Neural Crest Cell Contribution to the
Developing Circulatory System
Implications for Vascular Morphology?
Maarten Bergwerff, Marlies E. Verberne, Marco C. DeRuiter,
Robert E. Poelmann, Adriana C. Gittenberger-de Groot
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Abstract—In this study, the distribution patterns of neural crest (NC) cells (NCCs) in the developing vascular system of the
chick were thoroughly studied and examined for a correlation with smooth muscle cell differentiation and vascular
morphogenesis. For this purpose, we performed long-term lineage tracing using quail-chick chimera techniques and
premigratory NCC infection with a replication-incompetent retrovirus containing the LacZ reporter gene in combination
with immunohistochemistry. Results indicate that NCC deposition around endothelial tubes is influenced by anteroposterior positional information from the pharyngeal arterial system. NCCs were shown to be among the first cells to
differentiate into primary smooth muscle cells of the arch arteries. At later stages, NCCs eventually differentiated into
adventitial fibroblasts and smooth muscle cells and nonmuscular cells of the media and intima. NCCs were distributed in
the aortic arch and pulmonary arch arteries and in the brachiocephalic and carotid arteries. The coronary and pulmonary
arteries and the descending aorta, however, remained devoid of NCCs. A new finding was that the media of part of the
anterior cardinal veins was also determined to be NC-derived. NC-derived elastic arteries differed from non-NC elastic
vessels in their cellular constitution and elastic fiber organization, and the NC appeared not to be involved in designating
a muscular or elastic artery. Boundaries between NC-infested areas and mesodermal vessel structures were mostly very
sharp and tended to coincide with marked changes in vascular morphology, with the exception of an intriguing area in
the aortic and pulmonary trunks. (Circ Res. 1998;82:221-231.)
Key Words: quail-chick chimera n pharyngeal arch artery n smooth muscle cell n great artery n neural crest migration
N
eural crest cells have been studied extensively for their
migratory behavior and potential to differentiate into a
wide variety of cell types. In cardiovascular research, the crest
region located between the midotic placode and somite 3, also
called the cardiac NC,1 has received much attention because of
its major role in cardiovascular development. Participation in
outflow tract septation,1,2 media formation of the pharyngeal
arch arteries,3–5 and contribution to parasympathetic cardiac
innervation3 have been documented using quail-chick chimeras. The importance of the extensive contribution of NCCs to
such a wide variety of cardiovascular structures is made clear by
the occurrence of congenital malformations involving the NC
and presenting more or less severe cardiovascular anomalies.
Syndromes associated with 22q11 deletions and vitamin A
deficiency are known NC-associated disorders, including cardiovascular malformations.6,7 Experimental removal of the
cardiac crest in chick embryos,8,9 administration of excess
retinoids,10 –12 and deletion of retinoic acid receptor genes13 also
result in severe malformations like ventricular septal defects,
common arterial trunk, and aortic arch interruptions. In the
present study, the emphasis is not on malformations due to NC
disorders but on the actual role of the crest in normal
development, focusing on the ectomesodermal lineage that
differentiates into vascular SMCs to form the media of the
great thoracic arteries.
Vascular SMCs are reported to originate from either the
splanchnic mesoderm14,15 or the NC.3 Recently, a third possible origin was ascribed to the endothelial cells, which appear to
transdifferentiate into SMCs in the early avian dorsal aorta.16 Le
Lièvre and Le Douarin3 have reported on the distribution of
NCCs to the great arteries in the thorax. Other studies have
further elaborated on NC seeding of the pharyngeal arches
both in birds4,5,17–19 and in mammals.20,21 The tunica media of
the great vessels derived from the pharyngeal arch arteries (eg,
aortic arch, brachiocephalic arteries, and pulmonary arch
arteries) was shown to consist almost exclusively of NCCs,
whereas other thoracic vessels (eg, pulmonary arteries, subclavian arteries, and descending aorta) appeared devoid of NCCs
and thus entirely mesodermal in origin.3,5 This unequal distribution of cells of different embryonic origins may be of
influence on differential vascular patterning.
Recently, we reported on the differentiation of the thoracic
arteries during chick embryonic development and suggested
Received May 12, 1997; accepted October 21, 1997.
From the Department of Anatomy and Embryology, Leiden (the Netherlands) University Medical Centre.
Correspondence to A.C. Gittenberger-de Groot, Department of Anatomy and Embryology, Leiden University Medical Centre, PO Box 9602, 2300 RC
Leiden, Netherlands.
E-mail [email protected]
© 1998 American Heart Association, Inc.
