Download Embryonic vascular development: immunohistochemical

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Development 102, 735-748 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Embryonic vascular development: immunohistochemical identification of
the origin and subsequent morphogenesis of the major vessel primordia
in quail embryos
J. DOUGLAS COFFIN and THOMAS J. POOLE
Department of Anatomy and Cell Biology, SUNY Health Science Center at Syracuse, 766 Irving Avenue, Syracuse, NY 13210, USA
Summary
The development of the embryonic vasculature is
examined here using a monoclonal antibody, QH-1,
capable of labelling the presumptive endothelial cells
of Japanese quail embryos. Antibody labelling is first
seen within the embryo proper at the 1-somite stage.
Scattered labelling of single cells appears ventral to the
somites and at the lateral edges of the anterior
intestinal portal. The dorsal aorta soon forms a
continuous cord at the ventrolateral edge of the
somites and continues into the head to fuse with the
ventral aorta forming the first aortic arch by the 6somite stage. The rudiments of the endocardium fuse
at the midline above the anterior intestinal portal by
the 3-somite stage and the ventral aorta extends
craniad. Intersomitic arteries begin to sprout off of the
dorsal aorta at the 7-somite stage. The posterior
cardinal vein forms from single cells which segregate
from somatic mesoderm at the 7-somite stage to form a
loose plexus which moves niediad and wraps around
the developing Wolffian duct in later stages. These
studies suggest two modes of origin of embryonic
blood vessels. The dorsal aortae and cardinal veins
apparently arise in situ by the local segregation of
presumptive endothelial cells from the mesoderm. The
intersomitic arteries, vertebral arteries and cephalic
vasculature arise by sprouts from these early vessel
rudiments. There also seems to be some cell migration
in the morphogenesis of endocardium, ventral aorta
and aortic arches. The extent of presumptive endothelial migration in these cases, however, needs to be
clarified by microsurgical intervention.
Introduction
1912). Subsequent minor vessels are formed by
sprouting from preexisting vessels. The disadvantage
of these classic studies was that only patent vessels
could be visualized. Others have expressed the need
for an endothelial cell marker which could identify
presumptive endothelial cells just as they segregate
from the mesoderm and begin organizing into cords
(e.g. Hirakow & Hiruma, 1981). The MB-1 (Peault et
al. 1983; Labastie etal. 1986) and QH-1 (Pardanaud et
al. 1987) monoclonal antibodies fill this need as they
label vascular endothelial cells and cells of the haematopoietic lineage in embryos of the Japanese quail.
The sprouting form of angiogenesis has been much
more extensively studied recently as it is the mechanism by which tumours recruit a new vascular supply
(Folkman, 1985). Angiogenic factors have also been
identified in developing systems. Kidney (Risau &
The morphogenesis of the embryonic vasculature
commences with the accumulation of presumptive
endothelial cells (PECs) into loosely associated cords
following their segregation from the mesoderm (review, Wagner, 1980). Reagen (1915) showed that
blood vessels of the embryo originate within the body
proper, not by invasion from the highly vascular
extraembryonic yolk sac. The first major blood vessels, the dorsal aortae and posterior cardinal veins,
form in situ by the segregation of mesenchymal cells
from the mesoderm. This has been visualized by
scanning electron microscopy (Hirakow & Hiruma,
1981; Meier, 1980). Many other vessels form by the
modification of extensive capillary plexuses as shown
by the many ink injection studies of Evans (1909,
Key words: endothelium, monoclonal antibody,
vasculogenesis, QH-1, quail embryo.
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/. D. Coffin and T. J. Poole
Ekblom, 1986) and brain (Risau, 1986) produce
angiogenic factors which resemble tumour angiogenic
factors (Shing et al. 1984; Klagsbrun & Shing, 1985).
