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Smooth muscle cells of neural crest origin form the aorticopulmonary septum in the avian embryo.

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THE ANATOMICAL RECORD 226:360-366 (1990)
Smooth Muscle Cells of Neural Crest Origin
Form the Aorticopulmonary Septum n the
Avian Embryo
ARTHUR C. BEALL AND THOMAS H. ROSENQUIST
The Heart Development Group, Department of Anatomy, Medical College of Georgia,
Augusta, Georgia 30912-2000
ABSTRACT
Previous studies have shown that the cells of the aorticopulmonary (AP) septum are similar to the smooth muscle cells of the mediae of the great
vessels in their common origin from the cardiac neural crest and in their common
expression of a n elastic extracellular matrix. The purpose of this study was to test
the cells of the AP septum for the presence of certain cytoplasmic proteins, especially smooth muscle alpha-actin (SMAA) whose presence is definitive of smooth
muscle.
A monoclonal antibody against SMAA was applied to normal chicken embryos a t
3.5-8 days of incubation and to age-matched embryos from which the cardiac
neural crest had been ablated surgically. Antibodies against the intermediate
filaments desmin, cytokeratin, and vimentin also were applied.
The results showed that the AP septal cells expressed SMAA during the process
of septation, days 5-8; but when the cardiac neural crest was ablated and septation
was defective, no cells in the conotruncal connective tissue expressed SMAA. None
of the intermediate filament proteins were detected in the septum.
These results indicate that the AP septal cells are smooth muscle and therefore
may be hypothesized to have a n active role in septation.
The process of aorticopulmonary (AP) septation has
been the object of study in numerous laboratories over
many years. Excellent recent reviews of the process
have been written by Thompson et al. (1984,1987). The
AP septum proper in the chicken embryo is formed at
the confluence of a dorsal and a smaller ventral condensation of cells, each of which is in contact with the
myocardial cuff (also called the myocardial sheath or
mantle) of the truncus arteriosus. Each of these two
condensations extends downstream or cephalad within
the archaic connective tissue of the spiralling
conotruncal ridges and joins the other at its cephalic
terminus, between the lumina of aortic arches 4 and 6.
The conjoint condensation is the AP septum. After it is
formed, the AP septum moves relative to the surrounding tissues toward the heart, while its dorsal and ventral tributaries become relatively shortened. In this
way the AP septum is transferred upstream to the
heart. The mechanism by which the septum becomes
transferred upstream was traditionally considered a
“zipper-like’’ fusion of the dorsal and ventral condensations (reviewed by Thompson et al., 1984). In 1979
however Thompson and Fitzharris introduced a n attractive alternative mechanism, wherein the retracting myocardium pulled the septum upstream by the
conjoint dorsal and vectral condensations. They referred to this a s the “tractor and sling” concept. The
myocardial sheath provided the locomotion, while the
cellular aggregations and their extracellular matrix
provided a sling for passive upstream transfer of the
myocardial energy.
0 1990 WILEY-LISS, INC.
Later it was shown that the cells which condense to
form the AP septum were ectomesenchyme of neural
crest origin which migrate into the truncus via aortic
arches 3, 4, and 6 (Kirby e t al., 1983). When this
“cardiac” neural crest is ablated surgically from
chicken embryos at about stage 10 (Hamburger and
Hamilton, 1951), the embryos fail to produce the typical whorl-like cellular aggregation typical of the AP
septum, and septation is invariably abnormal. The embryos so treated develop congenital anomalies of the
heart, especially persistent truncus arteriosus (Kirby
et al., 1985; Nishibatake et al., 1987; Kirby, 1988). The
biological or molecular basis of this special role of the
cardiac ectomesenchyme is not completely understood.
However i t has been shown recently that one of the
features of the ectomesenchyme in the AP septum that
distinguishes i t from the contiguous non-neural crest
mesenchyme is the assumption by the aggregating ectomesenchyme of a n elastogenic phenotype (Rosenquist et al., 1988). Indeed the expression of the tropoelastins and the aldehyde-rich protein is a
characteristic unique to the cardiac ectomesenchyme
in the early chicken embryo, whether that ectomesenchyme is in the AP septum or in the walls of the great
Received March 16, 1989; accepted May 16, 1989.
Address reprint requests and correspondence to Dr. Thomas Rosenquist, Professor of Anatomy, Medical College of Georgia, Augusta,
Georgia 30912-2000
AORTICOPULMONARY SEPTATION AND SMOOTH MUSCLE
Fig. 1. Sham-operated embryo, incubation day 6. All sections, x 740.
