/. Embryol. exp. Morph. Vol. 43, pp. 123-136, 1978
Printed in Great Britain © Company of Biologists Limited 1978
J 23
Cell death in the posterior
necrotic zone (PNZ) of the chick wing-bud: a
stereoscan and ultrastructural survey of
autolysis and cell fragmentation
By J. H U R L E 1 AND J. R. H I N C H L I F F E 2
From the Zoology Department,
University College of Wales, Aberystwyth
SUMMARY
The participation of lysosomes and cell profile changes were studied during mesenchymal
cell death in the PNZ of forelimbs of the 4^-day chick embryo.
Lysosome participation was studied cytochemically using as substrate either /?-gIycerophosphate or/?-nitrophenylphosphate. Acid phosphatase (AP) is localized within the Golgi
cisternae of the prospective dying cells, and small AP-positive autophagic vacuoles appear
when degeneration commences. As degeneration by enzymic digestion proceeds, these
vacuoles increase in size and appear to become autolytic since part of the AP activity seems
to 'leak' out of the autophagic vacuoles. Next, the cells fragment and the fragments are then
enclosed in heterophagic vacuoles of macrophages where digestion is completed.
The SEM showed that these intracellular changes in degenerating cells are accompanied by
changes in the cell surface structure. First, the degenerating cells lose the stellate appearance
of healthy mesenchymal cells and become rounded and pitted, then constrictions appear
and finally the cytoplasm breaks up into many small pieces. This final fragmentation may be
an active process rather than a mere consequence of vacuolation.
INTRODUCTION
While cell death is now recognized as a major morphogenetic shaping
mechanism (Saunders, 1966), the question of lysosomal involvement is still
controversial (Lockshin & Beaulaton, 1974«). In some systems cell death seems
to result directly from increased lysosomal activity involving first autophagy
and then autolysis as acid hydrolases leak out of autophagic vacuoles and
damage the cytoplasm. Epithelial cell death provides clear examples of this
type of mechanism (Ericcson, 1969), as in mesonephros regression in the chick
(Haffen & Salzegeber, 1977; Salzegeber & Weber, 1966), insect salivary gland
regression (Jurand & Pa van, 1975) and uterine epithelial deterioration during
1
Author's address: Departamento de Anatomia, Facultad de Medicina, Santander, Spain.
Author's address {for reprints): Zoology Department, University College of Wales,
Penglais, Aberystwyth SY23 3DA, Dyfed, U.K.
2
124
J. HURLE AND J. R. H I N C H L I F F E
implantation of the mouse egg (El-Shershaby & Hinchliffe, 1975). Other examples are known where it has been difficult to implicate lysosomes at a
primary level, although it is clear that disintegrating cells are digested in the
heterophagosomes of macrophages which are often differentiated locally.
Examples of this are muscle regression in metamorphic tadpole tail (Weber,
1964) and mesenchymal cell death during amniote limb development (e.g.
interdigital cell death in rat and chick embryos (Ballard & Holt, 1968; Pautou
& Kieny, 1971); the 'opaque patch' in the central mesenchyme of the chick
wing (Dawd & Hinchliffe, 1971)).
Amniote limb development involves substantial cell death in a number of
regions (Saunders & Fallon, 1967; Pautou, 1975; Milaire, 1977), and the shaping role of some of these areas has been demonstrated by studying the consequences of expanding or contracting the areas by teratogens or mutant genes
(Pautou, 1976; Kieny, Pautou & Sengel, 1976; Hinchliffe & Ede, 1973; Hinchliffe, 1974; Hinchliffe & Thorogood, 1974). While such a shaping role has not
been established (Saunders, 1966) for the posterior necrotic zone (PNZ) of
the developing chick wing, this has been the subject of a classic experimental
study (Saunders, Gasseling & Saunders, 1962), analysing its progressive determination, while Pollak & Fallon (1974, 1976) investigated DNA, RNA and
protein synthesis in the prospectively necrotic PNZ cells. While electron
microscopic studies have been made of the process of mesenchymal cell deterioration and ingestion by macrophages (Saunders & Fallon, 1967), no
studies have analysed specifically the role of lysosomes. The present study
examined the PNZ for evidence of lysosomal participation in cell death. In
addition to the standard Gomori ^-glycerophosphate method for localization
of acid phosphatase ultrastructurally, /7-nitrophenyl phosphate was used as a
substrate (Ryder & Bowen, 1975), since it is believed to detect more isoenzymes. Since the view has recently been expressed that cell fragmentation or
'apoptosis' is a universal accompaniment of cell death or deletion (Kerr,
Wyllie & Currie, 1972), the PNZ was examined under the stereoscan (SEM)
for any evidence of this process.
