332
EUResearch Advances Series
Role of Neural Crest in
Congenital Heart Disease
Margaret L. Kirby, PhD, and Karen L. Waldo, MS
Historical Background
In the preface for his monograph on neural crest in
1950, Horstadius' wrote, "Most text-books of embry-
ology or anatomy make only passing mention of the
neural crest as the source of origin of one or two
structures in the vertebrate body. Few books devote
even as much as a page to this interesting structure."
Although the passage of 40 years has seen a revolu-
tion in our understanding of the neural crest in basic
and clinical sciences, Horstadius' statement is still
applicable to the paucity of information about neural
crest in textbooks. Unfortunately, among scientists
and clinicians not directly involved in neural crest
research, this has led to almost profound ignorance
about the neural crest and its role in development.
The neural crest is an unusual structure that
appears early in development and has only a tran-
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sient existence because the cells of the neural crest
quickly migrate throughout the body and differen-
tiate into other cell types. Its transience and wide-
spread migration have made neural crest derivatives
a technical challenge to study. Because of these
difficulties, knowledge of neural crest derivatives
progressed somewhat haltingly through the last half
of the 19th century and the first half of the 20th
century, dependent on development of novel tech-
niques with which to study this labile population of
cells. In the past 40 years, with the availability of
tissue culture, autoradiography, interspecies trans-
plantation, immunohistochemistry, and in situ hybrid-
ization, we have begun to understand much more
about neural crest derivatives.2
The neural crest has been recognized primarily for
its contribution to the peripheral nervous system,
although the largest portion of Horstadius' 1950
monograph1 is devoted to non-neural derivatives of
the neural crest in the-vertebrate head and branchial
region. In 1888, Kastschenko first claimed that some
mesenchyme of the head originated from the neural
crest.' Platt (1893-1897) showed that cartilage of the
visceral arches and dentine of the teeth were derived
From the Department of Anatomy, Medical College of Georgia,
Augusta.
Supported by Public Health Service grant HL-36059.
Address for correspondence: Margaret L. Kirby, PhD, Depart-
ment of Anatomy, Medical College of Georgia, Augusta, GA
30912-2000.
Received December 12, 1989; revision accepted April 24, 1990.
from neural crest ectoderm.1 She proposed the term
"mesectoderm" for mesenchyme that originates from
the ectodermal rather than the mesodermal germ
layer. This term has been revised to "ectomesen-
chyme" to emphasize the origin of the mesenchyme
from ectoderm rather than its usual source.' Under
normal circumstances, we know of no source of
ectomesenchyme other than the neural crest; how-
ever, as will be discussed below, when the neural
crest is removed, other areas of the ectoderm might
have the capability of producing ectomesenchyme.
The importance of neural crest in the cardiovascu-
lar system was first documented by Drs. Christine Le
Lievre and Nicole Le Douarin in 1975.3,4 These
investigators transplanted quail neural crest into
chick embryos and discovered that the entire muscu-
loconnective tissue wall of the large arteries arising
from the heart in the chick was made of ectomesen-
chymal cells of quail (neural crest) origin. They also
noted that "the transition zone between the bulbus
arteriosus and the aortic trunks...contains a mixture of
quail and host (neural crest and non-neural crest)
cells." Rychters and Thompson and Fitzharris6 also
presaged the discovery that neural crest is involved in
development of the cardiac outflow septation. Rychter5
noted that pigment placed between the third and
fourth pharyngeal arches migrated into the base of the
aorta and pulmonary trunk in the aorticopulmonary
septum. Thompson and Fitzharris6 showed the dual
origin of mesenchymal cells in the developing outflow
tract. One population of cells was derived from the
endocardium, and a second population migrated into
the outflow tract from the pharyngeal region.
The relation between neural crest and outflow
tract septation became clear when various parts of
the cranial premigratory neural crest were ablated in
chick embryos.7 This proved to be the critical step in
understanding the significance of the presence of
these cells in the outflow tract, in that removal of the
neural crest resulted in outflow tract malformations.7
This relation has been explored more thoroughly in
the last 7 years, and the findings will be reviewed
below. Practically all of the work on neural crest has
been performed on the chick embryo for three rea-
sons: The embryo is accessible for surgery early in
development; it has a short developmental period of
21 days; and interspecies transplantation can be
 Kirby and Waldo Neural Crest and Heart Development 333

IJ'
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FIGURE 1. Diagrammatic representation of the neural crest highlighting the region of the cardiac neural crest. Division between
trunk and cranial neural crest is at somite 5. Cranial neural crest has ectomesenchymal cells, whereas trunk neural crest has few
of these cells. The cardiac area is located between the midotic placode and somite 3. This area of neural crest migrates into
pharyngeal arches 3, 4, and 6 as designated.

