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Expression of HNK1 epitope by the cardiomyocytes of the early embryonic chickIn situ and in vitro studies.

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THE ANATOMICAL RECORD 263:326 –333 (2001)
Expression of HNK1 Epitope by the
Cardiomyocytes of the Early
Embryonic Chick: In Situ and In
Vitro Studies
YUJI NAKAJIMA,1* KAZUNORI YOSHIMURA,2 MASAHIKO NOMURA,2
1
AND HIROAKI NAKAMURA
1
Department of Anatomy, Saitama Medical School, Saitama, Japan
2
Department of Physiology, Saitama Medical School, Saitama, Japan
ABSTRACT
Monoclonal antibody HNK1 reacts with a carbohydrate epitope in cell
surface glycoproteins and glycolipids. During development, in various species the HNK1 epitopes are expressed in migrating neural crest cells and in
the developing conduction cardiomyocytes. The conduction system is generally thought to be developed from cardiomyocytes, but some investigators
have hypothesized that it is derived from the neural crest because conduction myocytes express neural antigens, including HNK1. Using immunohistochemistry, we examined the spatiotemporal expression of HNK1 in
early chick cardiogenesis (stages 4 to 18) and whether cultured precardiac
mesoderm does or does not express HNK1 as well as sarcomeric myosin
(MF20). HNK1 was first expressed in the premyocardium at stage 8. At
stage 10, HNK1-positive cardiomyocytes were scattered along the straight
heart tube. By stage 18, HNK1-positive cardiomyocytes had become restricted to the atrium and sinus venosus. Atrioventricular cushion mesenchyme also expressed an HNK1 epitope. Immunostaining of HNK1 and
MF20 in cultured precardiac mesoderm showed that there are at least three
types of cells: 1) cardiomyocytes without HNK1 expression, 2) cells possessing both HNK1- and MF20-immunoreactivity, and 3) mesenchymal cells
with HNK1. Immunogold electron microscopy showed that cardiomyocytes
containing sparsely distributed myofibrils associated with the Z-band react
with anti-HNK1 antibody. Our observations showed a direct evidence for
the first time that the precardiac mesoderm generates HNK1-positive cardiomyocytes with morphological features similar to those of conduction
cardiomyocytes. Anat Rec 263:326 –333, 2001. © 2001 Wiley-Liss, Inc.
Key words: HNK1; cardiogenesis; conduction system; chick
embryo
The monoclonal antibody HNK1 recognizes a carbohydrate moiety formed by a sulfated glucuronic acid sugar
originally identified on human natural killer cells (Abo
and Balch, 1981) and this antibody reacts with a carbohydrate epitope present in certain types of cell-surface glycoproteins and glycolipids (Ariga et al., 1987; Kruse et al.,
1984). It is widely accepted that during development, the
HNK1 epitope is expressed in migrating neural crest cells
as well as in other tissues (Bannerman et al., 1998; Luider
et al., 1993; Newgreen et al, 1990; Tucker et al., 1984;
Vincent and Thiery 1984). Studies of cardiogenesis have
revealed that the HNK1 epitope is expressed on the cell
©
2001 WILEY-LISS, INC.
surface of the developing conduction myocardium in a
variety of species (Aoyama et al., 1995; Chuck and Watanabe 1997; Gorza et al., 1988; Ikeda et al., 1990; Nakagawa et al., 1993). During chick cardiogenesis, the HNK1
*Correspondence to: Yuji Nakajima, Department of Anatomy,
Saitama Medical School, 38 Morohongo, Moroyamacho, Irumagun, Saitama, 350-0495 Japan. E-mail: yuji@saitama-med.ac.jp
Received 2 December 2000; Accepted 23 February 2001
Published online 00 Month 2001
HNK1 IN EARLY CARDIOGENESIS
epitope is expressed not only in the conduction myocardium but also on mesenchymal cells of the endocardial
cushion tissue, the primordium of the valvular tissue of
the adult heart (Luider et al., 1993). The spatiotemporal
expression of the HNK1 epitope in the developing heart
has been well examined in various species, including
chick. Little or nothing is known about the expression of
the HNK1 epitope in early cardiogenesis, during which
extensive morphogenesis is carried out such as formation
of the precardiac mesoderm, primitive heart tube, and
cardiac looping.
