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Spatiotemporally separated cardiac neural crest subpopulations that target the outflow tract septum and pharyngeal arch arteries.

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THE ANATOMICAL RECORD PART A 275A:1009 –1018 (2003)
Spatiotemporally Separated Cardiac
Neural Crest Subpopulations That
Target the Outflow Tract Septum and
Pharyngeal Arch Arteries
MARIT J. BOOT, ADRIANA C. GITTENBERGER-DE GROOT,
LIESBETH VAN IPEREN, BEEREND P. HIERCK, AND
ROBERT E. POELMANN*
Department of Anatomy and Embryology, Leiden University Medical Center,
Leiden, The Netherlands
ABSTRACT
We used lacZ-retrovirus labeling combined with neural crest ablation in chick embryos
to determine whether the cardiac neural crest cells constitute one group of multipotent cells,
or they emigrate from the neural tube in time-dependent groups with different fates in the
developing cardiovascular system. We demonstrated that early-migrating cardiac neural
crest cells (HH9 –10) massively target the aorticopulmonary septum and pharyngeal arch
arteries, while the late-migrating cardiac neural crest cells (HH12) are restricted to the
proximal part of the pharyngeal arch arteries. These results suggest a prominent role for
early-migrating cells in outflow tract septation, and a function for late-migrating cells in
pharyngeal arch artery remodeling. We demonstrated in cultures of neural tube explants an
intrinsic difference between the early and late populations. However, by performing heterochronic transplantations we showed that the late-migrating cardiac neural crest cells were
not developmentally restricted, and could contribute to the condensed mesenchyme of the
aorticopulmonary septum when transplanted to a younger environment. Our findings on the
exact timing and migratory behavior of cardiac neural crest cells will help narrow the range
of factors and genes that are involved in neural crest-related congenital heart diseases. Anat
Rec Part A 275A:1009 –1018, 2003. © 2003 Wiley-Liss, Inc.
Key words: chick embryo; heart development; migration; ablation; neural tube
cultures; heterochronic transplantation
The neural crest in the early avian embryo is formed at
the dorsal midline as the neural folds fuse. The migratory
behavior of neural crest cells is characteristic of each axial
level, and is dependent on the developmental stage of the
embryo (Tosney, 1982). The region of the neural crest
between the midotic placode and the caudal limit of somite
3 has been called the cardiac neural crest because of its
involvement in cardiac development (Kirby et al., 1985).
In studies of quail-chick chimeras, cardiac neural crest
cells were observed in the aorticopulmonary septum,
branchial arch arteries, truncus arteriosus, and cardiac
ganglia (Le Lièvre and Le Douarin, 1975; Kirby et al.,
1983). In studies using retroviral labeling, neural crest
cells were shown to be among the first cells to differentiate
into primary smooth muscle cells of the arch arteries, and
to contribute later in development to the adventitia and
media of the pharyngeal arch arteries (Bergwerff et al.,
1998). Preferential labeling has been detected along the
medial side of the vessel walls of the aorta and pulmonary
©
2003 WILEY-LISS, INC.
trunk, continuing into the condensed mesenchyme and
prongs of the aorticopulmonary septum (Bergwerff et al.,
1998; Waldo et al., 1998). The condensed neural crest cells
of the aorticopulmonary septal complex and the prongs
disperse into scattered neural crest cells within the conal
cushions, extending to the free rim of the right ventricular
outflow tract septum (Poelmann et al., 1998; Waldo et al.,
1998). The neural crest cells of the prongs display abun-
*Correspondence to: Prof. Dr. R.E. Poelmann, Dept. of Anatomy and Embryology, Leiden University Medical Center, P.O.
Box 9602, 2300 RC Leiden, The Netherlands. Fax:
⫹31.71.5276680. E-mail: R.E.Poelmann@lumc.nl
Received 29 April 2003; Accepted 15 August 2003
DOI 10.1002/ar.a.10099
1010
BOOT ET AL.
dant apoptosis, while only a low number of neural crest
cells located in the pharyngeal arch artery vessel walls are
apoptotic (Poelmann et al., 1998, 2000).
In other studies using retroviral labeling, the contribution of the cardiac neural crest to the autonomous nervous
system (Verberne et al., 1998, 2000), as well as to regions
of the prospective conduction system and a mesenchymal
population at the venous pole of the heart (Poelmann and
Gittenberger-de Groot, 1999), were described in detail.