performed from the Japanese quail embryo, which
has a distinctive nuclear marker that makes it easy to
trace after transplantation.
Cardiac Neural Crest
Normal Distribution of Neural Crest Cells
in the Heart
The neural crest is divided into two major regions
known as cranial and trunk neural crest (Figure 1).
The cranial neural crest extends from the mid-
diencephalon to the caudal limit of somite 5, whereas
the trunk neural crest proceeds from somite 5 to the
caudal extent of the embryo (Figure 1). The region of
the cranial neural fold between the midotic placode
and the caudal limit of somite 3 generates the neural
crest cells that migrate into the cardiac outflow tract.
This region of neural crest has been called "cardiac
neural crest" for convenience. The largest population
of neural crest cells in the outflow tract is derived
from the region of the neural fold that will populate
pharyngeal arch 4.8 The cardiac neural crest migrates
from the neural fold into pharyngeal arches 3, 4, and
6.9 In the pharyngeal arches, the cells derived from
the neural crest provide support for the endothelium
of the aortic arch arteries.10 Some cells migrate from
the pharyngeal arches into the outflow tract where
they form the aorticopulmonary septum and popu-
late the truncal folds (Figure 2). The neural crest-
derived ectomesenchymal cells are arranged in ven-
tral and dorsal columns in the truncal folds that are
elongations from the aorticopulmonary septum."1
Late in development, the ectomesenchymal cells are
located between the proximal aorta and pulmonary
trunk." A few ectomesenchymal cells are scattered
in the cusps of the arterial valves" (Kirby, unpub-
lished observation). The caudal extent of migration
of neural crest-derived ectomesenchymal cells is
unclear.8 Downstream from the semilunar valves, the
ectomesenchyme forms smooth muscle of the tunica
media of the large arteries derived from the aortic
arch arteries (Figure 2).4 At the same time, ectomes-
enchymal cells interact with the pharyngeal endo-
derm in formation of the other derivatives of the
pharyngeal apparatus such as the thymus and the
parathyroid and thyroid glands (Figure 2).2
 334 Circulation Vol 82, No 2 August 1990
To head
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B Parathyroids
That the cardiac neural crest is a unique popula-
tion of cells was shown recently. When the cardiac
neural crest is removed and replaced by mesenceph-
alic (cranial) or trunk (caudal) neural crest (Figure
3), persistent truncus arteriosus (nonseptation of the
outflow tract) results.12 This is a surprising result in
that other areas of the neural crest have been pre-
sumed to be plastic. This indicates that the cardiac
neural crest is predetermined early in development.
Noden'3 also showed early predetermination of the
cranial neural crest. When he replaced presumptive
arch 3 neural crest with arch 2 neural crest, he found
that arch 2 derivatives were repeated in the arch 3
region.13 This finding prompted Noden to suggest
that the ectomesenchymal derivatives of the cranial
neural crest may direct patterning in the head.
The addition of mesencephalic neural crest to the
cardiac neural crest also results in persistent truncus
FIGURE 2. Panel A: Diagram illustrating cranial neural
crest migratory pathway through the pharyngeal region.
Some of neural crest cells in pharyngeal arches 3, 4, and 6
continue their migration from the pharyngeal region into
the outflow tract of the developing heart where they partic-
ipate in the formation of the aorticopulmonary and truncal
septa. Panel B: Diagram illustrating the complex contribu-
tion of the neural crest to the pharyngeal apparatus and
related structures (right) and the outflow tract (left). AA,
aortic arch; DOA, dorsal aorta.
Support and Maintenace of
Wall of Aortic Arch Arteries
Ectoderm
lesenchyme
~4~Endothelium
Thymus Thyroid and
arteriosus (Figure 3), whereas heart development
proceeds normally after addition of trunk to cardiac
neural crest.12 This indicates that the mesencephalic
neural crest may actually interfere with the normal
development of the cardiac neural crest during out-
flow septation.
In another series of experiments,14 ectomesen-
chyme was shown to be generated from the nodose
placode after removal of the cardiac neural crest
(Figure 4). The placodes are a series of ectodermal
thickenings in the head region of the embryo that
generate portions of the sensory ganglia of the head
and viscera. One of the most prominent of these is
the otic placode that generates the vestibulocochlear
ganglion and portions of the inner ear. The nodose
placode is immediately caudal to the otic placode and
generates the neurons that carry sensory information
from the thoracic and abdominal viscera to the brain
 Kirby and Waldo Neural Crest and Heart Development 335

