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Vascular endothelial growth factorA regulator of vascular morphogenesis in the Japanese quail embryo.

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THE ANATOMICAL RECORD PART A 272A:403– 414 (2003)
Vascular Endothelial Growth Factor:
A Regulator of Vascular
Morphogenesis in the Japanese
Quail Embryo
ERIC B. FINKELSTEIN AND THOMAS J. POOLE*
Department of Cell and Developmental Biology, SUNY Upstate Medical University,
Syracuse, New York
ABSTRACT
Experiments in mouse embryos indicate that a critical level of VEGF is
required for normal vascular development, as mice lacking a single VEGF
allele die at midgestation. Thus VEGF concentration may be a determinant
of the size and location of major blood vessels during formation of the
primary capillary plexus. Ectopic VEGF delivery was used to examine the
effect of VEGF concentration on early vascular patterning in the quail
embryo. VEGF was delivered by implanting VEGF-soaked heparin chromatography beads at three rostral-caudal locations in embryos with six somite
pairs, which allowed us to study the effect of VEGF on different cellular
activities. Ectopic VEGF resulted in significant changes in the vascular
pattern at three rostral-caudal levels. Quantitation demonstrated an increased vascularity in the area of the implanted VEGF bead compared to
the vascular pattern of embryos with control beads. Areas lateral to the
dorsal aortae that are normally avascular became vascularized, and there
was an apparent fusion between the dorsal aorta and lateral capillary
plexus. Anat Rec Part A 272A:403– 414, 2003. © 2003 Wiley-Liss, Inc.
Key words: VEGF; vascular development; vasculogenesis; quail
embryo; heparin bead
Vascular development begins in situ during early
somite stages in the avian embryo when angioblasts (endothelial cell precursors) emerge parallel to the embryonic
midline (Pardanaud et al., 1987; Coffin and Poole, 1988;
Dieterlen-Lievre and Pardanaud, 1998). Angioblasts, apparently induced by fibroblast growth factor-2 (FGF-2)
(Flamme and Risau, 1992; Krah et al., 1994; Cox and
Poole, 2000), begin to express vascular endothelial growth
factor receptor-2 (VEGFR2) (Eichmann et al., 1993, 1996;
Yamaguchi et al., 1993). The primary capillary plexus
forms from the induced angioblasts by a poorly understood
morphogenetic process influenced by FGF, vascular endothelial growth factor (VEGF), adhesion molecules, and the
extracellular matrix (Tallquist et al., 1999; Poole et al.,
2001). In a mature blood vessel, smooth muscle cells and
pericytes surround an endothelial cell layer. This mature
vessel structure forms following remodeling of the primary capillary plexus (reviewed in Darland and D’Amore,
1999; Conway et al., 2001).
There are two major mechanisms of vascular development: vasculogenesis and angiogenesis (Pardanaud et al.,
©
2003 WILEY-LISS, INC.
1989; Poole and Coffin, 1989, 1991). Vasculogenesis is the
de novo formation of blood vessels through angioblast
migration and cohesion, and is the mechanism by which
the paired dorsal aortae form ventrolaterally to the
somites on either side of the embryonic midline (Coffin
and Poole, 1988; Poole and Coffin, 1988, 1989; Poole et al.,
2001). Angiogenesis is the formation of new blood vessels
from preexisting vessels, and is the mechanism by which
E.B. Finkelstein’s current address is Schepens Eye Research
Institute, 20 Staniford St., Boston, MA 02114.
*Correspondence to: Dr. Thomas J. Poole, Department of Cell
and Developmental Biology, SUNY Upstate Medical University,
766 Irving Ave., Syracuse, NY 13210. Fax: (315) 464-8535.
E-mail: poolet@upstate.edu
Received 25 July 2002; Accepted 15 December 2002
DOI 10.1002/ar.a.10047
404
FINKELSTEIN AND POOLE
the intersomitic arteries form by sprouting from the dorsal
aortae (Poole and Coffin, 1989, 1991; Poole et al., 2001).
Vasculogenesis predominates in the quail embryo until
the seven-somite stage (Hamburger-Hamilton (HH) stage
9 (Hamburger and Hamilton, 1951)), after which both
mechanisms contribute to vascular development as intersomitic arteries branch from the rostral dorsal aortae, and
free angioblasts undergo cohesion to expand the vascular
plexus in caudal regions.
One growth factor involved in vascular development is
VEGF (also known as VEGF-A) (Conway et al., 2001;
Poole et al., 2001). VEGF is identified by its mitogenic
effect on endothelial cells, and its ability to induce endothelial cell chemotaxis (Ferrara, 1999) and localized
edema (Senger et al., 1983). All embryonic cell types,
except endothelial cells, express VEGF. A particularly
high level of VEGF expression is observed in the embryonic endoderm (Flamme et al., 1995a; Aitkenhead et al.,
1998). In contrast, VEGF receptors are expressed exclusively on endothelial cells and angioblasts, often adjacent
to areas of VEGF expression (Breier et al., 1995; Flamme
et al., 1995a; Eichmann, 1996). VEGF and its receptors
are essential for vascular development (reviewed in Poole
et al., 2001). Mice that lack a single VEGF allele die at
embryonic day 11.5 (Carmeliet et al., 1996; Ferrara et al.,
1996), which suggests that regulation of VEGF concentration is required during development. Severe defects have
also been reported in embryos that are homozygous-null
for VEGF, with death on day 10.5 (Carmeliet et al., 1996).
In VEGF-null embryos, endothelial cell numbers are reduced and a vascular pattern fails to form. Embryos that
are deficient for VEGF receptors are also embryonically
lethal, with a disorganized vascular pattern (Fong et al.,
1995, 1999; Shalaby et al., 1995).
