Targeting of both mouse neuropilin-1 and
neuropilin-2 genes severely impairs
developmental yolk sac and
Seiji Takashima*, Masafumi Kitakaze*, Masanori Asakura*, Hiroshi Asanuma*, Shoji Sanada*, Fumi Tashiro†,
Hitoshi Niwa†, Jun-ichi Miyazaki†, Seiichi Hirota‡, Yukihiko Kitamura‡, Takashi Kitsukawa§,
Hajime Fujisawa¶, Michael Klagsbrun?**, and Masatsugu Hori*
*Department of Internal Medicine and Therapeutics,†Department of Nutrition and Physiological Chemistry, and‡Department of Pathology, Osaka
University Graduate School of Medicine, Suita, Osaka 565-0871, Japan;§Laboratory of Specification Mechanisms 1, National Institute for Basic
Biology, Myodaiji, Okazaki 444-0867, Japan;¶Group of Developmental Neurobiology, Division of Biological Science, Nagoya University
Graduate School of Science, Chikusa-ku, Nagoya 464-8602, Japan; and?Departments of Surgical Research and Pathology,
Children’s Hospital and Harvard Medical School, Boston, MA 02115
Communicated by M. Judah Folkman, Harvard Medical School, Boston, MA, January 10, 2002 (received for review October 19, 2001)
Neuropilins (NP1 and NP2) are vascular endothelial growth
factor (VEGF) receptors that mediate developmental and tumor
angiogenesis. Transgenic mice, in which both NP1 and NP2 were
targeted (NP1?/?NP2?/?) died in utero at E8.5. Their yolk sacs
were totally avascular. Mice deficient for NP2 but heterozygous
for NP1 (NP1?/?NP2?/?) or deficient for NP1 but heterozygous
for NP2 (NP1?/?NP2?/?) were also embryonic lethal and survived
to E10–E10.5. The E10 yolk sacs and embryos were easier to
analyze for vascular phenotype than the fragile poorly formed
8.5 embryos. The vascular phenotypes of these E10 mice were
very abnormal. The yolk sacs, although of normal size, lacked the
larger collecting vessels and had less dense capillary networks.
PECAM staining of yolk sac endothelial cells showed the absence
of branching arteries and veins, the absence of a capillary bed,
and the presence of large avascular spaces between the blood
vessels. The embryos displayed blood vessels heterogeneous in
size, large avascular regions in the head and trunk, and blood
vessel sprouts that were unconnected. The embryos were about
50% the length of wild-type mice and had multiple hemor-
rhages. These double NP1?NP2 knockout mice had a more severe
abnormal vascular phenotype than either NP1 or NP2 single
knockouts. Their abnormal vascular phenotype resembled those
of VEGF and VEGFR-2 knockouts. These results suggest that NRPs
are early genes in embryonic vessel development and that both
NP1 and NP2 are required.
vascular endothelial growth factor ? vascular endothelial growth factor
receptors ? blood vessels ? endothelial cells ? semaphorins
early developmental vasculogenesis and angiogenesis (2). VEGF
knockouts, even heterozygotes, are embryonic lethal, indicating
that VEGF vascular activity is tightly concentration-dependent
(3). A vascular role for VEGF has been implicated in collateral
vessel formation, wound healing, and in the response to ischemia
(4). VEGF is also a major contributor to tumor angiogenesis.
Tumor cells produce high levels of VEGF (5), which stimulate
tumor vascularization in response to hypoxia. VEGF antagonists
such as anti-VEGF antibodies and soluble VEGF receptors
inhibit tumor vascularization and significantly repress tumor
growth (6, 7). VEGF activities are mediated by two high affinity
receptor tyrosine kinases Flt-1 (VEGFR-1) and KDR?Flk-1
(VEGFR-2) (8, 9). VEGFR-2 knockouts result in impairment of
early stages of vasculogenesis and angiogenesis, whereas
ascular endothelial growth factor (VEGF) is a well charac-
terized angiogenesis factor. It induces the migration, pro-
VEGFR-1 knockouts affect later stages of angiogenesis such as
tube formation (10, 11).