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b-gal
DA
NC
NCC
SMC
TGF
Selected Abbreviations and Acronyms
5 b-galactosidase
5 ductus arteriosus
5 neural crest
5 neural crest cell
5 smooth muscle cell
5 transforming growth factor
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that morphogenesis of vessels described to be of NC origin was
different from that of pure mesodermal derivatives. The
presence of clearly distinguishable alternating SMC and nonmuscular medial cell layers appeared to be confined to NC
vessels. Furthermore, presumed NC vessels exhibited an elastic
fiber organization different from that of mesodermal vessels.22
Moreover, several studies recently mentioned different in vitro
characteristics of SMCs isolated from either mesodermal (abdominal aorta) or NC (thoracic aorta) vessels.23–25
In order to study both NCC distribution and NCC differentiation in the developing cardiovascular system at the same
time, we used a lineage tracing system with a replicationincompetent retrovirus containing the LacZ reporter gene.26
The particular retrovirus we used has been proven beneficial in
studying the avian NC lineage27–29 as well as several other cell
lineage systems.30 –32 Retroviral introduction of the LacZ gene
is, in contrast to fluorescent dye injections, beneficial in
detecting cell lineages over long time spans and is, at the same
time, much less invasive than chimera techniques, circumventing major interference with normal development. In addition,
clear whole-mount distribution patterns of NCCs can be
visualized using b-gal conversion. Using the retrovirus in
combination with immunohistochemistry, we have been able
to map distribution patterns of NCCs at any desired developmental stage and to superimpose vascular differentiation patterns in an attempt to relate NC contribution and vascular
morphogenesis. For comparison, quail-chick chimeras were
also included in the present study. Chimeras have the advantage of labeling virtually all cells emanating from the cardiac
crest.
Materials and Methods
Virus
The replication-incompetent virus, designated CXL, that was used in
the present study was kindly provided by Dr T. Mikawa (Cornell
University Medical College, New York, NY).26 The design of this
vector was based on spleen necrosis virus. It lacks the structural viral
proteins necessary for replication and carries the LacZ reporter gene.
Strong and stable expression of LacZ under control of the promoter in
the spleen necrosis virus long-terminal repeat has been established in
a wide range of avian embryonic cell types.26,27,30,31 The virus was
produced in canine packaging cells (D17.2G/CXL) cultured in
Iscove’s modified DMEM (IMDM, GIBCO) containing 5% FCS
(GIBCO) and penicillin/streptomycin.
On the day of the experiments, CXL was harvested from confluent
monolayers 24 hours after changing the medium. Cells were scraped from
the culture flasks and ground in their medium, followed by brief
centrifugation to remove cellular debris. CXL was subsequently pelleted
by centrifugation of the medium at 17 000g and 22°C for 21⁄2 hours and
resuspended in a small volume of medium containing 100 mg/mL
polybrene (Sigma Chemical Co) and indigo carmine blue. The harvested
virus batch was used for both infection of embryos and titration on cells.
Viral titers were determined in duplicate by infection of R2 rat fibroblasts
with dilutions of the virus batch on the day of the experiment. Titers
varied between 2 and 43106 transducing units/mL.
Infection of Embryos
Fertilized specified pathogen-free white leghorn eggs (ID-DLO) were
incubated for 36 to 42 hours at 37°C, windowed, and staged
according to Hamburger and Hamilton.33 Embryos between stages 8
and 10 were used for infection with freshly prepared CXL solution
containing polybrene. Using glass micropipettes and carefully exerted
pressure by a Hamilton syringe connected to an oil-filled system, the
neural groove/tube was filled with viral suspension from the somite-4
to -5 region in an anterior direction. Spilling of virus over the neural
folds or out of the anterior neuropore was minimized as much as
possible. After injection, the eggs were tightly sealed with Scotch tape
and returned to the incubator for further development.
Tissue Preparation and b-Gal Staining
Infected embryos were allowed to develop until stages 19 to 40,
collected, and further processed. The youngest embryos (stages 19 to
33) were fixed in toto by immersion in 4% paraformaldehyde in PBS
(4 hours, 4°C), whereas bigger embryos were first perfused with the
fixative via the right atrium, followed by immersion-fixation. After
extensive rinsing in PBS, the thoracic viscera were carefully removed
from fixed older embryos. Next, embryos and heart-lung specimens
were stained by immersion in X-gal solution (PBS containing
5 mmol/L potassium ferricyanide, 5 mmol/L potassium ferrocyanide,
2 mmol/L MgCl2, and 0.1% [wt/vol] X-gal [C14H15BrClNO6, Boehringer]) while being shaken at 37°C for 2 to 4 hours. Again, the
cardiovascular system of larger specimens was perfused with X-gal
solution before immersion-incubation. Stained embryos were thoroughly rinsed in PBS, evaluated macroscopically, and processed for
immunohistochemistry. The results are based on a total number of 56
successfully labeled embryos.
Immunohistochemistry
b-Gal–stained embryos and heart-lung specimens were swiftly dehydrated in graded ethanol, followed by Paraclear (Earth Safe Industries
Inc) and subsequent embedding in paraffin. Xylene-free Paraclear was
used to prevent the blue precipitate from dissolving. Sections of 5 mm
were cut and distributed over several series of glass slides, allowing for
various staining procedures of each embryo. Standard resorcin/fuchsin
staining was used to visualize elastic fibers, and monoclonal antibodies
against muscle-specific actin (HHF35,34 DAKO A/S) and HNK-135
enabled discernment of differential pathways of NC derivatives.
Before antibody incubation, endogenous peroxidase activity was
quenched by treatment with 0.3% H2O2 in PBS. Routine immunohistochemical staining was performed using overnight incubations
with the primary antibodies diluted in PBS with 0.05% Tween 20 and
1% chicken egg albumin (HHF35, 1:1000; HNK-1 medium, 1:10).
