Programmed cell death or apoptosis is a central feature of normal tissue development and is involved in many physiologic and pathologic processes. Apoptosis can modulate embryogenesis by sculpting structures, deleting unneeded tissues, and eliminating abnormal, senescent, nonfunctional, or harmful cells (Jacobson et al., 1997). Examples of the involvement of apoptosis in morphogenesis include the development of the limb bud (Garcia-Pelaez and Arteaga, 1993; Tone et al., 1994; Mori et al., 1995), the wing bud (Saunders et al., 1962), the tail bud (Sanders et al., 1986), the central nervous system (Raff et al., 1993; Naruse and Keino, 1995), the thymus (Surh and Sprent, 1994), neural crest cells (Jeffs et al., 1992), and the heart (Pexieder, 1975).
The embryonic chick heart is composed of different tissue types and changes shape dramatically throughout development. In examining programmed cell death in the embryonic chick heart, it is important to note that each tissue type develops at a different stage and in a different region; thus, each contributes to cardiac morphogenesis in a different way. The primitive embryonic chick heart consists of only two cell types, myocytes and endothelial cells, separated by the cardiac jelly, an extracellular matrix (Manasek, 1968; Markwald et al., 1977). All other tissue types, including the epicardium, coronary vessels, connective tissue, neural elements, and conduction tissues are either differentiated in situ or migrate from outside of the heart at later stages. As a new tissue type is recruited into the heart, complicated interactions between the new and preexisting tissues are expected, and tissue elimination and sculpting (thus, programmed cell death) become essential for organogenesis (Jacobson et al., 1997).
The potential importance of apoptosis in heart development had been noticed and investigated 30 years ago by Pexieder and others (Manasek, 1969; Pexieder, 1972a, b, 1975; Ojeda and Hurle, 1975; Hurle and Ojeda, 1979). In his classic work on apoptosis in the embryonic chick heart, by using mainly Nile blue sulfate histochemistry and electron microscopy, Pexieder reported 25 cell death foci inside and 6 foci outside of the embryonic chick heart between embryonic day (ED) 2 to ED 6. Recently, Watanabe et al. (1998) examined apoptosis in the outflow tract (OT) and found that apoptosis in the outflow tract is associated with caspase-3 activity. Poelmann et al. (1998) demonstrated that cardiac neural crest cells undergo apoptosis in the chick heart and appear to participate in septation of the outflow tract. These studies provided strong evidence that physiological cell death plays an important role in cardiac morphogenesis and functional adaptation throughout development.
In the present study, in situ labeling (TUNEL method) (Gavrieli et al., 1992) was used to describe the distribution of apoptosis in the embryonic chick heart, from early stages of development until hatching, to provide detailed, systematic information on apoptotic patterns during development of the chick heart. We examined tissue-specific distribution of apoptosis in the embryonic chick heart, by using in situ labeling of fragmented chromosomal DNA to detect apoptotic cells (Gavrieli et al., 1992), combined with tissue specific antibodies to colocalize apoptosis within specific tissues: (1) antibody MF20 (against atrial and ventricular myosin heavy chain protein) (Han et al., 1992) to delineate cardiomyocytes, (2) monoclonal antibody against alpha smooth muscle actin (SMA, Sugi and Lough, 1992) to identify vascular tissue, (3) EAP-300 antibody (McCabe et al., 1995) as a marker of Purkinje fibers.
We report here that the distribution of apoptosis in different tissue types varies greatly in the embryonic chick heart. At some stages of development (stages 32–34), apoptosis in the cushion tissues predominantly occur in the transitional areas where different tissue types adjoin each other, suggesting the roles of mechanical stress and cell–cell interactions in regulating apoptosis. Apoptosis in coronary vessels occurs late during cardiac development and may be associated with the switch from fetal circulation to mature circulation, whereas Purkinje fibers (located similarly as vascular smooth muscle cells) do not have much apoptosis at the same developmental stages.
TUNEL labeling is the method of choice in detecting apoptosis in situ. To ensure that apoptosis, not necrosis, is responsible for TUNEL labeling in sections of embryonic chick hearts, genomic DNA was extracted from subsampled tissues of stage 29 to 30 embryonic hearts in which TUNEL labeling indicated high levels of programmed cell death. These tissues included the outflow tract cushions (OTCs) and the atrioventricular cushions (AVCs). Similar negative control samples were prepared from the remaining myocardial tissues of these hearts in which relatively low levels of apoptosis were indicated by TUNEL labeling. These samples were ultracentrifuged to obtain low molecular weight genomic DNA. Upon electrophoresis, characteristic ladder patterns were found in the genomic DNA samples prepared from either the OTCs or AVCs but not in samples prepared from the myocardium or from the whole heart (Fig. 1), in which dying cells constitute a much smaller fraction of total cells.