Recently, we identified another VEGF receptor, neuropilin-1
(NP1), which is expressed by EC and tumor cells (12, 13). NP1 was
of neuronal guidance mediators. VEGF165binds to NP1 with a Kd
of 2 ? 10?10M, as does semaphorin 3A (Sema3A) (16). Other
members of the VEGF family, VEGF-B and placental growth
factor, are also ligands for NP1 (17, 18). Coexpression of NP1 and
the KDR-mediated chemotactic activity of VEGF165, suggesting
that NP1 is a coreceptor for VEGFR-2 (13). On the other hand, in
neurons NP1 seems to be a coreceptor for plexins, which are
transmembrane signal-transducing receptors (19).
There are two neuropilins, NP1 and NP2, and they localize to
a 45–50% amino acid sequence homology (20). There is ample
evidence that NPs play a role in angiogenesis. Both NP1 and NP2
are expressed by EC and bind VEGF165 to these cells (21).
Overexpression of NP1 in transgenic mice resulted in excess
capillary formation, dilated blood vessels, and extensive hem-
orrhage (22). NP1?/?mice die at E12.5–E13.5. Besides severe
fibers that express NP1, there were some vascular defects as well,
including partial impairment of neural vascularization, improper
development of brachial arch arteries and great vessels, and
disorganized vascular networks in the yolk sac (23). On the other
hand, no discernable abnormal vascular phenotype has been
reported for NP2-deficient mice (24, 25). Mice engineered to
express only VEGF120have fewer coronary vessels and a 4-fold
reduction in capillary density of the heart, one possible expla-
nation being that VEGF120 alone is insufficient for normal
angiogenesis because it cannot bind to NP1 (26). NP1 also has
a role in tumor angiogenesis. Conditional overexpression of NP1
in prostate carcinoma cells resulted in enhanced tumor angio-
genesis and growth characterized by high microvessel density,
dilated blood vessels, increased proliferating EC, and notably
less tumor cell and EC apoptosis, compared with noninduced
Abbreviations: NP1, neuropilin-1; NP2, neuropilin-2; VEGF, vascular endothelial growth
factor; EC, endothelial cell; SemaA, semaphorin A.
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March 19, 2002 ?
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controls (27), consistent with increased angiogenesis correlating
with decreased apoptosis (28).
NP1 and NP2 have some overlapping properties such as
expression by neurons, EC, and tumor cells. However, they differ
as well, for example, in the differential activation of NPs by their
ligands. Sema3A activates NP1, whereas Sem3F activates NP2
(20, 29). VEGF165binds NP1 and NP2 whereas VEGF145binds
only NP2 (30). These examples of differential properties suggest
that targeting both NP1 and NP2 genes together might yield a
more severe abnormal phenotype than targeting just one of
the two genes. Therefore, we generated transgenic mice in
which both NP1 and NP2 were targeted. We report here that
NP1?/?NP2?/?mice died very early in utero at E8.5 and exhib-
ited early defects in blood vessel development in the yolk sacs
and in the embryos. The double NP1?NP2 knockout mice had
a more severe abnormal vascular phenotype than either NP1 or
NP2 single knockouts. Interestingly, mice that were deficient for
NP2 but heterozygous for NP1 (NP1?/?NP2?/?) or deficient
for NP1 but heterozygous for NP2 (NP1?/?NP2?/?) were also
embryonic lethal but survived to E10–E10.5. These mice were
characterized by greatly diminished yolk sac vasculature, disor-
ganized blood vessels, and growth-retarded embryos. Together,
these results suggest that both NP1 and NP2 are required for
normal vasculogenesis and angiogenesis in early embryonic
Materials and Methods
Northern Blot Analysis.Northernblotwasperformedaspreviously
described (13). Briefly, E9.5 whole embryos were excised and
quickly minced in RNAzol (Tel-Test, Friendswood, TX). Total
RNA was isolated and electrophoresed on 1% formaldehyde-
agarose gels and transferred onto nylon membranes (Bio-Rad).
The membranes were hybridized with32P-labeled fragments of
mouse cDNA corresponding to nucleotides 12–530 of the NP1
ORF and nucleotides 5–620 of the NP2 ORF. A BAS photo-
imaging system (Fuji) was used for detection.