There is some controversy surrounding the origin of
embryonic endothelium. For example, Auerbach has
proposed that the specialized endothelium of the
brain differentiates in situ from mesenchymal stem
cells (Auerbach & Joseph, 1984); whereas, studies
with marked cells indicate the brain endothelium
derives from the invasion of proliferating capillary
sprouts (Stewart & Wiley, 1981). Transplantations
utilizing the nuclear differences between quail and
chick or mouse embryonic cells have revealed that the
endothelium of limb buds (Jotereau & LeDouarin,
1978; Wilson, 1983) and kidney (Ekblom et al. 1982)
clearly arises by invasive sprout penetration. The
segregation and directed migration of presumptive
endothelial cells and the importance of other largescale embryonic foldings, such as the anterior intestinal portal and lateral body folds, are here examined
using the monoclonal antibody QH-1 to stain quail
embryos of 0-22 somites. This descriptive analysis is
expanded in similar recent work (Pardanaud et al.
1987) and is a necessary prelude to an experimental
analysis using microsurgery of the relative contributions of cell migration and in situ differentiation to
the observed patterns of vascular morphogenesis.
Some of this work has been previously presented in
abstract form (Coffin & Poole, 1986).
Materials and methods
Immunocytochemistry
Whole mounts and sections of Japanese quail (Coturnix
coturnix japanica) were examined by using indirect immunofluorescence to label endothelial cells. A QH-1 monoclonal antibody (Ab) was used first at 1:400 for whole
mounts and 1:1000 for sections. This was followed by a goat
anti-mouse FITC-conjugated IgG secondary Ab (Accurate
Biochemical, Westbury, NY) at the same concentrations as
the primary. Both Ab were diluted in 3 % bovine serum
albumin (BSA) in phosphate-buffered saline (PBS).
Whole mounts
Whole mounts were prepared using techniques similar to
those reported by Pardanaud et al. (1987). Embryos were
removed from the yolk sac, rinsed in PBS and fixed in 4 %
formalin/PBS overnight at 4°C. The fixed embryos were
rinsed in PBS then permeabilized with successive changes
of (i) 30min - absolute methanol (aMeOH), (ii) 60min aMeOH and (iii) 30min- aMeOH; all at 4 °C with constant
agitation. After permeabilization, the embryos were rehydrated in an ethanol (EtOH) series (100%, 90%, 70%,
50%, 30%, 3min each) then rinsed in PBS. Nonspecificbinding sites were occupied by incubation in 3 % BSA at
4°C for 6-12h, followed by labelling of specific sites on
endothelial cells with the primary Ab at 4°C, 6-12 h.
Residual unbound primary Ab was washed away with three
PBS changes then the secondary Ab was applied for 6-12 h
at 4°C. Any unbound secondary Ab was removed with PBS
washes. Finally, the embryos were dehydrated in an EtOH
series, changed to toluene and mounted on slides in
Entellan® (VWR).
Sections
Sections were made by dissecting the embryos from the
yolk sac, rinsing them in PBS, then fixing them for 2h
minimum in Bouin's solution. After fixation, the embryos
were sequentially rinsed in PBS, dehydrated in an EtOH
series, changed to toluene and embedded in paraffin.
Sections were cut on a rotary microtome at 7/im and
mounted on albumin-coated slides. Paraffin was washed
from the slides with toluene, then the sections rehydrated in
an EtOH series, followed by.two changes in PBS and one in
3 % BSA for 30min. Next, the sections were stained for 2h
in primary Ab and washed for 30min in PBS, then stained
for 2h in secondary Ab and again washed in PBS. After
soaking overnight at 4°C in PBS, the slides were coverslipped with a 2 % A'-propyl gallate/80 % glycerol mixture
(pH8-5) and sealed for microscopy and photography.
Results
Whole mounts and sectioned embryos were examined for immunofluorescent labelling of quail endothelium that highlighted the developing vasculature.
Specifically, we studied development of the endocardium, the dorsal and ventral aortae, the first aortic
arch, the intersomitic arteries and the cardinal veins.
Results are summarized in Table 1.
(A) Endocardium
Construction of the endocardium from individual
endothelial cells is the first step in heart development.
Immunofluorescent labelling is first evident with the
appearance of PECs at the periphery of the embryo.
