/. Embryo!, exp. Morph. Vol. 33,3, pp. 523-534,1975 . 523 Printed in Great Britain

Cell death during the formation of tubular of the chick embryo

By J. L. OJEDA1 AND J. M. HURLE From the Department of Anatomy, Faculty of Medicine of Santander, and Department of Electron Microscopy, ' Marques de Valdecilla'' Medical Center, Santander, Spain

SUMMARY Light and electron microscopical examination of the heart of the chick embryo between stages 9 and .11 (Hamburger & Hamilton) revealed degenerating and dead cells specifically localized in the midline of endocardial tubes. The morphology of cell death in this system does not differ from that described in other embryonic tissues and organs. Phagocytosing cells are rarely seen. The results of this study show that a destruction of midline cells of both endocardial tubes takes place during the fusion of heart anlages. The possible roles of this cell death in the morphogenesis of the tubular chick heart are discussed.

INTRODUCTION Cell death and disintegration constitute a basic regulatory mechanism in the normal development of most tissues and organs. This concept, which assumes that cell death is a controlled process, is derived from the experimental analysis of necrotic limb areas of the chick embryo made by Saunders, Gasseling & Saunders (1962) and Fallon & Saunders (1968). As pointed out by Gliicksmann (1951) this programmed cell death (morphogenetic degeneration) is a frequent event in the fusion processes of paired anlagen. Observations of this kind have been reported by numerous authors in different species and organs (see review by Gliicksmann, 1951); however, few systematic studies of these phenomena have been made. Special attention has been focused on the fusion of palatine shelves (Mato, Aikawa & Katahira, 1967; Shapiro & Sweney, 1969; Pourtois, 1970; Matthiessen & Andersen, 1972) where epithelial cells from approaching palatal shelves degenerate and then die during the fusion process; however, the role of cell death in this fusion remains obscure (Baird & Verrusio, 1973; Tsai & Verrusio, 1973). As DeHaan (1967) pointed out there is little investigation about the possible role of cell death in heart morphogenesis; however, several papers were recently publishedonthistopic(Manasek, 1969; Pexieder, 1972; Kristic & Pexieder, 1973; 1 Author's address: Departamento de Anatomia, Facultad de Medicina, Santander, Spain.

33 EMB 33 524 J. L. OJEDA AND J. M. HURLE Hurle, 1974). During cardiac development there are many fusion processes where cell death may play an important role, but only the fusion of in the embryonic chick heart have been studied in detail (Hay & Low, 1972), and it was found that cell death is not present in this fusion process. A prominent step in heart morphogenesis is the fusion of the paired endo- cardial tubes. This process takes place in the of all vertebrates except man (Orts Llorca, Jimenez Collado & Ruano, 1960). In the chick embryo, fusion of the endocardial tubes begins at stage 9 + (Hamburger & Hamilton, 1951) and is completed at about stage 11 (Romanoff, 1960). Our preliminary observations (Ojeda & Hurle, 1973) suggested that cell death might be impli- cated in the fusion process of endocardial tubes. The work described in this paper was undertaken, using light and electron microscopy, to study in more detail the cell death that takes place in this process.

MATERIALS AND METHODS Fertile White Leghorn eggs were incubated at 38 °C to yield 28 normal embryos ranging from stage 9 to 11 of Hamburger & Hamilton (1951), and they were studied by means of light and electron microscopy. For electron microscopy, a portion of the shell was removed to expose the embryo which was flooded in situ with ice cold 3 % glutaraldehyde fixative solution made in 0-2 M cacodylate buffer, pH 7-3. The entire embryo was then removed and placed in a dish with cold fixative. The heart was dissected free under a binocular dissecting microscope and placed in fresh cold fixative during an additional period of 2 h. It was then rinsed in 0-2 M cacodylate buffer and postfixed in 1 % osmium tetroxide for another 2 h. Following dehydration in a graded series of acetones and propylene oxide, the heart was embedded in Araldite. To ensure that all embryos would be sectioned transversely, they were embedded in flat capsules and carefully oriented under the binocular dissecting microscope. Serial semithin sections were cut with a LKB ultratome III and stained with 1 % toluidine blue in 0-2 borax; these sections were used for precise localization of the border line of the tubular fusion, as well as for a first scanning of possible cell death areas. Ultrathin sections of selected areas were then made, mounted on uncoated copper grids, stained with lead citrate (Reynolds, 1963) and examined with Zeiss EM 9 and Philips EM 201 electron microscopes. For light microscopy ten embryos were fixed in situ with Carnoy solution. Afterwards, they were dehydrated, embedded in paraffin and serially sectioned transversely at 6/*m. The sections were stained using the Feulgen method.

