Table Of ContentErgebnisse der Anatomie und Entwickh:ultpgeschichte
Advances in Anatomy, Embryology md Cell Biology
Revues d' anatomie et de morphologie experimentale
51' 3
Editors
A.Brodal, Oslo' W. Hild, Galveston' J. van Limborgh, Amsterdam· R. Ortmann, Koln
T. H. Schiebler, W urzburg . G. Tondurv, Zurich' E. W ol/!, Paris
Tomas Pexieder
Cell death in the morphogenesis
and teratogenesis of the heart
With 52 figures
and 9 tables
Springer-Verlag Berlin Heidelberg New York 1975
Dr. mid. Tomas Pexieder
Protesseur-assistant
I nstitut d' H istologie et d' Embryologie
Faculte de M idecine
Universite de Lausanne
Rue du Bugnon 9
CH-IOll Lausanne
Suisse / Switzerland
Library of Congress Cataloging in Publications Data
Pexleder, Tomas, 1941-
Cell death in the morphogenesis and teratogenesis of the heart.
(Advances In anatomy, embryology, and cell biology: 51/3)
Bibliography: p.
Includes index.
1. Cell death. 2. Heart. 3. Morphogenesis. 4. Heart-Abnormities and deformities. I. Title. II. Series.
QL801.E67 vol. 51, fasc. 3 [QH671] 574.4'08s
ISBN-13:978-3-540-07270-6 [591.8'765] 75·11701
ISBN -13:978-3-540-07270-6 e-ISBN-13:978-3-642-66142-6
DOl: 10.1007/978-3·642-66142-6
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Contents
Preface . . . . . 6
I. Introduction 7
II. Bibliography updating (1967-1974) 9
III. Morphology of cell death. . . . . 12
IV. Topography of cell death in the chick embryo heart 17
V. Comparative topography of cell death zones in rat and human embryo
hearts. . . . . . . . . . . . . . . . . . . . 19
VI. Ultrastructure of the cell death in bulbar cushions 24
VII. Supravital Nile blue sulphate staining in the cell death studies 31
VIII. Cell death and the morphogenesis of the heart . . . . 37
IX. Interaction of hemodynamics with the cell death zones 47
X. Cell death and chemical teratogenesis of the heart. 58
Conclusions 72
Summary. 74
Acknowledgement. 76
References. . 76
Subject Index 100
5
Preface
In spite of the continuing progress of research in the fields of cellular and
molecular biology, which has oriented many embryologists towards molecular
biology, no concrete explanation of morphogenesis has yet been found. The
present state of knowledge of heart development is characterized by an enormous
discrepancy between the qualitative descriptions of what happens on the organ
level and the more or less quantitative information on subcellular and molecular
events. It is generally not understood how cells form tissues and how tissues
generate particular forms of an organ.
In an attempt to fill the gaps we systematically studied in the period 1968 to
1973 one of the general but rather neglected morphogenetic mechanisms which
integrates cells into tissues and organs-cell death. Only a small part of our
research on cell death in the development of chick, rat and human embryo hearts
has as yet been published in extenso. Most of it has been communicated in papers
delivered at different scientific meetings.
We would like to use the opportunity offered by Advances to present a syn
thesis and integrative review of our results. In this way the actual period of
discovery of the existence of cell death and of its morphogenetic role in the heart
development come to an end. This opens up the next phase of our research which
consists in studies of how cell death is integrated with other morphogenetic
mechanisms.
6
I. Introduction
The words "cell death" have a wide variety of meanings. For a pathologist,
for instance, there is an almost obligatory relationship between cell injury, cell
death and necrosis. For him the death of cells is a consequence of a pathogene
action (La Via and Hill, 1971). But there are in almost every living organism
thousands of cells that die spontaneously by a process which is called with some
ambiguity "physiological" or "spontaneous" cell death. This means that cells
are dying in healthy subjects under normal conditions without any exposure to
noxae, but merely as an expression of cellular turnover or as a result of normal
genetic and epigenetic regulations. Our investigations deal with this kind of cell
death.
The mere existence of this type of cell death implies for the pathologist that
not each cell identified as dying or dead is necessarily a consequence of injury.
Knowledge of the existence, localization and intensity of physiological cell death
should be a prerequisite for a correct diagnosis of pathological changes.