Apoptotic Patterns in the Chick Heart by Stages
In the developing heart, apoptosis takes two different forms: highly concentrated in a limited area (an apoptotic focus) or widely spread but at a very low level. In systematic examination of apoptosis in the embryonic chick heart, we found that at most stages, and in most regions, there always was a baseline level of apoptosis. This level varied somewhat with stage but, by TUNEL labeling, generally accounted for less than 0.1% of the total cell number in a specific area of tissue or in a given morphologic structure (e.g., in the interventricular septum), considerably lower than that observed by Pexieder (1975).
Before stage 24, we found no remarkable foci of TUNEL staining above background level in the heart. Occasionally, one or two apoptotic cells were found in either the atria or ventricles (Fig. 2A). At stage 24 (and also stage 25), the main loci of apoptosis in the embryo were found outside the heart, in regions of the dorsal mesocardium and in the lung buds (Fig. 2B).
In the outflow tract.
The first distinct focus of apoptotic activity in the embryonic chick heart was found at stage 25 in the OT, which formed a definite cell death zone by stage 26 (Fig. 2C). By stage 27, the number of apoptotic cells increased further in the OT. TUNEL-positive cells were most concentrated in the OT cushions, but a large number was also present in the myocardial layer (Fig. 2D). Both in cushions and myocardial tissues, more apoptotic cells were localized in the distal part than in the proximal part of the OT. After stage 27, the distribution of TUNEL-positive cells in the OT cushions shifted proximally, becoming more evenly distributed throughout the cushion tissues.
Apoptosis activity continued to increase in the OT at stage 28 and peaked by stage 29 (Figs. 2E–H, 7A), after which time the number of apoptotic cells declined quickly. Density of dying or dead cells in limited areas of the OT cushions were as high as 10% of total cell number at stages 28 and 29 (Fig. 2E–H). Cell death was more widely distributed in the OT than at any previous stages, involving most areas of the OT cushions but did not go beyond the semilunar valve level. This focus in the OT cushions represented the most active site of apoptosis found in the embryonic chick heart.
After stage 30, apoptotic activity in the OT decreased dramatically. This decline occurred mainly in the cushions (especially in the distal part), but was less remarkable in the myocardium. TUNEL-labeled foci in the cushions shrank as the OT as a whole shortened. By this stage, the right and left OT had separated, and a definitive muscular outlet septum was present.
Apoptosis activity in the OT declined further at stage 32, reaching background levels by stage 33 in most parts of the OT. Residual TUNEL-positive cells in the OT were almost completely confined to the basal regions of the conal cushions and became continuous with cell death zones in AV cushions-derived tissues (the mitral-aortic fibrous continuity) (Figs. 3C,H). In the fully formed muscular outlet septum, there were no apoptotic cells.
In the atrioventricular cushions.
Apoptotic cells began to appear in the AV cushions at stage 26. The density of dying cells at this site was much lower than that in the OT, and no discrete focus of cell death was seen at this stage. This more diffuse apoptotic activity was located mainly in the ventral AV cushions before fusion with the dorsal AV cushions. By stage 27, more TUNEL-positive cells were found in the ventral cushion and also at the fusion area of the dorsal and the ventral cushions.
Pronounced apoptotic zone was observed at stage 28 in the AV cushions (Fig. 2E), where it peaked by stages 29–31 (Figs. 2G,H, 3A,C). At stages 26 to 28, apoptosis in the OT dominated, whereas by stage 29, cell death levels in the OT and the AV cushions were equivalent in intensity. At stages 29–30, generally approximately 30–60 TUNEL-positive cells were found per section in the AV cushions, in more than 20 successive sections. These cells were located mainly in the recently fused AV cushions. The ventral cushion tended to contain more TUNEL-positive cells than the dorsal cushion. The lateral AV cushions had much lower levels of apoptotic signal than either the dorsal or ventral cushions.
By stage 30, the AV cushions had replaced the OT as the leading site of programmed cell death, and most apoptotic cells were located in the central part of the AV cushions. By stage 31, the patterns of apoptosis were not as discretely localized as those at stages 28–30, but, due to wider distribution of labeled cells and increased volume of the cushions, the overall levels of cell death were maintained at very high levels (Fig. 3C). Distinct from stage 30, numerous TUNEL-positive cells were found in the septal leaflet of the mitral valve and some of the apoptotic cells extended as far as the tip of the valve.
After stage 32, the level of apoptosis in the AV cushions also decreased obviously, although less dramatically than in the OTCs (Fig. 3D,H). Cell death in the septal leaflet of the mitral valve almost disappeared. TUNEL labeling in the fused cushions extended inferiorly and anteriorly to the medial part of the conal cushions, surrounding the proximal part of the great vessels. In effect, the disparate sites of apoptotic activity in the AV cushions and in the OT had now merged into a continuous complex.