The cardiac conduction system is composed of myocardium specialized for the generation and conduction of
electrical impulses to the working myocardium. The conduction myocyte shows specific morphological features at
the light and electron microscopic levels, as well as expression of specific genes, such as those for contractile proteins, intermediate filaments, cell adhesion molecules,
and connexins (Moorman et al., 1998). Conduction myocytes have been found to express proteins and epitopes
also expressed in neural tissue, including HNK1, acetylcholinesterase, the L/M subunit of neurofilaments and
GIN2 (Gorza et al., 1994). Furthermore, cardiac neural
crest cells migrate into the heart via the arterial pole and
are involved in the formation of the semilunar valves and
cardiac septa (Kirby et al., 1983). On the basis of these
observations, Gorza et al. (1988, 1994) proposed that conduction myocytes originate from the neural crest. There is
no hard evidence, however, to support the notion of an
extracardiac origin for the conduction system (Cheng et
al., 1999; Moorman et al., 1998). Other investigators have
considered that the conduction system develops from cardiomyocytes originating from the precardiac mesoderm
(DeHaan, 1965; Patten, 1956). Recent experiments of retroviral cell lineage studies in the embryonic chick heart
have shown that both peripheral and central conduction
tissues originate from cardiomyogenic progenitors of the
looped heart (Cheng et al., 1999; Gourdie et al., 1995).
In the present study, we used immunohistochemistry to
examine the spatiotemporal expression of the HNK1
epitope during early chick cardiogenesis, from stage 4
(trilaminar germ disk) (Hamburger and Hamilton, 1951)
through to stage 18 (at which neural crest cells enter the
heart) (Noden, 1991) and cultured precardiac mesoderm
obtained from stage 6 embryos and examined it immunohistochemically to determine whether mesoderm-derived
cardiomyocytes do or do not express the HNK1 epitope.
MATERIALS AND METHODS
Chick Embryos
Fertilized eggs from the domestic fowl (Gallus gallus)
were incubated for appropriate incubation times at 37.8°C
and 80% humidity. Embryos were collected on ice-cooled
phosphate-buffered saline (PBS) and staged according to
the criteria of Hamburger and Hamilton (1951). The
staged embryos were subjected to the experiments described below.
Antibodies
An HNK1 hybridoma was purchased from the American
Tissue Type Culture Collection (TIB200) (Abo and Balch,
1981). Cells were grown in RPMI1640 medium (GibcoBRL, Tokyo, Japan) supplemented with 20% fetal calf
serum (FCS, Gibco-BRL). Conditioned medium, contain-
327
ing HNK1 antibody (IgM), was harvested and used as a
primary antibody. The MF20 monoclonal antibody
(IgG2b), specific for sarcomeric myosin, was purchased
from the Developmental Studies Hybridoma Bank (IA,
USA) (Bader et al., 1982). Monoclonal antibody JB3
(IgG1), which recognizes chicken fibrillin-2 (Rongish et al.,
1998; Wunsch et al., 1994), was kindly provided by Dr. K.
Isokawa, Nihon University School of Dentistry.
Heart-Forming Mesoderm Culture
Heart-forming mesoderm from a Stage 6 chick embryo
was explanted in culture as described previously
(Imanaka-Yoshida et al., 1998). Briefly, the three germ
layers were separated in 0.25% trypsin (Gibco-BRL). The
resulting precardiac mesoderm was explanted onto fibronectin-coated (20 ␮g/ml in distilled water, incubated
for 12 hr at room temperature, then drained and air-dried;
Gibco-BRL) chamber slides (Nalge Nunc, Naperville, IL)
in Dulbecco’s modified Eagles medium (DMEM; GibcoBRL) supplemented with 10% FCS and streptomycin/penicillin (Gibco-BRL) under a humidified 95% air/5% CO2
atmosphere at 37°C.