Sections were then thoroughly rinsed, and bound primary antibodies
were visualized with horseradish peroxidase– conjugated rabbit antimouse antibodies (1:300, DAKO A/S) and treatment with 0.04%
diaminobenzidine tetrahydrochloride/0.06‰ H2O2 in 0.05 mol/L
TRIS-maleic acid (pH 7.6) for 10 minutes at room temperature. Then
the sections were counterstained with Mayer’s hematoxylin, dehydrated in ethanol and Paraclear, and mounted in Entellan (Merck).
Chimeras
Fertilized eggs of White Leghorn chicken and Japanese quail (Coturnix
coturnix) were incubated for 40 to 42 and 36 to 38 hours, respectively,
and windowed, and the embryos were staged after slight staining with
Nile blue sulfate. In stage-10 chicken and quail embryos, the cardiac
NC (midotic to somite 3) was ablated by removing the dorsal part of
the neural tube with a sharpened tungsten needle. Subsequently, quail
NC tissue was homotopically transplanted onto the graft site of the
chick embryo. After sealing the eggs, the chimeric embryos were
reincubated and killed between stages 28 and 40. Whole embryos and
dissected thorax segments were fixed in 2% acetic acid and 98%
ethanol (overnight at 4°C), embedded in paraffin, and further processed according to the aforementioned immunohistochemical proce-
Bergwerff et al
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Figure 1. a, Whole-mount b-gal–stained
stage-19 embryo showing NC-derived
cells in the head and pharyngeal region.
Within the arches (I, II, III, and IV), individual cells can be recognized as blue dots.
E indicates eye; H, heart. Bar5100 mm. b
and c, Light micrographs of a section
through the aortic sac region of the
stage-19 embryo, depicted in panel a,
showing presence of blue-stained NCCs
in the aortic sac and near the outflow
tract and double staining for musclespecific actin (cells combining blue and
brown staining, arrow) indicative of primary SMC differentiation. AoS indicates
aortic sac; OT, outflow tract. Bar550 mm
(b) and 7,5 mm (c). d, Whole-mount–
stained stage-24 embryo demonstrating
massive LacZ expression in the pharyngeal arch region. The dashed line indicates the clear dorsal and posterior
boundary of the NC-infested circumpharyngeal crest area. Bar5250 mm. e and f,
Micrographs of a stage-23 embryo showing periendothelial actin staining (brown)
of the arch arteries (III, IV, and VI) and
preferential localization of NCCs (blue) at
the most lateral side of the pharyngeal
arches. Part of the NCC population has
become clearly associated with the arteries (arrows). AoS indicates aortic sac; PC,
pharyngeal cleft; and T, trachea.
Bar5150 mm (e and f).
dures. In addition to antibodies revealing differentiation antigens
(HHF35, HNK-1, and smooth muscle a-actin [1A4, DAKO A/S]),
the antibody QCPN (a quail nuclear marker, Developmental Studies
Hybridoma Bank) was used to visualize distribution of quail NCderived cells. Twenty-two successfully transplanted chimeras were
used in the present study.
Results
Stages 19 to 21
NC-derived cells showing b-gal activity were shown to have
migrated into the entire pharyngeal arch region (Fig 1a). The
dense mesenchyme of the maxillary buds and branchial arches
that surrounds the arch arteries exhibited moderate to extensive blue staining. NCC distribution extended from the most
ventrally located arch mesenchyme, alongside the pharynx, and
generally up to the paired dorsal aortas and anterior cardinal
veins. The NC distribution pattern along the pharyngeal
arterial system is depicted in Fig 2a. In contrast to the
ectomesenchyme of the pharyngeal arches, the mesenchyme
that surrounds the aortic sac region is far less dense, and only a
few labeled cells positioned both dorsally and ventrally of the
aortic sac and in the endocardial cushion tissue of the outflow
tract were shown. Ectomesenchymal contribution to the aortic
sac therefore appeared limited at these stages. Endothelial cells
in the entire pharyngeal region never showed staining for b-gal
activity. By use of a double staining against muscle-specific
actins, part of the NCCs in the aortic sac revealed actin
expression indicative of early SMC differentiation (Fig 1b and
1c). The pharyngeal arch arteries, which are also aligned by
NC-derived ectomesenchyme, did not yet show signs of early
actin expression at this stage in development. The paired dorsal
aortas, on the other hand, showed a thin layer of actin-positive
cells directly adjacent to the endothelium. These primary
SMCs in the dorsal aorta were never shown to be of NC origin
in the region posterior to arch 4. However, the anterior dorsal
aortas (ie, ductus caroticus and early carotids) showed coexpression of actin and b-gal in a small number of cells,
suggesting the onset of SMC differentiation of NC-derived
cells.
Stages 23 and 24
Whole-mount–stained stage 23/24 embryos clearly demonstrated the disposition of the circumpharyngeal crest area (Fig
1d). Sections revealed massive NC infestation of the arches,
whereas further upstream toward the heart, the arch arteries
and aortic sac were surrounded by lower numbers of NCCs
(Fig 1e and 1f). The lateral sides of the arch arteries clearly
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Double staining for muscle-specific actins suggested that in
all aforementioned vessels, NCC located adjacent to the
endothelium had started differentiation into vascular SMCs,
whereas more peripheral NCC layers were still actin negative
(Fig 3).