Between stages 29 and 31, an apoptotic focus was found in the upper interventricular septum (IVS) (Figs. 2G, 3C, 4E). Approximately 10–20 TUNEL-positive cells per section (in approximately 10–15 successive sections) were found in the upper part of the septum, in a location corresponding to the position of the developing His bundle. The nature of the TUNEL-staining looked somewhat different from apoptotic patterns found elsewhere and also was different from that observed in the same area at other stages. The apoptotic cells at this locus were intense and transient, more clustered together, and some smearing of TUNEL-staining was noticed. This pattern of cell death was observed only between stages 29 to 31. After careful examination in many heart samples, we found that this phenomenon existed in approximately one third of all hearts investigated at each stage (5 of 11 in stage [ST] 29, 5 of 12 in ST 30, and 3 of 10 in ST 31). At stages 32 to 34, apoptosis was detected in the His bundle branches, especially in the left branch (Fig. 3D) but no longer in the His bundle area. Some TUNEL-positive cells were also observed in areas of the right AV ring (Fig. 3F).
A new apoptotic zone became evident at stages 32 and 33 in the ventricular trabeculae (Fig. 3D,F). Because trabeculae do not form a compact structure, the distribution of cell death appeared to be diffuse. However, due to the extent of the trabeculated myocardium at stages 32 to 33, the actual level of cell death in this tissue was close to that occurring earlier in the AV cushions or OT cushions. Interestingly, there always were more apoptotic cells in the right ventricle than in the left (Fig. 7C). This focus of cell death decreased since stage 34 (Fig. 3G,H) and disappeared by stage 36.
The dorsal mesocardium exhibited a high level of apoptotic activity from stage 24 to 30 (Fig. 2B). In the epicardium, most cells undergoing programmed cell death were localized in the conoventricular sulcus (Figs. 2C, 3B), the interventricular sulcus, and the AV sulcus regions (Fig. 3C,D,F,H). This signal also extended to the posterior inner curvature of the heart (mainly in the epicardium posterior to the OT and between the atria) and in the retroaortic epicardium (Figs. 2F, 3E). Other less prominent loci of TUNEL-positive cells included myocardium in the conoventricular region (Fig. 3B), the AV junction region (Fig. 3C), and the cardiac apex in the base of the interventricular septum.
In general, after stage 36, apoptosis in the embryonic chick heart dropped to background level in most tissues, including the OT and AV cushions. However, in the trabeculae, especially in the right ventricle and near the cardiac apex, some apoptotic cells were frequently noted, but no cell death zone was observed. A final focus of apoptosis emerged in wall of the coronary vessels and the aorta between stages 40 to 44 (see below).
Cushion Tissues Adjoining Cardiomyocytes Were Among the Major Foci of Apoptosis in the Developing Chick Heart
MF20 and TUNEL double staining were performed on serial sections from stages 28 through 34, when apoptosis was most remarkable in the embryonic chick heart. We found that apoptosis in cardiomyocytes was much less than in cushion tissues, and was located mainly in a few specific regions. The first was the conotruncus region of OFT from stages 27 through 29, which accompanied apoptosis activity in the outflow tract cushions (Fig. 4F). The second was the subconal myocardium at stages 32–34, near the atrioventricular junction areas (Fig. 4D,E). The third was the ventricular trabeculae, as well as in the papillary muscles. Cell death detected in the His bundle area and the bundle branches were also MF20 positive (Fig. 4E).
More important, MF20 and TUNEL double staining also revealed the localization of apoptotic cells in cushion tissues relative to nearby cardiomyocytes. At early stages (before stage 30), the volume of the atrioventricular cushions continues to enlarge to occupy a significant part of the heart. At this period, apoptosis was located in the central part of the atrioventricular cushions, away from cardiomyocytes below and above (Fig. 4A). Later (mainly stages 32–34), the relative volume of cushions shrank as the cardiomyocytes muscularize the atrioventricular regions. At this time, apoptotic cells were often scattered between the invading myocardial strips and fibroblast cells adjacent to invading cardiomyocytes in cushions were the major sites of apoptosis (Fig. 4B,C).
Apoptosis in the Vascular System Peaked in Late Stages of Development but Before Hatching
SMA antibody did not specifically stain any part of embryonic chick heart in paraformaldehyde-fixed paraffin-embedded sections until stage 40 (ED 14). After this stage, SMA stained vascular smooth muscle cells (including the aorta and pulmonary artery) intensely, especially in small blood vessels around stage 44.