Genotyping. Germ-line transmission of the targeted NP2 locus in
heterozygous mice was confirmed by Southern blotting by using
the probe shown in Fig. 1A. Genotyping was carried out by
genomic PCR of crude DNA samples prepared from a small
piece of yolk sac. Primers for Neo are 5?-GGCCTCTTCGC-
TATTACG-3? and 5?-GAGACTGGCCAAGCGGGTGTAAC-
3?. Primers for the replaced intronic fragment are 5?-TGG-
CTTCTCTCCATTAGCTGTCG-3? and 5?-GAGACTGGC-
CAAGCGGGTGTAAC-3?. NP1 genotyping has been previ-
ously described (15). The embryos that were genotyped included
50 embryos at E8.0, 76 embryos at E9.5, 60 embryos at E12.0,
and 54 embryos at birth.
PECAM-1 Immunostaining. Embryos and yolk sacs were removed
and fixed in cold 2% paraformaldehyde?20 mM sodium phos-
phate, pH 7.4, for 30 min. For PECAM staining, yolk sacs and
whole-mount embryos were incubated with anti-PECAM-1 an-
tibody (clone MEC13.3, PharMingen) and then with alkaline
phosphatase-conjugated secondary antibody (Promega) as pre-
viously described (11).
Production of NP1 and NP2 Single and Double Knockouts. VEGF
promotes the proliferation, migration, and survival of EC (1).
VEGF165crosslinking and Northern blot analysis demonstrated
NP1, and NP2 but neither VEGFR-1 nor VEGFR-3 (not
shown). Since both NP1 and NP2 are expressed by EC as
receptors for VEGF165, it was deemed necessary to target both
to determine accurately the function of NPs in blood vessel
To compare vascular phenotypes of NP1?/?and NP2?/?
knockouts, heterozygous mice obtained from Dr. H. Fujisawa
(15) were mated to produce NP1-deficient (NP1?/?) mice. To
generate NP2?/?mice, a targeting vector was constructed in
which the translated portion of the first coding exon and the
proximal part of the next intron of NP2 were replaced with a
promoterless Escherichia coli ?-galactosidase gene to produce
NP2?/lacZmice (Fig. 1A). These heterozygote mice (effectively
NP2?/?) were mated to produce NP2?/?mice. Balb?C mice
were mated five times to determine genetic background.
did not express NP1 (Fig. 1B Top, lane 1) or NP2 (Fig. 1B Middle,
lane 2), respectively. The lack of NP1 expression was not
compensated by NP2 overexpression (Fig. 1B Middle, lane 1
compared with lane 3) nor was lack of NP2 expression compen-
sated by NP1 overexpression (Fig. 1B Top, lane 2 compared with
lane 3). The NP1?/?genotype was embryonic lethal, and the
mice died at E12.5. They had vascular defects as previously
reported (23), for example, transposition of great vessels. On the
other hand, NP2?/?mice were viable, surviving up to 2 weeks
after birth and with no apparent abnormal vascular phenotype,
NP2 gene in mouse embryonic stem cells. (Top) Restriction map of a mouse
NP2 genomic fragment showing sites for the restriction enzymes EcoRI (E),
BamHI (B), HindIII (H), and SmaI (S). The small bar depicts the region used
as a probe for Southern blot analysis. (Middle) Targeting vector in which a
region encompassing the first coding exon and the proximal part of the
next intron of NP2 (1.7 kb) are replaced by a lacZ-neo cassette (4 kb).
(Bottom) Predicted structure of the targeted NP2 allele. (B) Northern blot
analysis of NP1 and NP2 mRNA expression levels at E9.5 for various NP1
and NP2 genotypes. Genotyping was carried out on littermates. Lane 1,
NP1?/?NP2?/?; lane 2, NP1?/?NP2?/?; lane 3, NP1?/?NP2?/?; lane 4,
NP1?/?NP2?/?. In heterozygotes, NP mRNA levels are less than one-half of wild-
type mRNA levels. Densitometer readings: NP1 Northern blot (Top), densities in
lanes 1–4 are 0, 350, 250, and 1,050 arbitrary units, respectively. NP2 Northern
NP1 and NP2 knockouts. (A) Targeting vector for disruption of the
www.pnas.org?cgi?doi?10.1073?pnas.022017899Takashima et al.