In chimeras and retrovirally infected embryos, both aortic
and pulmonary roots, now separated from each other by the
aorticopulmonary septum, showed a remarkably smaller contribution of NCCs than the media of the arch arteries further
downstream. Moreover, LacZ-positive and quail cells were
preferentially distributed along the medial vessel walls and
continued into the condensed mesenchyme and prongs of the
aorticopulmonary septum (Fig 4a and 4b). The lateral sides of
the bases of both vessels were devoid of NCCs, whereas
further downstream, before branching into the arch arteries,
NC distribution became entirely circumferential. At these
stages of development, the aortic and pulmonary roots and the
proximal parts of the arch arteries do not stain for musclespecific actins.
Figure 2. Schematic representation of NCC contribution to the
developing arterial system in the chick, as deduced from quailchick chimeras and retrovirally infected embryos. Density of
dots corresponds with relative contribution of NCCs to periendothelial tissue or the media. a, Stage 21. b, Stage 27. c, Stage
31. d, Stage 40. AoAr indicates aortic arch; AoS, aortic sac;
AsAo, ascending aorta; BA, brachiocephalic artery; CA, carotid
artery; CO, coronary artery; DA, ductus arteriosus; DAo, dorsal
aorta; DsAo, descending aorta; PA, pulmonary artery; PT, pulmonary trunk; SA, subclavian artery; and III, IV, and VI, pharyngeal arch arteries.
showed more seeding with NCCs than the median parts at
these stages. Moreover, no indication of a prospective aorticopulmonary septum was found in any of the embryos, as
NCC numbers were extremely low at the dorsal side of the
aortic sac area. NCCs extended well into the outflow tract
cushions, preferably in a dorsally located stretch of cells. In the
aortic sac and arch arteries, part of the NCC population was
double-stained for LacZ and muscle actin.
Stages 26 to 28
By stages 26 to 28, the third, fourth, and sixth pharyngeal arch
arteries are entirely ensheathed by NC-derived cells (Fig 2b).
Also, the remnants of the combined first and second arch
arteries, still present as small lumenized vessels branching from
the paired proximal third arches, are surrounded by NCCs.
The NCC-containing vessel wall surrounding the third arch
artery continues downstream along the paired anterior parts of
the dorsal aorta (also called carotid arteries at this stage36). In the
fourth and sixth arches, however, LacZ labeling stops abruptly
at the junction with the dorsal aortas. No NCCs could be
found in the posterior part of the dorsal aorta. Occasionally,
some LacZ-positive cells were localized in the proximal part of
the pulmonary artery (3 of 13 embryos), close to its branching
point with the sixth arch artery. No obvious differences in
NCC distribution were detected between the left or right part
of the pharyngeal arch region. The primary vessel wall of the
left fourth arch artery and of both left and right carotid ducts
(all of which obliterate during this relatively short time span)
was shown to possess a marked NCC contribution.
Stages 30 to 34
Compared with earlier stages (stages 26 to 28), the distribution
of NCCs in the vascular system remained basically unaltered.
The vascular tree, however, is subject to extensive remodeling
during these stages. Its morphology and the distribution of
NCCs within the system are depicted schematically in Fig 2c.
Most marked differences with earlier stages are those resulting
from the degeneration of the left fourth arch artery and of both
carotid ducts. In addition, the carotid arteries have lengthened
considerably as a result of the caudal positioning of the heart in
the thorax with respect to its initially more rostral position near
the head and also as a result of the lengthening of the neck
itself. Within persisting vessels, LacZ-labeled cells were localized at all possible positions in the NC-derived vessel walls; ie,
no preferential labeling could be detected in either peripheral
or adluminal cell layers. Blue staining of the condensed
mesenchyme of the aorticopulmonary septum was still seen to
be continuous with the median side of the pulmonary and
aortic outflows, whereas no staining was found at the lateral
side of the bases of these vessels.
Between stages 30 and 34, histology of the great arteries is
characterized by separation of cell layers of the media due to
deposition of matrix components. SMCs in the proximal part
of the arterial tree do not express muscle actin at these stages
(see also Reference 22). More distally, periendothelial cell
layers express muscle actin and are surrounded by a few actin
negative cell layers. NCCs were distributed both in proximal
actin-negative parts and in more distal parts showing periendothelial actin expression, and they assumed actin-negative as
well as actin-positive cellular phenotypes.
Stages 35 to 40
NCC distribution in stages 35 to 40 was confined to the aortic
and pulmonary trunks, aortic and pulmonary arch arteries, and
brachiocephalic and carotid arteries (Fig 2d). Both media and
adventitia were shown to be of NC origin. NC adventitial
fibroblasts and NC neural cell types could easily be distinguished by staining the latter with the HNK-1 antibody. The
Bergwerff et al
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Figure 3. a, Stage-26 pharyngeal arch artery demonstrates massive contribution of NCCs to the developing media, both the actinpositive periendothelial layer and more peripheral actin-negative layers. Bar550 mm. b, High magnification of a stage-26 arch artery
allows for clear colocalization of b-gal and a-actin staining (arrows). Bar510 mm.
coronary and pulmonary arteries and the descending aorta
were found consistently negative in our study.
In the proximal part of the arterial tree, part of the media cell
population starts reexpression of actin from around stage 35–36
onward. This wave of secondary actin expression starts at the
interface with the myocardium and results in a markedly
layered vessel wall consisting of an actin-negative intima-like
layer surrounded by a media that is composed of alternating
SMCs and nonmuscular cell layers. This rather thick lamellar
wall starts directly after Valsalva’s sinus, thereby narrowing the
lumen considerably. At the level of the sinus, both aortic and
pulmonary vessel walls were thin and nonlamellar but showed
muscle-specific actin positivity in peripheral cell layers up to
the connection with the outflow tract myocardium (Fig 4c).