Addition Neural Crest Replacement

PTA PTA

FIGURE 3. Diagram illustrating the lack ofplasticity of other areas of the neural crest in replacing the cardiac neural crest. In the
experiment shown, mesencephalic or trunk neural crest was used to replace or augmnent the cardiac neural crest. In the replacement
experiment, the cardiac neural crest from the chick host is removed and discarded, and then the quail or donor neural crest is grafted
into the same site. Neither trunk nor mesencephalic neural crest was capable of replacing the cardiac neural crest as evidenced by
the resulting persistent truncus arteriosus (PTA) after cardiac neural crest replacement. Addition of mesencephalic neural crest to
the cardiac neural crest also resulted in persistent truncus arteriosus indicating that the mesencephalic neural crest actually interferes
with the cardiac neural crest in outflow tract septation.

stem. Under ordinary circumstances, the nodose pla- entire cardiac neural crest is removed. In this para-
code has no non-neuronal derivatives. In the absence of digm, the persistent truncus arteriosus can take any
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the cardiac neural crest, the ectomesenchyme produced
by the nodose placode migrates to the aortic sac and
the truncal cushions, but it is not capable of causing
outflow septation. The cells derived from the nodose
placode mimic the cells derived from the neural crest in
that they ultimately assume smooth muscle phenotypes
in the walls of the great arteries.
The fact that these alternate populations of cells
are not capable of supporting outflow tract septation
strongly supports the uniqueness of the ectomesen-
chymal cell population in the cardiac neural crest.
Neural Crest-Related Malforznations
To appreciate the effects of neural crest ablation
completely, it is important to understand that the early
studies involved removal of the neural crest at 24-30
hours of incubation followed by reincubation until days
8-11 of incubation. At that point, the hearts are large
enough to analyze macroscopically.1516 The embryos
that survived until the time of analysis represent a
self-selected population because only a fraction of the
operated embryos actually lives to days 8-11. This same
situation is true clinically where the infants cared for by
pediatric cardiologists constitute a selected population;
that is, the embryos or fetuses that are able to compen-
sate for their defects live to the time of analysis or birth.
Outflow tract. Ablation of the premigratory cardiac
neural crest results in a variety of malformations of
the heart and great vessels (Figure 5), depending on
the extent of the ablation.1-17 For example, persis-
tent truncus arteriosus results very predictably if the
of a number of morphological manifestations. How-
ever, an obligatory component for the positive iden-
tification of persistent truncus arteriosus has been
the presence of a single outflow valve. This single
valve typically has four to six cusps. The presence or
absence of the aorticopulmonary septum appears to
depend on the completeness of the cardiac neural
crest ablation. On the other hand, a ventricular
septal defect is always a component of this defect.16
The position of the single outflow vessel with respect
to the ventricles is variable; that is, it can arise
entirely from the right or left ventricle or can straddle
the ventricular septum. In the most common config-
uration, the vessel arises entirely from the right
ventricle. This does not appear to correlate with the
presence of any other morphological abnormalities,
such as nonpersistence of the right or left aortic arch
arteries.'6
Ablation of a smaller area within the cardiac neural
crest, or of cranial neural crest outside the cardiac
area, results in a spectrum of defects that have been
classified under the generalized title of dextroposed
aorta.'6 These malformations include double outlet
right ventricle, tetralogy of Fallot and Eisenmenger's
complex. Transposition of the great arteries occurs
infrequently after neural crest ablation.
Aortic arch arteries. Associated with each of these
cardiac defects is a variable occurrence of interrupted
aortic arch, or anomalies of the other great arteries,
including persistence of some vessels that should oth-
erwise disappear (Figure 5). In the chicken, the defin-
 336 Circulation Vol 82, No 2, August 1990

and have not been seen in the detailed morphological

studies on days 8-11 of incubation.15-17
Inflow anomalies. Neural crest ablation also causes
changes in atrioventricular alignment (Figure 5). The
inflow tract anomalies that have been seen include
tricuspid atresia, tricuspid stenosis, straddling of the
Otic Cardiac Outflow tricuspid valve, and double inlet left ventricle. Atrio-
Placode
ventricular septal defects occur infrequently after
Nodose
Placode
-
i- Cardiac neural crest ablation.16 In the neural crest model of
N.C. heart malformations, there is an obligatory relation
between inflow and outflow anomalies. Chick embryos
with inflow anomalies caused by ablation of the neural
crest always have coexisting persistent truncus arterio-
LL-
sus or one of the dextroposed aorta anomalies. This is
quite different from the occurrence of these defects in
humans where outflow tract anomalies very rarely
appear with inflow anomalies. On the other hand,
outflow anomalies in the chick occur most commonly
with no signs of inflow anomalies.16 Atrial anomalies
A are very difficult to analyze in chick embryos, and a
systematic study of the atria after neural crest ablation
has not been performed to date. However, our impres-
sion is that the atrial septation and other details of
atrial morphology are normal.
Veins. Even though there are severe defects in the
heart and arteries derived from the aortic arch
arteries, neither systemic nor pulmonary venous
Persistent truncus
anomalies occur after cardiac neural crest ablation
arteriosus (Figure 5).19
Cardiac
Noncardiovascular defects. Removal of the cardiac
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Placode N.C. neural crest affects development of all the structures