These cells are concentrated in angiogenic sites near
the headfold on each side of a Zacchei stage-4
embryo (Fig. 1). At the 1-somite (IS) stage, the PECs
begin to aggregate into capillary plexuses at the
bilateral angiogenic sites and migrate mediad. Thus
by 2S (Fig. 2), the enlarging plexuses are connected
to the extraembryonic circulation laterad, while
mediad they grow into the pericardial coelom above
the anterior intestinal portal (AIP). At 2S these
plexuses are considered embryonic heart primordia
because their location on either side of the AIP is the
same as the future vitelline veins and sinus venosus.
The investing intraembryonic heart primordia fuse
at the midline of a 3S embryo. From this point of
fusion, directly above the AIP, the ventral aorta
elongates toward the head as in a 4S embryo (Fig. 3).
At 6S the ventral aorta splits, and each of the two
branches fuse with the dorsal aortae bilaterally to
Quail embryonic vascular development
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Fig. 1. A Zacchei stage-4 embryo whole mount. A
cluster of PECs and individual cells are seen around the
periphery of the head fold (HF) in this ventral view of
the right side. Bar, 100^m.
form the first aortic arches (Fig. 4). Thus by 7S, the
embryonic heart lies caudal to the paired ventral
aortae as the straight descending portion of a 'Y'
connected at its caudal extent to the omphalomesentric or vitelline veins (Fig. 5).
From 7S to 20S, our results agree with previous
reports (Evans, 1912). In the area surrounding the
pericardial coelom, large capillary strands are seen
extending from the sinus venosus to the extraembryonic circulation (Fig. 6). As the head and heart move
farther apart the strands appear to break away and
degenerate,
(B) Dorsal aortae
Dorsal aorta development begins concomitant with
heart formation. Just below the bilateral primitive
angiogenic sites, PECs migrate mediad independently of the forming heart. These cells are destined
to form the paired dorsal aortae. Therefore, by the IS
stage the PECs at each lateral angiogenic site have
already been segregated for two different fates, one
population directed toward heart development and
another toward the dorsal aortae. The latter aggregate into angiogenic islets that appear as isolated
capillary strands. At IS these vessels consist of a few
Fig. 2. Definitive heart primordia (HP) extend medially
through the pericardial coelom (PC) of a 2S embryo.
Notice the large PECs medially and how the HP form a
diffuse capillary plexus (CP) laterally. The neural tube
(NT) and notochord (NC) appear as grey unlabelled
background. Bar, 150/im.
PECs and islets lying over the segmental plate on
each side of the notochord (Fig. 7).
As development proceeds, more PECs are seen
migrating mediad and the dorsal aortae become
bilateral longitudinal lines of PECs and islets. At 4S
the aortae appear as vascular cords extending from
the head to just beyond the last pair of somites
(Fig. 3). This in situ coalescence of PECs continues as
the aortae form their lumens and extend further into
the head, eventually joining the first arch (Fig. 4).
Caudally, the dorsal aortae elongate with the body.
From the 7S to 12S stages, the dorsal aortae appear as
two large vessels bilaterally (Figs 5,8). They become
attached to the extraembryonic circulation by a
capillary plexus at 12S, which becomes the vitelline
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J. D. Coffin and T. J. Poole
artery at later stages (Figs 8, 9A). At 15S the dorsal
aortae move closer together as the lateral body folds
adhere to close the AIP (Fig. 9B). Later, a fusion
occurs between the two vessels just caudal to the
heart, forming the descending aorta.
(C) Aortic arches
First arch development occurs at 4S-6S by fusion of
the ventral and dorsal aortae in the head. As mentioned above, the ventral aorta bifurcates at 6S and
the paired dorsal aortae extend well into the head of
this stage embryo (Fig. 3). These two events lead to
the fusion of the two ventral aortic branches with the
paired dorsal aortae to form the first aortic arch. It is
poorly defined at 6S (Fig. 4), but quite obvious at 7S
(Fig. 5). Other nonspecific capillary strands can be
seen between the ventral and dorsal aortae at 12S
(Fig. 8) that might be primordia of any of the other
five aortic arches, but this could not be confirmed.
