1 Division of Biology 216-76, California Institute of Technology, 1201 E.
California Boulevard, Pasadena, CA 91125, USA
2 Howard Hughes Medical Institute, California Institute of Technology, 1201 E.
California Boulevard, Pasadena, CA 91125, USA
3 Department of Molecular Oncology, Genentech, South San Francisco, CA94080,
USA
4 Department of Neuroscience, The Johns Hopkins University School of Medicine,
725 North Wolfe Street, Baltimore, MA21205, USA
* Author for correspondence (e-mail: wuwei{at}caltech.edu)
Accepted 23 December 2004
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SUMMARY |
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Key words: VEGF, Neuropilin 1, Arterial differentiation, Mouse
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Introduction |
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These observations raised the question of the nature of the signal(s) that
control nerve-vessel alignment and arterial differentiation, and their
cellular source(s). In culture, VEGF-related factors derived from sensory
neurons or Schwann cells can induce arterial differentiation in endothelial
cell (EC) precursors (Mukouyama et al.,
2002). These studies, however, left unresolved the issue of
whether nerve-derived VEGFA is actually required for arterial differentiation
in the limb skin in vivo. Furthermore, the lack of an in vitro assay for
nerve-vessel alignment made it unclear whether VEGF, or rather some other
molecule, is involved in this patterning process.
Two recent studies have provided evidence of a role for VEGFA in promoting
arterial differentiation in vivo. In the zebrafish, Vegf is essential
for formation of the dorsal aorta, the major midline arterial vessel
(Lawson et al., 2002).
However, this vessel develops by de novo assembly of angioblasts, rather than
by remodeling of a capillary network. Therefore, these data do not address
whether Vegf is required to induce arterial differentiation from a
pre-existing capillary plexus, the mechanism by which most small arterial
vessels form in higher vertebrates. In mice, transgenic overexpression of
Vegfa in the heart promoted an increased number of cardiac arterial
vessels (Visconti et al.,
2002
). Although these gain-of-function data suggest that VEGFA can
promote or enhance arteriogenesis in vivo, they did not address whether it is
actually required for this process during normal development.
The early embryonic lethality of Vegfa mutants has made it
difficult to address whether nerve-derived VEGFA is required for nerve-vessel
alignment and arterial differentiation in vivo
(Carmeliet et al., 1996;
Ferrara et al., 1996
). To
circumvent this problem, we have now used several different lines of
transgenic mice expressing Cre recombinase, singly or in combination, to
specifically delete Vegfa in the major cell types comprising
peripheral nerve. In parallel, we have examined the role of neuropilin 1
(NRP1), a co-receptor for VEGF164 that is specifically expressed in
arteries. Our data indicate that nerve-derived VEGFA is indeed required for
arterial differentiation in vivo, and further suggest that arteriogenesis may
be promoted by an NRP1-mediated positive feedback loop. Surprisingly,
nerve-vessel alignment occurs normally in these conditional mutants, despite
the defect in arteriogenesis. This suggests that such alignment is mediated
either by a residual low level of VEGFA, or by a distinct nerve-derived
signal.
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Materials and methods |
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Genomic PCR
Cre-mediated recombination was assayed using genomic DNA essentially as
described (Inoue et al.,
2002). Ten dorsal root ganglia were isolated from one E15.5 embryo
and dissociated with Proteinase K (Sigma). Skin from both limbs was isolated
from one E15.5 embryo, and dissociated by digestion with type 3 collagenase
(Worthington) and dispase (Gibco BRL). The cells were incubated with rat
anti-mouse P75 monoclonal antibody (M. Rao and D.J.A., unpublished) and then
goat anti-rat IgG magnetic beads (Dynal). The magnet-selected P75+
cells were dissociated with Proteinase K. Genomic DNA was extracted and
analyzed for recombination by genomic PCR. using expand Taq DNA polymerase
(Roche) for 45 cycles. The PCR products were fractionated by electrophoresis.