Even if human logic and philosophy of science prefer creative and constructive
events like cell proliferation, to quote only one of the morphogenetic mechanisms,
the interest in spontaneous cell death can be traced back to the beginning of the
20th century (Loos, 1898; Schultz, 1908; Graper, 1914). The first review article
on the subject was written by Ernst (1926), whose attention was brought to this
problem by Kallius (1931). Another pupil of Kallius, Glucksmann, is considered
as the pioneer of modern research on cell death in developmental biology. He is
the author of the second most important review paper on the subject (Glucks
mann, 1951, 1965). Since 1950 there have been three major phases of research
dealing with physiological cell death. In the early fifties, as a result of the stimula
tion aroused by De Duve (1959) discovery of lysosomes, there have been many
papers on the biochemical aspects of spontaneous cell death (Scheib, 1963; Weber,
1963, 1965, 1966, 1969; Batra and Strehler, 1967; Hickey, 1971; Perriard and
Weber, 1971; Robinson, 1972; Ryffel and Weber, 1973).
In the sixties certain investigations (Zwilling, 1959, 1964) started to analyse
the role of cell death in teratogenesis. As a review of this particular aspect the
papers by Menkes et al. (1964, 1970) are recommended. With the Saunders' (1966)
review article on the morphogenetic significance of cell death comes the third
most recent period concerned with the regulation of cell death. Further fairly
complete survey of the literature with coverage up to 1967 can be found in Wendler
(1972).
In comparison with 5 papers published on physiological cell death in 1969
the actual number of communications on this subject has increased three times.
This is due not only to a nonspecific effect of a general publication explosion but
rather to a specific shift in research interest. We can understand this increase
better if we are aware of the recent emphasis of research on the mechanisms of
teratogenesis (Scott et al., 1971; Ritter et al., 1973; Wilson, 1973, 1974). This
7
rising interest and reevaluation is best documented in certain chapters devoted
to cell death in some recent monographs (Saxen and Rapola, 1969; Ebert and
Sussex, 1970; Bellairs, 1971; Hamilton et al., 1972).
The concept of spontaneous cell death does not limit its impact on develop
mental events. It is an important variable in all reasonable mathematical descrip
tions and models of cell population kinetics (Biggers, 1964; Heinmets, 1966;
Jagers, 1970; Shackney, 1973) and in considerations of normal cellular turnover
(Lala and Patt, 1966; Norby et al., 1967; Beyer, 1970; Eaves, 1973). Even cancer
research could be concerned with physiological cell death using it among other
things as an explanation for the discrepancy between the intensive proliferation
and the low macroscopic growth rates of some tumours (Steel, 1967; Searle et al.,
1973) or to solve the principal question of tumoral tran.'lformation (Norby and
Mellgren, 1971).
The selected subject on which we focus our research is the vertebrate heart.
The development of this organ is quite fascinating as it differs substantially from
the development of other embryonic systems in many aspects. The heart has to
develop at the same time as its function is of primordial importance for the
survival of the embryo. There is a high degree of interaction between the behaviour
of blood streams characterized by their respective hemodynamic parameters and
the heart organogenesis. Furthermore the study and understanding of heart
development is obscured by complicated patterns of spatial changes. Some prob
lems also arise from the non uniform terminology.
The descriptive studies of heart development (Born, 1888, 1889; Greil, 1903;
Keith, 1924; Odgers, 1938; De La Cruz and Da Rocha, 1956; Wilson, 1961;
Grant, 1962; Shaner, 1962; Los, 1966, 1968; Langman and van Mierop, 1968;
Asami, 1969, 1972; Goor et al., 1970, 1972; Goerttler, 1971; De La Cruz et al.,
1972; Anderson et al., 1974), prepared the way for experimental analyses on the
organ level (Stephan, 1948, 1952; Rychter, 1962; Rychter, Lemez, 1958b; Le
Douarin, 1964). Only re'cently has the interest turned to the underlying mechanisms
at the tissue and cellular level. From this approach it is especially the early
cardiogenesis (De Haan, 1965), the formation of the heart tube (Orts-Llorca,
1964; Stalsberg, 1970) and its bending (Manasek et al., 1972) that have been
investigated with some success. In the following phases of heart organogenesis,
one is confronted with a rather confusing situation characterized by De Haan's
(1968) statement: "there is not a single laboratory in this country or elsewhere
where experiments are being performed to test whether the fundamental process
responsible for closure of the interventricular foramen is mitosis, cell migration,
adhesion or death of the cells involved; nor is anyone investigating how a parti
cular genotype or a virus may disturb these processes to yield such a defect".