MATERIALS AND METHODS
Ultrastructural detection of acid phosphatase (AP) activity and stereoscan
studies of the PNZ of the forelimb were made, using chick embryos ranging
from stages 23 to 25 (Hamburger & Hamilton, 1951). The PNZ is readily
located and is easily penetrated by substrates during incubation: thus problems
associated with the preparation of frozen sections (Dawd & Hinchliffe, 1971)
can be by-passed.
For cytochemical studies, embryos were fixed for 3 h in 2-5 % glutaraldehyde
in 0-1 M cacodylate buffer at pH 7-3, and then transferred to buffer solution
where small pieces of the PNZ were dissected out under a binocular microscope
Cell death in the chick wing-bud
125
The fragments were incubated for 45-60 min either in the Gomori ^-glycerophosphate medium (Barka & Anderson, 1963) or in /7-nitrophenylphosphate
medium (Ryder & Bowen, 1975). After incubation the fragments were washed
in buffer, postfixed in osmium tetroxide, dehydrated and infiltrated in araldite.
Ultrathin sections stained with 5 % uranyl acetate and lead citrate (Reynolds,
1963) or, more rarely, unstained were observed under the electron microscope.
Fragments of non-necrotic regions of the limb were also processed for comparison with the PNZ. Controls of the reaction were made by omitting the
substrate from the incubating medium.
To prepare specimens for scanning electron microscopy, embryos were fixed
in glutaraldehyde as above, and rinsed in cacodylate buffer in which, the forelimbs were transversely sectioned through the level of PNZ. Both pieces of the
limbs were dehydrated through a series of acetones, dried by the critical point
method and gold-coated in a Polaron apparatus vapour coater. Specimens were
viewed using a Stereoscan 600 (Cambridge Scientific Instrument Ltd.) at
7-5 kV.
RESULTS
The PNZ is located at the posterior border of the forelimb during stages
23/4-24/5 and reaches maximum intensity at stage 24. In the vicinity of the
PNZ, and in the PNZ itself, viable mesenchyme cells in section have the typical
irregular profile of mesenchymal cells with prominent filopodia. Within the
cytoplasm most of the cells show a well-developed AP-positive Golgi body
with numerous AP-positive vesicles apparently in process of detachment from
its margin (Fig. 1). AP activity can also be found in some instances in cisternae
not closely associated with the Golgi which may represent GERL (Golgiassociated endoplasmic reticulum, from which lysosomes arise) cisternae.
These cells have abundant free ribosomes and mitochondria, sparse endoplasmic
reticulum, and the chromatin of the nuclei is not condensed. Often these cells
are dividing.
Cytoplasmic lesions in the mesenchyme of the PNZ
A number of different lesions can be found in PNZ mesenchymal cells,
which otherwise resemble viable cells as the nucleus and cell profile appear
normal. Such cells cannot be found in non-PNZ areas of the limb. The smallest
lesion present is found in PNZ cells resembling healthy mesenchymal cells but
having within the cytoplasm small AP-positive vesicles 1-2/tm in diameter,
sometimes containing membranous remnants of cytoplasmic organelles (Fig. 2).