No marked apoptotic foci were found in blood vessels before stage 40, including the aorta and the pulmonary artery. Later, as SMA staining became positive in vascular smooth muscle cells, many vascular smooth muscle cells were labeled by TUNEL as well. At stages 40 to 44, we found that coronary vessels were the major focus of apoptosis (Fig. 5A,B), sometimes also in tissues adjacent to blood vessels. Typically, in a cross-section of a small coronary vessels, we observed 1–5 TUNEL-positive cells and that number was significantly higher than apoptotic levels in non–SMA-positive cells (Fig. 7D).
The most dramatic apoptotic pattern at stages 40 to 44 was found in smooth muscle layers of the aorta, and to a much less extent, in the pulmonary artery. This finding was especially true at stage 44, when the smooth muscle cell death in the aorta was very high (Fig. 5C,D). The extent of apoptosis activity included all layers of the tunica media and full circumference. Such apoptotic activity ended abruptly near the semilunar valve area.
To address whether this change of apoptotic patterns in the aorta was due to hemodynamic changes from embryonic circulation to posthatching circulation in the embryonic chick heart, we further examined cell death in the vascular system of older chick hearts. Chick hearts were collected each day from ED 20 through 3 days after hatching. The aorta and pulmonary artery were carefully examined with TUNEL labeling. No remarkable apoptosis activity was found in the vascular system in the chick heart (including the vascular system) from ED 20 until 3 days after hatching.
Peripheral Cardiac Conduction Tissue Did Not Have Much Apoptosis During Late Development
The increased level of apoptosis activity in areas adjacent to coronary vasculature around stages 40–44 raised another question as to whether Purkinje's fibers contribute to apoptosis, as Purkinje's fibers also exist in the perivascular area. To address this question, EAP-300 and TUNEL double staining were performed in both ED 14 and ED 18 embryonic chick hearts. Because TUNEL labeling requires pretreating sections with proteinase K, which would block EAP-300 immunostaining, we did EAP-300 immunostaining first with alkaline phosphatase, then performed TUNEL labeling with horseradish peroxidase. The color development conditions were carefully controlled to reveal both signals.
EAP-300–positive cells were found in perivascular and subendocardial regions where Purkinje's fibers are located. In perivascular regions, apoptosis predominantly occurred in the vasculature per se (vascular smooth muscle cell layer, which were located inside of EAP-300–positive cell ring) at these stages, in both small and large coronary vessels (Fig. 6). Cell death in EAP-300–positive Purkinje's fibers was very uncommon, although we did observed a few TUNEL-positive cells in EAP-300–positive cells (Fig. 6B). Most SMA-negative perivascular apoptotic cells turned out to be contractile cardiomyocytes, as demonstrated by MF20-positive and EAP-300–negative staining (data not shown). This finding was especially true at later stages (ED 18). Similarly, apoptotic cells were even more rare in subendocardial Purkinje fibers. We were not able to discriminate these tissues with EAP-300 and TUNEL double staining at earlier stages (before stage 40) due to nonspecific expression of EAP-300.
Quantitative Analysis of Apoptosis in Chick Heart
The number of apoptotic cells were statistically analysed in the OT and AV cushions (from stage 24 through 33), in the ventricular trabeculae (stages 31 to 33) and in the coronary vessels in stages 40 and 44. Our data demonstrated that the number of apoptotic cells in both the OTCs and the AVCs increased quickly and steadily with the development of the heart, from unnoticeable level at stage 24 through peak level at stage 29 or 30, respectively (Fig. 7A,B). The increase of apoptotic cells in both the OTCs and the AVCs was more dramatic during early stages, more or less doubling with every stage from stage 24 through 27. The absolute number of apoptotic cells reached similar peak level in both the OTCs and the AVCs, although the peak level of apoptosis in the OTCs was a stage earlier than in the AVCs. After that, apoptosis activity in the OTCs decreased as quickly as it built up, approaching background level by stage 33 (Fig. 7A). The decline of apoptotic cells in the AVCs after stage 30 was also very significant but less dramatic compared with that in the OTCs (Fig. 4B), partly due to enlarged volume of AV cushion-derived tissues.
We also counted apoptotic cells in the ventricular trabeculae from stage 31 through 33 (Fig. 7C). The counting did not include the ventricular walls and the interventricular septum. Apoptotic cells in the ventricular trabeculae were observed as early as stage 27, but remained at very low level before stage 31. This changed sharply by stage 32, when apoptosis activity was significantly higher than its previous stage in both the left and the right ventricular trabeculae. In addition, apoptotic level in the right trabeculae was significantly higher than the left side in all cases examined (stages 31–33) (Fig. 7C).