A Transmitted light, phase-contrast image of a section through the
truncus arteriosus flanked by the two atria (A). Dorsal is to the left
and ventral to the right. A central condensation of cells forms the AP
septum (*) which separates the two great vessels. Arrows = the myocardial cuff. B: Same section as in a, green epifluorescence, antismooth muscle alpha actin (SMAA). All of the cells of the septum (*)
are positive but the SMAA-positive region ends abruptly ventrally.
There are a few cells in the lumina1 aspect of walls of the great vessels
36 1
that are positive, just dorsal to the septum. The myocardial cuff (arrows) is negative a t this level. c: Next more distal section from that
A and B, epifluorescence, polyclonal anti-desmin. All of the cells of
the septum (*) are positive. The most intense reaction is found in the
myocardial cuff (arrows); there is also binding of the antibody in the
flanking atria and in the proximalmost great vessel walls. D. Next
more distal section from C, epifluorescence, monoclonal anti-desmin.
The septum (*) bound no antibody while the myocardial cuff was
faintly positive (arrows).
362
A.C. BEALL AND T.H. ROSENQUIST
vessels (Rosenquist et al., 1988), both of which are
derived from the cardiac neural crest (LeLievre and
LeDouarin, 1975; Kirby et al., 1983).
Thus the ectomesenchymal cells in the cardiovascular outflow region appear to be a homogeneous and
continuous population that includes both the septum
and the great vessels. The distinct histologic similarity
between cells of the artery wall and the septum and the
clear distinction between those cells and all other mesenchyme in their vicinity was noted over 20 years ago
(Jaffee, 1967), before their common origin was established.
Because the AP septal cells are so like the medial
cells in their origin, general histologic features, and
early expression of elastin, we hypothesized that some
of the cells of the AP septum also would have the capacity to express a smooth muscle phenotype. (That a
significant fraction of the medial cells of the great vessels do not become smooth muscle was reported by
Hughes in 1943, and was confirmed recently by Skalli
et al., 1986.) To test this hypothesis we applied antibodies against smooth muscle alpha-actin to sections of
normal chicken embryos. The results show that smooth
muscle alpha-actin is a constant component of essentially all of the cells of the AP septum and suggest the
possibility of a n active role for these cells in the process
of septation. To be sure that the cells expressing the
muscle-specific protein in the septum were in fact of
neural crest origin, we compared control embryos with
experimental embryos from which the neural crest had
been ablated surgically. Smooth muscle alpha-actin
was not expressed by any mesenchymal cells in the
truncus arteriosus of the experimental embryos during
the septation period.
An earlier study by Sumida e t al. (1987) had reported
that the intermediate filament desmin was also found
in the AP septum of the chicken embryo. Therefore the
distribution of desmin was compared in the present
study with the distribution of smooth muscle alphaactin; however, the presence of desmin in the AP septum was not confirmed. The AP septum also was negative for two other intermediate filaments, vimentin
and cytokeratin.
MATERIALS AND METHODS
which the stain was applied and the vitelline membrane torn but no further manipulation of the embryo
was carried out. Shams were collected at the same intervals as the experimental embryos. Some of the
shams were collected after 12-14 days when most of
the cells had assumed their ultimate phenotype
(Schmid et al., 1979). A rooster of unknown breed,
weight 2 kg, was purchased from a local poultry dealer
and his great vessels were prepared as described below.
The late embryonic and adult great vessels were used
to check the validity of certain negative reactions that
were found in the early embryonic AP septum andlor
great vessels (see below).
Experimental and sham-operated embryos and the
adult great vessels were placed in a solution of methyl
alcohol, chloroform, and acetic acid (“methacarn”; Puchtler et al., 1970) at 4°C for 12-24 hr. Then they were
processed through alcohols and xylenes at 4°C into lowmelting-point Paraplast at 57°C. We have found the
best preservation of epitopes for immunohistochemistry, as well a s preservation of excellent histologic detail with this procedure (Rosenquist et al., 1988). Sections of 10 pm were cut and mounted serially on glass
slides.
lmmunohistochemistry
Sections generally were rehydrated rapidly with the
routine, xylene/xylene/lOO% ethano1/100% ethanollwatertwater, each for 3 min. If a particular antibody was
found not to bind to the embryo a t any site, the paraffin
removal step was lengthened to include 1 hr in the
initial xylene to be sure that the lack of reaction was
not a n artifact of retained paraffin. After rehydration
the sections were placed in phosphate-buffered saline,
pH 7.4, with 0.5% bovine serum albumin (PBS/BSA),
for 30 min at 37°C. Then they were incubated with 20
pllsection of the diluted primary antibodies detailed
below for 1 h r at 37”C, rinsed with PBSIBSA, and
incubated with 20 phection of the appropriate secondary antibody labelled in all cases with rhodamine.