The quail embryo has been used extensively for studies
of early events in vascular development. This has been
facilitated by the use of a monoclonal antibody, QH-1,
which specifically recognizes quail endothelial cells and
angioblasts (Pardanaud et al., 1987). By using the quail
embryo as an “in vivo vasculogenesis assay” (Drake et al.,
1992), growth factors can be assayed for their effects on
the vascular pattern. In one study (Drake and Little,
1995), microinjection of recombinant human VEGF in the
segmental plate of quail embryos with five to six somite
pairs resulted in hypervascularization. Other studies using retroviral delivery demonstrated that four isoforms of
quail VEGF could affect the vasculature by increasing
blood vessel permeability without altering vessel patterns.
To further examine the role of VEGF in quail vascular
pattern formation, microsurgical techniques were used to
deliver recombinant human VEGF165 (rhVEGF165) to
day 2 quail embryos. Since VEGF is a heparin-binding
protein, chemically linked heparin chromatography beads
were used for VEGF delivery. Human VEGF165 and its
quail counterpart, VEGF166, are 75% similar (Yue and
Tomanek, 2001), so it was expected that the effects of
human VEGF165 would be similar to that with native
quail VEGF. The use of beads to deliver growth factors has
several advantages over microinjection, including focal
delivery of growth factors, a slow-release source of growth
factor, and a traceable delivery location. This method has
been used to deliver FGF-2 (Cox and Poole, 2000) and
BMP-4 (Schmidt et al., 1998) to precise locations in avian
embryos, and to deliver VEGF to explanted lung tissue
(Healy et al., 2000).
The present study focuses on morphogenesis of the
paired dorsal aortae, which proceeds in a rostral to caudal
sequence in three distinct stages. The first stage is the
migration and cohesion of single angioblasts along the
embryonic midline to form a solid cord. The solid cord then
differentiates into an endothelial-lined tube as a lumen
forms (the mechanisms of which are unclear). Finally,
vessels branch from the tubular dorsal aorta to form the
intersomitic arteries. Data presented here show that
bead-delivered VEGF can affect all three stages of dorsal
aorta morphogenesis. Ectopic VEGF also resulted in increased vascularization lateral to the dorsal aortae, as
well as apparent fusion between the dorsal aortae and the
capillary plexus lateral to bead implantation sites.
MATERIALS AND METHODS
Microsurgery Methods
Fertile Japanese quail eggs (Coturnix coturnix japonica)
were purchased from the Cornell University Poultry Science Labs (Ithaca, NY). Eggs were incubated in a forcedair humidified incubator at 38°C and 60% relative humidity (Hamburger, 1960) for approximately 32 hr, until six or
seven somite pairs formed (HH stage 8-9 (Hamburger and
Hamilton, 1951)). Throughout this work, the number of
formed somite pairs (6S ⫽ six somite pairs) will be used to
indicate the developmental stage of the embryos. This is
more precise than using the HH staging system for early
development, since each HH stage contains a range of
somite numbers (for example, HH stage 8 has four to six
somites while HH stage 9 has seven to nine somites).
Bead Implantation
Lyophilized recombinant human VEGF 165 (rhVEGF165; PeproTech, Rocky Hill, NJ) was reconstituted
at a concentration of 1.0 mg/ml in sterile phosphate-buffered saline (PBS). An equal volume of growth factor and
PBS-washed heparin-Toyopearl chromatography beads
(Supelco, Bellefonte, PA) was mixed and incubated for 1 hr
at room temperature for approximately 0.5 mg/ml VEGF,
as previously described for other growth factors (Crossley
et al., 1996; Schmidt et al., 1998; Cox and Poole, 2000).
This high soaking concentration was used to saturate the
beads with growth factor (Eichele et al., 1984; Crossley et
al., 1996; Schmidt et al., 1998; Cox and Poole, 2000). After
the beads were soaked (referred to here as VEGF beads),
they were rinsed in sterile Howard’s saline (0.12 M NaCl,
1.53 mM CaCl2, 4.96 mM KCl) (Packard et al., 2000).
Beads 50 – 60 ␮m in diameter were visually selected and
implanted into somites of embryos with six or seven
somite pairs. Somites were used for bead delivery because
of their close proximity to the developing dorsal aortae,
and because somites give rise to fewer angioblasts than do
other mesodermally-derived tissues (Wilting et al., 1995;
Cox and Poole, 2000). Beads were implanted into the
somites with an electrolytically sharpened tungsten needle by making a small incision in the dorsal or lateral wall
of the somite and pushing a bead into the somite, without
removing any tissue. Beads soaked in sterile PBS without
growth factor were implanted into embryos as controls
(referred to as control beads). The VEGF and control
beads were implanted in three locations along the rostralcaudal axis to study the effect of ectopic VEGF on vascular
development. Somites within the embryo are at different
developmental stages along the rostral-caudal axis. They
405
VEGF AND VASCULAR PATTERN FORMATION
can be precisely staged using a nomenclature system in
which Roman numerals are used to number the somites
rostrally from the segmental plate (I being the most recently formed), and an Arabic numeral is used to indicate
the total number of somites in the embryo (Christ and
Ordahl, 1995; Stockdale et al., 2000). In embryos with six
somite pairs, beads were implanted in somites I/6 (referred to as the last-formed somite), IV/6 (referred to as
the middle somite), and VI/6 (referred to as the firstformed somite) (Fig. 1A). In embryos with seven somite
pairs, beads were implanted in somites I/7 (referred to as
the last-formed somite), IV/7 (referred to as the middle
somite), and VI/7 (a similar stage as the first-formed
somite in an embryo with six somite pairs).
For all experiments, the blastoderm was dissected from
the yolk in sterile Howard’s saline and cultured dorsal
side up on agar/albumen dishes (Packard et al., 2000).
Whole-egg supernatant with a 1:100 dilution of 29.2
mg/ml L-glutamine, 10,000 U/ml penicillin-G, and 10,000
mcg/ml streptomycin (Irvine Scientific, Santa Ana, CA)
added was used for media. After incubation for 4 or 7 hr at
38°C with 60% relative humidity, the embryos were removed from the culture dishes, rinsed in PBS, and fixed
for analysis.