The inner lining of Valsalva’s sinus was always actin negative
and was never shown to harbor NCCs. The outer actinpositive cell layers only showed NC contribution adjacent to
the aorticopulmonary septum, which showed clear smooth
muscle actin expression at these stages as well (Fig 4d to 4g).
Otherwise, the ascending aorta and pulmonary trunk were
found to contain an extensive mesodermal contribution in
contrast to the arch arteries. Preferential localization of NCCs
at the septal side of the bases of these vessel, as was already
determined from stage 26 onward, persisted in the older
embryos. Further downstream, the NC area expanded within
the aortic and pulmonary trunk walls and encircled them
entirely before branching into the arch arteries (Fig 4d).
Chimeras showed non-NC areas of variable extensions, making it difficult to delineate a true boundary. The coronary
arteries were connected with the aorta in this non-NC area
and never showed any NCCs in the media or adventitia. At the
site of this connection the periendothelial actin-stained muscular coronary arteries enter the lamellar aortic media in an
actin negative “window” (Fig 4c and 4d).
Double staining for b-gal and muscle actin revealed that
NCCs had differentiated into both SMCs and nonmuscular
cells in the media and intima and also into adventitial cells in
the great arteries (Fig 5a and 5b). The relative number of
b-gal–labeled cells was consistently greater in the media than in
intimal cell layers. Chimeric staining also suggested that the
media of the arch arteries was composed almost exclusively of
NCCs, whereas a number of nonquail cells was often encountered in the innermost part of the lamellar vessel walls. Within
the media, no preferential labeling was discerned between
SMCs and nonmuscular cells.
During later stages of development, the pulmonary arch
arteries can be divided into three distinct segments based on
morphological features.22 The first segment, the proximal
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Figure 4. Interestingly, the most proximal part of the arterial tree, ie, the ascending aorta and pulmonary trunk, remained largely devoid
of NCCs throughout development. a and b, Adjacent sections of a stage-28 chimera, stained with QCPN (a-quail) and HHF35 (a-actin),
respectively. Note in these sections, at the level of the pulmonary outflow, the markedly present condensed mesenchyme (CM) of the
aorticopulmonary septum and the area between the dashed lines, which is not seeded by NCCs. IV and VI indicate the fourth and sixth
arch artery, respectively; T, trachea. Bar5300 mm. c and d, Schematic representations of the outflow tract area at stage 40. Panel c
depicts smooth muscle actin expression patterns, clearly demonstrating a lamellar pattern of alternating SMCs and nonmuscular cells
in the elastic media of the ascending aorta (AsAo) and pulmonary trunk (PT), as well as typical periendothelial actin staining in the muscular coronary artery (CO), which connects to the aorta in an actin-negative “window.” M indicates myocardial cuff of the outflow tract.
Panel d depicts the presence of NCCs in this region, indicated by dotted areas. Note that in this region, actin expression patterns
appear to be independent of cell origin. e, f, and g, Adjacent sections of a stage-40 chimeric embryo stained with QCPN (e), HHF35 (f),
and 1A4 (g). Apart from the NC-derived aorticopulmonary septum, the aortic (Ao) and pulmonary roots are for the greater part of mesodermal origin. Bar5250 mm (e, f, and g).
elastic segment, revealing the aforementioned lamellar structure, harbored an extensive NC contribution, as did the second
segment, the muscular DA. The third segment, elastic segment, revealed seeding with NCCs except for its most downstream region. NCC contribution to the distal parts of the
aortic and pulmonary arches showed a sudden disappearance,
somewhat upstream from where they join to continue as the
descending aorta. This sharp boundary was markedly visible in
whole-mount b-gal–stained specimens (Fig 5d). Elastin staining revealed differences in the matrix organization between the
elastic arch arteries and the elastic descending aorta. NCderived arch derivatives harbored thin elastic fibers in both
longitudinal and circular directions, whereas the non-NC
descending aorta contained neatly assembled elastic lamellae,
which were circularly arranged in the vessel wall (Fig 6a and
6b). The transition between both elastin organization types was
gradual, consisting of thinning of the vascular wall, loss of thin
isolated fibers, and concurrent appearance of thick circular
elastic lamellae. This transition occurred in the distal parts of
the arch arteries, which still consist predominantly of NCderived cells.
Another sharp boundary of the NC area could be discerned
at the junction of the muscular pulmonary artery with the
elastic sixth arch artery. No NCCs were ever detected in the
pulmonary arteries at stages 35 to 40. In sharp contrast to the
arch arteries, the typical muscular pulmonary arteries showed
only very little elastin staining and dense periendothelial actin
staining instead of alternating SMC and nonmuscular cell layers
(Fig 6c and 6d).
The DA was the only part of the NC-derived arch arteries
that did not develop into an elastic artery but rather into a
typical muscular one. With low elastin levels and periendothelial actin staining, it resembled the pulmonary and coronary
arteries.