(Note: the intense yellow-green autofluorescence of
some embryonic proteins under blue epif luorescence
could be taken in error for the emission of a fluorescein-labelled secondary antibody, e.g., the illustrations
in Rosenquist and McCoy, 1987; or Rosenquist et al.,
Preparation of the Tissues
Fertile Arbor Acre chicken eggs (Central Soya of
Athens, GA) were incubated for 30 h r in a humidified
atmosphere a t 38°C. A window was made in the shell
and the embryos (approximately HH stage 9; Hamburger and Hamilton, 1951) were stained lightly with
neutral red. The vitelline membrane was torn over the
embryos, and all of the neural folds over somites 1-3
and two somite lengths above somite 1 (the “cardiac
neural crest”; Kirby et al., 1985) was removed bilaterally with a microcautery needle (Narayanan, 1970).
This technique causes minimal damage to surrounding
tissues (Bockman and Kirby, 1984). After surgery the
eggs were sealed and returned to the incubator for periods of time which, in the control or sham-operated
embryos (see below), yielded embryos of HH stages 2135, days 3.5-8. These incubation times bracketed completely the time of septation (Kirby et al., 1983).
Sham-operated embryos (“shams”) were prepared in
Fig. 2. The same sham-operated embryo shown in Figure 1, same
magnifications throughout. A. Transmitted light, phase-contrast image of a section through the truncus arteriosus at a point nearer the
heart than in Figure 1.The characteristic “whorl” conformation of the
AP septum (*) is obvious dorsally. The undivided lumen of the truncus
is traversed by fixed blood (B) which may give the impression of a
completed septum by its location in this section. The blood gives a
distinctive, intense autofluorescence (see A-C, below). A = atria;
arrows = myocardial cuff. B: Same section as in A, epifluorescence,
anti-SMAA. The dorsal limb of the septum (*) is well-marked by the
antibody. An infolding of the myocardial cuff in direct contact with
the septum and just to its left in this section also is positive. On the
other hand the rest of the myocardial cuff (arrows) is negative. C:
Section immediately proximal to that in A and B, epifluorescence,
polyclonal anti-desmin. The cells in the dorsal limb of the septum are
positive (*I, while the most intense reaction is in the myocardial cuff
(arrows); the atria show a lesser reaction. D: Section immediately
proximal to that in C, epifluorescence, monoclonal anti-desmin. The
dorsal limb of the septum is negative (*) while the myocardial cuff is
positive.
AORTICOPULMONARY SEPTATION AND SMOOTH MUSCLE
Fig. 2.
363
364
A.C. BEALL AND T.H. ROSENQUIST
Fig. 3. Sham-operated embryo, incubation day 8. x 1,480. A Transmitted light, phase-contrast image of a section through the proximal
truncus just above the heart a t the time of completion of septation,
same orientation as Figures 1 and 2. The dense aggregation of cells
which forms the septum (S) is obvious. Arrows = myocardial cuff. B:
Same section as A, green epifluorescence, anti-SMAA. The cells of the
septum (S) react intensely whereas all other areas including the entire myocardial cuff are negative.
1988; therefore it is essential that fluorescein be
avoided during epifluorescence studies of embryonic
tissues.) Each slide carried four or more sections. One
section on each slide received neither the primary nor
the secondary antibody; one section received only the
secondary antibody; and the two or more remaining
sections received both the primary and the secondary
antibody. Details of the primary antibodies are given
below; the ratios that are given are for our empirically
optimum working dilutions of the antibodies in PBSI
BSA.
The following monoclonal primary antibodies were
localized with the secondary antibody, rhodamine-labelled rabbit anti-mouse IgG l:lO, ICN Immunobiochemicals: 1) Anti-smooth muscle alpha-actin clone
1A4 (Skalli et al., 19861, ascites 1:200, Sigma; 2) Antidesmin clone DE-U-10 (Debus et al., 1983), ascites 1:
10, Sigma; 3) Anti-desmin clone DE-B-5 (Debus et al.,
19831, Sigma, ascites l:lO, Sigma; 4) Anti-vimentin
clone V9 (Osborn et al., 19841, ascites 1:4, Boehringer
Mannheim Biochemica; 5) Anti-cytokeratin clone K813 (Gigi et al., 1982), ascites l:lO, Sigma.