In counting of apoptotic cells in vascular smooth muscles, we used yoyo-1 nuclear staining to delineate individual smooth muscle cell and total cell number. Apoptotic figures constituted approximately 6% of vascular smooth muscle nuclei counted at both stages 40 and 44. That was significantly higher than the level of apoptotic cells among nonvascular smooth muscle cells (approximately 0.2–0.3%, the background level of apoptosis at these stages) and also significantly higher than the level of apoptotic cells among all cells (approximately 0.2–0.4%) at the same stages (Fig. 7D). In addition, among all the apoptotic cells in the ventricular myocardium, 26% (stage 40) to 41% (stage 44) of them were detected in the vascular smooth muscle. This finding was in clear contrast to the percentage of vascular smooth muscle cells in total cells of the ventricular wall, which accounted for only 1.5% (stage 40) to 2.6% (stage 44) of the total cell number (Fig. 7E).
Our findings confirm and extend those reported previously by Pexieder and others (Manasek, 1969; Pexieder, 1972a,b, 1975; Ojeda and Hurle, 1975; Hurle and Ojeda, 1979). However, there are important differences in apoptotic patterns discriminated in the present study and those described in earlier reports. In the current work, few apoptotic cells were detected between stages 17 and stage 25, which is quite different from Pexieder's work, which reported many cell death zones at these stages. In contrast, the earliest cell death zone reported here, the dorsal mesocardium, was not noted in Pexieder's report. In later developmental stages, the most distinctive cell death site identified by TUNEL in our study, the AV cushions, were reported negative at ED 6 and ED 8 by vital staining. Part of this discrepancy may be resolved by consideration of differences inherent to the histologic methods used. The Nile blue sulfate staining and electron microscopy used by Pexieder emphasized cytoplasmic lysosomal phagocytic or autolytic processes that may extend over periods longer than those few hours in which TUNEL labeling follows endonuclease activation (Bursch et al., 1990). Overall, the TUNEL-based method discriminated much lower levels of cell death than reported by Pexieder at all stages.
In this study, we have charted and illustrated the entire spatiotemporal distribution of apoptosis during development of the chick heart. A dynamic pattern of apoptotic activity is observed during embryogenesis of the heart. Primarily, these dynamic shifts in programmed cell death occur in close spatial and temporal proximity with the major remodeling events that accompany cardiac morphogenesis and septation. Consistent with the central role of cushion tissues in such remodeling (Markwald et al., 1998), the main foci of apoptosis shifts within different cushions in accordance with the principal morphogenetic events under way. This wave-like progression in the cushions initiates at the start of outflow tract division and culminates during fusion of the AV and conal cushions as ventricular septation concludes. Secondarily, programmed cell death seems correlated with the differentiation of specialized cardiac tissues, including differentiation of components of the central and peripheral conduction system. Our data suggest that apoptosis is crucial to the transformation of the heart from a simple tube to a complex multichambered pump. The high resolution information delineated here also provides a map for precise targeting of such activity in subsequent mechanistic and molecular studies of programmed cell death in development of the heart.
Programmed Cell Death Is Associated With Morphogenesis of the OT and AV Canal
Functional roles of apoptosis during development in the chick have been suggested both for the shortening or removal of outflow myocardium (Watanabe et al., 1998) and the removal or collapse of subjacent cushion material (Poelmann et al., 1998), processes that result normally in formation of a continuous muscular outlet septum between and below the semilunar valves. This process appears most prominently at stages 29–30, stages at which apoptosis peaks in these cushions, suggesting a potential correlation between these events. After stage 31, coinciding with the completion of these remodeling processes, apoptosis in the OT decreases quickly to background levels. Such a role for focal cell death in outlet septation has been suggested from study of human embryos, as well (Okamoto et al., 1981; Thompson et al., 1985). Recent mouse studies have associated reduced apoptosis with hypercellularized cushions and failure of the muscular outflow septum to form in the neurofibromin I knockout mouse (Lakkis and Epsein, 1998), in contrast with increased apoptosis with similar morphologic sequelae in the mouse lacking transforming growth factor beta-2 (Bartram et al., 2001).
Similarly, in AV cushions, apoptosis was not found during the cushion enlargement phase but most massively in cushion reshaping and shrinking phases. Before stage 27, the ventral and the dorsal AV cushions are not yet fused and few apoptotic cells were observed. At stage 28, as the AV cushions begin to fuse, apoptosis rises dramatically. This finding, together with the upward growth of the interventricular septum and the AV valve formation at stages 29 and 30, sets up the framework of the four-chamber heart, thus establishing the definitive prehatching blood flow patterns (Jaffee, 1970). After this period, cell death in the AV cushion-derived tissues declines. By stage 36, apoptosis in the AV cushions-derived tissues and most parts of the heart drops to background level, well in accord with the fact that tissue remodeling in the embryonic chick heart has largely completed by that time.