RESULTS Observations made on serial semithin sections by light microscopy permitted the fusion steps of the endocardial tubes to be clearly followed. The fusion Cell death during heart formation in chick 525

Fig. 1. Semi-schematic drawing taken from a semithin cross section of the heart of stage 10 chick embryo. The arrows show the place where dead cells and phago- cytes can be found. Dots represent the two strands of cardiac jelly, (e, ; s, incomplete sagittal septum; a, anterior gut; dm, developing myocardium.) of the heart rudiments begins at stage 9 or 9 +, starting at the anterior end of the future heart and progressing posteriorly, so that in stage 11 the two rudiments of the have not yet joined. Fusion begins in the ventral part of both endocardial tubes and progresses towards the dorsal part. After fusion of the ventral part, the primitive heart is formed by a single endocardial tube which presents an incomplete septum in the sagittal plane. This septum is formed by a double endocardial layer and two strands of the cardiac jelly that end in the thickened floor of the anterior gut. Light microscopy of the heart anlagen also reveals some pycnotic cells in the endocardial tube. The electron-microscopic observations reveal the precise distribution of the dying cells. Cell death in the developing reaches its maximum between stages 10— and 11, and it is restricted almost exclusively to the incomplete endocardial septum (Fig. 1). Stages in cell degeneration and death may be classified into the following cate- gories :

(a) Dead endocardial cells A few individual dead cells can be seen in the endocardial layer. They appeared on the ventral end of the incomplete sagittal septum, precisely at the site of fusion of the two endocardial tubes.

33-2 526 J. L. OJEDA AND J. M. HURLE

Fig. 2. (A) typical example of dead endocardial cell situated in the fusion point (pf) of both endocardial tubes. (B) Detail of area (indicated by arrow in A) showing ribosomal crystals. Note the parallel-row arrangement.

The morphological changes indicative of endocardial cell death are similar to those seen in other embryonic cells. The normal flat cell shape is altered; dead cells assume a rounded shape but frequently retain connexions with adjacent cells (Fig. 2A). All cellular elements show an increased electron density; the chromatin appears dense and marginal; small vacuoles are found and the mitochondria and endoplasmic reticulum are swollen. In all cases the perinuclear cisterna is widened and often shows beaded structures. In some endocardial dead cells distinct crystalline structures are seen which look like ribosome crystals; they are commonly arranged in parallel sheets (Fig. 2B) and in some cases are associated with microtubules. Finally some dead cells show glycogen pools which are not normal constituents of developing endo- cardial cells. Cell death during heart formation in chick 527

Fig. 3. Phagocyte in the endocardial layer with an ingested dead cell which shows little sign of digestion. Note the existence of a beaded structure (arrow) as well as some swollen mitochondria (m).

(b) Phagocytosis in the endocardial cells Phagocytosis is rarely seen in the endocardial cells. It generally appears in the most cephalic level of the heart tube, in the ventral boundary. Initially a dead cell is surrounded by arms of cytoplasm from one viable endocardial cell, and later a dead cell with no sign of digestion appears within the cyto- plasm of an endocardial cell (Fig. 3). The phagocytosing cell associates with its neighbours by focal tight junctions and has numerous free ribosomes but few organelles. In all cases the phagocytes contain only an ingested dead cell, and morphologically they seem to correspond to the stage I of the classification of Ballard & Holt (1968). In a large number of endocardial cells located in the sagittal septum, the electron microscope revealed vacuoles containing swollen mitochondria, ribo- somes, fat droplets and membranous material but no nuclear material. These structures might represent autophagic vacuoles.