The sequences of the PCR primers are: VEGF419F
(5'-CCTGGCCCTCAAGTACACCTT-3') and VEGFc5R2
(5'-ACATCTGCTGTGCTGTAGGAAG-3'). For genotyping, genomic DNA was
isolated from embryonic tail tissue and the presence of Cre transgenes was
detected by PCR using PLATINUM Taq DNA polymerase (GibcoBRL) for 28 cycles.
The sequence of the PCR primers were VEGF419F
(5'-CCTGGCCCTCAAGTACACCTT-3') and VEGF567R
(5'-TCCGTACGACGCATTTCTAG-3') for floxed Vegf allele;
Cre5'a (5'-ACCTTCCTCTTCTTCTTGGG-3') and Wnt1s
(5'-TAAGAGGCCTATAAGAGGCG-3') for Wnt1-Cre transgene;
IRES-N (5'-GCAAGGTCTGTTGAATGTCGTTGA-3') and IRES-C
(5'-GTACCTTCTGGGCATCCTTCAGC-3') for Isl1-Cre transgene;
and Cre1 (5'-GTTCGCAAGAACCTGATGGACA-3') Cre2
(5'-CTAGAGCCTGTTTTGCACGTTC-3') for Cre transgene.
Immunohistochemistry
Staining was performed essentially as described previously
(Mukouyama et al., 2002).
Embryos or limb skin tissue was fixed with 4% paraformaldehyde/PBS at 4°C
overnight, sunk in 30% sucrose/PBS at 4°C and then embedded in OCT
compound for frozen sectioning (15 µm). Staining was performed using
anti-ß-galactosidase antibodies (rabbit polyclonal antibody, 5-prime
3-prime, 1:1000, overnight at 4°C or goat polyclonal antibody, Bio Trend,
1:1000, overnight at 4°C) to detect lacZ expression; anti-GFP
antibody (rabbit polyclonal antibody, Molecular Probes; 1:500, overnight at
4°C); anti-BFABP antibody (rabbit polyclonal antibody, T. Müller,
1:1000, 3 hours at room temperature) to detect glial cells; Alexa
green-conjugated HuD antibody (mouse monoclonal antibody, clone 16A11,
Molecular Probe; 1:50, 1 hour at room temperature); anti-neuronal class III
ß-tubulin antibody (mouse monoclonal antibody, clone Tuj1, Covance,
1:500, 1 hour at room temperature) to detect neurons; anti-PECAM-1 antibody
(rat monoclonal antibody, clone MEC 13.3, BD Pharmingen, 1:300, overnight at
4°C) to detect endothelial cells; Cy3-conjugated anti-
SMA antibody
(mouse monoclonal antibody, clone 1A4, Sigma, 1:500, 1 hour at room
temperature) to detect smooth muscle cells; and anti-VEGF antibody (goat
polyclonal antibody, R&D, 1:200, overnight at 4°C). For
immunofluorescent detection, FITC-, Cy3-, Alexa-488-, Alexa-568-, or
Cy5-conjugated secondary antibodies (Jackson, 1:300, Southern Biotechnology
Associations, 1:300, and Molecular Probes, 1:250, 1 hour at room temperature)
were used. All confocal microscopy was carried out on a Leica SP confocal
(Leica).
Whole-mount immunohistochemical staining of limb skin was performed as
described previously (Mukouyama et al.,
2002). Embryos at E15.5 were dissected, fixed overnight in 4%
paraformaldehyde/PBS at 4°C, and dehydrated in 100% methanol at -20°C.