Immunostaining of Whole-Mount Embryos
For QH-1 whole-mount immunostaining, embryos were
fixed in 10% formalin as previously described (Coffin and
Poole, 1988; Cox and Poole, 2000). Nonspecific antibody
binding was blocked with 3% bovine serum albumin (BSA;
Fraction V powder; Sigma, St. Louis, MO), in PBT (PBS
with 1% BSA, 0.1% Triton X-100). Embryos were then
incubated with QH-1 monoclonal antibody tissue culture
supernatant (Developmental Hybridoma Studies Bank,
Iowa City, IA) diluted 1:10 in 3% BSA/PBT, and rocked
overnight at 4°C. After the primary antibody incubation,
the embryos were washed with PBT, blocked and incubated overnight with a cy2-conjugated anti-mouse IgG
F(ab⬘)2 secondary antibody (Jackson Immunoresearch,
West Grove, PA) in BSA/PBT at 4°C. The embryos were
washed in PBS for 4 hr or overnight at 4°C and dehydrated through graded ethanols, cleared in toluene, and
mounted on glass slides ventral side up with Entellan (EM
Sciences, Fort Washington, PA).
Image Collection and Analysis
Embryos stained as whole mounts were imaged using
laser scanning confocal microscopy with a 1024ES confocal imaging system (BioRad, Hercules, CA) mounted on a
Nikon Eclipse E600 microscope (Nikon, Melville, NY). Images were collected with a 10⫻ objective at 5.0-␮m intervals through the entire stained embryo as a z-series with
Laser Sharp Image Acquisition software (BioRad). Each
z-series consisted of 30 – 40 sections depending on thickness of embryos when mounted on slides. Individual zseries were collapsed into vertical projections by the use of
Laser Sharp Processing and Confocal Assistant software
(BioRad). A vertical projection combined all images collected through a three-dimensional embryo into a single
one-dimensional image. The locations of beads and the
stages of embryos at fixation were confirmed using differential interference contrast (DIC) microscopy.
Changes in vascular patterns that resulted from ectopic
growth factor delivery were quantified. The Image Pro-
cessing Tool Kit (Reindeer Games, Asheville, NC), a set of
plug-ins for Adobe Photoshop, was used for all analyses.
Color images were converted to grayscale, and then the
images were inverted such that stained areas were dark
on a light background. Peaks in the image histogram were
separated for further processing using an equalize function with linear emphasis. Stained vascular areas were
selected and copied to manually exclude background
staining in the neural tube and somites. Finally, multitoned images were converted to binary images using a set
threshold value. Binary images were visually inspected to
ensure that they accurately reflected the staining patterns
in the original confocal images (Fig. 1B and C).
Measurements were made from the binary images with
a sample area approximately 250 ␮m on a side, extending
from the medial dorsal aorta to the lateral edge of the
capillary plexus with the bead-implant site centered rostral-caudally. The use of this sample area enabled the
stained areas to be measured reproducibly between embryos. This sample area was optimal for measuring maximal effects at a radius of 125 ␮m from implanted beads.
The same sample area was used for measurements on the
bead-implanted and contralateral sides of the embryos,
ensuring that the measurements were at the same rostralcaudal level. The stained area fraction was measured utilizing the ‘‘global’’ measurement command in the plug-in.
The stained area fraction is the area occupied by stained
pixels (black) relative to the total sample area, expressed
as a percent:
Area fraction (%) ⫽
Stained area
.
Total sample area
The stained area fraction was recorded for the experimental and control sides of each embryo, and these data
were used in all subsequent analyses. In about 30% of the
embryos with beads implanted, a hole resulted from the
operation. This hole is a region devoid of tissue due to
beads protruding through the ventral side of the embryo,
and can be visualized as an unstained area. For embryos
with holes, the stained area fraction was measured using
the standard sample area, with areas of the holes subtracted out to yield a modified area fraction:
Stained area
Modified area
fraction (%) ⫽ [Total sample area- area of hole].
The area of the holes was approximately 10% of the sample area measured. The same area for the holes was subtracted from sample areas on both sides of the embryos.
Statistical Analysis
For analysis, stained area fractions as measured were
grouped by embryo starting stage and bead location. From
the grouped measurements, the mean, standard deviation
(S.D.), and standard error of the mean (S.E.M.) were calculated. The total number of embryos with control beads
was seven in the first-formed somite, nine in the middle
somite, and five in the last-formed somite. The total number of embryos with VEGF beads used in analyses was 11
in the first-formed somite, 18 in the middle somite, and 12
in the last-formed somite. For statistical analysis, P ⬍
0.01 was designated to be significant. The bead-implanted
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FINKELSTEIN AND POOLE
Fig. 1. Somite numbering and quantitation methods. A: Confocal
image of a normal (unoperated) embryo with six somite pairs (6S) to
show the extent of vascularization at the stage the VEGF was delivered.
QH-1 labeling was performed without Triton X-100 in the washes to
slightly increase background and provide a clear view of the somites.
Along the left side, the somites are numbered as described in Materials
and Methods. CP, capillary plexus; DA, dorsal aortae (cord beginning to
form); SOM, somites; NT, neural tube; PSM, presegmental mesoderm.
B-C: Quantitation of changes in vascular patterns resulting from delivery
of ectopic VEGF on heparin-Toyopearl chromatography beads. Parts B
and C are the same images as in Fig. 3C and D, with VEGF beads
implanted in the middle somite of embryos with six somite pairs. Em-
bryos were incubated for 7 hr and stained with the QH-1 monoclonal
antibody as whole mounts. Row 1 depicts grayscale confocal images.