Although molecular mechanisms leading to cell death in the developing heart remain to be established, the focal and synchronized patterns of cell death presented here codistribute with regions of myocardium or matrix suggested from other work to be sites of mechanical tension within the growing heart. Thus, apoptotic regions within myocardium (bracketed in Figs. 2C,D, 3B,C,F) conform to known zones of terminal differentiation and high alignment of myocardium (Thompson and Fitzharris, 1985; Thompson et al., 1995), all tracts of early muscle that either disappear or contribute to definitive conduction tissues. Similarly, regions of highly aligned cell and matrix fibers have been described within condensed mesenchymal tissue of the outflow tract (Thompson et al., 1984; Thompson and Fitzharris, 1985) consistent both with focal mechanical tension during truncal septation and with the regions and periods of dramatic cell death reported here. The same effect is also demonstrated in apoptosis of AV cushion tissues adjacent to the myocardium, as revealed in MF20 and TUNEL double labeling. For many morphologic changes, the myocardium is almost always the stress-producing tissue (the myocardium also contract spontaneously), and the cushions are the stress-bearing tissues (Manasek and Nakamura, 1985). Frequently, the stress produced by the myocardium transfers to the cushions to cause tissue remodeling, which is accompanied by a high level of apoptosis. Meanwhile, the initial focus of cells along the line of cushion fusion in the AV canal, in the absence of demonstrated mechanical tension, may indicate an active role of apoptosis itself in that fusion process. Speculation that mechanical tension may induce cell death is consistent with the seminal rescue experiments of Pexieder (1975), in which culture (and mechanical release) of isolated outflow segments before a critical period of development resulted in reduction of cell death foci 8–24 hr later. Finally, the occurrence of apoptosis in the outflow region before similar but protracted kinetics in the AV canal would seem to minimize potential roles of circulating factors in this process.
Association Between Apoptosis and Differentiation of the Cardiac Conduction System
In addition to linking programmed cell death to proper morphogenesis of the outflow tract and AV canal, this study also reveals important correlation between the distribution of apoptosis and the differentiation of cardiac conduction tissues. A large and very distributed focus of cell death identified was in the ventricular trabeculae between stages 32 and 34. Trabeculated muscle has been long known as rich in the cellular antecedents of specialized cardiac tissues (reviewed by Moorman et al., 1998). Previously, it has been demonstrated that contractile myocytes and cardiac conduction cells (Purkinje fiber cells) differentiate from common progenitors in the looped, tubular heart (Gourdie et al., 1995; Cheng et al., 1999). In prior experiments using pulse labeling with bromodeoxyuridine and [3H]thymidine, we reported that one of the earliest markers of cardiac specialized tissue differentiation is commitment to nonproliferation (Thompson et al., 1990, 1995). It was consequently posed that the decreases in relative abundance of Purkinje fibers within retroviral clones might be accounted for by decreases in mitotic rate within this cell population. The present data suggest that increased attritional rates may also be a factor in determining the density of conductive cells within clonal sectors—a strong indication that apoptosis is active in adaptive pruning of the cells composing the peripheral conductive network.
Another relatively transient but intense concentration of cell death was frequently noted in the His bundle region between stages 29 and 31. We found that approximately one third of the hearts examined at these stages had this phenomenon, which suggests that every embryonic chick heart undergoes this kind of apoptosis in the His bundle region during stages 29, 30, or 31. This phenomenon is of great significance. Recently, it has been shown that the chick ventricle undergoes a remarkable reversal in its activation pattern at precisely the same developmental period (Chuck et al., 1997). At present, there is no evidence that these two observations are directly related. Nonetheless, based on the patterns of recruitment to different parts of the conduction system revealed in retroviral lineage tracing studies (Gourdie et al., 1995; Cheng et al., 1999), we concluded in earlier studies that the peripheral conductive network organizes independently from the nodes and bundles of the central conduction system. The ventricular activation switch reported by Chuck and colleagues (1997) may represent an electrophysiological manifestation of the linkage between these independently forming central and peripheral compartments of specialized tissues. We might also speculate that such a sudden reversal in conduction pattern might itself trigger focal cell death, as a result of sudden increases in mechanical loading along the crests of central trabeculae as they coalesce to form the His bundle. Future work is required to determine whether, and how, programmed cell death is involved in expediting the breakdown of conductive barriers between different elements of the maturing conduction system and the establishment of preferential pathways of cellular activation in the heart. Scattered apoptosis found along bundles and branches of central conduction tissue may also represent the progressive attrition of invading neural crest derived populations along such sites (Poelmann and Gittenberger-de Groot, 1999), although possible roles of such cells or their death remain speculative. Although definitive neural elements are not found along central conduction fascicles in birds (Verberne et al., 2000), evidence for such innervation is found, to variable degree, in mammals, including rat (Petrecca and Shrier, 1998), guinea pig (Crick et al., 1996), rabbit (Anderson and Taylor, 1972), pig (Crick et al., 1999), cow (Forsgren, 1988), and human (Crick et al., 1994). At the level of resolution presented here, colocalization of TUNEL-positive nuclei or nucleosomes with tissue-specific antigenic markers does not necessarily prove that the dying cell was of that tissue type (Hurle and Ojeda, 1979).