Staining was performed using anti-PECAM-1 antibody to detect endothelial
cells; anti-ß-galactosidase antibody to detect lacZ expression;
anti-NRP1 antibody (rabbit polyclonal antibody, A.L. Kolodkin, 1:3000, 3 hours
at room temperature); and anti-CX40 antibody (rabbit polyclonal antibody,
Alpha Diagnostic International, 1:300, overnight at 4°C) as arterial
markers; Cy3-conjugated anti-
SMA antibody to detect smooth muscle
cells; anti-neurofilament antibody (mouse monoclonal antibody, clone 2H3,
Developmental Studies Hybridoma Bank, 1:200, 1 hour at room temperature);
anti-peripherin antibody (rabbit polyclonal antibody, Chemicon, 1:1000, 1 hour
at room temperature) to detect nerve fibers; anti-BFABP antibody to detect
Schwann cells; and anti-VEGF antibody. FITC-, Cy3-, Alexa-488-, Alexa-568- or
Cy5-conjugated secondary antibodies were from Jackson, Southern Biotechnology
Associations and Molecular Probes. TUNEL labeling was performed according to
the manufacturer's protocol (In Situ Cell Death Detection, Roche). The average
mean fluorescence (pixels/area) was analyzed using ImageJ software, and
statistical significance of samples (n>3) was assessed using
Student's t-test.
Whole-mount in situ hybridization of limb skin
Whole-mount in situ hybridization was carried out essentially as described
previously (Mukouyama et al.,
2002; Wang et al.,
1998
). E15.5 limbs were hybridized with a cRNA probe against the
ephrin B2 (Efnb2 - Mouse Genome Informatics) extracellular domain.
Flow cytometry and culture methods
The isolation and culture of ephrin B2-negative endothelial cells from
E10.5 Efnb2lacZ/+ embryos was performed as described
previously (Mukouyama et al.,
2002). All sorts and analyses were performed on a FACS Vantage
dual laser flow cytometer (BD Biosciences). The culture medium contained EMB-2
(Clonetics) with 15% FBS (Hyclone Laboratories), Penicillin/Streptomycin
(BioWhittaker) and 10 ng/ml bFGF (R&D). The freshly isolated endothelial
cells were challenged with serial dilutions (1-100 pg/ml) of
VEGF120 or VEGF164 isoforms (R&D), used at equal
mass/volume concentrations according to the manufacturer's technical
specifications. Cultures were incubated for 2 days in a reduced oxygen
environment to more closely approximate physiological oxygen levels
(Mukouyama et al., 2002
;
Studer et al., 2000
). X-gal
staining and immunohistochemistry on these cultures were performed as
described (Mukouyama et al.,
2002
).
The ephrin B2-lacZ-positive or negative cells were counted in PECAM1-positive cells and statistical significance was assessed using the Student's t-test.
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Results |
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As there is no single promoter-driven Cre line that simultaneously and
specifically targets all three of these cell types, we combined the Wnt1-Cre
driver with an Isl1-Cre driver, which is active in both sensory and
motoneurons, but not in Schwann cells (T. M. Jessell, unpublished)
(Srinivas et al., 2001;
Yang et al., 2001
). Analysis
of Isl1-Cre; R26R embryos using cell type-specific markers confirmed
that this Cre driver deletes in both sensory and motoneurons, but not in
Schwann cells (Fig. 1B;
Fig. 1H, inset;
Fig. 1J,K). Thus, the
combination of Wnt1-Cre and Isl1-Cre should suffice to
delete the conditional Vegfa allele (Vegfflox) in
sensory neurons, motoneurons and Schwann cells
(Fig. 1K; see Fig. S2 in the
supplementary material).
Peripheral nerve-derived VEGFA is required for proper arteriogenesis in the limb skin
To create embryos carrying both the Wnt1-Cre and Isl1-Cre
transgenes, as well as both `floxed' alleles of the Vegf gene
(Gerber et al., 1999), we
generated and then inter-crossed Wnt1-Cre; Vegfflox/+ with
Isl1-Cre; Vegfflox/+ mice. On average, one in 16 embryos
derived from this inter-cross inherited both the Wnt1-Cre and
Isl1-Cre transgenes, as well as both Vegf alleles
(Vegfflox/flox). The limb skin of such embryos was
subjected to whole-mount analysis of the expression of three independent
arterial markers: neuropilin 1 (NRP1), connexin 40 (CX40; GJA5 - Mouse Genome
Informatics) and ephrin B2. The expression of all three markers was greatly
reduced in small-diameter vessels of embryos of this genotype
(Fig. 2D,F,G,K,M, arrows;
Fig. 2N; data not shown). The
expression of smooth muscle markers was also reduced, albeit to a lesser
extent than that of the arterial endothelial markers, in such embryos (data
not shown). Embryos carrying either of the individual Cre transgenes
also showed a clear, but less extensive, reduction of arterial marker
expression (Wnt1-Cre; Fig.
