/. Embryol. exp. Morph. Vol. 41, pp. 161-173, 1977
Printed in Great Britain © Company of Biologists Limited 1977
Cytological and cytochemical studies
of the necrotic area of the bulbus of the chick
embryo heart: phagocytosis by developing
myocardial cells
By J. M. HURLE, 1 M. LAFARGA AND J. L. OJEDA
From the Departamento de Anatomia, Facultad de Medicina,
Santander, Spain
SUMMARY
The muscular wall of the bulbus cordis of the chick embryo was studied by electron microscopy during H. H. stages 27—31. Results show that the muscle cells of the bulbus undergo
necrosis during these stages. The morphology of dead cells in this area is similar to that
described in other embryonic myogenic tissues. Although dead myocytes appear acidphosphatase-negative, lysosomes may well be involved in this necrotic process as there is a
significant number of cells with autolytic lesions among the neighbouring healthy muscle
cells. The phagocytosis of the cell detritus resulting from the degenerative process appears to
be carried out by neighbouring myocytes and by macrophages. The significance of these
results is discussed both from the cytological and from the morphogenetic point of view.
INTRODUCTION
Physiological cell death is a common phenomenon during embryonic development in vertebrates (for review, see Gliicksmann, 1951; Saunders, 1966).
It occurs during the differentiation and morphogenesis of many organs, and in
the elimination of structures that are useful only during the embryonic period.
The mechanism of death at the cellular level has not been clarified so far.
However most of the studies suggest that necrotic processes take place in two
steps; the degeneration and spontaneous death of cells, followed by phagocytosis of the dead cells by neighbouring cells or by immigrating macrophages.
Areas of cell death, in most cases in the mesenchyme, have been reported
during development of the heart in a number of vertebrates: salamander
(Lemanski, 1973), rat (Pexieder, 1975; Okamoto & Satow, 1976) and man
(Pexieder, 1975). However most of the papers on the subject have been concerned
with the chick embryo, where cell death has been reported as taking place in
myocardial cells (Manasek, 1969; Pexieder, 1972; Hurle, 1974), mesenchyme
(Pexieder, 1972; Hurle, 1974) and endocardial epithelium (Ojeda & Hurle,
1
Author's address: Departamento de Anatomia, Facultad de Medicina, Santander, Spain.
162
J. M. HURLE, M. LAFARGA AND J. L. OJEDA
1975). In all the published accounts, phagocytosis of the resulting cell detritus
is carried out by undifferentiated cells. However, recent in vitro studies have
shown phagocytic activity by chick embryo myocardial cells (Garfield, Chacko
& Blose 1975).
In the present work we have studied the necrotic area present in the muscular
wall of the bulbus cordis of the chick embryo (Pexieder, 1972; Hurle, 1974) by
means of electron microscopy. Our results provide evidence suggesting that in
this necrotic area not only does cell death take place in differentiated cells but
also developing cardiac myocytes may participate in the elimination of cell
detritus.
MATERIAL AND METHODS
Fertile white Leghorn eggs were incubated at 38 °C to yield 20 normal
embryos ranging between stages 27 and 31 (Hamburger & Hamilton, 1951)
which were studied by means of electron microscopy.
Embryos were fixed in 3 % glutaraldehyde buffered in 0-1 M cacodylate at
pH 7-3. The heart was dissected free under a binocular dissecting microscope
and placed in fresh cold fixative during an additional period of 2 h. Half of
the total number of hearts were processed for electron microscopy (for details
see Ojeda & Hurle, 1975) and the other half were employed to detect acid
phosphatase activity. For this purpose after a prolonged rinse in cacodylate
buffer 100/*m sections of the bulbar region were prepared with the SmithFarquhar tissue sectioner (Sorvall and Company, Norwalk, Conn.). These
sections were incubated for 45-60 min in Gomori medium (Barka & Anderson,
1963) and then processed for electron microscopy. Some controls were made
omitting glycerophosphate from the incubating medium.
Both semithin and ultrathin sections of the araldite-embedded tissue were
made using the LKB ultratome III. The semithin sections were stained with
toluidine blue and used for orientation purposes. Ultrathin sections were
stained with lead citrate (Reynolds, 1963) and observed in an electron microscope (Philips EM 201).
RESULTS
The necrotic area of the muscular wall of the bulbus is easily recognized by
observation of toluidine blue-stained semithin sections. The shape and location
of this necrotic area show some changes during the stages in which it is present
(Hurle, 1974). However it is generally crescent-shaped and is located mainly
in the ventral and right side of the bulbus (Fig. 1). The intensity of necrosis
differs during the different stages in which the area can be found, reaching its
maximum between stages 29 (Fig. 2) and 31.