removed ama
aU.
R neural crest in the pharyngeal arches are affected.20
A~4
B M
FIGURE 4. Diagram representing the normal contribution of
the cardiac neural crest (N.C.) to the outflow tract (panel A)
and the situation when the cardiac neural crest is removed
(panel B). In this case, cells of the nodose placode migrate
into the areas where cardiac neural crest should be. These cells
are not capable of causing cardiac outflow septation but do
reinforce the walls of the great arteries to some extent.
itive aorta (fourth arch artery) is on the right rather
than on the left as it is in humans. After neural crest
ablation, the aorta on the left side occasionally persists
so that there are bilateral aortic arches or persisting
left-sided aorta with disappearance of the right-sided
aorta. The carotid vessels (third arch arteries) are also
frequently interrupted. In very severe cases, there is a
single persisting vessel that connects the heart with the
dorsal aorta.'8 These embryos die early in development
in the pharyngeal arches for which it is destined
(Figure 5). Hence, the thymus, parathyroid, and
thyroid glands, all of which obtain their stroma from
The neural crest migrating into arches 1 and 2, that
will develop into the lower face and upper neck, is
cranial to the cardiac region of neural crest13; thus,
facial structures are not usually affected by cardiac
neural crest removal.
Parasympathetic innervation. Although not men-
tioned previously, the cardiac parasympathetic inner-
vation is also derived from the cardiac neural crest.
One may expect a deficiency in parasympathetic
innervation of the heart after ablation of the cardiac
neural crest. This, however, is not the case because
cells from the nodose placodes migrate into the heart
and reconstitute the cholinergic cardiac plexus in the
absence of the cardiac neural crest.21
Early Changes in Cardiovascular Development
Induced by Neural Crest Ablation
Morphological Changes
Changes in heart and aortic arch artery morphol-
ogy occur while the heart is still in the looped tube
stage, long before septation of the outflow tract
normally occurs.22 Distinct changes are evident by
the third day of incubation, and these changes persist
in a manner that precludes formation of a normal
cardiovascular system.
 Kirby and Waldo Neural Crest and Heart Development 337

Cardiac lnflow Cardiac Neural Cardiac Outflow

Anomalies Crest Ablation Anomalies

Double Inlet
left ven(
MR Tricuspid
)
atresia Tetralogy of
Fal_ot
Eisenmengers
corplex

Straddling tricuspid
valve Persistent truncus Transpositon of the Double outet
artediosus great vessels right ventricle

Aortic Arch Arteries Non-Cardnovascular

Anomalies Anomalies
Veins
(Normal)

Interruption of Double aortic Thymus

the aorta arc
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Thyroid &
Variable absence of Left aortic parathyroids
the carotid arteres arch
FIGURE 5. Diagram illustrating the cardiovascular and noncardiovascular malformations induced by removal of the cardiac
neural crest.

Cardiac tube. At this stage in development, conotrun-
cal shape is altered even though the heart is a looped
tube and septation has not yet begun.2Z23 Leatherbury
et a23 further noted dilation of the primitive ventricle
and incomplete looping of the cardiac tube.
Aortic arch arteries. The quantity, as well as the
distribution, of mesenchyme is altered in the pha-
ryngeal arches surrounding the aortic arch arteries.
In normal embryos, the endothelial tubes of the
aortic arch arteries in the pharyngeal arches are
centrally located and completely ensheathed in
mesenchyme. In experimental animals, a large por-
tion of the endothelium of these vessels is directly
apposed to pharyngeal epithelium, without inter-
vening mesenchyme.10 At this time, the mechanism
by which reduced ectomesenchyme in the pharyn-
geal arches leads to abnormal development of the
arch vessels is not known.
Functional Changes
Functional, as well as morphological, changes
appear early in development subsequent to neural
crest removal. Microcinephotography has been used
to study ventricular function in chick embryos at
stage 18 with the result that a number of functional
deficiencies have been noted at this early stage.23
These include decreased emptying of the bulbus
cordis, incompetent truncal cushions, and lack of
blood flow in the fourth right aortic arch artery with
increased blood flow in the third right aortic arch
artery. At the same time, all of the indexes of
contractility, including shortening fraction, wall veloc-
ity, and ejection fraction are markedly depressed.
However, despite these functional changes, cardiac
output is normal because of the ventricular dilation.
This suggests that an embryo in the process of devel-
oping a heart defect is able to compensate for the
insult by dilation of the ventricle.
In a more recent study with a slightly altered
design, Tomita et a124 demonstrated some important
factors in the self-selection of experimental embryos
between days 3 and 11 of incubation. Heart rate,
ejection fraction, stroke volume, and cardiac output
were measured in all embryos on day 3 of incubation.
 338 Circulation Vol 82, No 2, August 1990

