Cell Death During the Formation of Tubular Heart of the Chick Embryo

/. Embryo!, exp. Morph. Vol. 33,3, pp. 523-534,1975 . 523 Printed in Great Britain Cell death during the formation of tubular heart 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 endocardial cushions 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 endocardial tubes. This process takes place in the heart development 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 phagocytes can be found. Dots represent the two strands of cardiac jelly, (e, endocardium; 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 sinus venosus 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 tubular heart 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 endocardial 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 aorta 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 cytoplasm 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, ribosomes, 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 endocardial 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.

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