Polyclonal rabbit anti-desmin primary antibody,
whole serum l:lO, was localized with the secondary
antibody, rhodamine-labelled goat anti-rabbit IgG 1:
10, 1"and 2" antibodies both Sigma.
Finally all sections were rinsed well with PBS/BSA
and coverslips were mounted over the sections with
Elvanol, Dupont.
Sections were observed and photographed in a Zeiss
Standard WL or a Zeiss Axioskop microscope under
green epifluorescence: exciter filter BP 510-560, dichromatic beam splitter FT 580, barrier filter DP 590.
Photographs were taken on Kodak Tri-X film.
TABLE 1. Results of antibody binding studies
Antigen
SMAA (Mab)
DES (Mab)
DES (PabI3
Vimentin (Mab)
Cytoker (Mab)
Location during septation
Arterial
Septal
Skeletal Cardiac
SM
SM
muscle
muscle
muscle
muscle
-
+
+
-
++-
++
-
-
-
-
-
-
+
+
f2
'Abbreviations: SMAA = smooth muscle alpha-actin; DES = desmin;
Cytoker = cytokeratin; Pab = polyclonal antibody; Mab = monoclonal antibody.
'Only in the myocardial cuff or sheath of the truncus arteriosus.
3Also bound by tracheo-esophageal mesenchyme.
AORTICOPULMONARY SEPTATION AND SMOOTH MUSCLE
RESULTS
The results of the antibody binding studies are summarized in Table 1.
Smooth Muscle Alpha-Actin (SMAA)
Cardiac neural crest cells migrate into the connective tissue space between the myocardial cuff and the
endocardium and aggregate in a peculiar whorl-like
configuration that is easily identified after about 5 d of
incubation (Figs. 1-3). This aggregation becomes the
definitive AP septum. Essentially all of the cells that
compose the septum are SMAA-positive, incubation d
6-8 (Figs. 1-3). Of the two condensations of ectomesenchyme which converge to form the definitive septum
(Thompson et al., 1984) (Fig. l),only the dorsal limb is
obviously SMAA-positive throughout its length (Fig.
2).
In the experimental embryos of 5-8 d, there were
either no cell aggregations typical of the formation of
the AP septum, or there was a small atypical dorsal
aggregation of cells like that described by Kirby (1988);
in neither case did any cells express SMAA in the
conotruncal connective tissue (between the myocardial
cuff and the endocardium).
The distalmost truncus arteriosus was SMAA-positive (Figs. 2, 3).
Desmin
The monoclonal anti-desmin antibodies were identical and showed no reaction in the AP septum (Figs. 1,
2). On the other hand, the AP septal cells bound the
polyclonal antibody against desmin a s soon as they
were found in their characteristic dense aggregation
(Figs. 1, 2).
The medial cells of the embryonic great vessel walls
failed to express desmin; however, the cells of the adult
great vessels were positive for the monoclonal antibodies (not shown).
The somites and the skeletal muscle derived from the
somites were reactive for the monoclonal anti-desmin
antibodies at all stages. The ventricles and the myocardial cuff of the heart also were reactive at all stages
(Figs. 1, 2). Visceral smooth muscle was initially negative but expressed desmin by 7 d (not shown).
The following regions all bound the polyclonal antidesmin a t all stages (not shown): the somites and all
skeletal muscle; all visceral and vascular smooth muscle cells; and the tracheoesophageal mesenchyme.
Vimentin was not detected in any embryonic or adult
tissue.
Cytokeratin was not detected in the AP septum or
any cells of the great vessels but was expressed by several embryonic tissues including the skin, epithelium
of the gut, and the pericardium (not shown).