2E,G,L, arrows; Fig.
2N); this was particularly evident for Isl1-Cre (data not
shown). TUNEL-labeling experiments indicated that the reduction of arterial
marker expression does not reflect a selective death of arterial endothelial
cells in these Vegf conditional knockout embryos
(Fig. 2O-U).
|
These data suggested that the arterial differentiation of small-diameter nerve-associated vessels, but not of large-diameter vessels, is compromised in Wnt1-Cre; Isl1-Cre; Vegfflox/flox embryos. One possible explanation for this dissociation is that autocrine or paracrine secretion of VEGFA by endothelial or smooth muscle cells in large-diameter vessels may compensate for the absence of nerve-derived VEGFA. In support of this, antibody staining revealed that VEGFA is expressed in both endothelial and smooth muscle cells of large-diameter blood vessels (see Fig. S4E-H in the supplementary material, arrows). Furthermore, expression of mRNA for VEGF164 was detected by RT-PCR in endothelial cells that were freshly isolated from E15.5 limb skin (data not shown).
The foregoing data suggested that nerve-derived VEGF is selectively required for arteriogenesis in small-diameter nerve-associated blood vessels within limb skin. To determine whether other aspects of neuro-vascularization might also depend on peripheral-nerve-derived VEGF, we examined blood vessel development in the dorsal root sensory ganglia (DRG) of the Vegfa conditional mutants. In control (Vegfflox/flox) embryos, blood vessels both surround the DRG (Fig. 3C, open arrowhead) and branch into it (Fig. 3C, arrow). By contrast, there was virtually a complete failure of such internal ganglionic vascular branching in the DRG of Wnt1-Cre; Isl1-Cre; Vegflox/lox or Wnt1-cre; Vegflox/lox embryos (Fig. 3B,D,G; data not shown), while the vessels peripheral to the ganglia appeared unaffected (Fig. 3C,D; open arrowheads). These data suggest that VEGFA derived from sensory neurons and/or glia is required for proper vascularization of the DRG, as well as for arteriogenesis in limb skin.
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Disruption of arteriogenesis, but not nerve-vessel alignment, by endothelial-specific deletion of the VEGF164 co-receptor NRP1
The residual low level of VEGFA in the peripheral nerves of our conditional
knockout embryos left open the possibility that such levels were sufficient
for the persistence of nerve-vessel alignment in these mutants. To address
this issue by an independent approach, we sought to selectively reduce the
VEGF sensitivity of endothelial cells. Because deletion of the core VEGFA
receptor FLK1 (VEGFR2) in endothelial cells would be expected to compromise
their viability (M. Hirashima and J. Rossant, personal communication), we
examined embryos with an endothelial cell-specific deletion of the
VEGF164 co-receptor NRP1
(Kawasaki et al., 1999;
Soker et al., 1998
).
Such a deletion was achieved by using the endothelial-specific
Tie2- promoter to drive Cre expression in a genetic background
containing a floxed allele of Nrp1 over a conventional null allele
(Gu et al., 2003). In
Tie2-Cre; Nrp1flox/- embryos, expression of arterial
markers, including CX40 and ephrin B2, was strongly reduced in limb vessels
(compare Fig. 6C with
6D, arrows;
Fig. 6E; data not shown).
Surprisingly, in these mutants even the large-diameter vessels exhibited a
strong reduction of arterial marker expression, as well as in smooth muscle
coverage (compare Fig. 6C with
6D; compare Fig.
6H with
6I, arrowheads). The defects in
limb skin arterial differentiation observed in mutants lacking endothelial
NRP1 were, therefore, similar but even more pronounced than those observed in
embryos deficient in peripheral nerve-derived VEGFA. Despite this more severe
failure of arteriogenesis in limb skin, the association of nerves with blood
vessels appeared normal in Tie2-Cre; Nrp1flox/- embryos
(Fig. 6B,G, compare arrows with
open arrowheads).
