1 Department of Biology, University of North Carolina at Chapel Hill, Chapel
Hill, NC 27599-3280, USA
2 Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA
15260, USA
3 Carolina Cardiovascular Biology Center, University of North Carolina at Chapel
Hill, Chapel Hill, NC 27599, USA
Author for correspondence (e-mail:
bautch{at}med.unc.edu)
Accepted 8 December 2003
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SUMMARY |
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Key words: Vascular pattern, Neural tube, Midline, VEGFA, Mouse-avian chimeras
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Introduction |
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Several well-characterized molecular pathways are implicated in vascular
patterning, including the VEGF, Notch, ephrin and EDG pathways, as well as
unidentified genes such as out-of-bounds (reviewed in
Hogan and Kolodziej, 2002)
(Fong et al., 1995
;
Shalaby et al., 1995
;
Carmeliet et al., 1996
;
Pereria et al., 1999
;
Zhong et al., 2000
;
Graef et al., 2001
;
Lawson et al., 2001
;
Zhong et al., 2001
;
Childs et al., 2002
).
Moreover, there are at least two classes of vascular patterning signals.
Short-range signals are produced locally and act via cell interactions at
short distances, whereas long-range signals affect target cells at a distance
from the source of the signal. The VEGF signaling pathway produces both short-
and long-range information to pattern blood vessels. VEGFA-coated beads induce
ectopic vessels both at the site of bead deposition and on the contralateral
side of avian embryos, indicating that both local and long-range signals are
set up by an exogenous source of VEGFA
(Bates et al., 2003
;
Finkelstein and Poole, 2003
).
Endogenous sources of VEGF also provide vascular patterning signals at
multiple levels. VEGF expression by retinal cells and neurons is associated
with local vessel patterning in the retina and limb, respectively
(Stone et al., 1995
;
Zhang et al., 1999
;
Mukouyama et al., 2002
;
Otani et al., 2002
). The
Vegfa locus produces several isoforms with different biochemical
properties, and analysis of embryos lacking heparin-binding VEGFA isoforms
suggests that matrix deposition of VEGFA is important to local patterning and
branching (Park et al., 1993
;
Keyt et al., 1996
;
Carmeliet et al., 1999
;
Ng et al., 2001
;
Ruhrberg et al., 2002
;
Stalmans et al., 2002
;
Zelzer et al., 2002
).
Additionally, endogenous VEGF is associated with long-range vascular
patterning in Xenopus, where VEGF secreted from a midline structure
called the hypochord is thought to induce angioblasts to migrate from lateral
areas to form the dorsal aorta (Cleaver
and Krieg, 1998
). However, the hypochord does not exist in avians
and mammals, and no embryonic structures have been identified as sources of
midline vascular patterning signals in higher vertebrates.
Nevertheless, important vascular beds are formed and patterned around the
midline of higher vertebrates. The perineural vascular plexus (PNVP) is a
capillary bed that forms around the developing brain and spinal cord. This
plexus provides essential nutrients and oxygen to the developing neural
tissue, and it is the source of vascular sprouts that subsequently invade and
metabolically support the neural tissue. These PNVP-derived vessels go on to
form the blood-brain barrier that is critical to proper CNS function in the
adult (Bar, 1980;
Risau, 1986
;
Risau and Wolburg, 1990
;
Bauer et al., 1993
). Although
the invasion of angiogenic sprouts into neural tissue has been described, the
developmental processes that pattern the PNVP have not been investigated. Both
quail-chick and mouse-quail chimera analysis identified somitederived
precursor cells as an important source of endothelial cells that comprise the
PNVP (Wilting et al., 1995
;
Klessinger and Christ, 1996
;
Pardanaud et al., 1996
;
Pardanaud and Dieterlen-Lievre,
1999
; Ambler et al.,
2001
). Moreover, our recent analysis of ES cell-derived grafts
showed that VEGF signaling is involved in vascular patterning around the
midline of higher vertebrates (Ambler et
al., 2003
). However, important questions remain unanswered,
including the source of the signal(s) that act on somite-derived angioblasts
to pattern the PNVP and the importance of VEGF in this process.