The internal carotid arteries are seen at 10S as
sprouts from the cranial portion of the first arch
(Fig. 6). These sprouts later extend rostrally into the
head to become the internal carotid arteries proper
(Fig. 10). Thus, the first arch is attached by the
internal carotid artery to a large capillary plexus that
forms over the developing brain. The anterior cardinal vein and the vertebral arteries also join this plexus
(Fig. 9C, D). Scattered PECs are seen over the
neural tube at 15S (Fig. 11), when the cephalic plexus
isfirstobvious (Fig. 12). By 19S the cephalic plexus is
well developed, extending over the dorsal surface of
Fig. 4. First arch formation at 6S. The ventral aorta
(VA) bifurcates and the branches fuse with the dorsal
aortae (DA) to form the first aortic arch (AA) on each
side of the neural tube (NT) in the embryonic head. Bar,
50/mi.
Fig. 3. A composite of a 4S embryo. The heart primordia
(HP) have fused at the midline above the AIP and are
joined laterally to the extraembryonic circulation. The
ventral aorta (VA) has grown cranially from the HP
fusion point. The dorsal aortae (DA) appear as paired
longitudinal broken lines of islets and PECs. The DA
extend through the AIP well into the head where they
are slightly out of focus in this photo. Caudally the
segmental plate (SP) shows some labelling. The neural
tube (NT), notochord (NC), and somites (S) are in the
background. Bar, 150ftm.
Quail embryonic vascular development
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Fig. 6. The cranial part of a 10S embryo. Broken
vascular strands (VS) that extended from the vitelline
veins (VV) and sinus venosus (SV) to the extraembryonic
circulation (EC) are seen. The well-developed first aortic
arches (AA) are obvious as are the embryonic heart
(EH), the ventral aortae (VA), and the dorsal aortae
(DA). The EH has begun to bend slightly to the right
forming the bulbus chordus (BC). Note the first small
sprouts of the internal carotid arteries (1CA) from the
apices of the aortic arches (AA). Bar, 75j<m.
the brain and forming a vascular ring around the
avascular optic cup (Fig. 13).
Fig. 5. A composite of a 7S embryo. The embryonic
heart (EH) is now developing between the ventral aortae
(VA) and the vitelline veins (VV, formerly the HP). The
dorsal aortae (DA) are large longitudinal vessels growing
caudally while maintaining a diffuse connection with the
extraembryonic circulation. The aortic arches (AA) are
obvious in the head but the cranial portions of the DA
are out of the focal plane. Bar, 200 jim.
(D) lntersomitic and vertebral arteries
lntersomitic arteries begin to sprout from the dorsal
aortae at about 7S. Once the intersomitic arteries
have reached the medial surface of the somite they
turn right angles to grow over that surface, fuse with
other sprouts and form the vertebral arteries adjacent
to the neural tube. Thus the vertebral arteries grow
caudad as longitudinal links between the intersomitic
arteries at their most medial extent. There were some
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J. D. Coffin and T. J. Poole
ICA
Fig. 7. A IS embryo showing PECs that segregated from
the primitive angiogenic sites laterally to migrate
medially, form angiogenic islets (ISL), and develop into
the dorsal aortae. Bar, 75 fim.
indications that vertebral artery rudiments already
exist cranially before the intersomitic arteries start to
form, but this could not be confirmed.
The first few intersomitic and interlocking vertebral arteries form between the second and third
somites of an 8S embryo (Fig. 14). In the head, at 10S
the vertebral arteries extend farther rostrally than the
most cranial somite. Other presumptive intersomitic
arteries are seen as incomplete sprouts from the aorta
between more caudal somites (Fig. 15). As development continues to the 15S stage, vertebral arteries
grow rapidly past the more caudal somites as their
corresponding intersomitic arteries reach the medial
edge of the somites (Figs 9B, 16). Meanwhile, at their
cranial extent, the vertebral arteries grow well into
the head and join the plexus there (Fig. 11).