Apoptosis in Vasculature Could Be Associated With Hemodynamic Changes in the Prehatching Period
On realization that a large fraction (26–41%) of all apoptosis in the ventricular walls (at stage 40 and 44, respectively) were localized in the SMA-positive cells and that SMA-positive cells constituted only 1.5 to 2.6% of the total cell population in the ventricular walls at these stages (Fig. 7E), the significance of apoptosis in blood vessels could not go unnoticed. Based on our data, apoptosis is not actively involved in early stages of angiogenesis but is important in later stages of vascular development, which involves coalescence of microvessels. Two mechanisms need to be considered here. One is that continuous augmentation of the vasculature system necessitates that some cells immediately outside of the vasculature must be removed to make room for new structures; thus, selective tissue elimination by cell death is likely. If this mechanism is true, then most apoptotic cells should be in the regions around the blood vessels. We did find some apoptotic cells in myocardium around blood vessels, but the majority of apoptosis was in the vascular smooth muscle cells. More important, perivascular Purkinje fibers, adjacent to the vascular smooth muscle cells, have very few apoptotic cells. An alternative explanation is that hemodynamic changes or other alterations in the circulation system trigger apoptosis in blood vessels either directly or indirectly.
Some dramatic events do happen during these stages. A closed coronary vasculature is completed soon after ED 14 (Rychter and Jelinek, 1971; Rychter et al., 1971; Rychter and Ostadal, 1971), which may alter the balance of mechanical stress on blood vessels. In addition, it should be emphasized that the maturation of the circulation system in the chick is different from mammals. In chick, noticeable respiration is observed at ED 19 (White, 1974), which is before hatching. This is so because in chick, without respiration, all gas exchange is accomplished by diffusion through the porous egg shell (in mammals, it is through placental circulation). At late stages of chick development, the surface area of the chorioallantois is increasingly insufficient to maintain the physiological need of the growing embryo (White, 1974; Burton and Tullett, 1985). Low blood oxygen was noticed as early as at ED 15, whereas blood CO2 tension increases steadily from ED 11 until ED 19 (Dawes and Simkiss, 1969), but the chorioallantois (the major site for gas exchange until the embryo breathes) in the chick embryo does not develop any further beyond ED 15 (Windle and Barcroft, 1938). These findings strongly suggest that ED 15–19 is a very important period for establishing pulmonary circulation and respiration and suggest a potential relationship with apoptosis during this period.
Monoclonal antibody MF20, QH-1 antibody, EAP-300 monoclonal antibody were all supernatants of cell culture. EAP-300 was kindly provided by Dr. Gregory J. Cole (Ohio State University, Columbus, OH). Monoclonal antibody against SMA was from Sigma (St. Louis, MO). Cy-5–labeled donkey anti-mouse immunoglobulin G (IgG) monoclonal antibody was from Jackson ImmunoResearch Lab (Philadelphia, PA). Rhodamine-conjugated antidigoxigenin antibody was from Boehringer Mannheim (Indianapolis, IN), yoyo-1 was from Molecular Probes (Eugene, OR), and Apoptag kit was from Intergen (Purchase, NY).
Genomic DNA Extraction and Electrophoresis
Low molecular weight DNA was extracted as follows: freshly isolated embryonic chick hearts were rinsed in Earle's balanced salt solution, and incubated with Dulbecco's phosphate buffered saline (PBS; from Gibco BRL) with 5mM ethylenediaminetetraacetic acid (EDTA) for 30 min at 4°C. Tissues were then trypsinized, (0.5% trypsin in D-PBS, 15 min), neutralized with chicken serum, digested with lysis buffer (5 mM Tris-HCl, pH8, 20 mM EDTA, 0.5% Triton X-100), and incubated on ice for 20 min. The samples were centrifuged at 27,000 × g for 20 min to separate fragmented DNA (supernatant) from intact DNA (pellet) (Cohen and Duke, 1984). The supernatants were extracted twice with phenol/chloroform and precipitated with ethanol. The pellets were then dissolved in 0.5 ml 0.1× SSC with 20 μg/ml RNase A, incubated at 37°C for 30 min, reextracted with phenol-chloroform, and precipitated with 70% ethanol. The DNA pellets were dissolved in 10 μl TE buffer, and electrophoresed in 1.5% agarose gel.