Embryos that lived until day 11 had significantly ELASTOGENESIS IN THE CHICK EMBRYO
higher ejection fraction, stroke volume, and cardiac
output on day 3 of incubation than embryos that did
not live to day 11.
These findings demonstrate that functional and
morphological changes occur before the time when
the predicted structural defects of septation would be
apparent in the heart. Cardiac function may be
maintained during development at the expense of
morphology. There may be physiological compensa-
tions in response to early morphological alterations
that maintain cardiac function within the normal
range.25 Leatherbury et a123 further suggest that the
mechanism of compensation for depressed contrac-
tility is ventricular dilation. This in turn interferes
with complete looping of the cardiac tube, which
hinders normal alignment of the developing inflow
and outflow tracts. The spectra of dextroposed aorta
and atrioventricular malalignment, discussed above,
are a result of these alterations.
Blood pressure has also been measured in the ven-
tricle and dorsal aorta.25 With the servonull technique
to measure intraventricular pressure in stage 18 chick
embryos, experimental animals displayed no significant
change in heart rate, peak ventricular pressure, or
end-diastolic pressure compared with controls. FIGURE 6. Flow chart illustrating the site of initiation of
Changes in Calcium Current elastogenesis in the conus cordis at stages 23-24 by the
ectomesenchyme and then the propagation of elastogenesis
Calcium currents have been assessed at 11 days of downstream along the developing great arteries.
incubation in hearts with persistent truncus arterio-
sus.26 Compared with normal hearts, hearts with per-
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sistent truncus arteriosus were enlarged (26% greater
ventricle to whole embryo weight) and had a twofold
reduction in the peak magnitude of the L-type or
dihydropyridine-sensitive calcium current. At the same
time, there was not a concomitant reduction in the
number of L channels detected in radioreceptor assays.
This indicates that protein synthesis is not affected in
production of the L channel but that there is a differ-
ence in post-translational modification of the channel
protein in experimental and control embryos.26 The
change could perhaps be due to a difference in the
phosphorylation status of the channel in myocardium
developing under the hemodynamic stresses outlined
above. Further exploration of the changes in the sub-
strates of electrical activity in the myocardium should
help cardiologists and surgeons in dealing with patients
who have congenital heart defects. Although the stud-
ies have not yet been performed, ventricular character-
istics at 3 days of development23 would lead to the
prediction that the same depression of calcium current
is present much earlier in development.
Extracellular Matrix
Rosenquist and colleagues27- 30 attempted to deter-
mine what factors are unique to the cardiac neural
crest. Elastogenesis is first found in foci at the
interface between the myocardial cuff and the ecto-
mesenchyme of neural crest origin on day 4 of
incubation (Figure 6). The foci coalesce as develop-
ment proceeds. Elastogenesis is then propagated
downstream along the developing great arteries in an
orderly proximodistal sequence by the ectomesen-
chyme (Figure 6). Ultimately, a laminated elastic
matrix continuum is present in the tunica media of all
of the elastic vessels.27 Rosenquist et a127 hypothe-
sized that elastogenesis is a critical event in outflow
septation.
When the cardiac neural crest is removed, the rate
of downstream propagation of the elastic matrix in
the great arteries is retarded, and the spatial config-
uration of the elastic matrix in older embryos is
disordered. At the same time, collagens I and III,
which are normally highly ordered in the walls of the
great arteries, are coarsened and lack any distinct
spatial order in arteries lacking the cardiac neural
crest.28,29
In addition to producing an elastic matrix, the mes-
enchymal cells of the outflow septation complex express
smooth muscle a-actin. Beall and Rosenquist30 hypoth-
esized that these cells may play a contractile role in
outflow septation. Whether or not these factors are
important in outflow septation, elastin, collagen I and
III, and smooth muscle a-actin probably all play a role