We have asked whether midline structures, specifically the neural tube, provide signals to pattern the PNVP of higher vertebrates. We show that the neural tube is the source of a vascular patterning signal that acts on the PNVP, and that VEGFA is a crucial component of this signal. These results provide the first identification of a midline signaling center that patterns vessels in higher vertebrates, and they suggest a model whereby embryonic structures with little or no capacity for angioblast generation act as a nexus for vessel patterning, and thus provide for their own sustenance.
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Materials and methods |
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Embryo manipulations
Noon of the day of the mouse vaginal plug was considered 0.5 days post
coitum (dpc). Mouse embryos were dissected at 8.5 dpc into M2 Medium
(Quinn et al., 1982)
containing pen/strep (penicillin (100 U/ml)/streptomycin (100 ng/ml)). Embryos
were pinned onto a Sylgard (Dow Corning) dish in M2 medium with tryp/panc [5
µg/ml trypsin (Sigma)/25 µg/ml pancreatin (Sigma)]. To loosen the
tissues, embryos were incubated in M2 medium with tryp/panc for 1 minute
before presomitic mesoderm or axial structures were surgically dissected using
a sharpened tungsten needle. Dissected tissues were placed in PBS with 10% FBS
containing pen/strep, then placed in collagen gels or quail hosts.
Fertilized Japanese quail eggs (CBT Farms, Chestertown, MD) were incubated
at 37°C for 48 hours to the HH 10-13 stage
(Hamburger and Hamilton,
1951). Quails were used (1) to provide a source of axial
structures (notochord or neural tube) for explant co-cultures, or (2) as hosts
for axial structures or mouse presomitic mesoderm. For explant co-cultures,
quail embryos were removed into Tyrodes buffer, then pinned onto a Sylgard
dish in Tyrodes buffer with tryp/panc. Caudal axial structures (at the level
of presomitic mesoderm) were surgically dissected using a sharpened tungsten
needle and the graft was transferred to PBS containing 10% heat-inactivated
FBS before placement in collagen. Quail host embryos were surgically prepared
according to graft type. For neural tube grafts, the intermediate mesoderm was
separated from the lateral plate mesoderm. For presomitic mesoderm grafts, the
lateral region of the quail presomitic mesoderm was removed. After graft
placement and addition of pen/strep, the eggs were sealed with Parafilm and
incubated for 48 or 72 hours at 37°C in a humidified incubator.
ß-Galactosidase detection and immunohistochemistry
Quail embryos containing either ROSA26 neural tube grafts or
Flk1+/ mouse presomitic mesoderm grafts were
processed for whole-mount ß-gal detection as previously described
(Ambler et al., 2001). Briefly,
embryos were sacrificed 48-72 hours post surgery, rinsed in PBS, and fixed in
4% paraformaldehyde (PFA) for 15-20 minutes at room temperature. Chimeras were
washed twice for 10 minutes in wash buffer [(0.1 M phosphate buffer, pH 7.3),
0.1% sodium desoxycholate, 0.02% NP40, 0.05% BSA (Sigma)], then transferred to
wash buffer containing 1 mg/ml X-gal (Sigma), 5 mM ferrocyanide and 5 mM
ferricyanide. After incubation for 14-18 hours at 37°C in the dark,
embryos were post fixed in 4% PFA and stored at 4°C until embedded.
Vegf-lacZ+/ or +/+ embryos were
processed for whole-mount ß-galactosidase detection similarly with one
exception these embryos were pre- and post-fixed in 0.2%
glutaraldehyde (in 0.1 M phosphate buffer containing 5 mM EGTA and 2 mM
MgCl2).