It was this later phase of heart formation and its mechanisms that attracted our
attention and research efforts.
In comparison with other areas of intense cell death studied rather extensively
like the posterior necrotic zone of wing buds (Saunders et al., 1962; Fallon and
Saunders, 1968), the interdigital zones (Menkes and Deleanu, 1964; Kieny, 1973),
the central nervous system (Hughes, 1961, 1973; Hughes and Fozzard, 1961;
Kallen, 1965; Prestige, 1965, 1974), the urogenital system (Bengmark and Fors
berg, 1959; Forsberg, 1961, 1967; Scheib, 1963; Forsberg and Olivecrona, 1963;
Salzgeber and Weber, 1966), the closing palatal shelves (Hughes et al., 1967;
Mato et al., 1967 a, b, 1968, 1972; AngeIici, 1968; Smiley and Dixon, 1968;
8
Sweney and Shapiro, 1970; Smiley and Koch, 1971, 1972; Holmsted, 1971;
Mathiessen and Andersen, 1972), data on cell death in the development of the
heart were at the beginning of our studies in 1968 extremely scarce, and no
systematic study existed. Only very careful reading will reveal some mention of
dead cells haphazardly observed by Goerttler (1957). The first cell death focus
so far described in heart development was in the region of the aorticopulmonary
septum (Menkes et al., 1965). Illies (1967) mentioned some dead cells in the hearts
of his series of human embryos. In 1969 Manasek, in one of his studies on the
histogenesis of the chick embryo heart, described the ultrastructure of dead cells
seen in the ventricular myocardium on the 7th day of incubation. Since the
beginning of our studies on the role of cell death in heart morphogenesis and
teratogenesis three reports have been published on the subject (Ojeda and Hurle,
1973; Satow et al., 1973, 1974).
We can now briefly resume the time schedule and experimental design in the
hope of answering questions on the existence of cell death in the developing heart
and its role in heart morphogenesis and teratogenesis.
In 1968 and 1969 we made a systematic study of the localization of cell death
foci in the heart of chick embryos (Pexieder, 1969 b, 1971 a, b, 1972 c, d; Krsti6 and
Pexieder, 1973b). Following the quantitative assessment of the intensity of cell
death phenomena (Pexieder, 1971 c, d) and its role in the cellular turnover of
developing hearts (Pexieder, 1973c, d; Wyss and Pexieder, 1973) we concentrated
on only some of the 31 zones of cell death appearing during the chick embryo
heart development-namely those occuring in the heart bulb. Using the vital
stainability of dead and dying cells with Nile blue sulphate, we studied the morpho
genetic role of cell death ~n the bulbar cushions (Pexieder, 1972 a). The dependency
of cell death patterns on hemodynamics was analysed by experimental pulmonary
stenosis (Pexieder, 1972b) and in organ culture (Pexieder, 1973a). To permit
some extrapolation of our observations on avian embryos the cell death foci were
looked for and found to' exist in rat, as well as in human embryos (Pexieder and
Paschoud, 1973). On the basis of all these observations we formulated the hypo
thesis that physiological cell death zones in the developing heart are a common
target for both chemical and biophysical teratogens (Pexieder, 1973 b). This
hypothesis was then verified in experiments where heart malformations were
produced using cyclophosphamide (Pexieder, 1974) and dexamethasone and the
corresponding cell death patterns were analysed.
In such a way the different theories on pathogenesis of congenital heart
anomalies formulated vaguely as "disorders of degeneration" (De La Cruz et al.,
1971), "inadequateandfor excessive resorption" (Van Mierop and Gessner, 1972)
receive a cellular substrate concrete in time, space and quantity-substantially
modifying the original ideas on heart morphogenesis and teratogenesis.