DISCUSSION
The expression of different muscle-specific proteins
and the expression of intermediate filament proteins
may vary greatly during development 1) geographically among the various cell types and 2) chronologically, within a given cell type. Furthermore a given cell
type in the embryo may express simultaneously more
than one isoform of a protein. The regulation and modulation of the expression of genes for the various mus-
365
cle proteins are not well-defined but are the topic of a
great deal of contemporary research (e.g., Hayward et
al., 1986; Minty et al., 1986; Ordahl, 1986; Carroll et
al., 1988; Ruzicka and Schwartz, 1988). The functional
meaning and consequence of the presence of a given
muscle-specific protein or intermediate filament protein is unclear. However it has been established that
smooth muscle alpha-actin (SMAA) is found only in
cells which have the capacity to contract (Skalli et al.,
1986). Vascular smooth muscle cells (SMC) which have
achieved phenotypic maturity and mitotic quiescence
express SMAA rather than the beta form of actin
(Clowes e t al., 1988). Thus SMAA is the actin isoform
characteristic of all vascular smooth muscle cells when
they are functional, and its expression in a n embryonic
cell is necessary and sufficient for its positive identification as smooth muscle (Gabbiani et al., 1981; Skalli
et al., 1987). It may be surmised therefore that the
expression of SMAA by the cells of the aorticopulmonary septum indicates their differentiation into smooth
muscle and implies their contractile capacity (although
contraction per se has neither been observed here nor
reported elsewhere). The presence of SMAA in these
cells of neural crest origin, taken with their elastogenic
capacity, appears to justify fully the conclusion that
yielded the title of this study: smooth muscle cells of
neural crest origin form the aorticopulmonary septum
in the avian embryo.
The absence of any cells that express SMAA in the
conotruncal connective tissue following ablation of the
cardiac neural crest and the failure of septation in the
same embryos constitute a bit of circumstantial evidence in favor of a n active role for septal cells.
The potential for active participation by the AP septal cells in the process of septation in the chicken embryo has been indicated also by data reported previously by others. Sumida e t al. (1989) have reported that
the mushroom-derived toxin phalloidin was bound to
the myocardium as well as to the cells of the AP septum
in the 5 d chicken embryo; and phalloidin has a specific
affinity for F-actin (reviewed by Sumida et al., 1989). A
polyclonal antibody against desmin that was bound by
cells of the AP septum suggested a contractile potential
to Sumida e t al. (1987). However in the present study
desmin was not detected in the AP septum with the
monoclonal antibodies although the validity of the antibodies was supported by their reaction in the same
embryos with other tissues that typically express
desmin. The binding in this study and in the study of
Sumida et al. (1987) of a polyclonal “anti-desmin” antibody by the AP septum may have been the result of
the presence of some epitope other than one of the
desmin epitopes, e.g., one of the fibronectin epitopes.
Indeed the similarity between the distribution of the
polyclonal anti-desmins used by us and by Sumida et
al. (1987) and the distribution of the monoclonal antifibronectin antibody used by Icardo (1985) is striking
and was remarked upon also by Sumida e t al. (1987,
1989). Whatever the basis for the discrepancy between
the results of the polyclonal compared with the monoclonal antibodies, the true functional roles of the intermediate filaments including vimentin, cytokeratin,
and desmin are not known, and any essential relationship that they may have to contractility has not been
clearly established. The fact that no intermediate-type
A.C. BEALL AND T.H. ROSENQUIST
366
filament was detected in the AP septum in the present
study cannot therefore be interpreted as evidence
against a contractile capacity for the septal cells.
A contribution by septal cell smooth muscle contraction to septation may be hypothesized from these data
and those of Sumida et al. (1987,1989); however, based
upon the work of Thompson and others (cited above), it
seems likely that the putative septal contraction would
be only one of several simultaneous, coordinated events
that result in successful septation. Nevertheless the
concept of active septal contraction may have a n impact on teratology, for example in the interpretation of
data from studies where beta-stimulating agents have
been used to produce cardiovascular malformations
that involve improper septation (reviewed by Ishikawa
e t al., 1980). In such cases a direct pharmacologic impact upon the septal smooth muscle by the drug in
question would have to be given consideration as a potential teratogenic agent, along with hemodynamic
change and other factors.
In summary, the present study has shown the presence of SMAA in essentially all of the cells of the AP
septum during the process of septation in the chicken
embryo. On the other hand, no cells which expressed
SMAA were observed in the conotruncal connective tissue in chicken embryos which had undergone surgical
ablation of the cardiac neural crest. These data support
the possibility that the ectomesenchymal cells may
contribute actively to the process of AP septation.
ACKNOWLEDGMENTS
This study was supported by grants HL 36059 and
HL 42164 from the National Institutes of Health, National Heart, Lung and Blood Institute, and by a grant
from the American Heart Association, Georgia Affiliate.
Technical assistants were Harriet Stadt and Donna
Kumiski, surgery; Charlotte Fray, histology; and Judy
McCoy, photography.
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crest, septum, forma, muscle, avian, smooth, embryo, neural, origin, aorticopulmonary, cells
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