The importance of the cardiac neural crest population for
normal heart development was confirmed in studies of
chick embryos in which this population was surgically
removed. These embryos displayed cardiovascular malformations, such as persistent truncus arteriosus, ventricular septal defects, inflow anomalies, and interruption or
persistence of the aortic arch arteries (Bockman et al.,
1989; Kirby and Waldo, 1990, 1995). The cardiovascular
phenotypic features described in embryos in which the
neural crest was ablated are similar to those found in
certain human diseases, such as DiGeorge syndrome and
velocardiofacial syndrome (Schiaffino et al., 1999). This
has led to the proposal that DiGeorge syndrome is a neural crest-related disease, the phenotype of which results
from the absence of a gene copy or from diminished doses
of specific gene products at critical time points during
early development (Goldmuntz and Emanuel, 1997; Garg
et al., 2001; Kochilas et al., 2002). It is necessary to understand the exact differentiation (Thomas et al., 1998),
timing, and migratory behavior of cardiac neural crest
cells in order to elucidate the underlying genetic mechanisms and environmental messages. In a previous study
(Boot et al., 2003), we demonstrated that the exact timing
of cardiac neural crest cell emigration from the neural
tube in chick embryos was between Hamburger and Hamilton (1951) stages HH9 and HH13–, and that at HH14
and HH15 no cells were emigrating from the dorsal or
ventral side of the neural tube between the level of the otic
placode and the caudal limit of somite 3.
The cardiac neural crest cells have been regarded as one
group of cells with the capacity to contribute to various
aspects of the developing cardiovascular system, ranging
from the tunica media of the aortic arch arteries to the
condensed mesenchyme in the aorticopulmonary septum
of the outflow tract (Kirby, 1993). However, a direct correlation between the occurrence of a persistent truncus
arteriosus and morphological abnormalities of the aortic
arch arteries has not been described, even though these
abnormalities have been found both together and separately after ablation of the neural crest (Kirby and Waldo,
1990). Whether this is due to variations in the execution of
the ablation technique, or reflects the fact that we are
dealing with different subpopulations of cardiac neural
crest cells is the subject of this study.
At the cardiac level, no division into early and late
neural crest cell migration patterns, nor any studies on
limited developmental potential have been described. The
first focus of this study was the migratory behavior of
time-dependent migratory groups within the cardiac neural crest population, which we examined by marking the
neural crest cells with a retroviral construct harboring the
LacZ reporter gene (Bergwerff et al., 1998; Poelmann et
al., 1998). Retrovirus was injected into the neural tube,
either early (HH9 –10) or late (HH12) during cardiac neural crest cell migration, and the contribution of the total
cardiac neural crest population was compared to the con-
tribution of the late-migrating neural crest only. By combined early neural crest cell labeling and removal of the
late cardiac neural crest cells, the contribution of the
early-migrating cardiac neural crest cell population was
studied separately. These embryos also showed the effects
of ablation of late-migrating cardiac neural crest cells on
cardiovascular development.
The second focus of this study was to determine whether
there are intrinsic differences between early- and latemigrating cardiac neural crest cells. Therefore, we cultured early (HH10) and late (HH12) neural tube explants,
and compared the in vitro migration and differentiation
patterns of the emigrating cardiac neural crest cells.
The third focus of this study was the influence of early
and late embryonic environments on neural crest cell migration, which we studied by performing heterochronic
transplantations. Late (HH12) quail cardiac neural crest
cells were transplanted into an early (HH10) chick embryo
in which the cardiac neural crest had been surgically
removed.
Using retroviral labeling, in vitro neural tube explant
cultures, and quail-chick chimera techniques, we demonstrated that the cardiac neural crest consists of early- and
late-migrating subpopulations that target the outflow
tract septum and the pharyngeal arch arteries, respectively.
MATERIALS AND METHODS
Retrovirus
The replication-incompetent spleen-necrosis retrovirus
that was used in this study contains a bacterial lacZ gene,
as described by Mikawa et al. (1991). The retrovirus was
produced in the canine cell line D17.2G/CXL and secreted
into the culture medium (Iscove’s DMEM containing 5%
fetal calf serum and 1% penicillin/streptomycin). After the
cell debris was removed, the virus was harvested by centrifugation (17,000 g for 21⁄2 hr) of the supernatant. The
pellet was resuspended in a small volume of medium
containing 100 ␮g/ml polybrene to improve transduction,
and 0.25 g/ml indigo carmine blue for visualization of the
virus solution during in ovo injection. The titer of the
retrovirus was determined on rat R2 fibroblasts. The titers of the virus solutions used in these experiments varied between approximately 1–5 ⫻ 106 transducing units
per milliliter.
Infection of Chicken Embryos
Fertilized, specified pathogen-free White Leghorn eggs
were incubated at 37°C for 33– 46 hr and windowed at
HH9 –12. The retrovirus solution was drawn into a glass
micropipette mounted on a micromanipulator, and then
carefully injected into the lumen of the neural tube using
a programmable microinjector (IM-300 Narishige, Japan).
Stage HH9 –12 embryos were injected into the neural tube
at somite levels 4 – 6, filling the neural tube in the anterior
direction until the virus solution reached the otic placode
level, to label the complete cardiac neural crest cell region.
In addition, part of the cranial and trunk neural crest cells
were labeled by diffusion of the retrovirus; however, these
cells do not contribute to the cardiovascular system (Kirby
et al., 1985; Artinger and Bronner-Fraser, 1992; Baker et
al., 1997) . After the embryos were injected, they were
washed with Locke’s solution. The eggs were sealed with
Scotch tape and returned to the incubator at 37°C for
further development.