II. Bibliography Updating
Review articles on cell death in embryonic development have been more or less regularly
published since 1926. The most important are those by Ernst (1926), Gliicksmann (1951),
Saunders (1966), Saunders'and Fallon (1966) and Wendler (1972), covering the corresponding
time periods. Instead of re-reviewing the literature already summarized in these reviews we
present a few tables listing the relevant papers published in the period 1967-1974. Last entries
are from August 1974. Table 1 indicates general articles. The references for ultrastructural
9
Table 1. Updating of bibliography on general aspects of cell death
A. Reviews and monographs Bolande, 1967
Forsberg and KiiJIen, 1968
Goerttler, 1958
Pol icard and Bessis, 1968
David, 1970
Menkes, Sandor and IlIies, 1970
Wendler, 1972
Cooper, 1973
B. Morphology Wendler, 1967
Trump and Ginn, 1969
Bessis, 1970
Farber, Verb in and Lieberman, 1971
Kerr, 1971
Scarpelli and Trump, 1971
Trump and Arstila, 1971
Macieira·Coelho, 1972
Basile, Wood and Braun, 1973
C. Cell population kinetics Odartchenko, Lewerenz, Sordat, Roos, and Cottier, 1967
Beyer, 1970
Jagers, 1970
Lala,1972
Shackney, 1973
D. Regulation Fallon and Saunders, 1968
Webster and Gross, 1970
Table 2. Updating of bibliography on morphology of physiological cell death
A. Light microscopy
Hughes, Furstman, Bernick, 1967 palatal processes rat
Alexandru, 1969 IV. ventricle chick
Dawd,1969 limb bud chick
Stanek, 1970 splenic cells chick
Modak and Perdue, 1970 lens cells chick
Holmsted, 1971 palatal processes mouse
Idoyaga.Vargas, Nasljeti and Azcurra, 1972 palatal processes mouse
Mato, Smiley and Dixon, 1972 secondary palate human
B. Histochemistry
Milaire,1967a limb bud mouse
Angelici and Pourtois, 1968 palatal processes rat
Ballard and Holt, 1968 interdigital spaces rat
Allenspach, 1970 oesophage chick
Center, 1970 limb bud mouse
Hammar and Mottet, 1971 interdigital spaces chick
Vermey-Keers, 1972 Hochstetter plate mouse
C. Ultrastructure
Chaudrhy and Shah, 1973 palatal processes hamster
Forsberg and Abro, 1973 uterovaginal anlage mouse
Kieny, Sengel and Pautou, 1973 limb bud chick
Schluter, 1973 neural tube mouse
Schweichel and Merker, 1973 divers rat, mouse
Waterman and Meller, 1973 palatal processes human
Watermann, Ross and Meller, 1973 palatal processes mouse
10
Table 3. Updating of bibliography on topography of physiological cell death
A. Central nervous system Maruyama and d'Agostino, 1967 rat
IIIies, 1969 human
Gamble, 1971 human
Alley and DuBrul, 1972 rat
Das and Hine, 1972 rat
O'Connor, 1972 chick
Reger, Holbrook and Pozos, 1972 divers
Chan-Palay, 1973 cat
Hughes, 1973 opossum
Stoeckel, Porte, Hindelang-Gertner rat
and Delmann, 1973
O'Connor and Wyttenbach, 1974 chick
B. Locomotory system Milaire, 1967 a mouse
IIIies, 1970 human
Kelley, 1970 human
Mitrovic, 1971 chick, rat
Tuffery, 1971 cat
Mitrovic, 1972 chick
Webb,1972 human
Kieny, 1973 chick
C. Digestive and respiratory Pourtois, 1970, a, b mouse, rat
systems Van Horn, 1971 chick
Andrew, 1972 mouse, rat
R0mert and Gauguin, 1973 mouse
D . Urogenital system Black,1971 guinea pig
Dorgan and Schultz, 1971 rat
Firth and Hicks, 1972 rat
Gondos, 1973 human
E. Lymphatic system Chen and Ruth, 1969 chick
Claesson, 1971 a mouse
Kirk, 1972 mouse
Abrunhosa, 1973 mouse
F. Skin and sensory organs IIIies, 1970 human
Thevenet and Sengel, 1971 chick
Modak,1972 chick
Table 4. Updating of bibliography on the cell death in morphogenesis
Dawd,1969 opaque patch chick
Alley and DuBrul, 1972 mesencephalon rat
HanzHkova and Gutmann, 1972 . pudendal nerve rat
Modak,1972 lens fiber cells chick
Cowan, 1973 nervous system chick
Gonzalo-Sanz and Llamas-Marcos, 1973 Wolffian duct chick
Kieny, 1973 limb bud chick
Mattanza, 1973a, b prosencephalon mouse, human
Silver and Hughes, 1973 eye rat
Alley, 1974 mesencephalon hamster
EI-Shershaby and Hinchliffe, 1974 blastocyst mouse
Jimbow, Szabo and Fitzpatrick, 1974 feather chick
Menkes and Mircov, 1974 face chick
11
