RESEARCH ARTICLE
671
Tie-1-directed expression of Cre recombinase in
endothelial cells of embryoid bodies and transgenic
mice
Erika Gustafsson1, Cord Brakebusch1, Kristiina Hietanen2 and Reinhard Fässler1,*
1Department of Experimental Pathology, Lund University, Lund, Sweden
2Molecular/Cancer Biology Laboratory, Hartmann Institute, University of Helsinki,
Finland
*Author for correspondence (e-mail: [email protected])
Accepted 1 December 2000
Journal of Cell Science 114, 671-676 © The Company of Biologists Ltd
SUMMARY
Tissue-specific gene inactivation using the Cre-loxP system
has become an important tool to unravel functions of genes
when the conventional null mutation is lethal. We report
here the generation of a transgenic mouse line expressing
Cre recombinase in endothelial cells. In order to avoid the
production and screening of multiple transgenic lines we
used embryonic stem cell and embryoid body technology to
identify recombinant embryonic stem cell clones with high,
endothelial-specific Cre activity. One embryonic stem cell
clone that showed high Cre activity in endothelial cells was
used to generate germline chimeras. The in vivo efficiency
and specificity of the transgenic Cre was analysed by
intercrossing the tie-1-Cre line with the ROSA26R reporter
mice. At initial stages of vascular formation (E8-9), LacZ
INTRODUCTION
The vascular system is the first organ that forms during
embryogenesis. Common progenitors of blood and endothelial
cells form at around E8 by differentiation of mesenchymal
precursor cells and are called hemangioblasts (Risau, 1995). In
the blood islands of the yolk sac, endothelial cell differentiation
occurs in close association with hematopoietic precursor cells,
while within the embryo endothelial cells mainly differentiate
from mesoderm as solitary angioblasts (Risau, 1995).
Endothelial cells of various organs have many common
morphological and functional properties, but also characteristic
differences (Garlanda and Dejana, 1997). A few examples are
the fenestrated endothelium in the kidney glomeruli, the
sinusoidal endothelium in the liver, and the highly specialised
endothelium in the brain microvasculature providing the bloodbrain barrier. Even within the same organ, heterogeneity exists
between large and small vessels, and between arteries and
veins. Endothelial cells play crucial roles in a wide variety of
specific functions, including tissue homeostasis, fibrinolysis
and coagulation, blood-tissue exchange, vasotonus regulation,
and in the activation and extravasation of blood cells in
physiological and pathological conditions (Risau, 1995). The
exact molecular mechanisms underlying these various
functions, however, are often unclear. Targeted inactivation of
genes expressed in endothelial cells is a powerful tool to tackle
these questions. If the gene of interest, however, is expressed
staining was detected in almost all cells of the forming
vasculature. Between E10 and birth, LacZ activity was
detected in most endothelial cells within the embryo and
of extra-embryonic tissues such as yolk sac and
chorioallantoic placenta. Ectopic expression of Cre was
observed in approximately 12-20% of the adult erythroid,
myeloid and lymphoid cells and in subregions of the adult
brain. These results show that the tie-1-Cre transgenic
strain can efficiently direct deletion of floxed genes in
endothelial cells in vivo.
Key words: Cre recombinase (Cre), Cre-loxP, β-Galactosidase
(LacZ), Endothelial cell, Transgenic mouse, tie-1 promoter
in many cell types, gene ablation might result in early
embryonic lethality preventing the analysis of its function in
endothelial cells. In such cases, an endothelial cell-restricted
gene inactivation would be desired. Tissue-specific gene
inactivation can be achieved using either the Cre-loxP or the
FRT/flip recombination system. Up to now, no mouse line
expressing Cre recombinase in endothelial cells was available
for such experiments.
Here we describe the generation and characterisation of a
transgenic mouse strain in which the Cre recombinase
expression is directed to endothelial cells by the mouse tie-1
promoter. The tie gene encodes a receptor tyrosine kinase
expressed at early stages of vascular development. It is first
detected at E8 in angioblasts of the head mesenchyme, in
endothelial cells of the dorsal aorta and in the blood islands of
the yolk sac (Korhonen et al., 1994). The same promoter
fragment has previously been used to direct expression of
reporter gene in endothelial cells undergoing vasculogenesis
and angiogenesis in vivo (Iljin et al., 1999; Korhonen et al.,
1995).