CARDIAC NEURAL CREST SUBPOPULATIONS
Labeling of Early Neural Crest and Ablation of
Late Cardiac Neural Crest
In double experiments, embryos were injected with retrovirus at HH10 as described above, and subsequently
reincubated for another 3– 4 hr at 37°C until they reached
HH11. At this stage the dorsal side of the neural tube was
microsurgically removed between the otic placode and the
caudal part of somite 3 (cardiac neural crest), using a
sharpened tungsten needle. The eggs were sealed and the
embryos survived until HH13–15 or HH30 –31.
Embryo Fixation and ␤-Galactosidase Staining
The embryos were allowed to survive for 8 hr to 7 days
after injection. HH11–28 embryos were immersion fixed in
toto. HH29 –32 embryos were decapitated and the thorax
was opened. In HH33–34 embryos the thorax was isolated
to optimally expose the heart and vessels to 2% paraformaldehyde in 0.2 M phosphate buffer for 1 hr, followed by
extensive rinsing in PBS. Subsequently, the embryos were
stained for the presence of lacZ by immersion in X-gal
solution (PBS containing 5 mM potassium ferricyanide, 5
mM potassium ferrocyanide, 2 mM MgCl2, and X-Gal (5bromo-4-chloro-3 indolyl ␤-D-galactopyranoside) for 2–5
hr at 37°C while being shaken. The stained embryos were
macroscopically evaluated for successful blue labeling of
lacZ-expressing cells. Infection grades vary among embryos of the same infection stage; therefore, we only selected intensely stained embryos. Similar numbers of intensely stained embryos were found in each group. The
group with HH9 or HH10 injected embryos was referred to
as the “early-injected group,” with labeling of early- and
late-migrating cardiac neural crest cells. HH12 injected
embryos were referred to as the “late-injected group,” with
labeling of the late-migrating cardiac neural crest cells.
The following conditions were studied: HH9 or 10 injection
(n ⫽ 19), HH10 injection combined with HH11 ablation
(n ⫽ 5), HH11 injection (n ⫽ 8), and HH12 injection (n ⫽
18).
Heterochronic Transplantation
Fertilized eggs from White Leghorn chickens and Japanese quails were incubated for 38 and 44 hr, respectively.
The eggs were windowed, and HH10 chick embryos and
HH12 quail embryos were selected according to the number of somites. Cardiac neural crest cell migration patterns in chick and quail embryos are very similar; therefore, quail-chick chimeras have been used in several
studies to describe neural crest cell migration (Kirby et al.,
1983; Verberne et al., 2000). For our heterochronic transplantation experiment, the chick cardiac neural crest region was removed with a tungsten needle. The cardiac
neural crest was excised from an HH12 quail embryo and
transplanted into the graft site of an HH10 chick embryo.
The eggs were sealed and the embryos (n ⫽ 5) survived
until HH30, followed by overnight immersion fixation in
4% paraformaldehyde in 0.1 M phosphate buffer and paraffin embedding.
In Vitro Culture System
HH10 and HH12 chick embryos were used for early and
late neural tube cultures, respectively. The embryos were
taken from the yolk sac and washed in Locke’s solution.
The neural tube region from the mesencephalon to the
1011
caudal level of the third somite pair was excised. The
somites and mesenchymal tissue were removed by placing
the neural tube region in a collagenase solution (0.5% in
Locke’s solution) for 10 min. Subsequently, the neural
tube region was washed with medium and transferred
onto a fibronectin-coated (20 ␮g/ml) coverslip in a 24-well
plate and grown in 1 mL Medium 199 containing 10% fetal
calf serum, 0.5% gentamicin, 1% penicillin/streptomycin,
1% ITS (Invitrogen Corporation, Paisley, UK). After 24 or
48 hr, the cultures were fixed for 30 min in 4% paraformaldehyde/0.1 M phosphate buffer and stored in 70% ethanol at 4°C before the early (n ⫽ 24) and late (n ⫽ 22)
neural tube explant cultures were immunohistochemically processed.
Immunohistochemistry
The experimental embryos were embedded in paraffin,
and serial sections (5 ␮m) were made. The fixed explant
cultures and alternate paraffin sections were immunohistochemically stained for muscle actin using the HHF35
antibody (DAKO, Glostrup, Denmark) (Tsukada et al.,
1987) and the HNK-1 epitope (Hybridoma Bank, Iowa
City, IA) (Abo and Balch, 1981) to mark muscle cells or
neural crest and differentiated nerve tissues (Poelmann et
al., 1998), respectively. Quail-derived cells were identified
using the QCPN antibody, a quail nuclear marker (Hybridoma Bank). Overnight incubations were performed
with the primary antibodies diluted (HHF35 1:1,000,
HNK-1 1:10, and QCPN 1:1 for sections; and HHF35 1:500
and HNK-1 1:50 for cultures) in PBS 0.05% Tween-20 and
1% chicken egg albumin. After the sections and cultures
were rinsed, they were incubated for 2 hr with peroxidaseconjugated rabbit anti-mouse antibodies (1:250, DAKO).