(c) Degenerating cells and cellular debris in the cardiac jelly Degenerating cells and cellular debris appear in the cardiac jelly located between the double endocardial layer of the sagittal septum (Fig. 4). There are two types of degenerating cells: (1) degenerating cells associated with endo- cardial viable cells, and (2) free degenerating cells. The first type of cells shows many signs of deterioration, but the nucleus and cytoplasm can be distinguished, the cytoplasmic matrix has disappeared (Fig. 5), the mitochondria are swollen and in many cases the cristae are free in the cytoplasm; ribosomes and swollen granular endoplasminc reticulum are still present. The endocardial viable cells have several protrusions of the surface membranes which make contact with neighbouring degenerative cells (Fig. 5). 528 J. L. OJEDA AND J. M. HURLE

Fig. 4. Electron micrograph of a transverse section through the incomplete endo- cardial septum of a stage 10 chick's heart. Some degenerating cells (dc) and cellular debris (cd) can be seen between the double endocardial layer (el). Note the close asso- ciation between the degenerating cells and the two strands of cardiac jelly (arrows).

The free degenerating cells can appear either as dark cells (Fig. 4) or more frequently as disrupted cells (Fig. 6). They are always at an advanced stage of deterioration, but the nucleus can still be distinguished. In the disrupted cells, the cytoplasm is represented by floccular material, fat droplets and fibrils, and Cell death during heart formation in chick 529

Fig. 5. Degenerating cell located in the cardiac jelly. Note the protrusions of the endocardial viable cells, which contact (arrows) the dead cell. The damage to the cytoplasm due to vacuolation is noticeable. the cell membrane is broken. These degenerating cells are often associated with the thinner filaments and collagen fibrils of the cardiac jelly (Fig. 6). Other elements frequently seen in the cardiac jelly are cellular debris.

(d) Degenerative lesions in viable endocardial cells Degenerative lesions are often found within the viable endothelial cells in the fusion zone. Sometimes, the cytoplasm shows in discrete areas a series of annular structures with loss of the normal organelles; the plasma membrane is detached in these areas. In other cases the lesions are protuberances of cyto- plasm with a delicate membranous structure which looks like a spider's web. In both cases, swollen mitochondria containing an amorphous material are also found. In some instances, part of the degenerating cytoplasm seems to be in process of being cut off from the rest of the cell towards the cardiac jelly or into the endocardial lumen. The viable endothelial cells contain numerous Golgi groups surrounded by many primary and secondary lysosomes. These cells have many free ribosomes and are attached to adjacent cells by desmosomes. Mitotic figures have not been observed either by light or electron microscopy in the viable endocardial cells of the sagittal septum. 530 J. L. OJEDA AND J. M. HURLE

Fig. 6. Free degenerating cell in the cardiac jelly. Note segregation of chromatic (c) material. Thinner filaments (/) and fibrils, some of which have a periodic cross striation (arrow), can be seen in the disrupted cytoplasm.

(e) Phagocytes in the developing myocardium In the chick embryo between stages 9 and 11 myocardial cell death is a rare event. Early phagocytes are the only degenerative changes observed in all of our studied embryos (Fig. 7). These phagocytes were located in the ventral zone of the developing myocardium.

DISCUSSION During the process of fusion of the two heart anlages, after the two endo- cardial tubes meet each other in the midline, the disappearance of the septal cells could be explained by liberation and change into another cellular type, or simply by cell death. Our observations suggest that cell death plays an impor- tant role in the elimination of the medial septum. However, it is possible that cell death has other roles in the early morphogenesis of the heart. Hughes (1943) has related the cell death with the appearance of the elastic fibers. According to this author, 'it may be that the cells which degenerate leave behind them a substance from which elastic fibers are formed'. Based upon this, the cellular debris located between the double endocardial layer of the sagittal septum can participate in the elaboration of cardiac jelly. Some facts support this hypothesis; in some cases there is a close relation between cellular debris, collagen fibers, Cell death during heart formation in chick 531