Indirect Immunofluorescence Microscopy
Paraformaldehyde-fixed stage 4 –18 chicken embryos
were embedded in OCT™ (Miles, Elkhart, IN), then frozen
in liquid nitrogen. Frozen sections were cut on a cryostat,
mounted onto 3-triethoxysilylpropylamine-coated slides,
then air-dried. After rinsing with PBS for 15 min, sections
were blocked with 1% bovine serum albumin (BSA) in PBS
for 1 hr, incubated with HNK1 antibody (hybridoma supernatant) in a moist chamber for 2 hr at room temperature, rinsed with PBS, incubated in fluorescein (FITC)conjugated rabbit anti-mouse IgM (Jackson Immuno
Research Laboratories, Inc. PA; 10 ␮g/ml in blocking solution) for 1 hr, then rinsed with PBS and mounted in
mounting medium (0.2 M n-propylgallate in 90% glycerol/
10% PBS). Specimens were observed under a conventional
fluorescence microscope (OLYMPUS-BX60, Tokyo, Japan)
and photographed (Tmax 400, Kodak). The objectives we
used were OLYMPUS-Uplan Apo for immunofluorescence
and Nomarski images.
Cultures were drained of medium, rinsed with PBS,
fixed with 4% paraformaldehyde in PBS (pH 7.4) for 1 hr
at room temperature, then rinsed with PBS. Specimens
were blocked for 1 hr with 1% BSA in PBS containing 0.1%
Triton X-100, incubated with primary antibody mixture
(hybridoma supernatants, HNK1/MF20 or HNK1/JB3; 1:1
solution) at 4°C overnight, rinsed with PBS, incubated
with secondary antibody mixture (FITC-conjugated rabbit
anti-mouse IgM plus tetramethylrhodamine-5-(and-6)-isothiocyanate (TRITC)-conjugated affinity purified goat antimouse IgG, Jackson ImmunoResearch Laboratories; 10
␮g/ml in blocking solution) for 1 hr at room temperature,
rinsed with PBS and coverslipped with mounting medium.
Samples were observed under the fluorescence microscope
using narrow-band mirror units (U-MNIBA and U-MNG)
and photographed (PROVIA 400, FUJI FILM).
Immunogold Electron Microscopy
Cultures were drained of medium and washed in PBS.
They were then fixed with 0.5% glutaraldehyde and 4%
paraformaldehyde in 0.1 M PBS with the aid of microwave
irradiation (150 W; 120 sec; maximum temperature, 37°C)
328
NAKAJIMA ET AL.
(Nakajima et al., 1999a). Samples were then fixed for an
additional 2 hr at 4°C and rinsed in 7% sucrose in PBS for
12 hr. Nonspecific binding sites were blocked using 1%
BSA in PBS for 30 min. Cultures were stained with HNK1
antibody for 12 hr at 4°C, then rinsed in PBS and incubated with a 10 nm colloidal-gold-conjugated secondary
antibody (Amersham, Buckinghamshire, UK) for 1 hr at
room temperature. After extensive washing in PBS, they
were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate
buffer for 2 hr at 4°C, then post-fixed in 1% OsO4/cacodylate buffer for 40 min at 4°C. Samples were dehydrated
through a graded ethanol series and embedded in Epon.
Ultra-thin sections were cut, mounted on 100-mesh grids
and stained with uranyl acetate and lead citrate. Samples
were observed using a transmission electron microscope
(JEM-1010, JEOL, Tokyo, Japan) operated at 80 kV.