They were then washed, and a DAB-H2O2 procedure was
performed with 0.04% diaminobenzidine tetrahydrochloride, 0.006% H2O2 in 0.05 M Tris-maleic acid (pH 7.6) for
10 min. The sections and cultures were counterstained
with Mayer’s hematoxylin, dehydrated, and mounted in
Entellan.
RESULTS
Comparison of the Migration Patterns of the
Total Cardiac Neural Crest Cell Population and
the Late-Migrating Cardiac Neural Crest Cell
Population
Embryos were injected with a retrovirus containing the
lacZ marker gene into the neural tube lumen at HH9 –12.
Embryos injected at HH9 or HH10 formed the early-injected group, in which the lacZ gene of the retrovirus was
integrated into replicating neuroepithelial cells that gave
rise to the total population of cardiac neural crest cells.
Embryos injected at HH12 comprised the late-injected
group, in which the late-migrating cardiac neural crest
cells that leave the neural tube around HH12 were exclusively labeled.
The migration patterns of labeled cardiac neural crest
cells in the early- and late-injected groups were very similar at the first days (HH15/16, HH22, and HH26) following retroviral labeling. Both the early- (Fig. 1a) and lateinjected (Fig. 1e) embryos displayed lacZ-labeled cardiac
neural crest cells outside the dorsal side of the neural tube
and in the mesenchyme caudal to the otic placode. The
lacZ-labeled cells were observed in the circumpharyngeal
region around the dorsal aorta as strings of HNK-1 posi-
Fig. 1. Injection of lacZ retrovirus into the neural tube lumen followed
by ␤-galactosidase staining. a– d: Embryos injected at HH9 or HH10
survived until HH11, HH15, or HH30. a: Blue-labeled neural crest cells
(arrows) outside the neural tube at the level of the cardiac neural crest.
b: Labeled neural crest cells are located in the III (arrows) and IV pharyngeal arches. Other cells, supposedly of neural crest origin, stained
brown with the HNK-1 antibody. c: Labeled neural crest cells are located
in the proximal and distal parts (arrows) of the pharyngeal arch arteries.
d: High numbers of labeled neural crest cells are found in the condensed
mesenchyme (CM) of the aorticopulmonary septum. e– h: Embryos injected at HH12 survived until HH16, HH30, or HH33 to trace the latemigrating cardiac neural crest cells. e: Labeled neural crest cells are
located in the head mesenchyme cranial and caudal of the otic placode
(OP). f: Late-migrating cardiac neural crest cells just posterior to the otic
placode. Note that the blue cells are double-stained for HNK-1 (arrow).
g: Many blue cells in the proximal, actin-negative part of the pharyngeal
arch arteries III, IV, and VI. h: Blue cells at the peripheral, ventral side of
the aortic wall. Note the absence of lacZ-positive cells in the condensed
mesenchyme (CM) of the aorticopulmonary septum. i–m: Embryos that
were injected at HH10, and ablated at HH11 to trace the early-migrating
cardiac neural crest cells, survived until HH31. i: The labeled cells are
scattered over the pharyngeal arch arteries, and are concentrated in the
condensed mesenchyme and prongs of the aorticopulmonary septum
(arrows). j: The distal part of the left third pharyngeal arch artery has an
irregularly-shaped lumen and shows labeled early-migrating cardiac
neural crest cells. k: A segment of the vessel wall of the right sixth arch
artery is abnormally organized (between arrows). l: The labeled earlymigrating neural crest cells are evenly distributed over both the actinnegative and -positive parts of the right sixth arch artery (RVI). m: The
centrally located condensed mesenchyme (CM) of the aorticopulmonary
septum contains a large number of blue-stained early-migrating cardiac
neural crest cells. Bar ⫽ (j) 75 ␮m, and (a–i, k–m) 150 ␮m. Abbreviations:
Ao, aorta; CM, condensed mesenchyme; DAo, dorsal aortae; N, notochord; OP, otic placode; P, pulmonary trunk; R, rhombencephalon; S1,
somite 1; II, III, IV, VI, second, third, fourth, and sixth pharyngeal arch
arteries; L, left; R, right.
CARDIAC NEURAL CREST SUBPOPULATIONS
tive cells, and in the mesenchyme of the arches (Fig. 1b
and f).
However, at stage HH30 the patterns of the labeled
cardiac neural crest cells in the early- and late-injected
groups were strikingly different. In the first group of embryos, which were injected at HH9 or 10, the labeled
neural crest cells were scattered over the vessel walls of
the third, fourth, and sixth pharyngeal arch arteries (Fig.