Row 2 depicts the same images after vascular areas were selected,
copied, and converted to binary images. Note that this matches the gray
staining in row 1. The boxed areas are those selected for quantitation of
QH-1 staining, shown in isolation in row 3. Row 3 shows the regions of
embryos in which the QH-1 stained area fractions (the proportion of
stained area relative to total selected area) around the bead were measured on the bead-implanted and contralateral sides of the embryos.
[Color figure can be viewed in the online issue, which is available at
www.interscience.wiley.com.]
VEGF AND VASCULAR PATTERN FORMATION
Fig. 2. Ectopic delivery of VEGF at the level of the last-formed
somite. Beads were incubated in (B) PBS (control) or in (C and D)
recombinant human VEGF165 (VEGF) and implanted in the last-formed
of 6S quail embryos, followed by a 7-hr incubation. All embryos were
stained as whole mounts with the QH-1 monoclonal antibody, followed
by confocal microscopy. A: A schematic of an embryo to indicate a bead
implanted in the last-formed somite (dark somite). B: An embryo with a
control bead, fixed at the 11S stage. C: An embryo with a VEGF bead,
fixed at the 12S stage. D: An embryo with a VEGF bead, fixed at the 10S
stage. Note the increased vascularization on (C) the bead-implanted side
and (D) both sides, compared to (A) an embryo with a control bead.
These are ventral views, with the rostral end up. Asterisks indicate bead
locations. AVA, avascular area; DA, dorsal aortae; NT, neural tube; CP,
capillary plexus; SOM, somites. The scale bar in D is representative of
100 ␮m and applies to B–D.
sides of embryos with beads on the left were compared to
those with beads on the right by means of Student’s t-test.
If differences were not statistically significant, data for
beads on both the left and right sides were combined for
further analyses. Complete analysis was carried out for
embryos with VEGF or control beads. The paired t-test
was used to compare bead-implanted and contralateral
sides within the same embryo. Student’s t-test was used to
compare bead-implanted sides of embryos with VEGF or
control beads at the same location with the same starting
stage. Student’s t-test was also used to compare contralateral sides of embryos with VEGF or control beads. All
407
Fig. 3. Ectopic VEGF delivery at the level of the middle somite.
(C and D) VEGF or (B) control beads were implanted in the middle somite
of 6S stage quail embryos, followed by a 7-hr incubation. All embryos
were stained as whole mounts with the QH-1 monoclonal antibody,
followed by confocal microscopy. A: The location of beads when implanted in the middle somite (dark somite), shown schematically. B: An
embryo with a control bead, fixed at the 11S stage. A normal avascular
area is present lateral to the dorsal aortae on either side of the embryo.
C: An embryo with a VEGF bead, fixed at the 10S stage. Note the
reduced avascular area adjacent to the bead, with numerous blood
vessel branches between the dorsal aorta and capillary plexus, compared to the contralateral side. D: An embryo with a VEGF bead, fixed at
the 12S stage. The normally avascular area adjacent to the bead is
extensively vascularized. These are ventral views, with the rostral end
up. Asterisks indicate bead locations. AVA, avascular area; DA, dorsal
aortae; NT, neural tube; CP, capillary plexus; SOM, somites. The scale
bar in D is representative of 100 ␮m and applies to B–D.
statistical analyses were carried out with Excel 2000 (Microsoft, Redmond, CA).
Immunostaining of Sections
For QH-1 staining of sections, embryos were fixed in 4%
paraformaldehyde (PFA) in PBS, and processed for embedding in Paraplast (Monoject, St. Louis, MO) following
standard procedures. Then 10-␮m transverse sections
were mounted on albumen-subbed slides. For immunostaining, sections were deparaffinized and rehydrated following standard procedures, and blocked with 10% fetal
408
FINKELSTEIN AND POOLE
TABLE 1. Control (PBS) beads implanted in six-somite stage quail embryos
Bead
location
n
Sidea
Stained area fraction (%)b
Comparison with control side
First-formed somite
Middle somite
7
Control
54.4 ⫾ 3.9
Last-formed somite
9
Bead
54.4 ⫾ 3.1
P⫽NS
Control
53.6 ⫾ 2.1
5
Bead
53.1 ⫾ 1.6
P⫽NS
Control
58.4 ⫾ 1.3
Bead
56.3⫾ 2.9
P⫽NS
a
Measurements were made on (bead)-implanted and contralateral (control) sides of embryos.
The mean stained area fraction of all embryos with control (PBS) beads at this location (expressed as a percent ⫾ SEM:
Standard Error of the Mean).
NS, not significant.
b
bovine serum (FBS; Summit Biotechnology, Fort Collins,
CO) in PBS. Sections were then incubated with a 1:50
dilution of QH-1 tissue culture supernatant followed by
rhodamine-red-X-conjugated anti-mouse IgG F(ab⬘)2 secondary antibody (Jackson Immunoresearch) each in 10%
FBS/PBS, for 1 hr at room temperature. After a final wash
in PBS, sections were dehydrated through graded ethanols, cleared in xylene, and mounted with coverslips with
Entellan (EM Sciences, Fort Washington, PA). Images
were collected by use of a 20⫻ objective on a Nikon Eclipse
E800 epifluorescence microscope (Nikon, Melville, NY)
equipped with a SPOT-RT digital camera (Diagnostic Instruments, Sterling Heights, MI), and processed with
Adobe Photoshop 5.5 (Adobe Systems, San Jose, CA).
RESULTS
VEGF Can Affect Initial Events of Dorsal Aorta
Development
Beads implanted in the last-formed somite (I/6) were
used to study the effect of VEGF on initial stages of dorsal
aorta formation, including angioblast cohesion (Fig. 2A). A
control bead (soaked in PBS) (Fig. 2B) did not have an
observable effect on the vascular pattern within the embryo. Both sides of the embryo developed a normal dorsal
aorta approximately 100 ␮m in diameter. The capillary
plexus had a reticular appearance and was separated from
each dorsal aorta by an avascular area. Statistics confirmed that the control bead implant produced no significant change in the vascular pattern (Table 1). Therefore,
the observed effects of the VEGF beads were due to ectopic
growth factor delivery.