The subclavian arteries remained devoid of NCCs during
the greater part of the developmental range we studied (Fig
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Figure 5. a, NCC-populated vessel wall
of a stage-38 pulmonary arch artery
showing NC contribution to both the
media (M) and the intima (I). L indicates
lumen. Bar530 mm. b, Stage-38 aortic
media revealing NC-derived cells, which
have differentiated into lamellar SMCs
(arrows) and interlamellar nonmuscular
cells (arrowheads). Bar525 mm. c, Ventral
view of a stage-40 chick heart. Apart
from major NC infestation of the great
arteries, the NC-derived cardiac innervation plexus is seen to spread over both
ventricles. Ao indicates aorta; BA, brachiocephalic artery; PT, pulmonary trunk;
RA, right atrium; and RV, right ventricle.
Bar5750 mm. d, Dorsal view of a
stage-40 heart-lung specimen. Note the
sharp NC boundaries in the aortic arch
(AoAr) and DA before they join to form
the descending aorta (DsAo). Arrowhead
indicates NC-derived neural tissue; the
DsAo itself is devoid of NCCs. LL and RL
indicate left and right lung, respectively.
Bar5250 mm.
6e). However, in the oldest embryos studied, we found
marked b-gal staining in a proximal cuff around the subclavian
artery. Intriguingly, this NCC-derived cuff corresponded with
a lamellar actin expression pattern. More distally in the
subclavian arteries, loss of b-gal staining coincided with a
change in actin patterns from the lamellar type to periendothelial expression.
From stage 35, the brachiocephalic, common carotid, and
inner/outer carotid arteries showed a marked NC contribution
along their entire length, possibly extending into the head
region (Fig 6f). In the carotid arteries, lamellar actin expression
patterns are replaced by periendothelial actin staining in a
cranial direction. The contribution of NCCs to these vessels,
however, remained unalteredly high along the entire tract well
up into the neck irrespective of changes in vascular
morphology.
In addition to the SMC population in the arterial system,
NCCs were also found to contribute to the developing
vascular walls of the anterior cardinal veins in the neck region
(Fig 6g). These venous NCCs had been present around the
anterior cardinal veins at earlier stages but had not yet been
clearly associated with them in terms of mesenchymal condensations and actin expression. Nascent actin expression in the
anterior cardinal veins occurred at around stage 31, and by
stage 35, contribution of NCCs to venous SMC layers could
easily be established. Smaller veins running alongside the
anterior cardinal veins and in the NC-infested thyroid region
were associated with NCCs as well, but these veins did not yet
develop a distinct primary media within the embryological
time span studied. In contrast to arterial NC-derived cells,
NCCs in the venous vessel wall did not reach the heart but
were present in an area with a posterior boundary at the level
of the thyroid.
Discussion
Early migratory behavior and mapping of hindbrain NC cells
within the pharyngeal arches have previously been studied in
both avian17,18,37 and mammalian20,21,38 embryos. However,
accurate mapping of the subsequent NCC contribution to the
vasculatory system that arises within the pharyngeal arch region
has been poorly illuminated for later phases of embryological
life. Results from the present study serve to fill this hiatus and
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Figure 6. a and b, Resorcin/fuchsin
staining for elastin revealed differences in
elastic fiber constitution between the NCderived aortic arch (a) and mesodermal
descending aorta (b) (stage 40). In the
aortic arch, thin longitudinal fibers prevail
nearest to the lumen (L); peripheral fibers
are more circularly arranged. The
descending aorta is filled with thick circular elastic lamellae. Bar5100 mm (a
and b). c and d, The pulmonary artery
(PA) develops entirely from the mesoderm. Its branching point from the NCderived pulmonary arch artery therefore
coincides with a sharp boundary in
QCPN (a-quail) staining (c) but also
includes a transition in actin staining (d)
from a lamellar pattern in the arch toward
periendothelial staining in the PA.
Bar5200 mm (c and d). e, Micrograph of
a stage-36 chimera shows the connection between the mesodermal subclavian
artery (SA) and QCPN-stained NC-derived brachiocephalic artery (BA).
Bar5100 mm. f, Media and adventitia of
the carotid arteries (CA) are stained with
QCPN, indicating their NC origin (stage
36). Bar5100 mm. g, QCPN-stained section of a stage-36 chimera clearly demonstrates that the anterior cardinal veins
(ACV), like the great arteries, receive an
extensive NCC contribution to the media
as well. The adjacent vagal nerve (VN)
harbors NC-derived satellite cells.
Bar5100 mm.
to complete detailed mapping throughout in ovo development. In addition, this study provides a direct link to the fate
of NC-derived vascular cells in terms of differentiation, both
on a cellular and tissue level, based on NCC behavior during
normal development.
NC Distribution in the Anterior Vascular
System: New Findings and Theories
In addition to the earlier reported NC contribution to the arch
arteries, we now show that the paired dorsal aortas harbor
NCCs as well, in the region anterior to arch 4 (ie, carotid ducts
and early carotid arteries). The anterior dorsal aortas reside in
the dorsalmost part of the circumpharyngeal crest area37 and
consequently develop a primary tunica media in which crest
cells will have been incorporated.