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In such cultures, both VEGF164 and VEGF120 induced expression of Efnb2-lacZ (Fig. 7A-F). At 100 pg/ml, both isoforms induced ephrin B2 expression to a similar extent (Fig. 7M, left panel). However, at lower concentrations (1-10 pg/ml), VEGF164 was at least twice as effective as VEGF120 at inducing ephrin B2-lacZ expression (Fig. 7M, green bars). There was no effect on cell survival at any of the concentrations tested, for both isoforms (Fig. 7M, right panel). These data suggest that NRP1-binding isoforms of VEGF may preferentially promote arterial differentiation, in comparison with non-NRP1-binding isoforms, at very low concentrations of the growth factor.
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Discussion |
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Despite their profound effects on arterial differentiation, neither the Vegfa nor the Nrp1 conditional mutants disrupted nerve-vessel alignment. This genetic uncoupling of arteriogenesis and nerve-vessel association suggests that guidance of the vessel branching pattern by the nerve requires either a very low concentration of VEGF, or more likely a second, as yet unidentified, signaling system (Fig. 8A).
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Recent data have demonstrated that the endothelial specific Notch ligand
Delta-like 4 (Dll4) is essential for arteriogenesis in vivo
(Duarte et al., 2004;
Gale et al., 2004
;
Krebs et al., 2004
). Studies
in zebrafish have suggested that dorsal aorta formation is controlled by VEGF
and Notch signaling, acting in series
(Lawson et al., 2001
;
Lawson et al., 2002
). In
preliminary experiments, we have found that arterial markers are induced in
cultured murine embryonic endothelial precursor cells not only by VEGF, as
previously reported (Mukouyama et al.,
2002
), but also by soluble Notch ligands. Interestingly, the
ability of VEGF to induce arterial marker expression is blocked by peptide
inhibitors of Notch activation, while a soluble VEGF receptor antagonist does
not block induction of such markers by Notch ligands (Y.M. and D.J.A.,
unpublished). These data suggest that in mouse, as in zebrafish, VEGF induces
arterial differentiation by acting through the Notch pathway. How the
interaction between these two signaling systems occurs is not yet clear.
What cell type(s) within peripheral nerve are the most important source(s)
of VEGFA? We observed the strongest reduction of arterial marker expression
when Vegfa was deleted in all three neural cell types
(Wnt1-Cre+Isl1-Cre), and a weaker phenotype using either
Wnt1-Cre or Isl1-Cre alone
(Fig. 2 and data not shown).
One explanation for this higher expression is that it reflects a requirement
to eliminate Vegfa expression in all three peripheral nerve cell
types, as Isl1-Cre is not expressed in Schwann cells, while
Wnt1-Cre is not expressed in motoneurons. Motoneurons are not the
primary source of VEGF in the nerve, however, as arterial marker expression is
normal in Olig1; Olig2 double mutants
(Zhou and Anderson, 2002),
which lack all spinal motoneurons, but have a normal complement of sensory
neurons (Y.M., Q. Zhou and D.J.A., unpublished). A Schwann cell-specific
knockout of Vegfa is currently not possible, because all available
Cre driver lines active in these peripheral glia, including Po-Cre
(Feltri et al., 1999
;
Giovannini et al., 2000
),
desert hedgehog (Dhh)-Cre
(Lindeboom et al., 2003
) and
BFABP-Cre (Anthony et al.,
2004
), also promote recombination in peripheral neurons (Y.M. and
D.J.A., unpublished). This most probably reflects the early expression of
these glial promoters in multipotent neural crest stem or progenitor cells
(Kim et al., 2003
).