RESULTS
Double Immuno-Labeling for HNK1 and MF20
in cultured precardiac mesoderm
To clarify whether precardiac mesoderm differentiates
to HNK1-positive cardiomyocytes, we cultured precardiac
mesoderm obtained from stage 6 embryos for 2–3 days and
then double-stained the resulting cultures with antiHNK1 antibody and MF20 (anti-sarcomeric myosin). As
described previously, all the precardiac mesoderm we cultured was beating spontaneously after 48 hr in culture
(Imanaka-Yoshida et al., 1998). Most of the cultured cells
(hereafter referred to as first type of cells) expressed sarcomeric myosin (MF20) and this was assembled into sarcomeres; however, these cardiomyocytes did not show any
apparent staining for HNK1 (Fig. 1, A1 and A2). Some
cells (the second type), which were located adjacent to the
first type, expressed both sarcomeric myosin and an
HNK1 epitope (Fig. 1, B1 and B2). The MF20 staining in
cells of the second type was seen as a cytoplasmic punctate
staining pattern, not as sarcomeric pattern (Fig 1, B1 and
B2). In an effort to locate a cell type possessing both the
HNK1 epitope and striated myofibrils, we next stained the
cultured precardiac mesoderm with anti-HNK1 antibody
and FITC-phalloidin. Using fluorescence microscopy, however, we could not find a cell type possessing both HNK1
epitope and striated myofibrils labeled with FITC phalloidin (not shown). Cells of the third type showed a typical
mesenchymal phenotype (characterized by cellular polarity and migratory appendages). These cells stained with
HNK1 antibody, but were not labeled with MF20 (Fig. 1,
C1 and C2). Thus, the results of double-immunostaining
experiments with HNK1 and MF20 indicated that at least
three types of cells exist in cultured precardiac mesoderm:
1) the first type, a typical working cardiomyocyte with
well-developed striation, does not express the HNK1
epitope (Fig. 1, A1 and A2); 2) the second type shows
labeling for both HNK1 epitope and MF20 (Fig. 1, B1 and
B2); and 3) the third type possesses a mesenchymal phenotype and expresses the HNK1 epitope (Fig. 1, C1 and
C2). After 7 days in culture, most of the cultured cells
expressed sarcomeric myosin that was assembled into
well-developed sarcomeres; however, these cardiomyocytes did not show HNK1 immunoreactivity (not shown).
In this long-term culture condition, some HNK1-positive
cells expressed sarcomeric myosin that was incorporated
into sparsely distributed myofibrils (arrows in Fig. 1, D2).
Fig. 1. Double immunofluorescence labeling of HNK1 and MF20
(sarcomeric myosin) in cultured precardiac mesoderm. Stage 6 precardiac mesoderm was cultured on a fibronectin-coated plastic dish for 48
hr (A–C) or 7 days (D). The resulting cultures were fixed and doublestained with HNK1 and MF20 antibodies. At least three types of cells
could be identified. A: Shows the first type, cells expressed sarcomeric
myosin (MF20), which is assembled into well-developed striated myofibrils, but lacking the HNK1 epitope. B: Shows the second type, cells
expressing both HNK1 and MF20. In this cell type, immunostaining of
MF20 did not reveal a sarcomeric staining pattern at 48 hr in culture. C:
Shows the third type, cells characterized by a mesenchymal phenotype
and expressing HNK1 alone. D: Shows the second type after 7 days in
culture, cells showing both HNK1 (arrowheads) and sarcomeric staining
of MF20 (arrows). Panels A1, B1, C1, and D1 show HNK1 staining;
panels A2, B2, C2, and D2 show MF20 staining. Scale bars ⫽ 20 ␮m (A,
B), 50 ␮m (C), 10 ␮m (D).
Immunogold Electron Microscopic Detection of
HNK1 Epitope in Cultured Precardiac
Mesoderm
Light microscopic double-immunocytochemical analysis
of cultured precardiac mesoderm revealed that cells of the
second type exhibited both HNK1 and anti-sarcomeric
myosin (MF20) immunoreactivities. Although this type of
cell exhibited an MF20 immunoreactivity, however, we
could not find striated myofibrils labeled with MF20 or
FITC phalloidin. This suggests that cells of the second
type contain sparsely or poorly distributed myofibrils that
resist detection by light microscopy. In an effort to deter-
HNK1 IN EARLY CARDIOGENESIS
329
Fig. 2. Immunogold electron microscopic detection of HNK1 epitope
in cultured precardiac mesoderm. Stage 6 precardiac mesoderm was
cultured for 72 hr, then fixed and stained with HNK1 antibody. A: A cell
containing sparsely distributed myofibrils (mf) associated with nascent
Z-bands (z) shows anti-HNK1-10 nm gold complexes on the cell surface
(arrowheads). A tiny intercellular junction is seen (ij). B: On the other
hand, a cell containing well-developed myofibrils (mf) in association with
Z-bands (z) does not react with HNK1 antibody. C: A cell possessing a
mesenchymal-cell phenotype shows anti-HNK1-10 nm gold complexes
on the cell surface (arrowheads). D: Staining with secondary antibody
alone. Scale bars ⫽ 500 nm (A, D), 1 ␮m (B, C).