in maintaining hemodynamic characteristics of the nor-
mally developing cardiovascular system. From the
hemodynamic studies on early embryos presented
above, we know that these functional parameters are
critical in determining the final phenotype of a cardio-
vascular malformation.
 Kirby and Waldo Neural Crest and Heart Development
Application in Humans
All of the heart defects described in chick embryos
after neural crest ablation have also been seen in
human infants. In the chick embryo, removal of the
cardiac neural crest also affects other structures to
which that area of neural crest contributes. Isolated
heart defects occur rarely, if ever, without changes in
thymus, parathyroid, and thyroid glands. It is too
early to say whether this is the case in humans. The
most obvious correlation in humans is the DiGeorge
syndrome. Typically, the DiGeorge syndrome
includes hypocalcemia, defective thymic-dependent
cellular immunity, anomalies of the face, ears, and
palate, and congenital cardiovascular malformations.
Van Mierop and Kutsche31 suggested that the
DiGeorge syndrome is indeed due to abnormal
development involving the neural crest. The interest-
ing observation by Wells et a132 that the thyroid
cartilage, a derivative of the fourth pharyngeal arch,
is hypoplastic in patients with DiGeorge syndrome or
tetralogy of Fallot indicates that other outflow anom-
alies may be associated with abnormalities of the
neural crest. Sulik et a133 suggested that fetal alcohol
syndrome should also be included among syndromes
in which the cranial neural crest is damaged. Fur-
thermore, she proposes that some cases of DiGeorge
syndrome can be directly attributed to ethanol or its
metabolites.
Other syndromes have been suggested as linking
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neural crest abnormalities with heart defects. These
include the CHARGE association34 and congenital
neuroblastoma with congenital heart disease.35
Retinoic acid embryopathy has also been attributed
to damage to the neural crest in humans and
animals.36-38
The evidence has become very strong that neural
crest is involved in a number of syndromes and is
affected by certain teratogens. Our current concern is
to determine what role, if any, neural crest plays in
generation of congenital heart defects that are not
part of a syndrome. A recent study by Todd and
Todd39 showed an increase in the rate of otitis media
associated with outflow tract anomalies compared
with other heart defects. The outflow defects
included transposition of the great arteries, tetralogy
of Fallot, and aortic stenosis. They suggested that
patients who are prone to otitis media have architec-
tural differences in their eustachian tubes. The eusta-
chian tube develops from pharyngeal arches 1 and 2
and is dependent on ectomesenchyme in those
arches: hence, the suggested association between
outflow tract defects and middle ear infections. An
alternate explanation is that resistance to infection in
these children is not adequate because of compro-
mised thymic-mediated immunity.40,41 It is also
becoming apparent that children with heart defects
have mild facial dysmorphogeneses.41-44
It is important to document changes in neural
crest-associated tissues in any case of heart defect to
339
begin to understand which of these defects is due to
malfunction of the neural crest during development.
Role of Neural Crest in Heart Development
Our current understanding of the cardiac neural
crest indicates that it plays a complex role in heart
development with at least two major components.
The simpler of these seems to be its participation in
outflow septation. Although the cellular and molec-
ular events that the neural crest directs have not been
elucidated, it is clear that the presence of a certain
number of these cells in the outflow tract is essential
for septation to occur. Below this critical number,
septation does not occur. Because the major defects
in DiGeorge syndrome are persistent truncus arteri-
osus and interrupted aortic arch type B, this syn-
drome can probably be attributed to a lack of the
critical number of cardiac neural crest cells in pha-
ryngeal apparatus and the outflow tract. This could
be caused by a failure of proper migration into the
area or by excessive cell death.
There are still a number of questions with regard