Fig. 7. A phagocyte in the developing myocardium, containing one dead cell, in which mucleus and cytoplasm can be distinguished. Note how the phagocyte is closely bound to their neighbouring cells. amorphous and filamentous material of the cardiac jelly; moreover, this material is more abundant in the space of the endocardial sagittal septum, where cell death takes place, than in the endo-mioepicardial space (Ojeda & Hurle, unpub- lished observations). However an experimental analysis is necessary to verify this attractive hypothesis. Our observations on the morphology of the morbid and dead cells with the electron microscope are similar to the ultrastructural pictures described by numerous authors. This is in agreement with Krstic & Pexieder's (1973) idea of the uniformity in the morphology of cell death at the ultrastructural level. The ribosome crystals have been described by numerous investigators on physiological cell death (see Mottet & Hammar, 1972). Our observations of the ribosome crystals associated with microtubules are similar to those described 532 J. L. OJEDA AND J. M. HURLE by Maraldi et al. (1973) induced by hypothermia in cells treated with colchicine or vinblastine. These observations could suggest that cell death might be produced by mitotic aberrations as Kallen (1965) pointed out. On the other hand, Byers (1967) has demonstrated that prolonged hypothermia is necessary for ribosome crystallization in interphasic cells, whereas rapid cooling is enough to induce ribosome crystallization in mitotic cells. However the fact that mitotic figures have not been observed in viable endocardial cells of the septum is not in agree- ment with a mitotic aberrations-cell death regulation. The finding that degenerative changes can take place in dying cells without digestion by a phagocyte and the rarity of phagocytosis images indicates that phagocytes are not the cause of cellular death. This is in agreement with the interpretation by Manasek (1969), Dawd & Hinchliffe (1971) and Krstic & Pexieder (1973) of their studies on cell death in several systems but is in dis- agreement with the assassination theory. Although the difficulty in deducing the correct sequence of events by electron microscope techniques is well known, our observations suggest that degenerating cells and cellular debris located in the cardiac jelly are derived from endocardial cells. Morbid endothelial cells and part of the degenerating cytoplasm would be released into the cardiac jelly and in this space the cellular disintegration would take place. This hypothetical sequence could explain the two types of degenera- ting cells present in the cardiac jelly. Furthermore, several investigators (Holt- freter, 1945; Manasek, 1969) have observed that degenerating cells are expelled from the surrounding normal tissue into the extracellular space.

RESUMEN El estudio por medio de la microscopia optica y electronica de corazones de embriones de polio comprendidos entre los estadios 9 a 11 de H.H., muestra celulas muertas y en degenera- cion. Estas celulas de localizan, preferentemente, en la linea media de los tubos endocardicos. La morfologia de la muerte celular que aparece en este sistema no difiere de la descrita en otros tejidos y organos. Los fagocitos son poco numerosos. Los resultados de este estudio, sugieren que durante la fusion de los esbozos cardiacos tiene lugar una destruction de las celulas mediales de ambos tubos endocardicos. Se discute el posible papel de la muerte celular en la morfogenesis del corazon tubular del embrion de polio.

The authors are grateful to' Fundacion M. Botin' for its help in the equipment of the electron microscopy laboratory in Valdecilla Medical Center. We wish to express our appreciation to Dr B. Herreros for his invaluable help in the preparation of this manuscript. Cell death during heart formation in chick 533