mine whether cells of the second type possess striated
myofibrils, we stained the cultured mesoderm with HNK1
antibody and observed it under the transmission electron
microscope. As shown in Figure 2, cells containing
sparsely distributed myofibrils associated with the nascent Z-band as well as glycogen granules were found to
have HNK1-gold particles on their cell surface (arrowheads in Fig. 2A). Myocardial cells that had well-developed striated myofibrils associated with the Z-band did
not show the HNK1 epitope (Fig. 2B). In contrast, cells
showing mesenchymal phenotype also expressed an
HNK1 epitope (Fig. 2C). Thus, immunogold electron microscopy suggests that cells possessing HNK1 epitopes
and containing sparsely distributed striated myofibrils
coincide with the cells of our second type.
that the HNK1-positive mesenchymal cells were surrounded with extracellular fibrillar deposition of JB3/
fibrillin-2, suggesting that the third type of cells appears
to represent either endocardial cushion mesenchymal
cells or subepicardial mesenchymal cells (Fig. 3).
Double Immunostaining of HNK1 and JB3/
Fibrillin-2 in Cultured Precardiac Mesoderm
To try to determine the predicted origin of the third type
of cells, we next examined whether cells of the third type
express JB3/fibrillin-2 (Rongish et al., 1998; Wunsch et al.,
1994). It has been reported that precardiac mesodermderived endocardium, endocardial cushion mesenchyme
and epicardial mesenchyme express JB3/fibrillin-2 as a
differentiation marker (Eisenberg and Markwald, 1995;
Nakajima et al., 1999b; Peretz-Pomares et al., 1998; Sugi
and Markwald, 1996). Double immunostaining revealed
Immunolocalization of HNK1 Epitope During
Early Cardiogenesis
Luider et al. (1993) reported an immunohistochemical
localization of HNK1 in the developing avian heart. They
did not, however, establish where the HNK1 antigen was
initially expressed within the heart or by which cell population. At stage 4, mesenchymal cells originating from
the epiblast migrate into the anterior lateral region and
form the precardiac mesoderm. At this stage, there is no
detectable staining for the HNK1 epitope within the precardiac mesoderm, whereas some endodermal cells, as
well as mesodermal cells subjacent to the primitive streak,
do express HNK1 epitope (Canning and Stern, 1988). After the trilaminar germ disk is completed, right and left
precardiac mesoderm, which are established in the anterior lateral region of the embryonic disk, migrate toward
the ventral midline of the body and fuse with each other,
resulting in the formation of the primitive heart tube.
During this early cardiogenesis, the HNK1 epitope was
first detectable in the premyocardium of the splanchnic
mesoderm at stage 8 (arrowhead in Fig. 4, A1). An expres-
330
NAKAJIMA ET AL.
Fig. 3. Double immunofluorescence labeling of HNK1 and JB3 (fibrillin-2) in cultured precardiac mesoderm. Stage 6 precardiac mesoderm
was cultured on a fibronectin-coated plastic dish for 48 hr. The resulting
cultures were double-stained with HNK1 and JB3 antibodies. Extracellular fibrillar staining for JB3/fibrillin-2 (B) is seen surrounding HNK1positive mesenchymal cells (A). Scale bar ⫽ 50 ␮m.
sion of the HNK1 epitope was also found in the endoderm
(arrow in Fig, 4, A1). After the completion of the primitive
heart tube, the heart begins to beat spontaneously and
generates a right-sided bend (D-loop) at Stage 10 –11. At
this stage, HNK1-positive cardiomyocytes were found
scattered along the tubed heart (Fig. 4, B1 and C1). At
stage 14, myocardial cells of the sinus venosus, atrium
and atrioventricular canal expressed the HNK1 epitope in
a region-specific manner (Fig. 5, A1 and B1). The myocardial cells of the distal region of the outflow tract also
expressed HNK1 epitope intermittently (data not shown).
Some of the endothelial cells in the atrioventricular canal,
where the formation of endocardial cushion tissue takes
place, exhibited the HNK1 epitope (arrowhead in Fig. 5,
B1). At stage 18, endothelial–mesenchymal transformation is carried out in the atrioventricular region, and the
migrating mesenchymal cells generate the endocardial
cushion tissue, the primordium of the valves and septa of
the adult heart. At this stage, mesenchymal cells in the
atrioventricular region expressed an HNK1 epitope extensively (arrowheads in Fig. 6, A1). On the other hand,
cushion mesenchymal cells in the proximal outflow tract
region (conus ridge) did not show an apparent HNK1immunoreactivity (* in Fig. 6A). In contrast, some cushion
mesenchymal cells in the distal outflow tract region expressed the HNK1 epitope extensively (arrow in Fig. 6A).