Whole-mount platelet endothelial cell-adhesion molecule (PECAM)
immunohistochemistry [adapted from Dent et al.
(Dent et al., 1989)], was
performed as described (Ambler et al.,
2001
; Ambler et al.,
2003
). Embryos were stored in PBS until photographed with an
Olympus SZH10 dissecting microscope. Embryos were embedded, sectioned and
mounted as previously described (Ambler et
al., 2001
). QH1 staining was performed on sections as previously
described (Ambler et al., 2001
)
and viewed with a Nikon Opitphot 2 microscope.
Collagen gel explants
Mouse presomitic mesoderm was either cultured alone or in combination with
quail axial structures. The collagen [final concentration 1.5 mg/ml (Sigma)]
contained one part 10x (1 M) HEPES, one part 10x minimal essential
media (MEM), one part sodium bicarbonate (11.76 mg/ml) and seven parts rat
tail collagen type I (2.14 mg/ml, resuspended in 3% sterile acetic acid). This
solution was stored on ice until incubated at 37°C for gelling. A base
layer of collagen was gelled into wells of a 96-well tissue culture plate and
stored at 37°C. Explants were added to liquid collagen (35 µl) and
placed over the gelled collagen in wells at 37°C for 10 minutes. After
gelling, basal medium [DMEM-H, 10% heat inactivated FBS (Hyclone), pen/strep
and gentamicin (50 ng/ml)], or basal medium with 30 ng/ml recombinant human
VEGF164 (R&D Systems), 25 ng/ml human recombinant bFGF (Sigma), 1 µg/ml
Flt/Fc (R&D Systems) or 10 µM SU5416 (SUGEN) was added. Explant
cultures were then incubated for 24-72 hours at 37°C in a humidified 5%
CO2 incubator. Cultures containing
eGFP+/ tissues were viewed with epiflourescence and
photographed before fixation. Cultures were fixed in 0.2% glutaraldehyde (in
0.1 M phosphate buffer containing 5 mM EGTA and 2 mM MgCl2) for 20
minutes before ß-gal staining (described above). Stained explants were
then photographed on an Olympus IX50 inverted microscope.
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Results |
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Discussion |
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What is the source of angioblasts that colonize the PNVP? We and others
have shown that somite-derived angioblasts reproducibly contribute to PNVP
formation (Wilting et al.,
1995; Klessinger and Christ,
1996
; Pardanaud et al.,
1996
; Pardanaud and
Dieterlen-Lievre, 1999
; Ambler
et al., 2001
), and in this study the neural tube was sufficient
for formation of a somite-derived vascular plexus in collagen co-cultures.
Presomitic mesoderm grafts placed next to a `buffer' of avian cells that
prevented direct contact with neural tube contributed to the PNVP, showing
that progenitor/endothelial cells can migrate over a distance in response to
neural tube vascular patterning signal(s). However, it is likely that other
embryonic tissues also contribute endothelial/progenitor cells to PNVP
formation. For example, the lateral plate mesoderm is a rich source of
migratory angioblasts, and lateral plate grafts can contribute vascular cells
to the PNVP (Noden, 1988
;
Poole and Coffin, 1989
;
Pardanaud et al., 1996
;
Cleaver and Krieg, 1998
;
Pardanaud and Dieterlen-Lievre,
1999
; Childs et al.,
2002
). When neural tubes were placed adjacent to lateral plate
mesoderm in our study, the ectopic PNVP that formed did not compromise PNVP
formation around the endogenous neural tube. Thus, it is likely that
angioblasts from the lateral plate mesoderm contributed to the ectopic PNVP,
and that both somite-derived and lateral plate-derived angioblasts can respond
to vascular patterning signal(s) from the neural tube. This idea is supported
by the finding that Tbx6 mutant embryos do not have posterior somites
(Chapman and Papaioannou,
1998
), yet they have three posterior neural tubes that each have a
PNVP.