Fig. 8. This composite of a 12S embryo shows the large
dorsal aortae (DA) running from the aortic arches (AA)
cranially to a level caudal to the last somite where they
show a firm attachment to the extraembryonic circulation
by capillary plexuses (CP) that will become the vitelline
arteries. Note the avascular area lateral to each DA that
contained many diffuse capillaries earlier (Figs 3, 5). The
embryonic heart has a distinct bulbus chordus (BC).
Internal carotid artery (ICA) sprouts are seen off the tops
of the aortic arches (AA). Also note the intersomitic
arteries (ISA) running between the DA and the vertebral
arteries (VTA). Bar, 250^m.
Quail embryonic vascular development
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PCV
VVLA
Fig. 9. Transverse sections of a 15S embryo. Bars, 150^m. A is from a caudal area through the vitelline arteries (VLA)
in the splanchnopleure. In the somatopleure, labelled capillary strands (CS) and plexuses are seen that will contribute to
the posterior cardinal vein. B is a plane just caudal to the heart. The large vitelline veins ( W ) are shown as are the
AIP, the posterior cardinal veins (PCV), the vertebral arteries (VTA), and the dorsal aortae (DA). In 9C, the section is
through the truncus arteriosus (TA) of the heart. The dorsal aortae (DA) and cephalic plexuses (CeP) are also shown.
Further cranially, D shows the ventral aortae (VA) after they exit from the heart. Also labelled are the DA that will
join the VA by the first aortic arch; and the cephalic plexus (CeP) into which the anterior cardinal vein, vertebral artery
and the internal carotid artery flow.
(E) Cardinal veins
Cardinal vein PECs can be seen early, at 5S in the
somatopleure, between the mesoderm and the ectoderm (Fig. 17). These cells slowly migrate mediad
from each side of the embryo toward the sites of the
future common cardinal vein or duct of Cuvier. Soon
after the first segment of the vertebral artery forms at
8S, processes are seen running from these PECs
between the somites (Fig. 14). These processes join
the vertebral artery mediad, presumably to become
the intersomitic veins dorsal to the intersomitic arteries (Fig. 18). Laterally, PECs continue to aggregate
as the common cardinal vein that forms in situ
(Fig. 16). By 20S the common cardinal vein is well
developed, as are the intersomitic veins and arteries
that connect the aorta, the vertebral artery and the
cardinal veins (Fig. 18).
Farther caudad at the level of the segmental plate,
nonspecific angiogenic islets form capillary strands on
the lateral plate beneath the ectoderm (Fig. 19A). As
the posterior cardinal vein grows caudad from the
common cardinal vein, these islets contribute to the
extension of it. At 20S the posterior vein is a fine
elongate capillary plexus closely associated with the
Wolffian duct (Fig. 19B).
Discussion
Use of the monoclonal antibody QH-1 has begun
reconsideration of classical models of embryonic
vascular development. Pardanaud et al. (1987) and
Poole & Coffin (1988) have recently reported on the
early formation of the heart, dorsal aorta and posterior cardinal veins. We have carried these studies
considerably farther to describe the origins of the
vitelline arteries and veins, ventral aorta, first aortic
arch, internal carotid arteries, cephalic plexus, vertebral arteries, intersomitic arteries and veins, and
the cardinal veins.
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/. D. Coffin and T. J. Poole
Fig. 10. Internal carotid artery (ICA) sprouts from the
first aortic arches (AA) that have begun to stretch into
the head of a 15S embryo. Notice the conspicuous ventral
aortae (VA). Bars, 50/im.
However, of equal import to this new descriptive
anatomy of the embryonic vasculature are the questions that arise about its morphogenesis. We envision
embryonic vascularization as a programmed process
in development where the principal blood vessels are
formed by differentiated PECs that undergo directed
cell migration to form definitive structures in situ.
Thus it is essential to understand how the PECs
undergo cell-cell recognition and adhesion, use cellmatrix interactions and differentially express certain
genes during angiogenesis to form vascular patterns.
We must, therefore, use modern techniqes to reexamine the origin and behaviour of PECs and the
interaction between other developmental processes
and neovascularization.