The myocardium of the sinus venosus and atria, especially
that of right atrium, expressed an HNK1 epitope (arrowheads in Fig. 6, B1 and C1). The endothelial cell lining in
the right and left atria also expressed HNK1. In the outflow tract region, the distal (cranial) end of the myocardium exhibited an HNK1 epitope (Fig. 6, B1 and C1).
Interestingly, some mesenchymal cells, which are thought
to originate from the cardiac neural crest and migrate into
the aortic pole, did not show an apparent HNK1 epitope (*
in Fig. 6, C1 and C2). Some of the endothelial cells in the
trabeculated ventricle exhibited an HNK1 epitope (arrowhead in Fig. 6, D1).
DISCUSSION
The present study demonstrates that at least three
types of cells are developed from the precardiac meso-
Fig. 4. Immunofluorescence localization of HNK1 epitope in stage
8 –11 hearts. A: At Stage 8, left and right cardiogenic regions begin to
fuse with each other. The HNK1 epitope is found on the cellular surface
of the premyocardial cell (arrowhead) in the premyocardium of the
splanchnic mesoderm (my). No apparent staining is seen within the
endothelial cells (e). Endoderm cells beneath the cardiogenic region
show an HNK1 epitope (arrow). Box region in A2 indicates the part of the
cross-section of the precardiac region that is shown in A1. B and C: At
Stage 11, the heart consists of two epithelial layers, an inner endocardium (e) and an outer myocardium (my) separated by an acellular extracellular matrix (cj). HNK1-positive cardiomyocytes are scattered along
the heart tube. In addition, some endothelial cells express an HNK1
epitope (arrowheads), as do endoderm (arrow in C1). B2 and C2 are
Nomarski microscopic images of panels B1 (cross-section) and C1
(coronal section), respectively. e, endocardium; cj, cardiac jelly; my,
myocardium. Scale bars ⫽ 50 ␮m (A1), 200 ␮m (A2, B, C).
derm. Using double immunostaining of HNK1 and MF20,
we identified the following types: cells of the first type
possess a striated sarcomeric myosin but not the HNK1
epitope; those of the second type possess both sarcomeric
myosin and the HNK1 epitope; the third type has a characteristic mesenchymal phenotype with HNK1.
Cells of the first type were characterized by a welldeveloped striated myofibril that was stained with antisarcomeric myosin (MF20). Immunogold electron microscopy revealed that cells possessing such well-developed
striated myofibrils did not react with anti-HNK1 antibody. These results indicate that cells of the first type are
HNK1 IN EARLY CARDIOGENESIS
Fig. 5. Immunofluorescence localization of HNK1 epitope in stage 14
heart. At stage 14, the HNK1 epitope is distributed in the myocardium of
the atrioventricular (av) canal and atrium (a) and also in the wall of the
sinus venosus (sv). Some endothelial cells in the atrioventricular (av)
region express an HNK1 epitope (arrowhead in B1). A: Coronal section
of stage 14 heart; A2, Nomarski microscopic image of A1; B: sagittal
section of stage 14 atrioventricular canal; B2, Nomarski microscopic
image of B1; a, atrium; av, atrioventricular canal; e, endocardium; l, liver
cells; my, myocardium; ot, outflow tract; sv, sinus venosus. Scale bar ⫽
100 ␮m.
working cardiomyocytes; however, there is no evidence
that all the working cardiomyocytes lack the HNK1
epitope during chick cardiogenesis. Cells of the second
type exhibited both HNK1 and anti-sarcomeric myosin
immunoreactivity, but no apparent striation at the light
microscopic level at around 2–3 days in culture. Immunogold electron microscopy revealed that cells containing
sparsely distributed myofibrils in association with
Z-bands were stained with anti-HNK1 antibody. Thus, we
consider that those cardiomyocytes having both myofibrils
and an HNK1 epitope coincide with our second type of
cells. In addition, some cells possessing HNK1 epitope
expressed sarcomeric myosin that was assembled into
sparsely distributed myofibrils after 7 days in culture.