which confirmed previous results (24, 25). NP1?/?heterozygotes
were mated with NP2?/?heterozygotes. F1 mice were born in a
normal Mendelian ratio. Of the F1 mice, NP1?/?NP2?/?male
and female mice were mated to generate double knockout
Vasculature Defects in the Yolk Sac. NP1?/?NP2?/?mice died at
about E8.5. No organized vessels were detected in the yolk sac
(Fig. 2A), and the embryos within the yolk sac were almost
completely resorbed. By contrast, in mice deficient only for NP1,
mostly normal blood vessel network formation occurred both in
the yolk sac (Fig. 2B) and embryo (not shown) at E10.0. Normal
yolk sac vasculature was observed in mice deficient for NP2 only
(Fig. 2C). Thus, the abnormal vascular phenotype in the yolk sac
of the double NP knockout was more severe than either NP
Interestingly, mice deficient for NP1 or NP2 but heterozygous
for the other NP gene died in utero but survived longer, to
E10–E10.5, making analysis of the embryo phenotype much
more feasible then analysis of E8.5 NP1?/?NP2?/?embryos,
which were poorly formed and very fragile. The yolk sacs of
NP1?/?NP2?/?mice at E10.0 had the same phenotype as
wild-type mice and served as controls (Fig. 2D). These normal
yolk sacs had a dense capillary plexus, large collection vessels,
and the embryo within the yolk sac was readily visible. On the
other hand, mice deficient for NP2 but heterozygous for NP1
(Fig. 2E) and mice deficient for NP1 but heterozygous for NP2
(Fig. 2F) had yolk sacs of normal size but lacked the larger
collecting vessels and contained irregular and much less dense
sacs and embryos collected and fixed separately. To analyze vas-
cular network formation, yolk sacs were stained with antibodies to
the specific EC marker PECAM. The yolk sacs of NP1?/?NP2?/?
embryos had branching arteries and veins as well as a fine capillary
bed (Fig. 3A). By contrast, arterial and venous branching was
not observed in the yolk sacs of NP1?/?NP2?/?(Fig. 3B) or
NP?/?NP2?/?(Fig. 3C) mice. Instead, the vasculature was char-
acterized by vessel heterogeneity, disorganization, and immaturity.
Thickened blood vessels, lack of capillaries, and large avascular
spaces between the blood vessels, perhaps caused by the absence of
capillaries, were observed. There were also occasional dead-ended
sprouts not connected to other sprouts.
Vasculature Defects in the Embryo. The embryos inside the poorly
vascularized yolk sacs also showed severe vascular impairment.
The control E10 embryo (NP1?/?NP2?/?) had well organized
blood vessels as determined by PECAM staining (Fig. 4A). For
example, large branching intracranial arteries, the dorsal aorta,
and the outflow tract were detected. Small capillary networks
were detected in the head and distal part of the body. In contrast,
the NP1?/?NP2?/?embryo (Fig. 4B) had severe vascular ab-
normalities. Blood vessels were disorganized, heterogeneous in
size, and in some cases thickened. Blood vessel density was low
in the head region, and there were avascular regions in both the
head and trunk. The dorsal aorta was poorly formed, and the
blood vessels can be detected. (B and C) E10 yolk sacs of NP1?/?NP2?/?and NP1?/?NP2?/?mice, respectively. Note presence of dense capillary plexus and large
(E) E10 yolk sac of NP1?/?NP2?/?mouse embryo. Larger collecting vessels seem to be absent, and capillary density is very diminished. (F) E10 yolk sac of
NP1?/?NP2?/?mouse. Blood vessel impairment is similar to that in E. The embryos in D and E (shown together) and F are littermates.
Impairment of yolk sac blood vessel formation in double NP knockout mouse embryos. (A) Yolk sac of E8.5 NP1?/?NP2?/?double knockout mouse. No
Takashima et al.
March 19, 2002 ?
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outflow tract was undetectable. There was some small vessel
sprouting, but unconnected to other sprouts and without for-
mation of a capillary plexus. Similar results including avascular
regions, blood vessel size heterogeneity, thickened blood vessels,
and abnormal formation of the dorsal aorta and outflow tract
were observed in NP1?/?NP2?/?embryos (Fig. 4C). However,
the blood vessel density in the head region (Fig. 4C) appeared
to be higher than in Fig. 4B.