Electron microscopy confirms that this area is located within the muscular
wall of the bulbus. This muscular wall consists of myofibril-rich developing
cardiac myocytes, joined by intercalary discs and desmosomes.
Cell death in the muscular wall of the bulbus
163
Fig. 1. Vital-stained chick heart of stage 28 H. H. showing the necrotic area of the
bulbus (B). (Staining with neutral red by the technique of Dawd & Hinchliffe,
1971.)
Three different kinds of degenerative events can be distinguished by the ultrastructural study: muscle cells with focal cytoplasmic degenerations, isolated
dead muscle cells and phagocytes.
The muscle cells with focal cytoplasmic degenerations remain joined to the
neighbouring muscle cells by intercalary discs. As can be seen in Fig. 3, the
degenerative lesions include vacuolated mitochondria and endoplasmic reticulum, disorganised myofibrils and aggregations of ribosomes. As this focal
164
J. M. HURLE, M. LAFARGA AND J. L. OJEDA
?*
^
I
Cell death in the muscular wall of the bulbus
165
degenerations proceeds, it appears that the damaged organelles are in process
of sequestration in a membrane-bounded vesicle which can thus be identified
positively as an autophagic vacuole.
The dead muscle cells, usually fusiform, appear free in the extracellular space.
Both the nucleus and the cytoplasm of these cells show degenerative features
(Fig. 4). The nucleus always shows the chromatin condensed and it often
appears fragmented. The cytoplasm shows a great increase in electron density
and within it, numerous disorganized myofilaments and electron-dense mitochondria can be recognized. Vacuolation of the endoplasmic reticulum is also
usual. As degeneration of these cells proceeds, cellular breakdown takes place,
in which round cellular fragments of high electron density can be found in the
extracellular space. Scattered myofilaments, degenerated cellular organoids
and in some instances nuclear material can be recognized within these fragments.
Very often these cellular fragments appear partially surrounded by cytoplasmic
processes of the neighbouring muscle cells (Fig. 5).
Phagocytic cells are numerous in this area of necrosis and their number increases in the older stages. Two different types of phagocytes can be differentiated by their cytological characteristics. Most of the phagocytic cells appear
to be muscle cells. One or two phagosomes, containing cellular fragments
identical to those seen in the extracellular space, can be recognized within the
cytoplasm of these cells (Fig. 6). The engulfed cellular fragments usually conserve the cellular membrane and show scattered myofilaments and degenerated
cellular organoids (Fig. 7). Occasionally nuclear material can also be recognized
(Fig. 8). The size of these heterophagic vacuoles is usually larger than the autophagic vacuoles. (Vacuoles or phagosomes enclosing material derived from the
same cell are described as 'autophagic' and where the engulfed material is
obtained from outside the cell as 'heterophagic'.) Sometimes the heterophagosomes present an advanced stage of digestion, and then only membranous
material can be recognized. In these cases heterophagosomes can no longer be
distinguished from autophagic vacuoles which present identical morphology.
The ultrastructure of these phagocytic cells reveals their myocytic nature. In
the large oval nucleus containing little condensed chromatin, one or two nucleoli
are usually observed. In the cytoplasm, myofibrils are abundant although their
FIGURES
2-4
Fig. 2. Transverse semi-thin section of the bulbus at stage 29, showing abundant
necrotic cells within the muscular layer. Notice the difference between large dead
cells and small rounded cell fragments. Bulbar cushion (B). Toluidin blue. Electron
micrographs are from the bulbus of stage 27-31 embryos.
Fig. 3. Electron micrograph of a muscle cell showing a large autolytic lesion.
Fig. 4. Dead myocardial cell free in the intercellular space. The confluence of large
vacuoles in the cytoplasm suggests that the cell is in the course of fragmentation.
Myofilaments can be recognized within the cytoplasm (arrows).
166
J. M. HURLE, M. LAFARGA AND J. L. OJEDA
Cell death in the muscular wall of the bulbus
167
number appears inferior to that of the neighbouring cells (Figs. 6-8). The Golgi
complex appears well developed and shows numerous vesicles in the course of
being pinched off. These cells are still linked by desmosomes to neighbouring
cells.
Besides these phagocytic myocytes, cells with the morphological characters
of mature macrophages can occasionally be found within this necrotic area
(Fig. 9). These cells contain up to five phagosomes in an advanced stage of
digestion. The nucleus appears peripherally displaced by the great number of
phagosomes, and contains abundant condensed chromatin. The cytoplasm
comprises a thin rim which surrounds the phagosomes. Myofilaments were
never found within the cytoplasm of these cells. Although these macrophages
usually appear in close contact with the muscle cells (Fig. 9), specific junctions
were never seen. In some instances degenerative features, such as nuclear condensation or cytoplasmic vacuolization, were seen in the macrophage itself.