We have developed an efficient and fast approach to generate
tie-1-Cre transgenic mice. The tie-1-Cre expression construct
was electroporated into embryonic stem (ES) cells which are
pluripotent cells that differentiate in embryoid bodies into a
wide variety of cell types including endothelial cells. This
differentiation route was employed to identify clones that
express a functionally active Cre enzyme in endothelial cells.
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JOURNAL OF CELL SCIENCE 114 (4)
One ES cell clone that showed high Cre activity in endothelial
cells was used to generate transgenic mice. They efficiently
delete loxP flanked DNA sequences in endothelial cells in vivo.
Therefore, the tie-1-Cre strain is an excellent tool for analysis
of gene functions in endothelial cells using the Cre-loxP
recombination system.
MATERIALS AND METHODS
Generation of the tie-1-Cre expression construct
The tie-1-Cre expression construct was generated in two steps. First,
a floxed neomycin cassette was introduced into a plasmid containing
a 1.15 kb HindIII-ApaI fragment of the mouse tie-1 promoter (Iljin et
al., 1999). In the second step, an EcoRI-XhoI fragment of pMC1-Cre
(Gu et al., 1993) containing a nuclear localisation signal, Cre cDNA
and a pA+ signal and a neomycin resistance cassette flanked by two
loxP sites was introduced into the construct.
ES cell culture, generation and analysis of embryoid
bodies and generation of transgenic mice
The expression construct was electroporated into R1 ES cells. ES cell
culture was performed as previously described (Fässler and Meyer,
1995). G418 resistant ES clones were screened by Southern blot
analysis of ES cell DNA. Genomic DNAs were digested with EcoRI
and HindIII, fractionated on 0.7% agarose gels, transferred to
Hybond-N+ filters (Amersham) and probed with a [32P]dCTP labeled
1.15kb HindIII-ApaI fragment of the tie-1 gene and a [32P]dCTP
labeled 1.7 kb EcoRI-XhoI fragment of the Cre gene to analyse the
integration of the transgene.
Wild-type RI and tie-1-Cre ES cells were used to generate
embryoid bodies. For in vitro differentiation in embryoid bodies, ES
cells were cultured in hanging drops as previously described (Bloch
et al., 1997). Briefly, 800 cells were cultured in 20 µl of DMEM,
supplemented with 20% fetal calf serum, non-essential amino acids,
and 0.1 mM β-mercaptoethanol, hanging from the lid of a Petri dish
for 2 days allowing the formation of embryoid bodies and then for 3
days in bacteriological dishes. Subsequently, the aggregates were
plated on Tissue Tek chambers precoated with 0.1% gelatine and
incubated for 9 days. The embryoid bodies were generated either in
the presence or absence of G418 throughout the procedure.
Immunofluorescent staining of embryoid bodies was performed as
previously described (Bloch et al., 1997) using rat anti-PECAM-1 as
a primary (Pharmingen) and Cy3-conjugated goat anti-rat secondary
antibody (Jackson).
The transgenic ES cell clone 9 was used to generate germline
chimeras according to standard procedures (Fässler and Meyer, 1995).
Mice heterozygous for the tie-1-Cre transgene were subsequently
crossed with the ROSA26R reporter mouse strain (Soriano, 1999).
LacZ staining
For whole-mount staining, dissected embryos and yolk sacs between
E8-E10.5 were fixed for 30 minutes at room temperature in fixation
buffer containing 0.2% glutaraldehyde, 5 mM EGTA, 2 mM MgCl2,
and 0.1 M sodium phosphate, pH 7.3. Fixed embryos were then
washed three times with 0.1 M sodium phosphate buffer containing 2
mM MgCl2, 0.2% NP-40 and 0.1% Na-deoxycholate. The staining
was carried out in washing buffer supplemented with 2.1 µg/ml
potassium ferrocyanide, 1.6 µg/ml ferricyanide, and 1 mg/ml X-gal.
Staining reactions were performed overnight at 37°C. Embryos were
subsequently embedded in paraffin, sectioned and counter-stained
with Chromotrop2R. Older embryos (E11.5-E17.5) and dissected
adult organs were fixed overnight in fixation buffer at 4°C, incubated
in 20% sucrose in 0.1 M phosphate buffer for 1 day and embedded in
Tissue Tek. Frozen sections (8-10 µm) were stained for LacZ activity
as previously described. In all experiments, embryos lacking the tie-
1-Cre transgene served as negative controls and demonstrated that the
staining was a specific indicator of Cre-mediated activation of the
reporter gene.