to the exact distribution of the neural crest in the
heart. These include the possibility and extent of
cardiac neural crest involvement with the sinoatrial
and atrioventricular nodes and conduction system,
the coronary arterial system, and the formation of the
semilunar valves.
The role of the cardiac neural crest that is more
difficult to understand is the one it plays in the
pharyngeal arch region. The interplay among all the
elements of the pharyngeal arches including endo-
thelium of the aortic arch arteries, endoderm of the
pharyngeal pouch, ectoderm of the pharyngeal
grooves and arches, and, finally, mesenchyme of
non-neural crest origin, must be quite complex. A
combination of interactions must determine which
arch arteries persist and which disappear. Perhaps
the same or a completely different set of interactions
determines the hemodynamic characteristics of indi-
vidual arch arteries that then influence the final
normal or abnormal phenotype of the developing
heart. The extracellular matrix probably plays a
major role in this developmental regulation; how-
ever, many other factors may be involved and are yet
to be elucidated.
References
1. Horstadius S: The Neural Crest: Its Properties and Derivatives in
the Light ofExperimental Research. London, Oxford University
Press, 1950
2. Le Douarin NM: The Neural Crest. Cambridge, Mass, Cam-
bridge University Press, 1981
3. LeLievre CS, Le Douarin NM: Mesenchymal derivatives of
the neural crest: Analysis of chimaeric quail and chick
embryos. J Embryol Exp Morphol 1975;34:125-154
4. Le Douarin NM, Teillet M-AM: Experimental analysis of the
migration and differentiation of neurectodermal mesenchymal
derivatives, using a biological cell marking technique. Dev Biol
1984;41:162-184
5. Rychter Z: Analysis of relations between aortic arches and
aorticopulmonary septation,. in Rosenquist GC, Bergsma D
 340 Circulation Vol 82, No 2, August 1990
(eds): Morphogenesis and Malformation of the Cardiovascular
System. New York, Alan R Liss, 1978, pp 443-448
6. Thompson RP, Fitzharris TP: Morphogenesis of the truncus
arteriosus of the chick embryo heart: The formation and
migration of mesenchymal tissue. Am JAnat 1979;154:545-555
7. Kirby ML, Gale TF, Stewart DE: Neural crest cells contribute
to aorticopulmonary septation. Science 1983;220:1059-1061
8. Phillips MT, Kirby ML, Forbes G: Analysis of cranial neural
crest distribution in the developing heart using quail-chick
chimeras. Circ Res 1987;60:27-30
9. Miyagawa ST, Waldo K, Tomita H, Kirby ML: Migration of
cardiac neural crest cells: A temporospatial study in early
quail-chick chimeras. NYAcad Sci 1989;588:427-429
10. Bockman DE, Redmond ME, Kirby ML: Alteration of early
vascular development after ablation of cranial neural crest.
Anat Rec 1989;225:209-217
11. Sumida H, Akimoto N, Nakamura H: Distribution of the
neural crest cells in the heart of birds: A three dimensional
analysis. Anat Embryol (Berl) 1989;180:29-35
12. Kirby ML: Plasticity and predetermination of mesencephalic
and trunk neural crest transplanted into the region of the
cardiac neural crest. Dev Biol 1989;134:401-412
13. Noden DM: The role of the neural crest in patterning of avian
cranial skeletal, connective, and muscle tissues. Dev Biol
1983;96:144-165
14. Kirby ML: Nodose placode provides ectomesenchyme to the
developing heart in the absence of cardiac neural crest. Cell
Tissue Res 1988;252:17-22
15. Besson WT III, Kirby ML, Van Mierop LHS, Teabeaut JR II:
Effects of cardiac neural crest lesion size at various embryonic
ages on incidence and type of cardiac defects. Circulation
1986;73:360-364
16. Nishibatake M. Kirby ML, Van Mierop LHS: Pathogenesis of
persistent truncus arteriosus and dextroposed aorta in the
chick embryo after neural crest ablation. Circulation 1987;
Downloaded from http://ahajournals.org by on March 13, 2020
75:255-264
17. Kirby ML, Turnage KL, Hays BM: Characterization of
conotruncal malformations following ablation of "cardiac"
neural crest. Anat Rec 1985;213:87-93
18. Rosenquist TH, Kirby ML, Van Mierop LHS: Solitary aortic
arch artery. A result of surgical ablation of cardiac neural crest
and nodose placode in the avian embryo. Circulation 1989;
80:1469-1475
19. Phillips MT, Waldo KL, Kirby ML: Neural crest ablation does
not alter pulmonary vein development in the chick embryo.
Anat Rec 1989;223:292-298
20. Bockman DE, Kirby ML: Dependence of thymus development
