the arterial endothelium,
suggesting that in this context both DLL4 and JAG1 cannot
activate Notch1. Consistent with our observation, endotheliumspecific loss of JAG1 was shown to disrupt vascular smooth muscle
cell development but did not affect endothelial Notch activation
and arterial-venous differentiation.46 It has been proposed that, in
E10.5 embryos, endothelial JAG1 acts by signaling to adjacent
smooth muscle precursors to promote their differentiation, whereas
DLL4 signals to adjacent endothelial cells to influence angiogenesis and arterial specification.46 Here, we report that signaling by
DLL1 and Notch plays a critical and indispensable role in the
differentiation of fetal arteries.
Loss of Notch1 activation was accompanied by downregulation of genes specifically expressed in arterial, but not
venous, endothelial cells. Previously, it was shown that expression
of an activated Notch4 transgene in adult mice could induce venous
expression of the ephrinB2 gene,47 indicating that Notch signaling
can arterialize blood vessels. Our data demonstrate that also
constant Notch activity is essential to maintain artery-specific gene
expression. In addition to the down-regulation of arterial endothelium-specific genes, expression of smoothelin, a gene predominantly
expressed in arterial vascular smooth muscle cells,48 was virtually
absent from Dll1 mutant arteries. In addition, in mice mutant for
Notch3, which is expressed in vascular smooth muscle cells of
arteries but not of veins, smoothelin expression was severely
down-regulated, but arterial endothelial markers were expressed
normally,49 which suggests that the arterial identity of vascular
smooth muscle cells is established independently from the underlying endothelium. Thus, the loss of smoothelin expression in Dll1
mutants might indicate that DLL1 is also a critical ligand for
Notch3, rather than reflecting a secondary event arising as a
consequence of altered endothelial identity. Loss of DLL1 activity
in fetal vessels and down-regulation of arterial markers had
remarkably mild morphologic consequences that we could detect.
At present, the reason for the observed perinatal lethality of fetuses
lacking DLL1 in the vascular endothelium is not completely clear,
although the reduced arterial lumen might impair blood transport
into the periphery. The DLL1 phenotype is also much milder than
sustained ectopic Notch activity in vascular endothelium, which led
to enlarged vessels, shunting, and eventually death.47 Loss of
Notch(1) activity in early postnatal angiogenesis might reveal its
significance only after longer time periods than we analyzed, or
under pathophysiologic conditions, as has been observed for
heterozygous Dll1 and Notch1 mutants.29,45
Dll1-mediated Notch activation is required for reception of
VEGF signals
Studies in zebrafish have first indicated that Notch acts downstream
of VEGF-A during formation of the large axial blood vessels of the
trunk, determines arterial and venous cell fates in these vessels,25,28
and regulates angiogenesis.50,51 Similarly, studies of mammalian
endothelial cells in culture have shown that VEGF-A administration induces Notch1 and Dll4 expression and thus placed the Notch
pathway downstream of the VEGF-A pathway.52 During angiogenesis in the postnatal retina in response to VEGF, Dll4 expression is
induced in the tip cell, whereas the activation of Notch signaling in
neighboring endothelial cells is thought to suppress sprouting of
these cells by suppressing the expression of Vegfr2 and thereby
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BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
Dll1 FUNCTION IN FETAL ARTERIES
5687
responsive to VEGF-A signals. This does not exclude that under
hypoxic or other physiologic conditions VEGF signals can further
increase expression of Notch components and enhance Notch
signals as observed in postnatal vessels.29,42,45,53
In summary, our results define a specific role for the Notch
ligand DLL1 in arterial vascular endothelial cells during fetal
stages of development. DLL1 acts nonredundantly to DLL4 and is
required to maintain arterial identity, and in this developmental
context, acts upstream of VEGF.
Acknowledgments
Figure 7. Proposed interrelation of Notch and VEGF-A signaling in fetal
arteries. Dll1-mediated Notch activity maintains the responsiveness of arterial
endothelial cells for VEGF-A. For details, see “Discussion.”
limiting the response to VEGF-A.42,53 In addition, Notch mediates
the angiogenic effect of VEGF as response to hypoxia.45 Collectively, these data indicate that, in various contexts, Notch mediates
VEGF-A signals in the vascular endothelium and, in part, acts in a
negative feedback loop with VEGF signaling.
