Expression of HNK1 epitope by the cardiomyocytes of the early embryonic chickIn situ and in vitro studies.код для вставкиСкачать
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: firstname.lastname@example.org 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). REFERENCES Abo T, Balch CM. 1981. A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK-1). J Immunol 127: 1024 –1029. Aoyama N, Kikawada R, Yamashina S. 1993. Immunohistochemical study on the development of the rat heart conduction system using anti-leu-7 antibody. Arch Histol Cytol 56:303–315. Aoyama N, Tamaki H, Kikawada R, Yamashina S. 1995. Development of the conduction system in the rat heart as determined by Leu7(HNK-1) immunohistochemistry and computer graphics reconstruction. Lab Invest 72:355–366. Arguello C, Alanis J, Valenzuela B. 1988. The early development of the atrioventricular node and bundle of His in the embryonic chick heart. An electrophysiological and morphological study. Development 102:623– 637. Ariga T, Kohriyama T, Freddo L, Latov N, Saito M, Kon K, Ando S, Suzuki M. 1987. Characterization of sulfated glucuronic acid containing glycolipids reacting with IgM M-proteins in patients with neuropathy. J Biol Chem 262:848 – 853. Bannerman PG, Oliver TM, Nichols WL Jr, Xu Z. 1998. The spatial and temporal expression of HNK-1 by myogenic and skeletogenic cells in the embryonic rat. Cell Tissue Res 294:289 –295. Bader D, Masaki T, Fischman DA. 1992. Immunochemical analysis of myosin heavy chain during avian myogenesis in vivo and vitro. J Cell Biol 95:763–770. Canning DA, Stern CD. 1988. Changes in the expression of the carbohydrate epitope HNK-1 associated with mesoderm induction in the chick embryo. Development 104:643– 655. Cheng G, Litchenberg WH, Cole GJ, Mikawa T, Thompson RP, Gourdie RG. 1999. Development of the conduction system involves recruitment within a multipotent cardiomyogenic lineage. Development 126:5041–5049. Chuck ET, Watanabe M. 1997. Differential expression of PSA-NCAM and HNK-1 epitopes in the developing cardiac conduction system of the chick. Dev Dyn 209:182–195. DeHaan RL. 1965. Development of pacemaker tissue in the embryonic heart. Ann NY Acad Sci 127:7–18. Edelman GM. 1988. Morphoregulatory molecules. Biochemistry 27: 3533–3543. Eisenberg LM, Markwald RR. 1995. Molecular regulation of atrioventricular valvuloseptal morphogenesis. Circ Res 77:1– 6. Gorza L, Schiaffino S, Vitadello M. 1988. Heart conduction system: a neural crest derivative? Brain Res 457:360 –366. Gorza L, Vettore S, Vitadello M. 1994. Molecular and cellular diversity of heart conduction system myocytes. Trends Cardiovasc Med 4:153–159. Gourdie RG, Mima T, Thompson RB, Mikawa T. 1995. Terminal diversification of the myocyte lineage generates Purkinje fibers of the cardiac conduction system. Development 121:1423–1431. Hamburger V, Hamilton HL. 1951. A series of normal stages in the development of the chick embryo. J Morphol 88:49 –92. Ikeda T, Iwasaki K, Shimokawa I, Sakai H, Ito H, Matsuo T. 1990. Leu-7 immunoreactivity in human and rat embryonic hearts, with special reference to the development of the conduction tissue. Anat Embryol 182:553–562. Imanaka-Yoshida K, Knudsen KA, Linask KK. 1998. N-cadherin is required for the differentiation and initial myofibrillogenesis of chick cardiomyocytes. Cell Motil Cytoskeleton 39:52– 62. 