Flow cytometry and antibodies
Single cell suspensions from adult double-transgenic thymus, spleen
and bone marrow were investigated by flow cytometry. The cells were
loaded with fluorescein di-β-D-galactopyranoside (FDG) to detect
LacZ-positive cells (Nolan et al., 1988) and immunostained with
markers for the erythroid, myeloid and lymphoid lineages. The
following biotinylated antibodies were used as lineage markers: antiCD3 (2C11), anti-CD45R/B220 (RA3-6B2), Ter119, Gr-1 (RB6-8C59)
and anti-CD11b (Mac-1, M1/70) (all Pharmingen). Biotinylated
antibodies were visualised with streptavidin-PE (Southern
Biotechnologies). Dead cells were excluded from analysis on a
FACScalibur (Becton Dickinson) by propidium iodide (1 µg/ml,
Sigma) counter-staining.
RESULTS AND DISCUSSION
The tie-1-Cre expression construct (Fig. 1A) was
electroporated into R1 ES cells. G418-resistant ES clones were
screened by Southern blot using probes specific for the tie-1
promoter and the Cre cDNA to verify the integration of the
transgene (Fig. 1B). Clones expressing a functional tie-1-Cre
transgene in endothelial cells were identified using an in vitro
assay. ES cells were differentiated in embryoid bodies to obtain
endothelial cells. In clones where the transgene is expressed,
endothelial cells should lose their floxed neomycin resistance
cassette due to the Cre activity. As a result, these cells become
neo-sensitive and die in the presence of G418. Since expression
of PECAM-1 precedes that of tie-1 in embryoid bodies (Vittet
et al., 1996), the differentiated endothelial cells were visualised
by PECAM-1 staining. An extensive vascular network was
evident by PECAM-1 staining in embryoid bodies derived
from wild-type ES cells as well as cells from clone 9 (Fig. 1C).
On the contrary, when embryoid bodies from clone 9 were
cultured in the presence of G418, only very poorly developed
capillary-like structures and scattered PECAM-positive cells
were present suggesting that the endothelial cells die soon after
differentiation and activation of the tie-1-Cre transgene (Fig.
1C). Out of five clones tested, two showed a behaviour similar
to clone 9, i.e. few PECAM-positive cells and no visible
endothelial network in embryoid bodies cultured in the
presence of G418. This in vitro screening procedure has
significant advantages in terms of time, effort and expense
reduction when compared to the generation and analysis of
several conventional transgenic mouse strains. The assay is
easy to perform and is applicable to investigate transgenic
expression in any cell type easily obtained in embryoid bodies
such as endothelia, muscle, nerve, and epithelia.
ES clone 9 carried one copy of the integrated transgene (Fig.
1B) as judged by comparing hybridisation signals obtained
from the transgene to that obtained for the endogenous tie-1
promoter. This clone was used to generate germline chimeras
according to standard procedures (Fässler and Meyer, 1995).
Analysis of the progeny of founder animals showed that the
transgene was transmitted at a frequency of 50%. Moreover,
mice homozygous for the transgene were viable, fertile and
showed no overt abnormalities indicating that the random
insertion of the transgene did not disrupt an essential gene.
Endothelial cell-specific Cre mouse strain
673
Fig. 1. Tie-1-Cre transgene, Southern blot analysis and in vitro differentiation analysis of transfected ES cells. (A) The expression construct is
comprised of a 1.15 kb fragment of the mouse tie-1 promoter linked to a nuclear localisation signal, the coding sequence of Cre recombinase
followed by a polyadenylation signal. In addition the construct contains a floxed neomycin resistance cassette. (B) After transfection, DNA
from G418-resistant ES colonies was genotyped by Southern blot using tie-1- and Cre-specific probes. (C) Functional in vitro analysis of
endothelial cell Cre expression. PECAM-1 staining of wild-type and ES cells from clone no 9 differentiated in embryoid bodies for 5 days in
suspension culture and 9 days in tissue culture chambers. A prominent capillary network is present in embryoid bodies derived from wild-type
ES cells. An equally well developed vascular network is evident in embryoid bodies derived from clone 9 grown in normal media. When
embryoid bodies derived from clone 9 are cultured in the presence of G418, only very poorly developed vessel-tubes and scattered PECAMpositive cells are present due to activation of Cre in endothelial cells leading to excision of the neomycin resistance cassette and loss of G418resistance. Bar, 250 µm.