The definitive embryonic heart consists of three
layers: an inner endocardium made of endothelial
cells, a muscular myocardium and an outer epicardium. In the chick, the endocardium has been shown
to develop first, followed by the myocardium and
Fig. 11. Some individual angiogenic islets (ISL) are
labelled sitting on the dorsal surface of the embryonic
brain (EB) at 15S. These may contribute to a developing
cephalic plexus that forms in the head. Notice the
vertebral arteries (VTA) adjacent to the neural tube
(NT), the anterior cardinal vein (ACV) more laterally
and the optic cups (OC) destined to form the eyes. Bar,
75 /zm.
then the epicardium (Manasek, 1968). It appears that
the endothelium establishes the pattern for the developing heart and blood vessels. The QH-1 monoclonal antibody proved quite useful as an endothelial
marker. The QH-1 epitope is expressed soon after the
PECs segregate from the lateral plate. Our results
show that blood vessels, individual cells and angiogenic islets, are conspicuously labelled. The definitive
vasculature then results from the growth and modification (i.e. regression, selective cell death, etc.) of
the early vessels described here. Hence the basic
morphological data, illustrated in Fig. 20, that are
necessary for further studies on embryonic vascular
development have been established.
Ink injection studies had shown the embryonic
heart forming at the 6S-8S stages by the fusion of
Quail embryonic vascular development
743
large patent blood vessels at the midline (Evans,
1912). The dorsal and ventral aortae and other
embryonic blood vessels were thought to form shortly
thereafter, originating from these initial vessels or by
the modification of existing capillary plexuses (Evans,
1909). The data presented here and by Pardanaud et
al. (1987) and by Poole & Coffin (1988) indicate that
the heart and dorsal aorta form early. Heart primordia are undergoing directed growth toward the midline of the pericardial coelom at IS. Other PECs
destined for the dorsal aorta segregated from the
initial cluster of individual PECs as early as Zacchei
stage 4.
We are not certain, however, as to what caused the
separation of PECs for the early heart and for the
Fig. 12. In this 15S whole mount, the anterior cardinal
veins (ACV) are seen growing up over the dorsal surface
of the brain to contribute to the cephalic plexus that will
cover the entire surface at later stages. At the bottom of
the photo, a flexure (F) between the metencephalon and
the rhombencephalon is noticeable. Bar, 15 (im.
Fig. 14. A high magnification dorsal view of an 8S whole
mount near the site of the forming common cardinal vein.
PECs migrate in the somatopleure toward this focal point
and send processes between the somites that join the
vertebral artery (VA). We presume that these processes
form the intersomitic veins dorsal to the intersomitic
arteries. Bar,
Fig. 13. This lateral view of the head of a 19S embryo
shows the well-developed cephalic plexus (CeP) that
surrounds the optic cup (OC) and extends up over the
dorsal surface of the brain. Bar, 50^m.
dorsal aorta from each other. It could be that the two
populations differentially express an epitope that
leads to altered behaviour between them. Alternatively, differences in the surrounding extracellular
matrix may direct the PECs onto separate migratory
pathways. The extent of migration of PECs is unclear
and we are currently examining this question by using
blockages and transplants. The physical force of the
forming body folds could also present barriers that
favour migration in certain directions. The mechanism for the directed migration of PECs in embryonic
vasculogenesis is unknown, but it probably involves a
combination of some or all or the forementioned
events.
Another area where populations of PECs separate
is in the development of the dorsal aortae and
posterior cardinal veins. The cells of the dorsal aortae
744
/. D. Coffin and T. J. Poole
Fig. 15. A ventral view of a 10S embryo. Lateral PECs
are migrating toward an area where the common cardinal
veins are forming. Intersomitic arteries (ISA) are slightly
out of focus as are some forming intersomitic veins (ISV).