1c) at HH30. The cells were observed both in the actinnegative proximal part of the pharyngeal arch arteries
close to the aortic sac region, and in the more distal,
actin-positive part of the pharyngeal arch arteries close to
the descending aorta (Fig. 1c). The cardiac neural crest
cells did not contribute to the descending aorta. The labeled neural crest cells were incorporated into the periendothelial layer, as well as the peripheral layers of the
vessel walls. Furthermore, the labeled cells contributed
intensely to the condensed mesenchyme and the prongs of
the aorticopulmonary septum (Fig. 1d). In contrast, the
second group of embryos injected at HH12 demonstrated
that the population of late-migrating cardiac neural crest
cells was located almost exclusively in the proximal part of
the third, fourth, and sixth pharyngeal arch arteries at
HH30 –HH33 (Fig. 1g). The position of the late-migrating
cardiac neural crest cells colocalized largely with the actin-negative (HHF35) boundaries at these stages of development. The late-migrating neural crest cells were not
limited to a specific position in the vessel wall, but were
encountered in both the periendothelial layer and the
more peripheral layers. The late-migrating cardiac neural
crest cells contributed to the medial side of the pulmonary
trunk and the ascending aorta, as well as to the ventral
side of the ascending aorta (Fig. 1h). The latter group of
positive cells extended with a low number of cells into the
outflow tract cushion tissue. Interestingly, the late-migrating cardiac neural crest cells were not found in the
prongs, and were nearly absent in the condensed mesenchyme of the aorticopulmonary septum (Fig. 1h). By subtracting the expression pattern of the late-migrating cardiac neural crest cells (injection HH12) from the
expression pattern of the total cardiac neural crest cell
population (injection HH9 –10), we expected the earlymigrating cardiac neural crest cells to migrate to the pharyngeal arch arteries and, more specifically, to the condensed mesenchyme of the aorticopulmonary septum.
Migration Pattern of the Early-Migrating
Cardiac Neural Crest Cell Population
We exclusively studied the early-migrating cardiac neural crest cells that leave the neural tube around HH10 by
injecting retrovirus into the neural tube lumen at HH10,
reincubating the egg, and subsequently ablating the latemigrating cardiac neural crest at HH11. At HH30 –31 the
distribution of the late-migrating cardiac neural crest and
the effect of the late-ablation on the heart and pharyngeal
arch arteries (Fig. 1i) were assessed. The ablation of the
late-migrating cardiac neural crest resulted in malformed
pharyngeal arch arteries III, IV, and VI with an irregular
lumen structure (Fig. 1j) and abnormal vessel walls in
which the layers were stacked abnormally (Fig. 1k). Furthermore, the proximal part of the left third arch artery
was hypoplastic. It must be stressed that the aorticopulmonary septum was formed normally in these late-ablated
embryos (Fig. 1m). The labeled early-migrating neural
crest cells were present in the pharyngeal arch mesen-
1013
chyme and scattered in the media of the third, fourth, and
sixth pharyngeal arch arteries. Cells were noted in the
periendothelial layer and the more peripheral layers of
the arch artery vessel walls in both the proximal parts
(which are actin-negative) and the more distal parts
(which are actin-positive) (Fig. 1l) of the arteries. Early
neural crest cells were found at the medial side of the
pulmonary trunk and ascending aorta immediately downstream of valve level. Furthermore, they contribute
heavily to the condensed mesenchyme of the aorticopulmonary septum (Fig. 1m), with a few cells scattered in the
prongs. The experimental procedure and the results are
summarized in Fig. 2a and b. It must be noted that the
differences in pharyngeal arch artery structure shown in
Fig. 2a and b are partly due to the difference in developmental stage (HH30 vs. HH31). However, the irregular
shape of the pharyngeal arch arteries and the hypoplasia
of the LIII were not related to the developmental stage;
rather, they were caused by the ablation of the late-cardiac neural crest.
In Vitro Cultures of Early and Late Neural
Tube Explants
We evaluated the intrinsic differences between the total
neural crest cell population and the late cardiac neural
crest cell population by culturing early (HH10) and late
(HH12) neural tube explants. In the first hours after the
explants were placed in culture medium, we observed
proliferating neuroepithelial cells (identified by their distinct morphology of compactly organized epithelial cells)
and migrating neural crest cells (recognizable by their
protrusions and web-shaped appearance). After 24 hr in
culture, the early and late explants all showed high numbers of neural crest cells. Staining for HNK-1 showed that
in the early-explant cultures almost all neural crest cells
were positive for HNK-1 (Fig. 3a), whereas in the lateexplant cultures a number of cells with the typical neural
crest cell morphology were negative for HNK-1 (Fig. 3e).
After 48 hr in culture, this difference was still present and
even more obvious. In early-explant cultures, most of the
neural crest cells stained positive for HNK-1 (Fig. 3b),
while this percentage was much lower in the late explants
(Fig. 3f). Early- and late-explant cultures were also
stained for HHF35 to display actin expression. On average, 20% of the neural crest cells in the early explants
showed actin staining (Fig. 3c and d). This is in contrast to
the late explants, in which up to 90% of the cells that
showed neural crest cell characteristics stained positive
for actin (Fig. 3g and h).