To determine the expression of a functional Cre in vivo, tie1-Cre transgenic mice were intercrossed with the ROSA26R
indicator strain in which the LacZ reporter gene is activated
Fig. 2. Temporal and spatial expression of
LacZ in tie-1-Cre and ROSA26R double
transgenic embryos and yolk sacs detected
by whole-mount LacZ-staining. (A) At
E8.5, LacZ-positive cells are located in the
developing heart tube, dorsal aorta and the
head mesenchyme. (B) Ventral view of the
same embryo, showing strong LacZ
staining in the paired dorsal aorta
(arrowheads). (C) In the E8.5 yolk sacs
LacZ-positive cells mark the forming
vasculature. (D) By E10.5, LacZ staining
is more pronounced and nearly continuous
throughout the embryonic vascular system.
(E) A higher magnification of an E10.5
embryo showing the LacZ-positive
capillary plexus of the head. (F) At E10.5,
the continuous vascular network of the
yolk sac is positive for LacZ staining. At
all stages analysed, ROSA embryos
lacking the tie-1-Cre transgene were
negative for LacZ staining (not shown).
after Cre-mediated excision of a loxP flanked neomycin
cassette (Soriano, 1999). Embryos resulting from such matings
were dissected at E8-E17.5 during development and at adult
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JOURNAL OF CELL SCIENCE 114 (4)
Fig. 3. LacZ staining of tissues derived from
E10 (A and B) and E14.5 (C-H) tie-1-Cre and
ROSA26R double transgenic embryos.
(A) At E10, the endocardium and the
endocardial cushion tissue mesenchyme are
strongly LacZ-positive. (B) The endothelium
of the dorsal aorta and a few hematopoietic
cells show positive staining. (C) At E14.5,
the prominent capillary network of the lung is
strongly stained whereas the bronchioli are
negative. (D) In liver, LacZ staining is
detected in the large veins, while the dot-like
staining most likely stain the nuclei of the
sinusoidal endothelial cells. In addition,
scattered cells within the liver possibly of
hematopoietic origin are LacZ-positive
(arrows). (E) Vessels of the developing
kidney glomeruli and those between the
tubuli are LacZ-positive. (F) Strong LacZ
staining is present in the intersomitic vessels.
(G) The capillaries within the brain are LacZpositive while the brain tissue is devoid of
staining. (H) Capillaries in the spinal cord
and the skin (arrows) are LacZ-positive.
Abbreviations: a, atrium; v, ventricle; ect,
endocardial cushion tissue; da, dorsal aorta.
Bars: 500 µm (A,B); 250 µm (C,E-H); 125
µm (D).
stages and examined by LacZ staining. At
all stages analysed, no LacZ activity was
detected in ROSA26R control embryos
lacking the tie-1-Cre transgene.
In the tie-1-Cre and ROSA26R double
transgenic embryos LacZ activity was
detectable already at E8-E8.5 (Fig. 2A).
Most cells in the developing heart tube
and in the dorsal aorta were LacZpositive (Fig. 2A and B). In the yolk sac,
cells of the forming vessels were stained
(Fig. 2C). By E9.5, LacZ-positive cells
were observed in the developing heart,
dorsal aorta, head mesenchyme and in
the yolk sac (not shown). At E10, LacZ
staining was detected in most cells of the
embryonic vascular system (Fig. 2D and
E). Positive cells were found in the
developing heart, developing plexus of
the head (Fig. 2E), intersomitic vessels
and the carotid arteries. In yolk sac, the
vascular tree showed a distinct LacZ
staining (Fig. 2F). On histological
sections, strong LacZ signals were visible in the heart
endocardium and in the endocardial cushions (Fig. 3A) that
forms via transdifferentiation of endothelium into mesenchyme
(Sinning et al., 1992). Since the Cre-mediated activation of
the reporter gene occurs before the transdifferentiation, the
resulting mesenchymal cells express the activated reporter
gene. The endothelial lining of the dorsal aorta was strongly
stained, and a few positive hematopoietic cells were visible in
the lumen (Fig. 3B). At E14.5, the prominent capillary network
of the lung was strongly stained while the epithelium of the
bronchioli was negative (Fig. 3C). In liver, LacZ staining was
detected in the large veins, in the nuclei of the sinusoidal
endothelial cells and in a few scattered cells possibly of
hematopoietic origin (arrow in Fig. 3D). The vessels of the
developing kidney glomeruli and those between the tubuli were
LacZ-positive (Fig. 3E) and a strong LacZ staining was present
in the intersomitic vessels (Fig. 3G). The capillaries within the
brain were LacZ-positive while the neuronal tissue was devoid
of staining (Fig. 3G). Furthermore, capillaries in spinal cord
and skin were LacZ-positive (Fig. 3H). LacZ activity was also
detected in the fetal blood vessels in the labyrinthine layer of
the chorioallantoic placenta (not shown).