VEGF beads implanted in the last-formed somite (I/6) of
quail embryos with six somite pairs resulted in an increased degree of vascularization lateral to the dorsal
aorta (Fig. 2C). This effect was observed directly lateral to
the implanted beads, extending several hundred microns
along the rostral-caudal axis. Regions of the embryo that
are normally avascular became heavily vascularized in
the majority of samples (92%; Table 2 and Fig. 2C). An
apparent fusion occurred between the dorsal aorta and
capillary plexus (Fig. 2C). In the remaining samples (8%),
a VEGF bead produced an increased number of blood
vessel branches near the bead site (Table 2; Fig. 2D). The
effect of a VEGF bead was not always restricted to the side
of an embryo where the bead was implanted. A decreased
avascular area was sometimes observed on the unoperated sides of embryos with VEGF beads (Fig. 2D).
No statistical difference between VEGF beads implanted on the left and right sides occurred in 6 somite
stage embryos at the level of the last-formed somite (Table
3). Therefore, embryos with beads on the left or right side
were pooled for further analysis. With beads implanted at
the level of the last-formed somite, there was no statistical
difference between QH-1-stained areas on bead-implanted
and contralateral sides within individual embryos (Table
3). This was supported by observed morphological effects
on the vasculature of both sides in half of the embryos
analyzed (6/12; 50%), even though beads had been implanted on only one side in each embryo. The effect on both
sides of the embryos was further examined by comparison
of QH-1 staining on control sides of embryos with control
or VEGF beads. Our analysis revealed a significant difference between control sides of embryos with VEGF or control beads at the level of the last-formed somite (Table 3).
A significant difference between the bead-implanted sides
of embryos with VEGF or control beads indicated that all
observed effects resulted from ectopic VEGF (Table 3).
Similar results were obtained for beads implanted in the
last-formed somite of embryos with seven somite pairs
(data not shown).
Ectopic VEGF Has an Effect When Delivered at
a Level of Active Lumen Formation
No morphological effects were observed when control
beads were implanted in the middle somite (Fig. 3B). A
normal avascular area lateral to the dorsal aorta was
crossed by only small-caliber blood vessels, and the capillary plexus was distinct from the dorsal aorta on the
bead-implanted and control sides of the embryos (Fig. 3A).
As with the control beads in the last-formed somite, no
significant difference resulted from implanting a control
bead in the middle somite (as determined by a comparison
of bead-implanted and control sides (Table 1)).
When a VEGF bead was implanted in the middle somite
of 6S embryos (Fig. 3C and D), observable changes in the
vascular pattern included increased vascularization lateral to the dorsal aorta (Fig. 3C and D), with an apparent
fusion between the dorsal aorta and capillary plexus (Fig.
3D). The dorsal aorta was expanded compared to that in
an embryo with a control bead implanted at the same level
(Fig. 3B). While loss of avascular areas was limited to
bead-implanted sides, there was an observable dorsal
aorta enlargement on control sides as well (Fig. 3D). In the
most extreme examples (67%; Table 2 and Fig. 3D), areas
normally devoid of angioblasts were completely vascularized. In other cases (33%; Fig. 3C) the number of blood
vessel branches crossing these areas adjacent to the bead
increased (Table 2). In addition to being more numerous,
the branches on the bead-implanted sides were larger
than those on the control sides (Fig. 3C). The effect ex-
409
VEGF AND VASCULAR PATTERN FORMATION
TABLE 2. Changes in the QH-1- stained areas resulting from VEGF delivery in six-somite stage embryos
Bead location
n
Sidea
Stained area
fraction (%)b
Increased
branching
Lost avascular
areas
First-formed somite
Middle somite
11
Control
Lc57.9 ⫾ 5.8
Rd62.5 ⫾ 5.0
Last-formed somite
18
Bead
81.7 ⫾ 3.2
Control
68.6 ⫾ 3.1
55%
(6/11)
45%
(5/11)
12
Bead
80.3 ⫾ 3.5
Control
77.0 ⫾ 2.7
33%
(6/18)
67%
(12/18)
Bead
77.8 ⫾ 3.1
8%
(1/12)
92%
(11/12)
a
Measurements were made on (bead)-implanted and (control) contralateral sides.
Data represent the mean stained area fraction (expressed as a percent ⫾ SEM: Standard Error of the Mean).
c
Mean stained area fraction for beads implanted on the left side (⫾ SEM: Standard Error of the Mean).
d
Mean stained area for beads implanted on the right side (⫾ SEM: Standard Error of the Mean).
b
TABLE 3. Analysis of the effect of ectopic VEGF at three rostral-caudal positions in six-somite stage embryos*
Bead location
Bead sidea
n
Comparison of
VEGF-delivery
sidesb
Bead-implanted vs.
contralateral
sidesc
Bead-implanted
sidesd
Contralateral
sidese
First-formed somite
Left
6
Right
5
Middle somite
Left
10
P ⬍ 0.01
Right
8
Last-formed somite
Left
2
P ⫽ NS
Right
10
P ⫽ NS
P ⬍ 0.01
P ⫽ NS
P ⬍ 0.01
P ⫽ NS
P ⬍ 0.01
P ⫽ NS
P ⬍ 0.01
P ⬍ 0.01
P ⫽ NS
P ⫽ NS
P ⬍ 0.01
P ⬍ 0.01
*For all analyses, embryos with beads implanted on left and right sides were combined if there was no statistical difference
between the sides of bead implantation (P ⬎ 0.01).
The side of an embryo where a bead was implanted.
b
Comparison of beads implanted on left or right sides of embryos.
c
Comparison between bead-implanted and contralateral sides within the same embryo.
d
Comparison of bead-implanted sides in embryos with control (PBS) or VEGF beads
e
Comparison of contralateral sides in embryos with control (PBS) or VEGF beads.