A striking feature of this necrotic area is the presence of numerous myocytes
undergoing mitosis (Fig. 10). The detail of the ultrastructure of these cardiac
myocytes in mitosis has been described by Hay and Low (1972).
Acid phosphatase
In normal muscle cells, acid phosphatase activity was demonstrated in Golgi
cisternae and in associated vesicles (Fig. 11), which are well developed in the
cardiac muscle cells of the bulbus. The acid-phosphatase-containing vesicles
appear in process of being budded off from the marginal portions of Golgi
cisternae. Probably such small vesicles represent one type of primary lyosome.
Acid-phosphatase-positive vesicles with the morphology of dense bodies are
sparse in these cells.
Autophagic vacuoles possess strong acid phosphatase activity (Fig. 12).
By contrast, the isolated dead cells appear acid-phosphatase-negative. Only
occasionally can low acid phosphatase activity be found within these cells.
In the phagocytic muscle cells, heterophagosom.es with undigested contents
do not show acid phosphatase activity. However, phagosomes with welldigested contents, which show strong enzyme activity as pointed out above, are
not easily classified as either auto- or heterophagic. Finally, as can be seen in
Fig. 13, macrophages have an intense acid phosphatase activity located mainly
in the phagosomes in an intermediate stage of digestion.
FIGURES 5 AND 6
Fig. 5. Muscle cell fragment in close contact with the neighbouring myocardial cells.
Note the presence of filopodia containing myofilaments surrounding the fragment
(F). A few scattered myofilaments are visible within the fragment (arrows).
Fig. 6. Electron micrograph of a myocardial cell showing a phagocytosed cell
fragment. Compare the morphology of the ingested fragment to the one shown in
Fig. 5. The Golgi complex appears very developed in the muscle cell (G).
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J. M. HURLE, M. LAFARGA AND J. L. OJEDA
Cell death in the muscular wall of the bulbus
169
DISCUSSION
Our results confirm previous work reporting the presence of a necrotic area
in the muscular wall of the bulbus of the chick embryo's heart (Pexieder, 1972;
Hurle, 1974).
From this cytochemical and cytological study no conclusive evidence concerning the mechanism of cell death has been found. The only precocious lesion
seen in this necrotic area was the formation of autophagic vacuoles in apparently
healthy muscle cells. The mechanism of formation of these autophagic vacuoles
appears identical to that reported in other tissues (Fox, 1973), and the degradation of the contents must be very rapid as they always show strong acid phosphatase activity. The formation of autophagic vacuoles has been interpreted
as the mechanism of death in most of the necrotic areas of epithelial tissues
(Scheib, 1965; Salzgeber & Weber, 1966; El-Shershaby & Hinchliffe, 1975)
and also in the degeneration of anuran tail muscle at metamorphic climax (Fox,
1975). However, autophagic vacuoles have also been observed in normal cells
in which they are interpreted as a defence mechanism avoiding the spread of a
local degenerative process to the rest of the cell (Ericsson, 1969). In our study,
considering the small number of these vacuoles per cell and the lack of intermediate stages between these cells and the isolated dead cells, the evidence is
not conclusive that autophagocytosis is the mechanism of degeneration. Krstic
and Pexieder (1973) found similar results in the necrosis of the mesenchyme of
the bulbar cushions and suggested that two different mechanisms of death were
taking place.
Although the capacity for mitotic division in developing cardiac myocytes
is widely known (Manasek, 1968; Hay & Low, 1972; Rumyantsev, 1972), the
presence of abundant mitosis within this necrotic area appears a discordant
feature. This fact could indicate a relation between cell degeneration and
mitosis, as suggested in other necrotic processes (Kallen, 1965; Gondos &
Hobel, 1973). However no morphological evidence supporting this hypothesis
was found in the present study.
FIGURES
7-9
Fig. 7. Cellular fragment phagocytosed by a muscle cell. Notice the conservation of
the cellular membrane and the presence of myofibrils with conservation of Z lines
(arrows).
Fig. 8. Muscle cell showing a large phagosome containing a dead cell. Although the
phagocytosed cell appears very deteriorated, the nuclear remnants can be recognized (N).
Fig. 9. Electron micrograph showing a large macrophage (M) within the muscular
wall of the bulbus. There is close contact between the macrophage and the muscle
cells. Note the well differentiated Z bands (arrows) and organized myofibrils in the
developing myocardiocytes.