A second explanation is that, since both Wnt1-Cre and Isl1-Cre are expressed in sensory neurons, the stronger phenotype of Wnt1-Cre, Isl1-Cre embryos reflects a higher level of Cre expression in these cells, leading to more efficient recombination at the Vegfa locus. The structure of the conditional Vegfa allele makes it impossible to determine whether individual sensory neurons have undergone Cre-mediated recombination in one or both alleles. However, the reduction of VEGFA immunoreactivity in the DRG appeared greater in Wnt1-Cre; Isl1-Cre mutants than in Wnt1-Cre mutants (data not shown), suggesting a more efficient recombination with both Cre drivers.
NRP1 is important for arteriogenesis
An endothelial-specific knockout of Nrp1 produced an even more
severe defect in arteriogenesis than was observed in the nerve-specific
Vegfa conditional knockouts. There are several possible explanations
for this difference. First, cardiac defects in the endothelial Nrp1
mutants (Gu et al., 2003)
might affect peripheral arteriogenesis via impaired blood flow; by contrast
cardiac development in the nerve-Vegfa mutants appears to be normal.
We think this is unlikely, because dorsal aorta arteriogenesis is normal in
conventional Nrp1-null mice (Y.M., D.J.A. and H. Fujisawa,
unpublished). Second, the Tie2-Cre; Nrp1 phenotype could reflect a
requirement for a ligand of NRP1 other than VEGF164, such as a
semaphorin. In avian embryos, implantation of Sema3a protein or of a
neutralizing anti-Sema3a antibody, caused a failure of proper vascularization
(Bates et al., 2003
). Others
have reported angiogenesis defects in Sema3a mutant mice on a CD-1
genetic background (Serini et al.,
2003
). However, we observed no defects in limb skin arteriogenesis
in Sema3a-null mice (Taniguchi et
al., 1997
), on the C57Bl6 background used here
(Mukouyama et al., 2002
).
Third, the stronger arteriogenesis defects in Tie2-Cre; Nrp1
embryonic limb skin vasculature may reflect a more complete interruption of
VEGF signaling than is obtained in Wnt1-Cre; Isl1-Cre;
Vegfflox/flox embryos. This could occur either because
deletion of a crucial coreceptor in endothelial cells reduces responsiveness
to VEGF164 derived from all local tissue sources, neural or
otherwise, and/or because Cre-mediated recombination of Nrp1 is
required in only one allele to achieve reduction to homozygosity, whereas
recombination of both Vegfflox alleles is required.
What might be the function(s) of NRP1 in arteriogenesis? Because NRP1 is
arterial specific (Moyon et al.,
2001; Mukouyama et al.,
2002
) and is itself induced by VEGF (Y.M., H.-P.G., N.F. and
D.J.A., unpublished) (Fig. 7)
(Oh et al., 2002
), it could
mediate a positive-feedback loop, that increases the sensitivity of nascent
arterial cells to VEGF. This feedback loop could be initiated by
non-NRP1-binding isoforms of VEGF (e.g. VEGF120), which we have
shown induce NRP1 in vitro, and maintained or further amplified by the
NRP1-binding isoform. These considerations suggest a model in which a
`winner-takes-all' competition for VEGF may control arterial differentiation,
with the outcome biased by a VEGF164-NRP1 positive-feedback loop
(Fig. 8B). In addition, because
NRP1 is selective for the heparin-binding isoform of VEGF, it could also
function to restrict induction of arterial markers to those vessels in close
proximity to nerves.
This model predicts that nerve-dependent arteriogenesis should be
compromised in mice selectively lacking the NRP1-binding isoform. But in such
mice, e.g. those expressing only VEGF120, arterial differentiation
is normal in both the retina (Stalmans et
al., 2002) and developing limb (Y.M., D.J.A. and P. Carmeliet,
unpublished). The levels of VEGF120 expression in these mutant
mice, however, are similar to the combined levels of VEGF120,
VEGF164 and VEGF188 expression in wild-type mice, i.e.
almost three-fold higher than normal
(Carmeliet et al., 1999
). As we
have shown in vitro, at higher concentrations (>10 pg/ml),
VEGF120 is equally effective as VEGF164 at inducing
arterial differentiation. Therefore the increased levels of VEGF120
in Vegf120/120 mice may obscure the requirement for the
VEGF164 isoform in arteriogenesis. Such an explanation would be
consistent with the fact that in zebrafish, overexpression of either
Vegf121 or Vegf165 can rescue arterial
differentiation blocked by a deficiency of Shh signaling
(Lawson et al., 2002
).