Notice the vertebral arteries (VTA) medially and the ISA
sprouts out of the focal plane caudally between the
somites (S). Bar, 100nm.
migrate between the endoderm and mesoderm in the
splanchnopleure, while cardinal vein PECs migrate
between the ectoderm and mesoderm in the somatopleure. On a whole mount of 4S-10S embryos, the
two populations are seen in the same field, but in
different focal planes. The question remains as to
when these populations separate. Before the formation of the first somite, the lateral plate mesoderm is
divided by the intraembryonic coelom, creating a
space between the two layers with medial and lateral
points of communication. We do not know whether
PECs are trapped in their respective layers when the
mesoderm splits, or if they differentiate from the
mesodermal layers after the coelom forms. The PECs
could also segregate at the periphery and differentially migrate into the splanchnopleure for the aorta,
and into the somatopleure for the cardinal vein. In
Fig. 16. Ventral view of a 15S embryo showing the
developing common cardinal veins (CCV), the vertebral
arteries (VTA), some intersomitic arteries (ISA) and ISA
sprouts. Bar, 60 ^m.
addition, we often noticed labelling in the segmental
plate, which could have some significance in aorta,
intersomitic artery or cardinal vein development.
Vascular morphogenesis in the embryo seems to
occur via two different mechanisms, by sprouting
from existing blood vessels or capillary plexuses, or
by the in situ aggregation of migrating PECs. These
two types of blood vessel formation are evident in
early stage embryos (1S-3S). The heart develops by
sprouting of the lateral heart primordia and the dorsal
aorta by the in situ aggregation of migrating PECs.
We report here that the patterns for the embryonic
heart, the ventral aortae, the first aortic arch and the
dorsal aortae are all established by the 6S stage of
development. Other embryonic vessels will subsequently develop from these vessels. Intersomitic
arteries sprout from the dorsal aortae, beginning at
the 7S stage. Internal carotid arteries sprout from the
apices of the first aortic arch at about 10S. However,
Quail embryonic vascular development
the cardinal veins may or may not depend on these
vessels for their development.
The above discussion alluded to the segregation of
cardinal vein and aortic PECs. But cardinal vein
formation proceeds much more slowly than the development of the dorsal aortae. The cardinal vein
pnmordia appear as large islets over the lateral plate
mesoderm until the 7S stage. They then form the
common cardinal vein near the AIP as described, but
Table 1. Sequence of embryonic vascular
development
Stage
(somites, S)
0
IS
2S
3S
4S
5S
6S
7S
8S
9S
Fig. 17. A dorsal view of a 5S embryo showing cardinal
vein PECs and angiogenic islets (ISL) in the
somatopleure. In the background are the developing
dorsal aortae and the extraembryonic circulation. Bar,
175 fim.
10S
12S
15S
20S
Fig. 18. Lateral view of a 20S embryo at the level of the
common cardinal vein (CCV) that is well developed at
this stage. The head would be to the right of this photo.
Attached to the CCV are some intersomitic veins (ISV)
that extend to the vertebral artery (VTA) above. Out of
the focal plane are some intersomitic arteries (ISA) in the
background. Bar, 50 ^m.
745
Description
PECs appear at bilateral angiogenic sites near
headfolds.
PECs aggregate into angiogenic islets then into
plexuses at each site as heart primordia; other
PECs from site migrate caudomedially for dorsal
aortae.
Heart primordia begin medial sprouting from each
side through pericardial coelom above AIP;
caudally migrating PECs begin to form islets that
align as bilateral craniocaudal dorsal aortae
primordia.
Heart primordia reach midline to fuse; dorsal
aortae as broken lines of islets extend into
headfold.
Ventral aorta sprouts from point of fusion of heart
primordia and grows craniad; dorsal aortae appear
as solid lines from head to segmental plate.
Ventral aorta has bifurcated at its cranial extent
and grows toward dorsal aortae; cardinal vein
PECs are apparent and begin to migrate medially.
Ventral and dorsal aortae fuse in head to form
first arches; cardinal vein PECs migrate to
primitive angiogenic sites.
Intersomitic arteries begin to sprout from dorsal
aorta between cranial somites; cardinal vein PECs
reach area dorsal to intersomitic artery sprouts.
First and second intersomitic arteries link medially
to vertebral arteries; cardinal vein PECs send
processes between somites to link with vertebral
artery segments.