Luider et al. (1993) reported that a narrow band of HNK1
immunoreactivity was found in the chick embryonic atrioventricular junction, from which an action potential typical of the conduction myocardium can be recorded (Arguello et al., 1988). Electron microscopic observations
aided by an electrophysiological technique have shown
that cardiomyocytes located in the atrioventricular node
and the bundle of His contain a few irregularly arranged
myofibrils (Arguello et al., 1988). The adult conduction
system in the chicken heart contains P-cells that characteristically contain sparse myofibrils (Lu et al., 1993).
Furthermore, recent retroviral cell lineage experiments
showed that both central and peripheral conduction systems originate from cardiomyocytes of the tubed heart
(Cheng et al., 1999). Thus, these observations, together
with our results, suggest that the second type of cells has
characteristics similar to those of conduction cardiomyo-
331
cytes. Cells of the third type characteristically showed a
mesenchymal phenotype with an expression of HNK1.
Another experiment showed that HNK1-positive mesenchymal cells expressed JB3/fibrillin-2. It has been reported that precardiac mesoderm-derived endocardium,
endocardial cushion mesenchyme and epicardial mesenchyme express JB3/fibrillin-2 (Eisenberg and Markwald,
1995; Nakajima et al., 1999b; Perez-Pomares et al., 1998;
Sugi and Markwald 1996). In addition, the endocardial
cushion mesenchyme and proepicardial organ also express
HNK1 epitope (Luider et al., 1993; in this report). Recent
retroviral cell-lineage experiments have shown that injected viruses into precardiac mesoderm are not detectable within epicardium/subepicardial mesenchyme at
later stage. (Mikawa et al., 1992). Thus, the third type of
cells is likely to be endocardial cushion mesenchymal cells.
The present study provides the first direct evidence that
the precardiac mesoderm gives rise to HNK1-positive cardiomyocytes that are morphologically coincident with conduction cardiomyocytes.
For some years, there has been a controversy as to the
origin of the conduction myocardium, because it coexpresses both neural and muscle genes. In addition, neural
crest cells migrate into the heart during development.
These observations suggest one of two possible origins for
the conduction myocardium: myogenic (Patten, 1956) or
neural crest (Gorza et al., 1994). In the present study, we
showed that the precardiac mesoderm generates HNK1positive cardiomyocytes with morphological features similar to those of the conduction myocardium. As yet, there
is no hard evidence to support the notion of an extracardiac origin for the conduction myocardium (Cheng et al.,
1999; Moorman et al, 1998). In vitro clonal analysis has
shown that the cardiac neural crest, from which some cells
migrate into the heart via the arterial pole, differentiates
into four types of cells including smooth muscle cells,
connective tissue cells, pigment cells, and cells of sensory
neuron lineage, but does not generate cardiomyocytes (Ito
and Sieber-Blum, 1991). Recent experiments on heterospecific chicken-quail chimeras and others involving
retroviral infection of stem cells by reporter gene LacZ
have shown that cardiac neural crest cells differentiate
into mesenchymal cells of the aorticopulmonary septum,
cardiac ganglion cells and smooth muscle cells of the pharyngeal arch arteries, but not conduction cardiomyocytes
(Poelmann et al., 1998). Another population of cardiac
neural crest cells, one that employs the venous pole as its
entrance to the heart, migrates to locations surrounding
the prospective conduction system, such as the atrioventricular node area, the retroaortic root bundle, the bundle
of His, the left and right bundle branches and the right
atrioventricular ring bundle (Poelmann and Gittenberger-de Groot, 1999). Thus, both in vivo and in vitro
fate-mapping experiments have failed to produce evidence
to support the notion of a neural crest origin for the
conduction myocardium, even if the conduction system
does express neural-tissue-associated substances.
Using an HNK1 antibody, several investigators have examined the development of the cardiac conduction system in
different species (Aoyama et al., 1993, 1995; Chuck and
Watanabe 1997; Gorza et al., 1988; Ikeda et al., 1990; Luider
et al., 1993; Nakagawa et al., 1993). During rat cardiogenesis, HNK1 immunoreactivity is first found in the ventricular myocytes of the looped heart, in which the conduction
myocytes will soon develop; at a later stage, the HNK1
epitope is expressed in the developing sinoatrial node, atrio-
332
NAKAJIMA ET AL.