Heterochronic Transplantations
The cardiac neural crest of an HH12 quail donor was
transplanted into an HH10 chick embryo host in which
the cardiac neural crest had been ablated. At HH30 the
late-migrating cardiac neural crest cells were stained for
the quail marker QCPN, and were shown to be located in
the vessel walls of the third, fourth, and sixth pharyngeal
arch arteries (Fig. 4a). The cells were found in the proximal and distal parts of the pharyngeal arch arteries. Quail
cells were also noted in the nerve bundles surrounding the
vessels (Fig. 4a). Many quail cells were observed in the
condensed mesenchyme and the prongs of the aorticopulmonary septum at HH30 (Fig. 4b), as well as in the outflow tract cushions (Fig. 4c).
1014
BOOT ET AL.
Figures 2– 4.
CARDIAC NEURAL CREST SUBPOPULATIONS
1015
Differences in distribution patterns between early- and
late-migrating cardiac neural crest cells were clearly visible by HH30. The first difference noted was that a high
number of early-migrating cardiac neural crest cells were
located in the condensed mesenchyme and the prongs of
the aorticopulmonary septum, while the late-migrating
cells were nearly absent in the septum. Removal of the
late-migrating cardiac neural crest resulted in normal
outflow tract septation, which suggests that the late-migrating cardiac neural crest cells are dispensable in the
septation process, and the early-migrating cardiac neural
crest cells play an essential role in outflow tract septation.
The notion that cardiac neural crest cells can be divided
into two subpopulations may explain the results found in
previous ablation experiments. These experiments
showed that abnormal patterning of the aortic arch arteries was found in combination with normal cardiac outflow
tract development (Kirby et al., 1997), as well as in embryos with a combination of persistent truncus arteriosus
and relatively normal aortic arches (Nishibatake et al.,
1987). This demonstrates that pharyngeal arterial arch
and outflow tract morphogenesis must be considered as
two separate mechanisms. Our findings indicate that the
cardiac neural crest population should be separated into
two groups with specific migratory behavior.
The second interesting observation was that the earlymigrating cardiac neural crest cells were scattered over
the proximal and distal parts of the pharyngeal arch arteries (with the exception of the dorsal aortae). This is in
contrast to the late-migrating cardiac neural crest cells,
which were concentrated in the proximal, actin-negative
area of the pharyngeal arch arteries, close to the heart.
The periendothelial actin-staining pattern shifts at
around HH27–HH31 from the proximal part of the pharyngeal arch arteries to the distal part of the vessels
(Bergwerff et al., 1996). Subsequently, at around HH30,
elastin expression is initiated at the proximal part of the
pharyngeal arch arteries, followed by the lamellar actin
expression that is also initiated at the proximal part of the
pharyngeal arteries. The borderline between periendothelial actin-negative and -positive parts of the arteries nicely
coincides with the boundary describing the area of the
late-migrating cardiac neural crest cells. The contribution
of the late-migrating cardiac neural crest cells to the proximal part of the pharyngeal arch arteries may indicate a
role for these cells in remodeling of the pharyngeal arch
arteries. This role is supported by the results from the
embryos in which the late-migrating cardiac neural crest
was removed. These embryos showed hypoplasia, as well
as abnormal vessel wall integrity of the pharyngeal arch
arteries (especially in the proximal part of the arteries
close to the heart).
The third difference between the early- and late-migrating populations was observed in the wall of the aorta and
the pulmonary trunk just downstream of valve level. The
early-migrating cardiac neural crest cells were found at
the medial side of these vessels, as described previously in
quail-chick chimeras (Waldo et al., 1998). Surprisingly,
the late-migrating cardiac neural crest cells were also
contributing to the peripheral, ventral side of the base of
the aorta, extending with a few cells into the outflow tract
cushions. The contribution of cardiac neural crest cells to
both the medial and peripheral sides of the aorta just
above valve level has been described for Cx43-lacZ transgenic mice, but has not been reported in chick embryos
(Waldo et al., 1999).
Studies with various neural crest reporter mice (Cx43lacZ (Waldo et al., 1999), Pax3-Cre/LacZ (Epstein et al.,
2000), and Wnt1-Cre/R26R (Jiang et al., 2000)) have
shown differences in neural crest distribution patterns.
For example, neural crest cells were observed in the ectoderm covering the pharyngeal arches in the Cx43-lacZ
Fig. 2. The experimental procedures used to discriminate between
early- and late-migrating cardiac neural crest cells. a: Representation of
the experimental procedure: injection of lacZ-retrovirus into neural tube
lumen at HH10, ablation of the cardiac neural crest at HH11, and the
position of the early-migrating cardiac neural crest cells in the heart and
vessels at HH31. The labeled cells are located in the proximal and distal
parts of the pharyngeal arch arteries and the aorticopulmonary septum.
b: Representation of the experimental procedure: injection of lacZ-retrovirus into neural tube lumen at HH12 and the position of the latemigrating cardiac neural crest cells in the heart and vessels at HH30.
Note that labeled cells are nearly absent in the condensed mesenchyme
and prongs of the aorticopulmonary septum (yellow structure), and in the
distal part of the pharyngeal arch arteries. Abbreviations: III, IV, VI, third,
fourth, and sixth pharyngeal arch arteries; L, left; R, right.