Heart begins to bend to the right to form bulbus
chordus; more intersomitic arteries form and
vertebral artery lengthens; intersomitic veins begin
to form from cardinal vein PEC processes;
common cardinal vein plexuses form.
Heart bends farther to right; dorsal aortae
lengthen; vitelline artery formation begins;
internal carotid arteries sprout from first arch.
Heart has complete right bend and begins left
bend more craniad; dorsal aorta attached to
extraembryonic circulation by vitelline artery
plexus; common cardinal veins are capillary
plexuses.
Heart is 'S' shaped with right and left bends;
dorsal aortae begin to fuse midline under AIP;
common cardinal veins as large plexuses that
anterior and posterior cardinal veins are extending
from; several nonspecific islets have moved
medially toward forming posterior cardinal veins;
vertebral arteries extend well into head to attach
to cephalic plexus.
Heart is convoluted and compact in pericardial
coelom; dorsal aortae are fused in abdomen; large
plexus over brain connected to internal carotid
arteries; posterior cardinal veins are thin plexuses
near pronephros that extend caudad from common
cardinal veins; caudal islets attaching to cardinal
veins.
746
J. D. Coffin and T. J. Poole
Fig. 19. Dorsal views of a 15S (A, bar, 100^m) and of a 20S (B, bar, 50/.im) embryo, at the level of the caudal extent
of the developing posterior cardinal vein, near the segmental plate (SP). As shown in A, the posterior cardinal vein
elongates caudally from the common cardinal vein and is joined by PECs and islets (ISL) that migrate medially from the
lateral plate in the somatopleure. A fine cardinal vein plexus (CVP) is thus formed, shown in B, that surrounds the
pronephros and sends strands between the somites to contribute to the vasculature there.
the islets remain unincorporated farther caudad.
However, when the pronephric duct begins to move
caudad, these islets form plexuses near the duct, and
then merge with the developing posterior cardinal
vein. There are probably morphogenetic interactions
between the cardinal vein and pronephros, but their
role in the formation of either structure is uncertain.
Studies using scanning electron microscopy in conjunction with immunofluorescence have yielded some
preliminary data on PEC behaviour (Poole & Coffin,
1988). It seems that the PECs migrate over the1
surface of the mesoderm until they recognize a
particular stimulus that causes them to become sedentary and contribute to a blood vessel. They may
migrate as individual cells, or as angiogenic islets.
These islets appear as small clusters of PECs that are
capable of migration as a group. It is unknown
whether the islet cells are clonal, the result of PEC
mitosis during migration or whether the migrating
PEC recruits other mesenchymal cells as it migrates.
But the PECs seem capable of forming islets either
before or after they reach their definitive position.
Whole mounts and sectioned tissues of early-stage
embryos stained with QH-1 proved useful for describing the early events of embryonic vascular development. This process involves two mechanisms for
neovascularization. One method is by in situ localization of migrating PECs to a vascular cord that then
enlarges and forms a lumen as a definitive blood
vessel, e.g. the dorsal aortae. The second method is
by sprouting of existing vessels, e.g. the intersomitic
arteries. These methods employ directed cell migration and selective cell-cell and cell-matrix
phenomena to guide the migration of PECs and
determine the location of the vessels. However, many
questions remain for future studies. Use of microsurgery is proving useful for understanding how the
Quail embryonic vascular development
141
B
Fig. 20. Diagrams summarizing the morphogenesis of the major vessel primordia: (A) Zacchei stage 4, (B) one pair of
somites, (C) two pairs of somites, (D) four pairs of somites, (E) six pairs of somites, (F) twelve pairs of somites.
patterns are formed and what types of interactions
are involved in embryonic angiogenesis.
Biology of Endothelial Cells (ed. E. A. Jaffe), pp.
393-400. Boston: Martinus Nijhoff.
COFFIN, J. D. & POOLE, T. J. (1986). Embryonic vascular
We thank Paul Kitos (Kansas University) and Clayton A.
Buck (Wistar Institute) for the QH-1 monoclonal antibody,
and Marisa Martini for technical assistance. This work was
supported in part by a Grant-in-Aid from the American
Heart Association, Upstate New York Chapter.
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