Our analysis has uncovered an additional interrelation between
the VEGF-A and Notch pathways in fetal arterial endothelium
(Figure 7). Here, Notch activity appears to be required for VEGF-A
signaling through VEGFR2 and its coreceptor NRP1, because
expression of both receptor and coreceptor was rapidly lost after
Notch1 activation ceased. Conversely, inhibition of VEGF-A
signaling had virtually no effect on expression of Notch1 and its
ligands DLL1 and DLL4, and the generation of NICD, indicating
that VEGF-A signals in large fetal arteries are not required for
endothelial Notch1 activity. Instead, DLL1-mediated Notch activity appears to be required to render these endothelial cells
We thank Katsuto Hozumi for providing the floxed Dll1 mice,
Dietmar Vestweber for the anti-Endomucin antibodies, and Rui
Benedito for help with the retina stains.
This work is supported by funding from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) for the
Cluster of Excellence REBIRTH (From Regenerative Biology to
Reconstructive Therapy).
Authorship
Contribution: I.S. performed research and collected, analyzed, and
interpreted data; R.H.A. contributed vital new reagents; and A.G.
designed research, analyzed and interpreted data, and wrote the
manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Achim Gossler, Institute for Molecular Biology OE5250, Medizinische Hochschule Hannover, Hannover,
Germany; e-mail: [email protected].
References
1. Risau W, Flamme I. Vasculogenesis. Annu Rev
Cell Dev Biol. 1995;11:73-91.
2. Risau W. Mechanisms of angiogenesis. Nature.
1997;386:671-674.
3. Rossant J, Howard L. Signaling pathways in vascular development. Annu Rev Cell Dev Biol.
2002;18:541-573.
4. Wang HU, Chen ZF, Anderson DJ. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2
and its receptor Eph-B4. Cell. 1998;93:741-753.
5. Adams RH, Wilkinson GA, Weiss C, et al. Roles
of ephrinB ligands and EphB receptors in cardiovascular development: Demarcation of arterial/
venous domains, vascular morphogenesis, and
sprouting angiogenesis. Genes Dev. 1999;13:
295-306.
6. Gerety SS, Wang HU, Chen ZF, Anderson DJ.
Symmetrical mutant phenotypes of the receptor
EphB4 and its specific transmembrane ligand
ephrin-B2 in cardiovascular development. Mol
Cell. 1999;4:403-414.
7. Gridley T. Notch signaling in vertebrate development and disease. Mol Cell Neurosci. 1997;9:
103-108.
8. Greenwald I, Rubin GM. Making a difference: The
role of cell-cell interactions in establishing separate identities for equivalent cells. Cell. 1992;68:
271-281.
9. Blaumueller CM, Artavanis-Tsakonas S. Comparative aspects of Notch signaling in lower and
higher eukaryotes. Perspect Dev Neurobiol.
1997;4:325-343.
10. Thomas U, Speicher SA, Knust E. The Drosophila gene Serrate encodes an EGF-like transmem-
brane protein with a complex expression pattern
in embryos and wing discs. Development. 1991;
111:749-761.
11. Fortini ME, Artavanis-Tsakonas S. The suppressor of hairless protein participates in notch receptor signaling. Cell. 1994;79:273-282.
12. Jarriault S, Brou C, Logeat F, Schroeter EH,
Kopan R, Israel A. Signalling downstream of activated mammalian Notch. Nature. 1995;377:355358.
13. Jarriault S, Le Bail O, Hirsinger E, et al. Delta-1
activation of notch-1 signaling results in HES-1
transactivation. Mol Cell Biol. 1998;18:74237431.
14. Villa N, Walker L, Lindsell CE, Gasson J, Iruela-Arispe
ML, Weinmaster G. Vascular expression of Notch
pathway receptors and ligands is restricted to arterial vessels. Mech Dev. 2001;108:161-164.
vasculature: insights from zebrafish, mouse and
man. Bioessays. 2004;26:225-234.
20. Adams RH, Alitalo K. Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol
Cell Biol. 2007;8:464-478.