Fig. 3. In vitro culture. a– d: Early (HH10) neural tube explants. e– h:
Late (HH12) neural tube explants. a: After 24 hr almost all neural crest
cells (NC) surrounding the early neural tube explant (E) stain positive for
HNK-1. b: The 48-hr culture of an early explant shows a high number of
HNK-1 positive cells. c: The early explant displays a low number of
actin-expressing cells after 48 hr in culture. d: The actin-expressing cells
of part c shown in higher magnification. e: Only a few neural crest (NC)
cells are positive for HNK-1; however, a high number of cells with the
neural crest morphology are negative for HNK-1 in this late-explant
culture after 24 hr (arrows). f: The number of HNK-1 expressing cells in
the late-explant culture is low after 48 hr g: Staining for actin displayed
high numbers of smooth muscle cells in the 48-hr late-explant cultures.
h: The actin-expressing cells in the late explants shown in higher magnification. Abbreviations: E, explant; N, neuroepithelial cells; NC, neural
crest cells. Bar ⫽ (d and h) 250 ␮m, and (a– c, e– g) 500 ␮m.
Fig. 4. Heterochronic transplantations. A quail HH12 late cardiac
neural crest was transplanted into an HH10 chick embryo and survived
to HH30. Late-migrating quail cardiac neural crest cells are observed in
(a) the vessel walls of the pharyngeal arch arteries (arrows) and nerve
tissue (arrowheads), as well as in (b) the condensed mesenchyme (CM)
of the aorticopulmonary septum (arrows) and (c) the outflow tract cushions (arrows). Bar ⫽ 150 ␮m. Abbreviations: Ao, aorta; P, pulmonary
trunk; III, IV, VI, third, fourth, and sixth pharyngeal arch arteries; L, left; R,
right.
DISCUSSION
The cardiac neural crest has been considered to be a
contiguous group of cells with full potential to contribute
to the various parts of the developing heart and pharyngeal arch arteries. As described in the present study, the
cardiac neural crest can be temporally separated into at
least two subpopulations. The early-migrating cardiac
neural crest cells leave the dorsal midline of the neural
tube at about HH10, and the late-migrating population
emigrates from the dorsal side of the neural tube about 6
hr later (HH12).
Differences in Fate and Function of Early- and
Late-Migrating Cardiac Neural Crest Cells
1016
BOOT ET AL.
mice, but they were not seen in the Wnt1-Cre/R26R neural
crest reporter mice. In Wnt1-Cre/R26R mice, an asymmetrical labeling pattern of neural crest cells was seen in the
pharyngeal arteries at E12.0, which has not been described in other neural crest cell reporter mice. The differences in neural crest staining in these mouse models
may be explained by a temporal-spatial expression of the
neural crest-related genes Wnt1, Cx43, and Pax3. We hypothesize that timing differences in gene expression could
separate the neural crest cell population in early- and
late-migrating neural crest cells, which target different
positions in the developing cardiovascular system.
Restricted Developmental Fate of LateMigrating Cardiac Neural Crest Cells
At HH30 the early-migrating cardiac neural crest cells
contributed to the outflow tract, and both the proximal
and distal parts of the pharyngeal arch arteries, whereas
the late-migrating cardiac neural crest cells appeared to
be restricted to the proximal part of the pharyngeal arch
arteries.
Differences in patterning of the early- and late-migrating cardiac neural crest cells may be due to intrinsic
differences. Studies by Newgreen and Thiery (1980)
showed differences in fibronectin expression between early- and late-migrating cranial and sacral crest cells. The
early crest cells were able to synthesize fibronectin, while
the later population was not, which suggests that the
neural crest cells became segregated over time. In our in
vitro experiments (this study) on the intrinsic differences
between early- and late-migrating cardiac neural crest
cells, we found that the number of late-migrating cardiac
neural crest cells that differentiated into smooth muscle
cells was higher than that found in early-neural tube
explant cultures. By combining our findings that 1) latemigrating cardiac neural crest cells predominantly differentiate into actin-expressing cells, and 2) late-migrating
cardiac neural crest cells migrate to a location within the
pharyngeal arch arteries where elastogenesis and lamellar actin expression are initiated, we could hypothesize
that late-migrating cardiac neural crest cells are involved
in the induction of lamellar actin expression. The in vitro
results suggested that the early- and late-migrating cardiac neural crest cells have intrinsic differences. However,
we questioned whether these differences should be considered as restrictions in developmental potential, as described for the trunk neural crest cells. Trunk neural crest
cultures and studies of DiI-labeled neural crest cells
(Artinger and Bronner-Fraser, 1992) have shown that early-migrating trunk neural crest cells differentiate into
many derivatives, including pigment cells, neurons, and
adrenergic cells, while late-migrating cells do not differentiate into adrenergic cells. This suggests that late-migrating trunk neural crest cells have a restricted developmental potential.
In our heterochronic transplantations at the cardiac
neural crest level, we showed that the late-migrating cardiac neural crest cells are not fully restricted in their
developmental potential. The late-migrating cardiac neural crest cells migrated in an early (HH10) environment,
in the absence of early-migrating cardiac neural crest
cells, to the condensed mesenchyme and prongs of the
aorticopulmonary septum, indicating the strong influences of the early-migrating cardiac neural crest cells and
the tissues surrounding the migrating cardiac neural crest
cells.