Whole mount (non-PECAM stained) embryos that were
within the yolk sacs in Fig. 2 D–F are shown in Fig. 4 D–F,
respectively. NP1?/?NP2?/?(Fig. 4E) and NP1?/?NP2?/?(Fig.
4F) embryos were much smaller than control littermates (Fig.
4D), about 50% less in length. In addition, multiple hemorrhag-
ing occurred in both of these embryos (Fig. 4 E and F).
Taken together, analyses of embryos and their yolk sacs
demonstrate that double NP knockouts display severe vascular
defects in early embryogenesis.
Our results suggest that targeting both NP1 and NP2 genes
results in a severe impairment of embryonic blood vessel devel-
opment. The double knockout abnormal vascular phenotype is
more severe than that obtained by targeting either NP gene
alone. Double NP knockouts (NP1?/?NP2?/?) survived only
until E8.5 and were characterized by lack of blood vessel
formation in the yolk sac, blood vessels abnormalities in the
embryo, and impaired embryo development. On the other hand,
NP1 or NP2 deficient mice, which survive to E12–E13 and to
postnatal life, respectively, were comparable to wild-type mice in
the density of yolk sac blood vessels, embryo size, and blood
It was difficult to analyze the embryos at E8.5 histologically
However, an abnormal vascular phenotype occurred even when
one of the NPs was lacking but the other NP had one wild-type
allele. These mice, NP1?/?NP2?/?and NP1?/?NP2?/?, survived
until about E10.5, and at this stage the yolk sacs and embryos
could be analyzed more readily. PECAM staining of yolk sacs
demonstrated a striking lack of large collecting blood vessels and
capillaries. The lack of capillaries might reflect a defect in
branching, resulting from the inability of EC to migrate and
proliferate because of diminished VEGF signaling. Many of the
blood vessels were thickened, possibly arterial–venous malfor-
mations that resulted from direct artery–vein interactions in the
absence of capillaries. There were large avascular regions be-
tween blood vessels.
PECAM analysis of whole mount knockout embryos showed
regions of avascularity, for example, in the head and trunk
regions. The intracranial arteries, dorsal aorta, and outflow tract
were poorly formed. Blood vessel density was sparse, and there
were large avascular spaces between the blood vessels, some of
which were thickened. Small sprouts were seen but not con-
nected to other sprouts. The embryos were relatively small,
about 40–50% reduced in length compared with wild type,
possibly as a result of severe anemia, hypoxia, or lack of
nourishment because of an improperly vascularized yolk sac. It
is not clear why having one wild-type copy of NP1 or NP2 when
the other gene is totally absent enhances embryo viability by
several days. It could be because of an NP dose effect in which
the NP ligands, VEGF165 and?or semaphorins, can function
partially when one NP gene copy is expressed. An adverse effect
on angiogenesis with the loss of one gene but heterozygous
in a related gene has been reported for the Id family (31).
Id1?/?Id3?/?mice survived to day E10.5 and displayed vascular
malformations of the brain. However, Id1?/?Id3?/?mice
were born but did not support tumor growth because of poor
Lack of yolk sac vasculature in NP double knockouts at E8.5
is one of the earliest abnormal vascular phenotypes reported to
date. The abnormal vascular phenotype of NP1?/?NP2?/?mice
resembled that of VEGFR-2 deficient mice, which were embry-
onic lethal and died in utero at E8.5–E9.5 (10). In these mice,
blood islands were absent, and embryonic and yolk sac blood
vessels were not observed. The double NP knockout also re-
sembles the vascular phenotype of VEGF-deficient mice, which
even as heterozygotes died in utero at E9.5–E10.5 because of
abnormalities in blood island development and angiogenesis (3).