So far, it is unclear why NCCs would populate the larger
part of the thoracic arterial system except for certain areas. In
contrast to NCCs that differentiate into, for example, the
enteric nervous cells,27,39 NCCs in the vasculature populate
only those areas that during their stage of anlage reside in an
anterior region corresponding with the position of the premigratory cardiac crest cells themselves. In both the arterial and
venous systems, the NC area has a clear posterior boundary,
which, because of remodeling processes, becomes harder to
delineate as a clear anterior-posterior axis–related boundary
during later development. Hox gene– encoded positional information in NCCs is proven to be involved in patterning of
the branchial arches40 – 42 and most likely is involved in determining NC distribution among the arch arteries as well. The
fact that the descending aorta and subclavian and pulmonary
arteries do not receive NCCs appears to be related to their
posterior positions in early development,43 whereas the coronary arteries have not yet formed their endothelial scaffolding
properly at the time of major NCC migration in the pharyngeal arch region, which occurs in a cranial-caudal sequence
between stages 15 and 25.4,37,44 Once NCCs have stopped
migration and started differentiation to some extent, they are
less likely to migrate further toward still unpopulated areas at a
later point in development.45 This might explain why thoracic
arteries that arise later in development, like the coronary
arteries, recruit their primary SMCs from the local mesenchyme, which is mesodermal in origin.
In the present study, older chimeric embryos generally
showed very few chicken cells in the vessel wall, located
mainly in the intima-like layer. This suggests that the relative
contribution of mesoderm-derived cells at final stages in
development is very small in these vessels, except for the
intima. These findings suggest a role of mesoderm-derived cells
in the genesis of this intima-like layer in conjunction with
NCCs during avian embryonic development. These mesodermal cells are virtually absent at earlier stages, suggesting
selective proliferation of a small number of progenitor media
cells, or they might arise via endothelial-mesenchymal transformation in a process similar to that described in the early
avian endocardial cushions46 and dorsal aorta.16
Other intriguing parts of the pharyngeal arch complex
concerning the NCC contribution are both the aortic and
pulmonary trunks. In retrovirus and chimera experiments,
Bergwerff et al
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both vascular roots appeared to have a dual origin in which
mesodermal cells clearly outnumbered NCCs. Moreover,
NCCs generally turned out to be spatially organized toward
the median (septal) side at the very bases, gradually substituting
mesodermal cells in a downstream direction; a somewhat
similar phenomenon was described by Takamura et al.2 The
chick ascending aorta and pulmonary trunk, or at least their
very bases, are generally hypothesized to originate from the
septated aortic sac. In contrast to designated derivatives of the
arch arteries (aortic arch, brachiocephalic arteries, and pulmonary arch arteries), the aortic sac appears to develop mainly
from the mesoderm, incorporating only a minor NC contribution. Actual translation of these chick NCC patterns into the
fully remodeled mammalian arterial tree remains difficult. The
aortic sac in mammals is considered to develop into the entire
ascending aorta, brachiocephalic artery, and pulmonary
trunk.9,47 However, the extension of the proximal cuff, which
we found largely devoid of NCCs, is unknown in these major
mammalian vessels. Unfortunately, long-term tracing studies,
similar to those in avian models, cannot be performed in
intrauterine mammalian development, and specific markers for
mesenchymal cells and SMCs derived from the NC, which are
expressed for a prolonged period of time, have not been
identified yet.
Our present finding of the contribution of NCCs to the
media of the anterior cardinal veins is the first so far. This
finding was accomplished by use of both retrovirus and
chimera experiments and strengthens the hypothesis that vascular NCC distribution is dictated by the presence of endothelial tubes at the early hindbrain level in both the arterial and
venous system. Unlike the outflow tract of the heart and the
great arteries, however, the venous system was never described
to be affected after NC ablation.48 To date, however, the
venous system has received relatively little attention in vascular
research, and our results suggest that it should be given more
consideration in NC-related experimental and clinical
research.
Implications for Cellular Phenotype and
Vascular Morphology?
Using retroviral NC tracing in combination with the HHF35antibody, we have been able to show that NCCs are among
the first cells to differentiate into primary SMCs in the
developing pharyngeal arterial system. This finding is in
contradiction with earlier reports by Rosenquist and Beall.49
Using an anti–smooth muscle actin antibody (1A4) on chimeric embryos and without the benefit of the QCPN antibody, they concluded that primary actin expression in the
arterial system occurred in mesodermal cells. This primary
mesodermal tunica media was to be replaced at a later stage by
actin-negative NCCs, which in turn expressed actin from
around day 12 onward, resulting in a wave of secondary actin
expression. We have recently reported on similar actin expression patterns22 but now reveal that NCCs are involved in both
primary and secondary expression waves.
Adventitial fibroblasts as well as SMCs and nonmuscular
cells of the media, which arise after the secondary actin
expression wave within the lamellar vessel wall,22,49 were in the
present study shown to be NC-derived. NCCs did not show
229
preferential differentiation into any of these cell types. Although true analogs of avian nonmuscular cells do not appear
to be present in mammalian vessels, the latter do harbor distinct
SMC phenotypes, which are usually referred to as contractile
and synthetic cells.50,51 Yablonka-Reuveni et al52 reported that
in chicks NCCs have the potential to differentiate into typical
SMCs, which express desmin and a-smooth muscle actin and
are surrounded by a laminin and collagen IV basement
membrane, and into cells lacking those characteristics (nonmuscular cells). Cellular phenotype in the pharyngeal arch
derivatives, therefore, appears not to be lineage-related.