21. Siekmann AF, Covassin L, Lawson ND. Modulation of VEGF signalling output by the Notch pathway. Bioessays. 2008;30:303-313.
22. Xue Y, Gao X, Lindsell CE, et al. Embryonic lethality and vascular defects in mice lacking the
Notch ligand Jagged1. Hum Mol Genet. 1999;8:
723-730.
23. Krebs LT, Xue Y, Norton CR, et al. Notch signaling is essential for vascular morphogenesis in
mice. Genes Dev. 2000;14:1343-1352.
24. Zhong TP, Childs S, Leu JP, Fishman MC. Gridlock signaling pathway fashions the first embryonic artery. Nature. 2001;414:216-220.
15. Beckers J, Clark A, Wunsch K, Hrabe De Angelis
M, Gossler A. Expression of the mouse Delta1
gene during organogenesis and fetal development. Mech Dev. 1999;84:165-168.
25. Lawson ND, Scheer N, Pham VN, et al. Notch
signaling is required for arterial-venous differentiation during embryonic vascular development.
Development. 2001;128:3675-3683.
16. Shutter JR, Scully S, Fan W, et al. Dll4, a novel
Notch ligand expressed in arterial endothelium.
Genes Dev. 2000;14:1313-1318.
26. Duarte A, Hirashima M, Benedito R, et al. Dosage-sensitive requirement for mouse Dll4 in artery development. Genes Dev. 2004;18:24742478.
17. Uyttendaele H, Marazzi G, Wu G, Yan Q,
Sassoon D, Kitajewski J. Notch4/int-3, a mammary proto-oncogene, is an endothelial cellspecific mammalian Notch gene. Development.
1996;122:2251-2259.
18. Alva JA, Iruela-Arispe ML. Notch signaling in vascular morphogenesis. Curr Opin Hematol. 2004;
11:278-283.
19. Shawber CJ, Kitajewski J. Notch function in the
27. Krebs LT, Shutter JR, Tanigaki K, Honjo T, Stark
KL, Gridley T. Haploinsufficient lethality and formation of arteriovenous malformations in Notch
pathway mutants. Genes Dev. 2004;18:24692473.
28. Lawson ND, Vogel AM, Weinstein BM. Sonic
hedgehog and vascular endothelial growth factor
act upstream of the Notch pathway during arterial
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
5688
BLOOD, 28 MAY 2009 䡠 VOLUME 113, NUMBER 22
SÖRENSEN et al
endothelial differentiation. Dev Cell. 2002;3:127136.
29. Limbourg A, Ploom M, Elligsen D, et al. The
Notch ligand Delta-like 1 is essential for postnatal
arteriogenesis. Circ Res. 2007;100:363-371.
30. You LR, Lin FJ, Lee CT, DeMayo FJ, Tsai MJ,
Tsai SY. Suppression of Notch signalling by the
COUP-TFII transcription factor regulates vein
identity. Nature. 2005;435:98-104.
31. Hrabe de Angelis M, McIntyre J II, Gossler A.
Maintenance of somite borders in mice requires
the Delta homologue DII1. Nature. 1997;386:717721.
32. Schuster-Gossler K, Cordes R, Gossler A. Premature myogenic differentiation and depletion of
progenitor cells cause severe muscle hypotrophy
in Delta1 mutants. Proc Natl Acad Sci U S A.
2007;104:537-542.
Gessler M. The Notch target genes Hey1 and
Hey2 are required for embryonic vascular development. Genes Dev. 2004;18:901-911.
38. Minoguchi S, Taniguchi Y, Kato H, et al. RBP-L, a
transcription factor related to RBP-Jkappa. Mol
Cell Biol. 1997;17:2679-2687.
39. Adam F, Sourisseau T, Metivier R, et al. COUP-TFI
(chicken ovalbumin upstream promoter-transcription
factor I) regulates cell migration and axogenesis in
differentiating P19 embryonal carcinoma cells. Mol
Endocrinol. 2000;14:1918-1933.
40. Kroll J, Waltenberger J. VEGF-A induces expression of eNOS and iNOS in endothelial cells via
VEGF receptor-2 (KDR). Biochem Biophys Res
Commun. 1998;252:743-746.