The effects of regional differences on the migratory behavior of individual neural crest cells and development of
cell depletion areas along the rostrocaudal axis in the
head have been demonstrated by in ovo time-lapse microscopy (Kulesa and Fraser, 1998). Furthermore, the importance of the spatial distribution of influential factors and
genes (e.g., Msx2 and Bmp4) has been shown in the neural
crest migration environment (Graham et al., 1994). Tbx1
is another gene that has been described as an environmental gene that affects neural crest migration. Tbx1 is
expressed in the pharyngeal endoderm surrounding the
pharyngeal arch arteries, and the Tbx1–/– mutant displays early growth defects of the pharyngeal arches in
combination with disturbed neural crest cell migration
(Vitelli et al., 2002a,b).
We postulate that the migration pathways of the latemigrating cardiac neural crest cells are affected by the
presence of the early-migrating cardiac neural crest cells.
The early-migrating cardiac neural crest cells may send
inhibitory or guiding signals to the late-migrating cardiac
neural crest cell population, thereby either restraining
them in the pharyngeal arch arteries, or preventing them
from migrating into the already established aorticopulmonary septum and diverting them to the lateral side of the
aorta and the outflow tract cushions. The hypothesis that
the early-migrating cells have guiding functions is supported by studies on the mesencephalic crest (Baker et al.,
1997), in which the late-migrating cranial neural crest
cells form neurons in cranial ganglia but not in cartilage of
the jaw. By ablating the early-migrating cells and heterochronic (late-to-early) quail-to-chick neural crest grafting,
it has been shown that the late-migrating cells are not
lineage-restricted and are able to contribute to the jaw
skeleton after ablation of the early-migrating cells. This
suggests that the absence of late-migrating cells in the
jaws may be regulated by the presence of earlier-migrating cells. The notion that neural crest cell groups influence
each other was also suggested by an analysis of the hindbrain, in which cell– cell contact was shown in streams of
neural crest cells that were bound for different branchial
arches (Kulesa and Fraser, 2000).
Our retroviral and ablation studies showed that the
differences in early- and late-migrating cardiac neural
crest cell patterning were not obvious before HH30, indicating that at around HH30 there is probably another
regulatory mechanism in the area of the pharyngeal arch
arteries and aorticopulmonary septum. It is conceivable
that the late-migrating cells receive their information in
the pharyngeal arches and close to the aorticopulmonary
septum.
In addition to environmental effects and signaling by
cell populations, the genes expressed by the cardiac neural
crest cells themselves play an important role in the differentiation and migration differences between the earlyand late-migrating cardiac neural crest cells. The neural
crest cells are probably separated into subpopulations by
the expression of different neural crest-related genes,
such as Pax3, Wnt1, BMP2, and BMP4. The idea that
cardiac neural crest subpopulations have their own specific gene expression patterns is supported by the differences in neural crest distribution shown in reporter mice
(Waldo et al., 1999; Epstein et al., 2000; Jiang et al., 2000).
Previous findings in Xenopus (Borchers et al., 2001) like-
CARDIAC NEURAL CREST SUBPOPULATIONS
wise showed expression patterns of Xcad-11, AP-2, twist,
and Snail that only partly overlapped in the migrating
cranial neural crest cell population. Borchers et al. (2001)
also postulated the existence of time-induced subpopulations in neural crest migration.
Finally, we have to keep in mind the extensive remodeling of the outflow tract. This involves elongation from
the secondary heart field (Waldo et al., 2001), and several
cell populations that define the tissue boundaries in this
complex region (Noden et al., 1995). The 6-hr time difference between the time points at which the early- and
late-migrating cardiac neural crest cells leave the neural
tube might result in the late-migrating cardiac neural
crest cells lagging behind. This would imply that earlymigrating cells reach the condensed mesenchyme and
prongs of aorticopulmonary septum, while most of the
late-migrating cells stay anterior to the elongating
conotruncus.
Implications of Time-Induced Subpopulations
of Cardiac Neural Crest Cells
The exact timing of the migratory cardiac neural crest
cell subpopulations is essential for normal cardiovascular
development. The finding that aorticopulmonary septum
formation depends on early-migrating (but not on latemigrating) cardiac neural crest cells in chick embryos may
indicate that the focus of the development of outflow tract
septation malformations, such as persistent truncus arteriosus, should be directed toward the early stages of cardiac neural crest cell migration. By narrowing the developmental range in which congenital heart diseases arise,
we may in the near future be able to exclude a number of
supposed candidate genes involved in neural crest-related
congenital heart diseases, such as DiGeorge syndrome
and velocardiofacial syndrome.
ACKNOWLEDGMENTS
We thank Kim van der Heiden for technical assistance.
The artwork and photographic contributions by Ron
Slagter, Bas Blankenvoort, and Jan Lens were indispensable. The retroviral construct was kindly provided by Dr.
Takashi Mikawa.
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