On the basis of (immuno)histological examination only, we
recently suggested that NC-derived thoracic arteries differ
from mesodermal arteries.22 In the present study, we confirm
this hypothesis. In general, it turned out that the typical
lamellar vessel wall structure only developed in vessels that
have an NC contribution. Interestingly, the boundaries of NC
areas tend to coincide with marked changes in vascular
morphology. At least three good examples of this phenomenon
were seen: (1) the junction between pulmonary arch artery
(NC-derived) and pulmonary artery (mesoderm), (2) the
proximal subclavian artery (NC)– distal subclavian artery (mesoderm), and (3) the junction of the fourth and sixth arch
arteries (NC) with the descending aorta (mesoderm).
Although these coinciding spatial boundaries are very suggestive of lineage-related morphogenesis, other findings partly
contradict this hypothesis. In both aortic and pulmonary roots,
for instance, we demonstrated a spatially organized dual origin
of the vessel wall. However, the areas in these vessels that were
shown to be of mesodermal origin did not differ morphologically from the NC areas nearby. An NC boundary, the exact
position of which could not be established by us, is certainly
not reflected in the histology of this part of the arterial tree,
suggesting the major influence of other factors, such as
hemodynamics. This area nonetheless remains very interesting
because of matrix-related pathologies, which predominantly
affect this region. Fibrillin-1–associated Marfan’s syndrome53,54
often presents dilatation or dissection of the ascending aorta,
whereas the elastin-related Williams syndrome involves supravalvular aortic stenosis.55
In addition, the sixth arch derivative, the DA, was shown to
receive an extensive NC contribution but did not join the
other arch derivatives in their elastogenic differentiation.
NC-derived arteries therefore can differentiate into both elastic
and muscular (DA) vessels. Although the DA shares the same
origin and presumably comparable hemodynamic and environmental factors with the other arch derivatives, its differentiation deviates rather early in development. Since NCCs carry
Hox gene– encoded positional information into the pharyngeal
arches,40,41 one could speculate on a further refinement of
lineage-dependent morphogenesis, as cardiac crest cells differ
in Hox gene expression according to their original position on
the anteroposterior axis. However, cardiac NCCs originating
from different rhombomeric origins were shown to mix rather
extensively within the circumpharyngeal crest before finishing
migration into the forming pharyngeal arches.4,18 In this way,
the sixth arch receives NCCs emanating from the somite 1 to
somite 3 level and therefore largely shares its cellular composition with that of arch 4. Yet regardless of these similarities,
230
Neural Crest and Vascular Differentiation
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only the sixth arch was shown to retain HOX 2.1 (HOXB5)
expression,56 thereby emphasizing its unique identity.
To date, an increasing number of in vitro experiments favor
the hypothesis of lineage-related SMC differentiation. SMCs
isolated from normally developed NC vessels were shown to
differ in vitro from “surrogate” SMCs that infest the arteries
after experimental NC ablation. SMCs (of undetermined
origin) that replaced the ablated NCCs showed much higher
levels of a-actin, tropoelastin, and expression of c-jun during in
vitro assays.57 In addition, in vitro experiments also elaborated
on differential responses to TGF-b1 by either thoracic or
abdominal aortic SMCs. TGF-b was shown to be growth
inhibitory when administered to abdominal (mesodermal)
SMCs, whereas it increased DNA synthesis in SMCs isolated
from the thoracic (NC) aorta.24,58 Topouzis and Majesky24
suggested that this differential TGF-b responsiveness might
result from lineage-dependent differences in glycosylation of
the type II TGF-b receptor. However, the implications of
their findings for vessel differentiation in vivo remain to be
elucidated, since cellular morphology and protein expression
levels of cultured cells, oddly enough, were similar in both
groups. Actin expression in vivo, however, was shown to be
restricted to only a subpopulation of media cells in the
NC-derived aortic arch. All other cells were of a nonmuscular
actin-negative phenotype.22,49,52 Nonetheless, differences in
potency unraveled by in vitro studies will aid evaluation of
morphological and physiological heterogeneity among vessels
that so far are considered to be similar. If SMC origin and
topographical localization indeed are of major importance in
defining the characteristics and performance of the cell, then
this should be taken into account in design and evaluation of in
vitro experiments as well as in surgical vessel grafting.
Last, origin-related SMC features may well be of significance in vascular pathogenesis. Intriguingly, spontaneous aortic
plaques in the chicken were only found in the descending part
of this vessel.59 The proximal part, showing the intricate
cellular lamellar organization, which we now know is predominantly of NC origin, does not develop such arteriosclerotic
pathology in the adult chicken. Because a proper correlation of
human and chick patterning is still lacking and because this
phenomenon cannot yet be translated to human arteriosclerosis-prone areas, more research into the NC in mammals will be
needed to elucidate its role in vascular biology.
Acknowledgments
This study was financially supported by grant 93.111 from the
Netherlands Heart Foundation. The excellent technical assistance of
Benno Vrolijk and Marie-Jozé Verdonk and graphic support by Jan
Lens and Bas Blankevoort are greatly appreciated.
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Neural Crest Cell Contribution to the Developing Circulatory System: Implications for
Vascular Morphology?
Maarten Bergwerff, Marlies E. Verberne, Marco C. DeRuiter, Robert E. Poelmann and Adriana
C. Gittenberger-de-Groot
Circ Res. 1998;82:221-231
doi: 10.1161/01.RES.82.2.221
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1998 American Heart Association, Inc. All rights reserved.
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