The abnormal vascular phenotype of NP double knockout mice
to the yolk sacs in Fig. 2 D–F, respectively. (A) The NP1?/?NP2?/?yolk sac has large branching vessels and a dense capillary network. (B) The NP1?/?NP2?/?yolk
sac lacks large branching vessels and lacks a capillary network. Some blood vessels are fused (white arrow). Large avascular spaces are found between the blood
vessels (black arrows). (C) NP1?/?NP2?/?yolk sac. Blood vessel impairment is severe with lack of large branching vessels and capillaries, and with fused vessels
(white arrow) and avascular regions (black arrow).
PECAM staining of E10 yolk sacs shown in Fig. 2. Blood vessel yolk sacs were stained with the EC specific marker PECAM. The yolk sacs in A–C correspond
www.pnas.org?cgi?doi?10.1073?pnas.022017899Takashima et al.
manifested itself earlier than that of the EC receptors VEGFR-1
and Tie-2, which mediate later stages of blood vessel develop-
ment, such as the ability of differentiated EC to assemble and
form capillary-like tubes (11, 32).
Our results demonstrate that both NP1 and NP2 genes are
functional in the vasculature and that both genes are needed to
mediate normal vasculogenesis and angiogenesis in the devel-
oping yolk sac and embryo. These two receptors must provide
some as yet undetermined nonoverlapping function in blood
vessel formation so that both are needed. For example, NP1 is
activated by Sema3A but not Sema3F, whereas NP2 is activated
by Sema3F but not Sema3A (20, 29). Thus, double knockouts in
whereas Sema3A would still be active in an NP2 knockout and
Sema3F in an NP1 knockout.
NPs are atypical in that they are specific high affinity
receptors for two structurally disparate ligands, members of
the VEGF family (13) and members of the semaphorin family
(16, 29). NPs do not seem to be receptor tyrosine kinases but
might act as adaptor proteins for receptor tyrosine kinases,
VEGFR-2 in the case of VEGF (13), and plexins in the case
of semaphorins (33). One question is whether the vascular
phenotype of NP double knockout mice is due to impairment
of VEGF interactions with NPs, semaphorin interactions with
NPs, or both interactions. It is plausible that knocking out NPs
affects VEGF activity as NP1 mediates KDR activity (13). In
addition, the vascular phenotype of NP1?/?NP2?/?mice re-
sembles that of VEGF and VEGFR-2 knockout mice. Sema-
phorins may also be affected by lack of NPs. Sema3A inhibited
in vitro angiogenesis via NP1 (34). However, no vascular
abnormalities were reported in sema3A-deficient mice (35,
36). Sema3A?NP interactions might mediate embryonic car-
diovascular development. The cardiovascular anomalies in
NP1?/?mice (23), such as aortic arch defects, were attributed
to inappropriate migration or differentiation of cardiac neural
crest cells (37). These cells express NP1, and their migration
that do not reach the distal regions. The outflow tract and dorsal aorta are not readily detectable. (C) E10 NP1?/?NP2?/?embryo. The vascular phenotype is similar to
in the vasculature was washed out by perfusion of the heart with PBS. Multiple hemorrhages occurred in the both embryos (black arrows).
Takashima et al.
March 19, 2002 ?
vol. 99 ?
no. 6 ?
is regulated by Sema3A (38), which is strongly expressed in Download full-text
embryonic heart (36). It is plausible that NPs mediate both
VEGF and semaphorin signal transduction pathways in vivo,
with VEGF?NP interactions mediating angiogenesis and
semaphorin?NP interactions mediating cardiovascular
In summary, the data presented here strongly suggest that
both NP1 and NP2 are necessary for normal yolk sac and
embryonic blood vessel development.
We thank Dr. H. Yoshida for whole mount embryo PECAM staining,
Dr. R. Otani for animal feeding, and Dr. A. Ogai and T. Fukushima for
technical assistance. We thank Eric Santiestevan for preparing the
figures and Drs. Roni Mamluk, Gerhard Raab, Shay Soker (Children’s
Hospital, Boston), and Dr. Patricia D’Amore (Schepens Institute, Bos-
ton) for reading the manuscript. This study was supported by Grants-
in-aid for Scientific Research Nos. 12470153 and 12877107 from the
Ministry of Education, Science, Sports and Culture, Japan, and by grants
from the Smoking Research Foundation in Japan (to S.T.) and National
Cancer Institute Grants CA37392 and CA 45548 (to M.K.).
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