Endothelial cell-specific Cre mouse strain
675
Fig. 4. LacZ staining of tissues derived from adult tie-1-Cre and
ROSA26 double transgenic mice. (A) In the kidney, capillaries of
the glomeruli and vessels surrounding the tubuli are LacZpositive. (B) LacZ staining is also detected in alveolar capillaries
of the lung while the epithelium of the bronchioli are devoid of
staining. (C) Small vessels and capillaries between the
myocardial muscle fibers are LacZ positive. (D) A higher
magnification shows LacZ staining of the thin endocardial cells
lining the ventricles. (E) In liver, the central venule and the nuclei
of the endothelial cells lining the sinusoidal spaces are LacZpositive. (F) The capillaries of the brain are LacZ positive.
(G) Several areas of the brain show a strong LacZ signal.
(H) Higher magnification of the cerebral cortex showing that
neurons of all layers are LacZ positive. (I) Higher magnification
of the hippocampus showing LacZ positive neurons of the
dentate gyrus and CA3, and positive staining of a proportion of
cells in CA2, while cells of CA1 are devoid of staining.
Abbreviations: m, myocardial muscle layer; e, endocardial lining;
cv, central vein; s, sinusoidal spaces; dg, dentate gyrus. Bars: 250
µm (A-C, F); 125 µm (D,E); 2 mm (D); 500 µm (H,I).
Taken together, between E10 and until birth, most
endothelial cells showed a positive LacZ staining. These
data are mainly in agreement with previously published
data on the expression of the endogenous tie-1 gene
(Dumont et al., 1995; Korhonen et al., 1994). The tie-1
promoter is not active in endothelial cells of the liver
sinusoids (Korhonen et al., 1995), while we detected LacZ
staining in these cells. This is most likely due to Cremediated activation of the LacZ gene in endothelial cells
before the sinusoidal spaces form. The tie-1-Cre induced
LacZ expression follows that of the endogenous tie-1 gene
expression (Dumont et al., 1995; Korhonen et al., 1994)
also at the initial stages of vascular formation, indicating
that Cre quickly deleted the floxed DNA sequence and
activated the LacZ reporter gene.
To locate LacZ-positive cells in adult tie-1-Cre and
ROSA26R double transgenic mice, kidney, lung, heart,
liver and brain were investigated by LacZ staining.
In kidney, capillaries of the glomeruli and vessels
surrounding the tubuli were LacZ-positive (Fig. 4A).
Strong LacZ staining was also detected in capillaries of
the alveolar network of the lung while the epithelium of
the bronchioli were devoid of staining (Fig. 4B). Small
vessels and capillaries between the myocardial muscle
fibers showed LacZ staining (Fig. 4C), and the same was
observed for the thin endocardial lining. In liver, the
central venule and the nuclei of the endothelial cells
lining the sinusoidal spaces were LacZ-positive (Fig. 4E).
Moreover, capillaries throughout the brain showed positive
staining for LacZ (Fig. 4F). In a few mice, not all endothelial
cells of adult organs were LacZ-positive (70-90% in
kidney, liver and brain, and 90-95% in lung and heart).