Fig. 6. Immunofluorescence localization of HNK1 epitope in stage 18
heart. A: Coronal section of stage 18 heart shows the HNK1 epitope
expressed extensively in atrioventricular (av) cushion mesenchymal cells
(arrowheads). A large majority of the mesenchymal cells in the outflow
tract (ot) failed to show an HNK1 epitope (*), whereas a few mesenchymal cells in the distal outflow tract did have an HNK1 epitope (arrow). A2
is a Nomarski microscopic image of A1. B and C: Coronal section of
Stage 18 heart shows the HNK1 epitope in the myocardium (arrowheads
in B1 and C1) of the sinus venosus (sv) and atrium (a). The endocardium
of the atrium expresses HNK1 (arrowheads in B1, C1). Liver cells also
express an HNK1 epitope (l). Some mesenchymal cells from the neural
crest that have migrated into the aortic sac do not show an HNK1
epitope (* in C). B2 and C2 are Nomarski microscopic images of B1 and
C1, respectively. D: High magnification view of the ventricle shows that
HNK1 epitope (arrowhead) is found in the endocardium of the trabeculated myocardium. Box region in D2 indicates the region shown in D1. a,
atrium; av, atrioventricular canal; e, endocardium; l, liver cells; my,
myocardium; ot, outflow tract; sv, sinus venosus. Scale bars ⫽ 50 ␮m
(D1), 100 ␮m (A), 200 ␮m (B, C, D2).
ventricular node, bundle of His, and Purkinje fibers (Aoyama
et al., 1993, 1995; Nakagawa et al., 1993). Immunoelectronmicroscopic observations in the developing rat heart have
shown that the HNK1 epitope is predominantly found on the
cell surface and in the extracellular matrix of cells in the
atrioventricular node and bundle of His (Aoyama et al.,
1993; Sakai et al., 1994). In the present study, HNK1-positive cardiomyocytes were scattered along the primitive heart
tube, only later becoming localized to the sinus venosus and
atrium by Stage 18 (Fig. 6). A number of cell–surface glycoproteins and extracellular molecules mediating cell– cell and
cell–substratum interactions are most likely involved in the
coordination of the process that contributes to the tissue
remodeling associated with tissue specification (Edelman,
1988). The spatiotemporal expression of HNK1 during early
cardiogenesis may reflect that the certain molecules carrying
the HNK1 epitope are involved in a critical process necessary for the establishment of the conduction myocardium.
Although the significance of the different HNK1 expression
patterns seen at different developmental stages and in different species remains unknown, the finding that the expression of the HNK1 epitope in the conduction myocardium is
conserved across species suggests that HNK1 expression
may play an important role in the formation of the conduction system (Chuck and Watanabe, 1997).
In the present study, we have demonstrated that the
precardiac mesoderm has the potential to differentiate to
form three types of cells including HNK1-positive cardiomyocyte with morphological features similar to those of
conduction cardiomyocytes. In addition, early in chick cardiogenesis, the HNK1 epitope is scattered along the primitive heart tube, only later becoming restricted to the
myocardium of the sinus venosus and atrium in which the
central conduction system will be developed at a later
stage. Further questions are whether the precardiac mesoderm contains three originally different types of cells or
the earliest cardiac primordial cells are pluripotent and
can differentiate to form conduction tissue, and whether
the HNK1-positive cardiomyocyte is a progenitor of the
conduction system at a stage in the development of the
chick heart before conduction system is firmly established.
ACKNOWLEDGMENTS
The authors thank Ms. K. Yoneyama for technical assistance. The monoclonal antibody JB3/fibrillin-2 developed by
Drs. A.M. Wunsch and R.R. Markwald, Medical College of
HNK1 IN EARLY CARDIOGENESIS
Wisconsin, was donated by Dr. K. Isokawa, Nihon University School of Dentistry. The monoclonal antibody MF20
developed by Dr. D.A. Fischman, Cornell University Medical
School, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD
and maintained by the University of Iowa, Department of
Biological Sciences, Iowa City, IA 52242. This work was
supported by a Grant-in-aid from the Ministry of Education,
Science and Culture of Japan (10670027 to YN).
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