VEGFA, a neural tube-derived molecular mediator of vascular pattern
The neural tube is a signaling center that organizes embryonic structures
around the midline. Along with the notochord, somites, and overlying ectoderm,
the neural tube produces molecular signals that pattern the neural tube
itself, and the somites that form adjacent to the neural tube. Moreover, many
of the molecules involved in these signals have been identified. For example,
sclerotome is induced by Shh production from the floor plate
(Fan and Tessier-Lavigne,
1994; Johnson et al.,
1994
), dermatome is patterned by secretion of neurotrophin 3 and
Wnt1 from the neural tube (Brill et al.,
1995
; Olivera-Martinez et al.,
2001
), and epaxial myoblasts are patterned by a combination of
Shh, Wnt1 and Wnt3a (Munsterberg et al.,
1995
; Stern et al.,
1995
; Ikeya and Takada,
1998
).
What molecular signals contribute to the neural tube-derived vascular
patterning signal? The temporal and spatial expression pattern of VEGFA is
consistent with it participating in a vascular patterning signal from the
neural tube. To further dissect the neural tube vascular patterning signal(s),
we adapted a co-culture system using a three-dimensional collagen matrix. This
approach was successfully used to dissect the molecular components of neural
pathfinding (Fan and Tessier-Lavigne,
1994), but has not been previously applied to the analysis of
vascular patterning signals. The co-culture of neural tube with presomitic
mesoderm showed that the neural tube is sufficient to induce a vascular plexus
from somitic tissue, and VEGFA can substitute for the neural tube and induce a
vascular plexus from presomitic mesoderm in this model.
Our pharmacological and genetic manipulations of the VEGF signaling pathway
indicate that it is a required component of the neural tube vascular
patterning signal. Moreover, our results indicate that the signal is VEGFA,
and that the relevant receptor is FLK1. SU5416 specifically inhibits signaling
of the FLK1 receptor, and its presence in co-cultures resulted in a complete
blockade of vascular plexus formation, as did genetic inactivation of
Flk1. Flt/Fc is a soluble form of the FLT1 receptor that also
completely blocked neural tube-dependent vascular plexus formation. Flt/Fc
binds PLGF, VEGFA and VEGFB (Barleon et
al., 1997; Olofsson et al.,
1998
), but of these three family members only VEGFA binds FLK1
(for a review, see Yancopoulos et al.,
2000
). Thus, the most likely molecular components of the neural
tube vascular patterning signal are VEGFA and FLK1. We recently showed that
VEGF signaling through FLK1 is crucial for patterning stem cell-derived
endothelial cells/progenitors around the midline by analysis of mutant ES
cell-derived grafts in avian hosts (Ambler
et al., 2003
). This work extends those findings to a requirement
for FLK1 expression in somite-derived endothelial cells/progenitors to form
and pattern a vascular plexus.
One caveat to the interpretation that VEGFA is a crucial neural
tube-derived vascular patterning signal is that the VEGF signaling pathway is
required for multiple steps in vascular development, including
differentiation. Thus it could be argued that in the absence of VEGF
signaling, the population of cells that respond to patterning signals is not
present. Although we cannot formally exclude an exclusive role for VEGF in
early specification of the vascular lineage, several lines of evidence suggest
that VEGF signaling is not an absolute requirement for angioblast
differentiation. Initial formation of murine endothelial cells and some
vessels occurs in the absence of FLK1 in vivo and during ES cell
differentiation, and zebrafish lacking FLK1 form many vessels but are
defective in sprouting angiogenesis
(Shalaby et al., 1995;
Shalaby et al., 1997
;
Schuh et al., 1999
;
Habeck et al., 2002
;
Ambler et al., 2003
). In the
avian embryo, exogenous bFGF induces angioblasts, while VEGFA promotes
angioblast migration and assembly (Cox and
Poole, 2001
; Poole et al.,
2001
; Finkelstein and Poole,
2003
). In our study, VEGF signaling is not required to activate
the FLK1 locus in a subset of presomitic mesoderm cells in the presence of
neural tube (Fig. 8D),
suggesting that cells can be specified to the vascular differentiation pathway
in the absence of VEGF. Moreover, VEGF signaling clearly imparts information
over and above that required for angioblast differentiation, as VEGFA could
substitute for the neural tube in forming a presomitic mesoderm-derived
vascular plexus, whereas bFGF, a second signal associated with angioblast
induction, increased the number of vascular cells but did not induce assembly
of a vascular plexus. It will be interesting to further dissect the
requirement for VEGF signaling in vascular patterning using conditional
targeting strategies and site-directed mutagenesis of the FLK1 receptor.