Although a commercial source of both VEGF isoforms was used according to
the manufacturer's technical specifications (R&D), we recognize the
possibility that these two preparations might have differences in specific
activity that could account for their differential ephrin B2-inducing
activities in our assay. It is difficult to compare the activities of these
two isoforms independently of the presence of NRP1, because both isoforms
induce this co-receptor at concentrations well below those at which they
promote survival and proliferation. However, using porcine aortic endothelial
cells, which do not express NRP1, Soker et al.
(Soker et al., 1998)
demonstrated that both VEGF121 and VEGF165 had equal
activities in a chemotaxis assay when the cells were reconstituted with
exogenous VEGFR2 (Soker et al.,
1998
). Furthermore the concentrations of VEGF120 used
in our assay were 1.4-fold higher, on a molar basis, than the concentration of
VEGF164 used at each serial dilution. The fact that the latter
isoform exhibited more potent ephrin B2-inducing activity, under conditions
where there was a bias in favor of VEGF120, makes it more likely
that the difference observed is indeed intrinsic to the two isoforms.
Relationship between arteriogenesis and nerve-vessel alignment
During normal development, arteriogenesis is immediately preceded by
nerve-vessel alignment and is dependent on the presence of nerves
(Mukouyama et al., 2002). Our
results demonstrate that it is possible to genetically uncouple these
sequential processes: in mutants that disrupt VEGF signaling from the nerve to
the vessels, arteriogenesis is disrupted, while nerve-vessel alignment is
apparently unperturbed. The simplest explanation for this uncoupling is that a
nerve-derived factor distinct from VEGF mediates the alignment process.
However, the residual VEGF detected in the conditional Vegf knockout
embryos leaves open the possibility that nerve-vessel alignment simply
requires a lower threshold level of VEGF, than does arteriogenesis.
Nevertheless, the fact that the arteriogenesis defect is even stronger in the
endothelial-specific Nrp1 knockout, while nerve-vessel alignment
remains intact, argues against the dual-threshold model. At the very least,
for such a model to be tenable, the Tie2-Cre; Nrp1 data would require
that alignment depend only on the non-heparin-binding, more diffusible VEGF
isoforms. This is reasonable given that alignment presumably requires
action-at-a-distance to attract the vessels to the nerves. However, we did not
see any significant defects in nerve-vessel alignment in the limb skin of
mutants that lack the VEGF120 isoform (Y.M., D.J.A. and P.
Carmeliet, unpublished). Therefore, the available data favor the notion that
VEGF and a separate, as yet unidentified signal, mediate arteriogenesis and
nerve-vessel alignment, respectively.
Is nerve-vessel alignment stochastic or deterministic?
What determines which blood vessels in skin become aligned with nerves and
undergo arteriogenesis? One explanation is that the process is stochastic: all
vessels are equally capable of becoming aligned with nerves, and the selection
of a subset for arteriogenesis is simply determined by their initial proximity
to nerves at the time of innervation. A mechanism involving a
`winner-takes-all' competition for limiting amounts of nerve-derived VEGF,
biased by the VEGF164-NRP1 positive-feedback loop discussed
earlier, would be well suited to such a mechanism
(Fig. 8B). Alternatively, a
subset of vessels may be pre-specified for association with the nerve. For
example, if nerves release a signal that recruits vessels to align with them,
then a subset of vessels might express higher levels of a receptor for this
alignment signal, before the nerves ever arrive. Such a pre-specification
mechanism would, however, require precise matching to ensure a sufficient
supply of presumptive arterial vessels for developing nerves. The
identification of nerve-derived signals for vessel alignment may help to
distinguish between these alternatives.
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Supplementary material |
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ACKNOWLEDGMENTS |
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