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J. M. HURLE, M. LAFARGA AND J. L. OJEDA
Cell death in the muscular wall of the bulbus
171
The elimination of dead cells in this area differs from that reported by Manasek (1969) in the embryonic ventricle of the chick. This author found most of the
phagocytosed dead cells in the subepicardial space and suggested that dead
cells were removed from the myocardium by immigrating phagocytes. In our
study of the bulbus, the pictures of muscle cell processes surrounding the fragmented dead cells (Fig. 5, 10) and the eventual presence of identical cell fragments within phagosomes in muscle cells, suggest a phagocytic capacity of the
muscle cells themselves. The preservation of the cellular membrane of the
ingested dead cell and/or the presence of nuclear material within the phagosome
allow us to differentiate these phagosomes from autophagosomes (Ericsson,
1969). However, this difference can only be established during the initial events
of phagocytosis. When digestion of the heterophagosom.es takes place, the
phagosomal contents lose structural details and turn into membranous material,
so that both kinds of phagosome become morphologically identical. Only the
size of the phagosome can be taken as a weak indication of the type of differentiation, as heterophagic vacuoles are usually larger than autophagosomes.
As in other necrotic areas, such heterophagosomes do not show acid phosphatase activity initially (Ballard, 1965; Dawd & Hinchliffe, 1971). This cytochemical observation confirms the morphological difference between these
vesicles and autophagosomes which always have a strong acid phosphatase
activity (Ericsson, 1969). Phagocytic capacity in muscle cells has been demonstrated in chick embryo cardiac myocytes in tissue culture (Garfield et al. 1975).
Our results suggest that this observation in vitro can be extended to the
embryonic tissue in vivo.
The presence of macrophages with abundant phagolysosomes is a constant
feature in most of the studies on physiological cell death. In the necrotic processes taking place in differentiated tissues, macrophages are usually considered
as differentiated from, neighbouring mesenchymal cells (Weber, 1964; Seinsch &
Schweichel, 1974; Fox, 1975). In our study, although such a hypothesis of
mesenchymal origin cannot be ruled out, some facts suggest that macrophages
may differentiate from the phagocytic muscle cells. Firstly, like Manasek (1968)
we were unable to find undifferentiated mesenchyme cells within the muscular
FIGURES
10-13
Fig. 10. Myocardial cell in metaphase. Besides the mitotic cell a cellular fragment
containing mainly nuclear material (N) is in the course of being surrounded by a
cytoplasmic process of a muscle cell (arrow). Metaphase chromosomes (C).
Fig. 11. Acid phosphatase activity in the Golgi complex and small vesicles in a healthy
myocardial cell of the bulbus.
Fig. 12. Myocardial cell showing an autolytic vacuole rich in acid phosphatase activity
(V). Notice the loss of organization of the fibrillar material.
Fig. 13. Electron micrograph showing the presence of acid phosphatase activity
in some of the vacuoles of a macrophage (arrows).
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J. M. HURLE, M. LAFARGA AND J. L. OJEDA
wall of the bulbus; secondly, it is usually accepted that there is a sequence in the
phagocytic process in most of the cases of physiological necrosis. The initial
phagocytes have very few ingested dead cells and the phagosomes are acidphosphatase-negative. In successive stages the number of phagosomes and the
acid phosphatase activity increase and the last stage in this differentiation process is the mature macrophage (Ballard, 1965; Dawd & Hinchliffe, 1971). In
this necrotic area, the young phagocytes can always be identified as myocytes,
and as undifferentiated mesenchymal cells are not present in the muscular wall
of the bulbus, it could be presumed that the macrophages are differentiated
from the phagocytic muscle cells. However labelling experiments should be
carried out to eliminate a further possibility, that phagocytic myocytes digest
the phagocytosed material without modifying their pathway of differentiation,
and that macrophages migrate into the cell death site from the blood. Saunders
(1966) showed that macrophages were present in the blood of 4-day chick
embryos.
The interpretation of the significance of this necrotic area in morphogenetic
terms is difficult, as the heart is a complex organ and other necrotic processes
are taking place at the same stages (Pexieder, 1975). The location of this area,
mainly in the right side of the wall of the bulbus, could be a factor participating
in the displacement of the bulbus towards the left side, which occurs after the
fourth day of incubation (Romanoff, 1960). However it seems most likely that
this area has a phylogenetic significance (following the terminology of Gliicksmann, 1951). The distal portion of the bulbus is converted into the proximal
parts of the aorta and pulmonary artery (Romanoff, 1960) so that the area of
cell death studied here may well represent the elimination of the muscular wall
of this part of the bulbus.
We wish to thank Dr J. R. Hinchliffe for his invaluable help and suggestions during the
preparation of this manuscript.
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{Received 10 February 1977, revised 10 May 1977)
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