on derivatives of the neural crest. Science 1984;223:498-500
21. Kirby ML: Nodose placode contributes autonomic neurons to
the heart in the absence of cardiac neural crest. J Neurosci
1988;8:1089-1095
22. Bockman DE, Redmond ME, Waldo KL, Davis H, Kirby ML:
Effect of neural crest ablation on development of the heart
and arch arteries in the chick. Am JAnat 1987;180:332-341
23. Leatherbury L, Gauldin HE, Waldo K, Kirby ML: Microcine-
photography of the developing heart in neural crest-ablated
chick embryos. Circulation 1990;81:1047-1057
24. Tomita H, Kirby ML, Leatherbury L: Hemodynamic changes
precede development of persistent truncus arteriosus in neural
crest-ablated chick embryos. JAm Coll Cardiol 1990;15:131A
25. Jackson WF, Gauldin H, Tomita H, Leatherbury L: Neural
crest ablation does not alter ventricular pressure or estimated
cardiac output despite altered morphology. Ann N YAcad Sci
1989 (in press)
26. Creazzo TL: Reduced L type calcium current in the embryonic
chick heart with persistent truncus arteriosus. Circ Res 1990 (in
press)
27. Rosenquist TH, McCoy JR, Waldo KL, Kirby ML: Origin and
propagation of elastogenesis in the developing cardiovascular
system. Anat Rec 1988;221:860-871
28. Rosenquist TH, Modis L: Spatial disorder of interstitial
collagens in the great vessels, associated with congenital heart
defects. Anat Rec 1989 (in press)
29. Rosenquist TH, Beall AC, Modis L, Fishman R: Impaired
elastic matrix development in the great arteries after ablation
of the cardiac neural crest. Anat Rec 1989;226:347-359
30. Beall AC, Rosenquist TH: Smooth muscle cells of neural crest
origin form the aorticopulmonary septum in the avian embryo.
Anat Rec 1989;226:360-366
31. Van Mierop LHS, Kutsche LM: Cardiovascular anomalies in
DiGeorge syndrome and importance of neural crest as a
possible pathogenetic factor. Am J Cardiol 1986;58:133-137
32. Wells TR, Landing BH, Galliani CA, Thomas RA: Abnormal
growth of the thyroid cartilage in the DiGeorge Syndrome.
Pediatr Pathol 1986;6:209-225
33. Sulik KK, Johnston MC, Daft PA, Russell WE, Dehart DB: Fetal
alcohol syndrome and DiGeorge anomaly: Critical ethanol expo-
sure periods for craniofacial malformations as illustrated in an
animal model. Am J Med Genet 1986;2(suppl):97-112
34. Siebert JR, Graham JM, MacDonald C: Pathologic features of
the CHARGE association: Support for involvement of the
neural crest. Teratology 1985;31:331-336
35. Bellah R. Andrea AD, Darillis, Fellows KE: The association
of congenital neuroblastoma and congenital heart disease. Is
there a common embryologic bases? Pediatr Radiol 1989;
19:119-121
36. Lammer EJ, Chen DT, Hoar RM, Agnish ND, Benke PJ,
Braun JT, Curry CJ, Fernhoff PM, Grix AW Jr, Lott IT,
Richard JM, Sun SC: Retinoic acid embryopathy. N Engl J
Med 1985;313:837-841
37. Pratt RM, Goulding EH, Abbott BD: Retinoic acid inhibits
migration of cranial neural crest cells in the cultured mouse
embryo. J Craniofac Genet Dev Biol 1987;7:205-217
38. Smith-Thomas L, Lott I, Bronner-Fraser M: Effects of
isotretinoin on the behavior of neural crest cells in vitro. Dev
Biol 1987;123:276-281
39. Todd JL, Todd NW: Congenital cardiac outflow tract anom-
alies and otitis media. Int J Pediatr Otorhinolaryngol 1988;
16:183-188
40. Guill MF, Arensman FW, Liffell MF, Ussry TW: Immunologic
defects associated with tetralogy of Fallot and truncus arteri-
osus. NER Allergy Proc 1988;9:309
41. Ussry TW, Arensman FW, Leatherbury L, Flannery DB,
Harmon JD, Guill MF, Lefelle MS, Bell RA, Strong WB:
Non-cardiac defects associated with conotruncal abnormali-
ties - Further implications of neural crest influence. JAm Coll
Cardiol 1989;13:118A
42. Takao A, Ando M, Cho K, Kinouchi A, Murakami Y: Etio-
logic categorization of common congenital heart disease, in
Van Praagh R, Takao A (eds): Etiology and Morphogenesis of
Congenital Heart Disease. Mt Kisco, NY, Futura Publishing Co,
1984, pp 253-269
43. Shimizu T, Takao A, Ando M, Hirayama A: Conotruncal
anomaly face syndrome: Its heterogeneity and association with
thymus involution, in Nora JJ, Takao A (eds): Congenital Heart
Disease, Causes and Processes. Mt Kisco, NY, Futura Publish-
ing Co, pp 29-41
44. Kinouchi A, Mori K, Ando M, Takao A: Facial appearance of
patients with conotruncal anomalies. Pediatr Jap 1976;17:84
KEY WORDS ectomesenchyme * chick embryos * heart
development outflow tract