Tissue Preparation for Immunohistrochemistry
Fertilized White Leghorn chicken eggs were incubated at high humidity at 37.5°C. Chick embryos (at least three embryos at each stage) were harvested at Hamburger and Hamilton stage 17 through hatching (Hamburger and Hamilton, 1951), fixed in 4% paraformaldehyde, and embedded in Polyfin (Triangle Biomedical Sciences, Durham, NC). Three sets of embryonic chick hearts were examined by regular TUNEL staining, including every section and every stage from stage 17 to 36, selected sections for every other stage for stages 38–48, and selected sections of new born chick heart (each day until 3 days after hatching). Serial sections (7 μm) of chick hearts before stage 36 or selected sections (every 100 μm) for later stages were used for this study. For double fluorescence immunostaining, five to eight hearts for each stage were processed similarly for quantitative analysis as described below. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
In Situ Detection of Apoptosis
Chick heart sections were treated as instructed by Apoptag kit. First, sections were deparaffinized with xylene and rehydrated through serial ethanol, digested with proteinase K (10 μg/ml for 15 min at room temperature), and rinsed in PBS. Endogenous peroxidase activity was blocked by 2% H2O2 (5 min at room temperature). Heart sections were subsequently incubated with terminal deoxynucleotide transferase (TdT) and digoxigenin-dUTP at 37°C for 1 hr. The incorporated digoxigenin-dUTP was detected by peroxidase-conjugated antidigoxigenin antibody and signal developed by incubation with 3, 3′-diamino-benzidine (DAB) in the presence of H2O2. Transmission mode confocal microscopy (BioRad MRC-1000) was used for photography.
For fluorescent TUNEL labeling, rhodamine-conjugated antidigoxigenin antibody was used instead of peroxidase-conjugated antibody, diluted 1:100 in PBSA-BSA (PBS containing 0.25% sodium azide and 1% bovine serum albumin). After incubation for 2 hr, sections were rinsed with PBS three times and incubated with fluorescent secondary antibody.
MF20 Antibody and Apoptosis Double Staining
For MF20 immunostaining in paraformaldehyde-fixed sections, slides were first treated by heating up to 95°C for 10 min in 50% glycerol-PBS solution, followed by rhodamine fluorescent TUNEL staining (described above), then incubated with MF20 antibody for 3 hr at room temperature, washed three times with PBS, and finally incubated with Cy-5–labeled donkey anti-mouse IgG secondary antibody (1 to 100 dilution in PBSA-BSA) for another 2 hr.
SMA Antibody and TUNEL Double Staining
After rhodamine-tagged TUNEL labeling, sections were incubated with SMA antibody (1:1,000 dilution in PBSA-BSA) at room temperature for 2 hr, washed with PBS, then stained with Cy-5 labeled donkey anti-mouse secondary antibody for another 2 hr.
EAP-300 and TUNEL Double Staining
No proteinase K pretreatment for EAP-300 immunostaining. Sections of stage 40–44 chick heart were incubated with monoclonal EAP-300 (1:1,000 in TBS) and then with alkaline phosphatase (AP) -tagged sheep anti-mouse antibody (from Sigma) (1:200 dilution in Tris buffer); after AP staining (NBT/BCIP) for EAP-300, the sections were digested with proteinase K (10 mg/ml for 10 min at RT) and went through peroxidase-mediated TUNEL labeling and stained with DAB.
Yoyo-1 Nuclear Staining
After double fluorescence immunostaining of SMA and TUNEL, heart sections were immersed in 1 N HCl for 5 min, incubated with 1:500 yoyo-1 in PBS for 5 min, rinsed in PBS, and covered with coverslips with PBS-50% glycerol with 0.1% sodium azide.
Quantitative Analysis of Apoptotic Cells
Quantitative analysis of apoptosis activity was based on counting of TUNEL-positive cells in serial sections of three sets of embryonic chick hearts from stages 24 through 33. Alternate sections were counted to avoid duplication in counting. Apoptotic cell counts at each stage were compared with its previous stage, and the results analyzed by Student's t-test. Similarly, we analyzed apoptotic cells in the right ventricular trabeculae vs. the left ventricular trabeculae.
Apoptotic cells in vascular smooth muscle cells in the ventricular walls at stages 40 and 44 were counted from five different hearts for each stage. yoyo-1 nuclear staining was used to delineate the morphology of nuclei and for counting of smooth muscle cells and total cell number in a section. TUNEL-positive cells, yoyo-1–positive nuclei, and smooth muscle cells from 10 sections across the middle ventricle of each heart were counted, values averaged as one measurement for each heart sample, and data analyzed by Student's t-test.
R.P.T., A.W., and R.G.G. received funding from the National Institutes of Health. R.G.G. is an Early Career Scholar of the National Science Foundation. Preliminary results were presented in at the Weinstein Cardiovascular Development Conference in 1995 in Rochester, NY.