333 Ito K, Sieber-Blum M. 1991. In vitro clonal analysis of quail cardiac neural crest development. Dev Biol 148:95–106. Kirby ML, Gale TF, Stewart DE. 1983. Neural crest cells contribute to normal aorticopulmonary septation. Science 220:1059 –1061. Kruse J, Mailhammer R, Wernecke H, Faissner A, Sommer I, Goridis C, Schachner M. 1984. Neural cell adhesion molecules and myelin-associated glycoprotein share a common carbohydrate moiety recognized by monoclonal antibodies L2 and HNK-1. Nature 311:153–155. Lu Y, James TN, Yamamoto S, Terasaki F. 1993. Cardiac conduction system in the chicken: gross anatomy plus light and electron microscopy. Anat Rec 236:493–510. Luider TM, Bravenboer N, Meijers C, van der Kamp AWM, Tibboel D, Poelmann RE. 1993. The distribution and characterization of HNK-1 antigens in the developing avian heart. Anat Embryol 188:307–316. Mikawa T, Borisov A, Brown AMC, Fischman DA. 1992. Clonal analysis of cardiac morphogenesis in the chicken embryo using a replication-defective retrovirus: I. formation of the ventricular myocardium. Dev Dyn 193:11–23. Moorman AFM, de Jong F, Denyn MMFJ, Lamers WH. 1998. Development of the cardiac conduction system. Circ Res 82:629 – 644. Nakagawa M, Thompson RP, Terracio L, Borg TK. 1993. Developmental anatomy of HNK-1 immunoreactivity in the embryonic rat heart: co-distribution with early conduction tissue. Anat Embryol 187:445– 460. Nakajima Y, Miyazono K, Nakamura H. 1999a. Immunolocalization of latent transforming growth factor-␤ binding protein-1 (LTBP1) during mouse development: possible roles in epithelial and mesenchymal cytodifferentiation. Cell Tissue Res 295:257–267. Nakajima Y, Yamagishi T, Yoshimura K, Nomura M, Nakamura H. 1999b. Antisense oligodeoxynucleotides complementary to smooth muscle ␣-actin inhibits endothelial-mesenchymal transformation during chick cardiogenesis. Dev Dyn 216:489 – 498. Newgreen DF, Powel ME, Moser B. 1990. Spatiotemporal changes in HNK-1/L2 glycoconjugates on avian embryo somite and neural crest cells. Dev Biol 139:100 –120. Noden DM. 1991. Origins and patterning of avian outflow tract endocardium. Development 111:867– 876. Patten BM. 1956. The development of the sinoventricular conduction system. Univ Mich Bull 22:1–21. Perez-Pomares JM, Macias D, Garcia-Garrido L, Munoz-Chapuli R. 1997. The origin of the subepicardial mesenchyme in the avian embryo: an immunohistochemical and quail-chick chimera study. Dev Biol 200:57– 68. Poelmann RE, Mikawa T, Gittenberger-de Groot AC. 1998. Neural crest cells in outflow tract septation of the embryonic chicken heart: differentiation and apoptosis. Dev Dyn 212:373–384. Poelmann RE, Gittenberger-de Groot AC. 1999. A subpopulation of apoptosis-prone neural crest cells targets to the venous pole: multiple functions in heart development? Dev Biol 207:271–286. Rongish BJ, Drake CJ, Argraves WS, Little CD. 1998. Identification of the developmental marker, JB3-antigen, as fibrillin-2 and its de novo organization into embryonic microfibrous arrays. Dev Dyn 212:461– 471. Sakai H, Ikeda T, Ito H, Nakamura T, Shimokawa I, Matsuo T. 1994. Immunoelectron microscopic localization of HNK-1 in the embryonic rat heart. Anat Embryol 190:13–20. Sugi Y, Markwald RR. 1996. Formation and early morphogenesis of endocardial endothelial precursor cells and role of endoderm. Dev Biol 175:66 – 83. Tucker GC, Aoyama H Lipinski M, Tursz T, Thiery JP. 1984. Identical activity of monoclonal antibodies HNK-1 and NC-1: conservation in vertebrates on cells derived from the neural primordium and some leucocytes. Cell Differ 14:223–230. Vincent M, Thiery JP. 1984. A cell surface marker for neural crest and placodal cells: further evolution of the peripheral and central nervous system. Dev Biol 103:468 – 481. Wunsch AM, Little CD, Markwald RR. 1994. Cardiac endothelial heterogeneity defines valvular development as demonstrated by the diverse expression of JB3, an antigen of the endocardial cushion tissue. Dev Biol 165:585– 601.