NS, not significant.
Statistically significant, P ⬍ 0.01.
a
tended 50 –75 ␮m in both rostral and caudal directions
from the bead sites.
Following quantification, the extent of changes in the
vasculature was the same regardless of which side the
bead was implanted on (Table 2). Therefore, all embryos
with a bead implanted in the middle somite were combined for analysis. Implantation of VEGF beads resulted
in an increased QH-1-stained area (Table 2), which was
statistically significant when experimental and control
sides of the same embryo were compared (Table 2). The
mean QH-1 stained area on the experimental VEGF sides
was 80.3%, while on the control sides it was 68.6%. There
was no observed effect resulting from control beads implanted in the middle somite (Table 1). However, there
was a significant difference between the experimental
sides of the embryos with control or VEGF beads, as well
as between their contralateral sides (Table 3). The effect of
VEGF beads was also significant when experimental and
control sides were compared within the same embryo. For
bead-implanted sides, significance (Table 3) indicated that
changes in the vascular pattern resulted from VEGF on
the beads, rather than the beads themselves or the heparin bound to them.
The effect on control sides of embryos can be observed by
comparing unoperated sides in Fig. 3D and B. A control
bead (Fig. 3B) did not affect the vascular pattern, so the
vascular pattern on both sides of the embryo is essentially
equivalent. With VEGF beads (Fig. 3C and D), the number
and size of blood vessel branches increased on unoperated
sides, so more anastomosing connections abnormally
bridged the gap between the dorsal aorta and the lateral
capillary plexus. With beads in the middle somite, fewer
embryos (28%, 5/28) had observable effects on control
sides than when the beads were implanted in the lastformed somite (50%; Table 2). A similar effect was observed with beads implanted in embryos with seven
somite pairs (data not shown).
Effect of Ectopic VEGF on Sprouting From the
Dorsal Aortae
Finally, to study the role of VEGF on sprouting from a
lumenized dorsal aorta, beads were implanted into the
first-formed somite (Fig. 4A). At this anatomical level, the
dorsal aorta first becomes an endothelial cell tube. Intersomitic arteries begin to branch from this tube at the 8S
stage. As with other anatomic locations, there were no
observable effects from control beads in the first-formed
somite (Table 1; Fig. 4B). Normally avascular areas were
traversed by only small-caliber blood vessels on the experimental and control sides of the embryos. Unlike other
410
FINKELSTEIN AND POOLE
many embryos, the size of the dorsal aorta also increased
(compare the embryo in Fig. 4C to the embryo in Fig. 4B).
Unlike beads implanted at other locations, there was a
significant difference between the effect of VEGF on the
left and right sides of the embryo (Table 2). Therefore,
subsequent analyses were performed separately for embryos with beads implanted on the left or right. With
VEGF beads on the left, the mean area fraction stained
with QH-1 was 81.7% on the experimental side and 57.9%
on the control side (P ⬍ 0.01; Table 2). With VEGF beads
on the right, the mean area fraction stained with QH-1
was 63.6% on experimental sides and 62.5% on control
sides (NS; Table 1). VEGF beads on the left resulted in
significant changes in QH-1-staining, while beads on the
right did not (Table 2). Similar effects were observed for
7S embryos with beads implanted at the level of a rostral
somite (somite VI/7 or VII/7; data not shown).
Ectopic VEGF Delivery Results in an Enlarged,
Patent Dorsal Aorta
Fig. 4. Ectopic VEGF delivery at the level of the first-formed somite.
(B) Control or (C and D) VEGF beads were implanted in the first-formed
somite of 6S embryos, followed by a 7-hr incubation. All embryos were
stained as whole mounts with the QH-1 monoclonal antibody, followed
by confocal microscopy. A: Bead implantation in the first-formed somite
(dark somite) is shown schematically. B: An embryo with a control bead,
fixed at the 11S stage. There is a normal vascular pattern on both the
bead-implanted and contralateral sides of this embryo. C: An embryo
with a VEGF bead, fixed at the 10S stage. Note the increased vascularization only on the bead-implanted side. D: An embryo with a VEGF
bead, fixed at the 9S stage. There is an increased vascularization only on
the bead-implanted side, with numerous blood vessel branches between the dorsal aorta and capillary plexus. These are ventral views, with
the rostral end up. Asterisks indicate bead locations. AVA, avascular
area; DA, dorsal aortae; NT, neural tube; CP, capillary plexus; SOM,
somites. The scale bar in D is representative of 100 ␮m and applies to
B–D.
To further analyze the effect of VEGF on the developing
vasculature, transverse sections of embryos with VEGF
beads were stained with QH-1. On the experimental sides,
the dorsal aorta was enlarged compared to the control
sides (Fig. 5A and B). The dorsal aorta increased in size
two- to threefold when VEGF beads were implanted in the
middle somite (compare the left and right sides of Fig. 5A
and B). The increase in dorsal aorta girth corresponded to
the loss of normal avascular areas observed in whole
mounts. The effect was observed for several sections from
the bead in a rostral-caudal direction indicating that
VEGF may diffuse several cell diameters. Fusion between
the normal dorsal aortae and the capillary plexus may
result in the observed dorsal aorta enlargement. In embryos with VEGF beads (Fig. 5A–C), the small vessels of
the capillary plexus are absent, compared to normal embryos (Fig. 5D). From transverse sections, it can be concluded that delivery of ectopic VEGF does not produce a
solid mass of QH-1-stained cells, but rather a large blood
vessel with lumen.
VEGF Induces Changes in the Vasculature
After Short Incubation Times
Experiments were carried out to determine the time
required for VEGF to affect the vasculature. Four-hour
incubations with VEGF resulted in an effect similar to
that observed after 7-hr incubations. Ectopic VEGF resulted in increased density of blood vessel branches or
vascularization of normally avascular areas in approximately 40% of the samples (Fig. 6A and B). For other
samples (Fig. 6C), no effect was observed after 4 hr.