41. Grego-Bessa J, Luna-Zurita L, del Monte G, et al.
Notch signaling is essential for ventricular chamber development. Dev Cell. 2007;12:415-429.
33. Hozumi K, Negishi N, Suzuki D, et al. Delta-like 1
is necessary for the generation of marginal zone
B cells but not T cells in vivo. Nat Immunol. 2004;
5:638-644.
42. Suchting S, Freitas C, le Noble F, et al. The Notch
ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching. Proc
Natl Acad Sci U S A. 2007;104:3225-3230.
34. Murtaugh LC, Stanger BZ, Kwan KM, Melton DA.
Notch signaling controls multiple steps of pancreatic differentiation. Proc Natl Acad Sci U S A.
2003;100:14920-14925.
43. Lobov IB, Renard RA, Papadopoulos N, et al.
Delta-like ligand 4 (Dll4) is induced by VEGF as a
negative regulator of angiogenic sprouting. Proc
Natl Acad Sci U S A. 2007;104:3219-3224.
35. Hicks C, Johnston SH, diSibio G, Collazo A, Vogt
TF, Weinmaster G. Fringe differentially modulates
Jagged1 and Delta1 signalling through Notch1
and Notch2. Nat Cell Biol. 2000;2:515-520.
44. Limbourg FP, Takeshita K, Radtke F, Bronson RT,
Chin MT, Liao JK. Essential role of endothelial
Notch1 in angiogenesis. Circulation. 2005;111:
1826-1832.
36. Diez H, Fischer A, Winkler A, et al. Hypoxiamediated activation of Dll4-Notch-Hey2 signaling
in endothelial progenitor cells and adoption of
arterial cell fate. Exp Cell Res. 2007;313:1-9.
45. Takeshita K, Satoh M, Ii M, et al. Critical role of
endothelial Notch1 signaling in postnatal angiogenesis. Circ Res. 2007;100:70-78.
37. Fischer A, Schumacher N, Maier M, Sendtner M,
46. High FA, Lu MM, Pear WS, Loomes KM, Kaestner
KH, Epstein JA. Endothelial expression of the Notch
ligand Jagged1 is required for vascular smooth
muscle development. Proc Natl Acad Sci U S A.
2008;105:1955-1959.
47. Carlson TR, Yan Y, Wu X, et al. Endothelial expression of constitutively active Notch4 elicits reversible arteriovenous malformations in adult
mice. Proc Natl Acad Sci U S A. 2005;102:98849889.
48. van der Loop FT, Gabbiani G, Kohnen G,
Ramaekers FC, van Eys GJ. Differentiation of
smooth muscle cells in human blood vessels as
defined by smoothelin, a novel marker for the
contractile phenotype. Arterioscler Thromb Vasc
Biol. 1997;17:665-671.
49. Domenga V, Fardoux P, Lacombe P, et al. Notch3
is required for arterial identity and maturation of
vascular smooth muscle cells. Genes Dev. 2004;
18:2730-2735.
50. Leslie JD, Ariza-McNaughton L, Bermange AL,
McAdow R, Johnson SL, Lewis J. Endothelial signaling by the Notch ligand Delta-like 4 restricts
angiogenesis. Development. 2007;134:839-844.
51. Siekmann AF, Lawson ND. Notch signalling limits
angiogenic cell behaviour in developing zebrafish
arteries. Nature. 2007;445:781-784.
52. Liu ZJ, Shirakawa T, Li Y, et al. Regulation of
Notch1 and Dll4 by vascular endothelial growth
factor in arterial endothelial cells: Implications for
modulating arteriogenesis and angiogenesis. Mol
Cell Biol. 2003;23:14-25.
53. Hellstrom M, Phng LK, Hofmann JJ, et al. Dll4
signalling through Notch1 regulates formation of
tip cells during angiogenesis. Nature. 2007;445:
776-780.
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2009 113: 5680-5688
doi:10.1182/blood-2008-08-174508 originally published
online January 14, 2009
DLL1-mediated Notch activation regulates endothelial identity in mouse
fetal arteries
Inga Sörensen, Ralf H. Adams and Achim Gossler
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