Surprisingly, in adult brain an intense LacZ staining was also
evident in certain neuronal populations (Fig. 4G). LacZ
positive cells mark most cortical areas (Fig. 4G and H)
including ventral/lateral cortical structures. LacZ staining
was also detected in some neuronal populations of the
hippocampus (Fig. 4G and I). Neurons of the dentate gyrus
and CA3 were strongly LacZ positive, and so was a
proportion of cells in CA2, while cells of CA1 were almost
completely devoid of staining (Fig. 4I). Ventral thalamus
lacked LacZ staining while dorsal thalamus including medial
and lateral geniculate were LacZ positive (not shown). In
midbrain, a few scattered LacZ positive cells were present. In
cerebellum, the LacZ positive cells had a patchy distribution
and some Purkinje cells, granule cells and deep nuclei
showed LacZ staining (not shown). During early
embryogenesis, no LacZ positive cells were detected in the
developing brain while a very weak and diffuse LacZ staining
was first evident at E17.5 (not shown) suggesting that the Cre
expression most likely is turned on in postmitotic neurons.
The tie-1 promoter had not previously been reported to be
active in neuronal cells and therefore, this expression of the
tie-1-Cre transgene most likely is ectopic. This unexpected
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JOURNAL OF CELL SCIENCE 114 (4)
Table 1. Double staining for LacZ activity (FDG as
fluorescent substrate) and markers for erythroid, myeloid
and lymphoid lineages of cells from adult tie-1-Cre ×
ROSA26R mice analysed by flow cytometry
LacZ-positive
Organ
Cell type
Marker
Total population
Thymus
T cells
CD3
12%
Spleen
B cells
B220
13%
Bone marrow
B cells
Erythrocytes
Granulocytes
Granulocytes and
macrophages
B220
Ter119
Gr-1
Mac-1
17%
14%
20%
19%
expression pattern of the tie-1-Cre transgene can be used to
delete floxed genes in certain neuronal subpopulations at
postnatal stages.
Apart from the wide LacZ staining of endothelial cells, also
a proportion of the hematopoietic cells were blue (Fig. 3C).
Counting on tissue sections revealed that 93 out of 700 blood
cells (13%) were LacZ−positive at E10. This is not very
surprising considering that the endothelial and hematopoietic
lineages are derived from a common precursor, the
hemangioblast (Choi et al., 1998) and that tie-1 has been
suggested to be expressed already at this stage (Korhonen et
al., 1992; Korhonen et al., 1994). It is not known, however, if
and where the tie-1 promoter is active at later stages of
hematopoiesis. Tie-1 expression has been reported in
hematopoietic stem cells (Iwama et al., 1993; Yano et al.,
1997), in some B cells (Hashiyama et al., 1996), and in several
leukemic cell lines (Armstrong et al., 1993; Kukk et al., 1997).
To further characterise Cre expression in the hematopoietic
lineage, cell suspensions from adult double transgenic
thymus, spleen and bone marrow were investigated by flow
cytometry. The cells were loaded with fluorescein di-β-Dgalactopyranoside (FDG) to detect LacZ-positive cells (Nolan
et al., 1988) and immunostained with markers for the erythroid,
myeloid and lymphoid lineages. These results are summarised
in Table 1. In thymus, 12% of the CD3-positive T cells were
LacZ-positive. In spleen, 13% of the B220-positive B cells
were positive for LacZ. In bone marrow, the numbers of LacZpositive cells were 17% of the B220-positive B cell population,
14% of the Ter119-positive erythrocytes, 20% of the Gr-1positive granulocytes and 19% of the Mac-1-positive
granulocyte and monocyte population. Taken together, between
12-20% of cells from these hematopoietic lineages were
positive for LacZ indicating that the tie-1-Cre transgene is not
preferentially expressed in a certain population at later stages
of hematopoiesis but rather in a common precursor cell.
In summary, we describe the generation of a tie-1-Cre
transgenic mouse strain that efficiently mediate excision of
floxed genes in endothelial cells in vivo. The availability of this
strain will be a valuable tool for assessing gene functions in
vascular development and disease, as well as dissecting
endothelial cell biology through the use of the Cre/loxP
recombination system. This will be particularly important
if constitutive gene ablation experiments result in early
embryonic lethality or lead to complex phenotypes that include
vascular defects as well as defects in other organ systems.
We acknowledge Dr Kenneth Campbell for helpful advice and
Catarina Cramnert for skilful technical assistance. This work was
supported by grants from the Crafoord Foundation, Network for
Inflammation Research, Kocks Foundation, Swedish Cancer
Foundation, Swedish Medical Research Council, and the Österlund
Foundation.
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