DISCUSSION
delivery locations, there was no significant difference between the control sides of the embryos with VEGF or
control beads implanted in the first somite.
As with the VEGF beads at other locations, beads implanted in the first-formed somite of embryos with six
somite pairs resulted in morphological changes (Fig. 4B
and D). The most dramatic effect (45%) was complete
vascularization lateral to the dorsal aorta in areas normally devoid of large vessels (note the fusion of DA and CP
in Fig. 5C). In other examples (55%), the density of blood
vessel branches increased near bead locations (Fig. 4D). In
Multiple stages of vascular development occur simultaneously along the rostral-caudal axis of an embryo (Coffin
and Poole, 1988; Poole and Coffin, 1989, 1991; Poole et al.,
2001). To determine the effect of ectopic VEGF on these
stages, we implanted heparin chromatography beads
soaked in recombinant human VEGF165 (VEGF beads) in
three rostral-caudal locations of quail embryos with six
somite pairs. To examine the effect of ectopic VEGF on
initial angioblast cohesion, we implanted VEGF beads in
the last-formed somite. Ectopic VEGF resulted in increased vascularization (apparently both vasculogenesis
VEGF AND VASCULAR PATTERN FORMATION
411
Fig. 5. Ectopic VEGF results in an enlarged lumenized dorsal aorta.
VEGF beads were implanted into quail embryos with six or seven somite
pairs, followed by 7-hr incubations. Representative 10-␮m paraffin sections from embryos with VEGF beads stained with QH-1 are shown. A:
A bead was implanted in somite 3 at the 6S stage and fixed at the 12S
stage. This section is 20 ␮m caudal to the last with a visible bead. The
area occupied by the bead is marked by a large arrow. B: A second
embryo with a VEGF bead in somite 3 at the 6S stage, fixed at the 11S
stage. C: A VEGF bead was implanted in somite 2 at the 7S stage, and
the embryo was fixed at the 13S stage. D: A normal 12S stage embryo
without a bead. Note the lateral blood vessels of the capillary plexus. In
all sections, note the larger dorsal aorta on the bead-implanted side
compared to the contralateral side. The paired dorsal aortae in panel D
(normal embryo) are of uniform size. Asterisks indicate bead locations. S,
somite; NT, neural tube; DA, dorsal aorta; CP, capillary plexus. The scale
bars in C and D represent 100 ␮m.
Fig. 6. Four-hour incubations with ectopic VEGF affected the vascular pattern. Embryos in panels A–C were incubated for 4 hr following
VEGF bead implantation. All embryos were stained as whole mounts
with the QH-1 monoclonal antibody, followed by confocal microscopy.
A: A VEGF bead was implanted in somite 4 at the 7S stage, and the
embryo was fixed with nine somites. There was an increased vascularization on the bead-implanted side compared with the contralateral side.
B: A VEGF bead was implanted in somite 2 at the 7S stage, and the
embryo was fixed with nine somites. This bead resulted in blood vessel
branches developing between the dorsal aorta and capillary plexus,
which did not occur on the contralateral side. C: A VEGF bead was
implanted in somite 4 at the 7S stage, and the embryo was fixed with 10
somites. In this example, there was little effect from VEGF after 4 hr.
These are ventral views, with the rostral end up. Asterisks indicate bead
locations. AVA, avascular area; DA, dorsal aortae; NT, neural tube; CP,
capillary plexus; SOM, somites. The scale bar in C is representative of
100 ␮m and applies to A–C.
and angiogenesis) lateral to the dorsal aortae. Changes in
the vascular pattern were significant on the bead-implanted and control sides of the embryos. The significant
change on the control sides suggests that VEGF from
beads diffuses across the embryonic midline.
VEGF beads were implanted in the middle somite so
that we could examine the effect of VEGF on formation of
a lumenized dorsal aorta from a solid endothelial cell cord.
In this case, there was a significant increase in vascular
density lateral to the dorsal aortae. In some samples,
areas normally devoid of vessels became filled with QH-1
stained tissue. In other samples, numerous blood vessel
sprouts crossed normally avascular areas. Finally, we implanted beads in the first-formed somite to examine the
412
FINKELSTEIN AND POOLE
effect of VEGF on sprouting from the patent dorsal aorta.
At this level, the major effect was an increased number of
vessel sprouts between the capillary plexus and dorsal
aorta. There were significant effects of VEGF on control
sides of embryos with VEGF beads in the middle or lastformed somites. This suggests that VEGF diffuses between bead-implanted and control sides of the embryo. A
significant effect on the vascular pattern was observed on
only the left side of embryos with VEGF beads in the
first-formed somite. At all three anatomical levels, ectopic
VEGF resulted in hypervascularization to different degrees, especially between the dorsal aorta and capillary
plexus. The degree of VEGF effects along the rostralcaudal axis may be explained by the differentiation state
of the somites in which the beads were implanted, or by
the response of mesodermal cells to ectopic VEGF.
Somites were used for bead-implant sites because they
give rise to a relatively low number of angioblasts (Wilting
et al., 1995) and closely appose the developing dorsal
aortae. The developmental stage of the somites could affect the diffusion of VEGF in all directions from implanted
beads. The last-formed somite pair has not yet developed
an epithelial layer surrounding the somitocoel (Hirsinger,
et al., 2000). Without this epithelial layer, VEGF could
diffuse from implanted beads more readily than at other
rostral-caudal levels. More mature somites become surrounded by a layer of extracellular matrix (ECM) (Hirsinger et al., 2000), to further reduce diffusion from beads.
At the level of the first-formed somite, further differentiation takes place (Hirsinger et al., 2000) and there are
tighter cellular connections and additional matrix. This
additional matrix may minimize diffusion of VEGF from
the beads, and explain the reduced diffusion of VEGF at
this axial level.