As discussed above, the finding that
Flk1/ presomitic mesoderm explants have more
ß-gal-positive cells when cultured with a neural tube than with exogenous
VEGFA, suggests that a second neural tube signal contributes to induction of
FLK1 expression (Fig. 9C).
VEGF-independent Flk1 expression is also seen in hemizygous
(Flk1+/) presomitic mesoderm neural tube
co-cultures incubated with Flt/Fc (K.A.H. and V.L.B., unpublished). Thus, it
is plausible that a VEGF-independent neural tube signal induces Flk1
expression in a subset of somitic cells. Flk1-expressing cells would
then be competent to respond to the signal that requires VEGFA, which is known
to induce further differentiation, as followed by expression of vascular
markers such as FLT1. VEGFA also induces migration of endothelial
cells/progenitors and assembly of a vascular plexus
(Fig. 9D). The ability of VEGFA
to substitute for the neural tube in the co-culture experiments may be dose
dependent, as preliminary results indicate that low concentrations of VEGFA do
not support robust vascular plexus formation from presomitic mesoderm (K.A.H.
and V.L.B., unpublished). This finding is consistent with a correlative study
that associated tissues expressing high levels of VEGFA with vasculogenic
colonization and tissues expressing lower levels of VEGFA with angiogenic
colonization (Miquerol et al.,
1999). It will be interesting to determine if neural tube signals
other than VEGF play important roles in vivo.
The hypothesis that additional signals emanate from the neural tube also
may explain a conundrum the murine gut endoderm lies almost
immediately ventral to the neural tube at E8.5, and expression of VEGFA RNA is
significantly stronger from this embryonic structure than from the neural
tube. Although the endoderm may be the source of a midline signal earlier in
development that induces formation of the dorsal aorta
(Cleaver and Krieg, 1998;
Miquerol et al., 1999
), at
this stage the endoderm does not recruit a vascular plexus and the neural tube
does recruit the PNVP. This suggests that VEGFA expression is not sufficient
to induce a competition between endoderm and neural tube for somite-derived
angioblasts. Thus, the neural tube may emit additional inductive and/or
vascular patterning signals that co-operate with VEGFA to recruit the
PNVP.
Conclusions
The identification of the neural tube as the source of a vascular
patterning signal from the midline of the vertebrate embryo enhances our
understanding of the basic processes of blood vessel formation, as vessels do
not form and expand randomly, but in response to specific environmental cues.
It also shows that embryonic tissues without endogenous capacity for vessel
formation can nevertheless communicate with other embryonic compartments in a
coordinated way, and recruit a vascular plexus to provide for metabolic needs.
The placement of VEGFA in this signal pathway provides the beginnings of a
mechanistic analysis of this process. A better understanding of how vascular
pattern is controlled also has multiple ramifications in medical therapies and
disease treatments. Specifically, our ability to induce appropriate
neovascularization in patients to treat blockage of coronary and other vessels
depends on understanding what cues normally pattern vessels, and how
endothelial cells and their precursors respond to these cues. Likewise, the
therapeutic goal of reconstituting blood vessels outside the body for grafting
purposes will be facilitated with a better understanding of vascular
patterning events.
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ACKNOWLEDGMENTS |
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Footnotes |
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