REFERENCES BAIRD,G. &VERRUSIO, A.C. (1973). Inhibition of palatal fusion in v/7n?byb-2-Theinylalamine. Teratology 7, 37-47. BALLARD, K. J. & HOLT, S. J. (1968). Cytological and cytochemical studies on cell death and digestion in the foetal rat foot: the role of macrophages and the hydrolytic enzymes. /. Cell Sci. 3, 245-262. BYERS, B. (1967). Structure and formation of ribosome crystals in hypothermic chick embryo cells. /. molec. Biol. 26, 155-167. DAWD, D. S. & HINCHLIFFE, J. R. (1971). Cell death in the 'opaque patch' in the central mesenchyme of the developing chick limb: a cytological, cytochemical and electron microscopic analysis. /. Embryol. exp. Morph. 26, 401-424. DEHAAN, R. L. (1967). Development of form in the embryonic heart. An experimental approach. Circulation XXXV, 821-833. FALLON, J. & SAUNDERS, J. W. JR. (1968). In vitro analysis of the control of cell death in a zone of prospective necrosis from the chick wing bud. Devi Biol. 18, 553-570. GLUCKSMANN, A. (1951). Cell deaths in normal vertebrate ontogeny. Biol. Rev. 26, 59-86. HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. /. Morph. 88, 49-92. HAY, D. A. & Low, F. N. (1972). The fusion of dorsal and ventral endocardial cushions in the embryonic chick heart. A study in fine structure. Am. J. Anat. 133, 1-24. HOLTFRETER, J. (1945). Neuralization and epidermization of gastrula ectoderm. /. exp. Zool. 98, 161-209. HUGHES, A. F. W. ( 1943). The histogenesis of the arteries of the chick embryo. /. Anat. 77, 266-287. HURLE, J. (1974). Estudio topografico, eitoquimico y ultraestructural de las areas de muerte celular durante la morfogenesis cardiaca en el embrion de polio. Doctoral thesis. University of Valladolid (Spain). KALLEN, B. (1965). Degeneration and regeneration in the vertebrate central nervous system during embryogenesis. In Progress in Brain Research 14, pp. 77-96. Amsterdam: Elsevier Publishing Company. KRSTIC, R. & PEXIEDER, T. (1973). Ultrastructure of cell death in bulbar cushions of chick embryo heart. Z. Anat. EntwGesh. 140, 337-350. LEMANSKY, L. (1973). Heart development in the Mexican Salamander Ambystoma Mexicanum. II. Ultrastructure. Am. J. Anat. 136, 487-526. MANASEK, F. J. (1969). Myocardial cell death in the embryonic chick ventricule. J. Embryol. exp. Morph. 21, 271-284. MARALDI, N., BIAGINI, G., SIMONI, P., BARBIERI, M., MARINI, M. & BERSANI, F. (1973). Ultrastructural investigation of the effect of DNA, RNA and protein synthesis inhibitors on ribosome crystalization. /. Ultrastruct. Res. 44, 265-278. MATO, M., AIKAWA, E., &KATAHIRA, M. (1967). Alteration of fine structure of the epithelium of the lateral palatine shelf during the secondary palate formation. Gunma. J. med. Sci. 16, 79-99. MATTHIESSEN, M. & ANDERSEN, H. (1972). Disintegration of the junctional epithelium of human fetal hard palate. Z. Anat. EntwGesch. 137, 153-169. MOTTET, N. K. & HAMMAR, S. P. (1972). Ribosome crystals in necrotizing cells from the posterior necrotic zone of developing chick limb. /. Cell Sci. 11, 403-414. OJEDA, J. L. & HURLE, J. M. (1973). Cell death during the fusion of the endocardial tubes in the chick embryo. I.R.C.S. Med. Sci. 5, 1-4. ORTS LLORCA, F., JIMENEZ COLLADO, J. & RUANO, D. (1960). La fase plexiforme del desar- rollo cardiaco en el hombre. An. Desarrollo 8, 79-98. PEXIEDER, T. (1972). The tissue dynamics of heart morphogenesis. I. The phenomena of cell death. B. Topography. Z. Anat. EntwGesch. 138, 241-254. POURTOIS, M. (1970). La resorption des murs epitheliaux au cours de la formation du palais primaire et du palais secondaire. Bull Grpmt. int. Rech. scient. Stomat. 13, 465-488. 534 J. L. OJEDA AND J. M. HURLE REYNOLDS, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. /. Cell Biol. 17, 208-212. ROMANOFF, A. L. (1960). The Avian Embryo. New York: Macmillan. SAUNDERS, J. W. JR., GASSELING, M. & SAUNDERS, L. (1962). Cellular death in morphogenesis of avian wing. Devi Biol. 5, 147-178. SHAPIRO, B. L. & SWENEY, L. (1969). Electron microscopic and histochemical examination of oral epithelial-mesenchymal interaction (programmed cell death). /. dent. Res. 48, 652- 660. TSAI, H. M. & VERRUSIO, A. C. (1973). Epithelial breakdown in single palatal shelves. Tera- tology 7, 29 (Abstract). {Received 2 April 1974)