The different effects of VEGF at three axial locations
could also be explained by the response of angioblasts to
VEGF. VEGF beads at all three axial locations studied
resulted in significant increases in QH-1 labeling when
bead-implanted sides of embryos with VEGF or control
beads were compared. At the level of the first-formed
somite, angioblasts on the right side of the embryo elicited
no observable effect in response to ectopic VEGF. With
beads implanted in the middle or last-formed somites,
there was no difference in the effect of VEGF beads implanted on either the left or right side. At the level of the
first-formed somite, significant effects of ectopic VEGF
only on the left side may be explained by asymmetrical
development of the heart just rostral to this somite (Sugi
and Markwald, 1996). It was previously demonstrated
that a higher level of QH-1 on the right side corresponds to
more endocardial precursor cells on that side (Coffin and
Poole, 1991; Sugi and Markwald, 1996). As such, angioblasts at this level may be precommitted to the endocardial lineage and may not be capable of being incorporated
in the dorsal aorta in response to ectopic VEGF.
The three stages of dorsal aorta development involve
distinct cellular behaviors of angioblasts along the rostralcaudal axis, including angioblast cohesion and migration,
cellular changes of lumen formation, and sprouting angiogenesis. Ectopic VEGF has effects at all three rostralcaudal levels, which suggests that it is active in three
distinct cellular behaviors during vascular development.
Activity in early embryos contrasts with that in older
embryos, in which VEGF induces edema (Flamme et al.,
1995b; Schmidt and Flamme, 1998). Thus, VEGF effects
may be reflective of the developmental stage of the vasculature, with edema occurring predominantly in more mature blood vessels.
Morphologically, the number of QH-1 positive cells increased near areas of ectopic VEGF delivery. The area
lateral to the dorsal aorta, which normally has a lower
level of QH-1 staining compared to the remainder of the
embryo, became highly vascularized, and the number and
size of blood vessel branches increased. In cross-section,
an enlarged dorsal aorta with a lumen was observed,
which is suggestive of an expanded blood vessel rather
than a mass of QH-1-positive cells formed by vasculogenesis. This may have resulted from an increase in angioblast numbers through induction, proliferation, or chemotaxis from the lateral plate mesoderm, an area rich in
angioblasts (Ferrara, 1999; Zachary and Gliki, 2001). The
observed effects are probably not a result of angioblast
induction, since it has been demonstrated that VEGF has
a different effect on the vasculature compared to fibroblast
growth factor-2 (FGF2), which can induce an angioblast
phenotype (Cox and Poole, 2000; Poole et al., 2001). FGF-2
induces an angioblast phenotype from mesodermal tissues
that normally do not have large numbers of angioblasts
(Cox and Poole, 2000). Following induction, angioblasts
are characterized by their expression of VEGF receptors,
which must be present for VEGF activity (Eichmann et
al., 1993; Yamaguchi et al., 1993). Therefore, angioblast
induction occurs at an earlier developmental stage than
does VEGF activity. Finally, ectopic VEGF resulted in
changes in the vascular pattern after very short time
periods, with similar results observed following 4- or 7-hr
incubations. This suggests that VEGF does not upregulate
angioblast and endothelial cell proliferation, since the cell
cycle time in embryos with six or seven somite pairs is
approximately 10 hr (Sanders et al., 1993). More likely,
VEGF acts by mediating chemotaxis of angioblasts and
endothelial cells during embryonic vascular development,
and perturbing the balance between cell-cell cohesion and
cell-substrate adhesion.
Data (Table 3) indicate that ectopic VEGF can diffuse
across the embryonic midline to affect the contralateral
side. There is evidence of growth factor diffusion from
beads implanted in embryos (Storey et al., 1998). Increasing the growth factor concentrations used to soak beads
led to diffusion distances of 3– 4 cell diameters with 50
␮g/ml growth factor (Storey et al., 1998). The same is
expected to hold true for VEGF. By image analysis, it was
determined that observed effects were not due to embryo
manipulations or the beads themselves. There were no
observable effects on the vasculature resulting from control beads, indicating that heparin on beads does not remove endogenous VEGF from embryonic tissues. To investigate the mechanisms by which VEGF can affect the
developing vasculature, focal delivery of VEGF on chromatography beads is superior to previous methods used in
microinjection studies (Drake and Little, 1995). Combined
with focal delivery of VEGF inhibitors, such as truncated
forms of VEGF receptors (Drake et al., 2000), bead implantation will be useful for studying the mechanism of
VEGF action in distinct cellular processes during vascular
development.
The embryos studied differed by a small developmental
window, but VEGF delivery at different anatomical levels
tested a broad window of activity in vascular development.
The effect of ectopic VEGF was rapid (observed in as little
VEGF AND VASCULAR PATTERN FORMATION
as 4 hr), which suggests that a mechanism other than
increased proliferation is involved. Ectopic VEGF can disrupt the normal vascular pattern, indicating that a critical
level of VEGF is required for normal vascular development. This correlates with previous studies in mouse embryos, in which decreased (Carmeliet et al., 1996; Ferrara
et al., 1996) or increased (Miquerol et al., 2000) VEGF
levels led to abnormal development. The present data
indicate that the role of precisely controlled VEGF levels
within the embryo may be to determine which areas become vascularized and which remain avascular, by influencing angioblast migration and adhesion.
ACKNOWLEDGMENTS
We thank Dr. Douglas Robertson (SUNY Upstate Medical University, Syracuse, NY) for assistance with statistical analysis, and Dr. Christopher Cox (University of
Arizona) for helpful discussion. The QH-1 monoclonal antibody developed by Pardanaud et al. (1987) was obtained
from the Developmental Hybridoma Studies Bank under
the auspices of the NICHD, and maintained by the Department of Biological Sciences, University of Iowa, Iowa
City, Iowa.
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