AP activity may spread from these small necrotic foci into the adjacent cytoplasm.
Other cells, presumably more deteriorated, contain larger autophagic vacuoles
2-4 jam in diameter (Fig. 3) with high enzymic activity. The micrographs again
suggest some spreading of the local degenerative process (Fig. 4), since in these
9
EMB 43
126
J. HURLE AND J. R. H I N C H L I F F E
Cell death in the chick
wing-bud
127
cells at successive levels a well-defined vacuole may change into an ill-defined
AP-positive region of the cytoplasm (Figs. 3, 4). While the nucleus remains
normal (Fig. 3), within the cytoplasm vacuolization of organelles (largely
mitochondria) is frequent and cytoplasmic necrotic foci as described above are
also present.
Dying cells
In other cells the most remarkable feature is a vacuolization of most of the
cytoplasmic organelles, so intense as to suggest that the cytoplasm is fragmenting
(Fig. 5). The AP activity within these cells does not appear very high, but small
intracytoplasmic necrotic foci can often be found. The nucleus, still apparently
normal, is displaced from the cell centre and is surrounded by only a thin rim
of cytoplasm, which is undeteriorated but sparse in organelles. Between the
nuclear region and the rest of the cytoplasm appears a constriction, suggesting
that the two cellular regions are detaching from each other.
Finally, some cells show signs of both cellular fragmentation and large autophagic vacuoles (Fig. 6). They are characterized by several constrictions in the
cytoplasm, so that large areas are joined only by narrow bridges. As in the
preceding cells, the nucleus is located in one of the cell fragments surrounded
only by a thin rim of cytoplasm. Increased chromatin condensation is usual.
Fragmenting cells sometimes have microfilament bundles at the point of
constriction.
Isolated cell fragments
As the process of cell death advances, the integrity of the dying cells is lost
and only cell fragments or cells with unequivocal signs of necrosis can be found.
These fragments correspond with Kerr's apoptotic bodies.
Two different kinds of cell fragments are seen. One kind (2-5 jum) consists
All TEM micrographs are of PNZ material treated for AP. Figs. 6 and 7 with
jP-nitrophenylphopshate, remainder with ^-glycerophosphate as a substrate.
FIGURES
1-4
Fig. 1. Electron micrograph showing a healthy non-PNZ mesenchymal cell. Note
AP activity in the Golgi complex.
Fig. 2. Mesenchymal cell of PNZ, showing early cytoplasmic alterations. AP
activity appears both within the Golgi cisternae and associated vesicles (G), and
also in small autophagic vesicles (V). Note the normal appearance of the nucleus
and the cell profile.
Fig. 3. Mesenchymal cell of PNZ, showing a large AP positive autophagic vacuole
and in addition some small autophagic vesicles (V). Note the normal appearance of
the nucleus and the rounded profile of the cell.
Fig. 4. Same cell as that of Fig. 3 showing the large autophagic vacuole in different
section. Note the spreading of AP activity into the surrounding cytoplasm.
9-2
128
J. HURLE AND J. R. H I N C H L I F F E
Cell death in the chick wing-bud
129
only of electron-dense cytoplasm, with most of the organelles vacuolized and
the ribosomes often aggregated as banded granules. AP activity can be detected in some of the vacuoles, and it has sometimes spread into other areas of
the fragment (Fig. 7). P-nitrophenylphosphate appears to give a more positive
indication of AP activity in these fragments. The other kind of fragment
(4-6 fim) contains mainly the nuclear part, greatly increased in electron density.
The degree of alteration of these fragments is variable : when degeneration is
less advanced, the nucleus can be clearly differentiated from the thin rim of
surrounding cytoplasm. In more advanced deterioration there is no clear
nuclear/cytoplasmic separation (Fig. 8). In these fragments AP activity was
not found using either glycerophosphate or /?-nitrophenylphosphate as substrate.
Phagocytosis
The cellular fragments lying free in the extracellular space are soon surrounded
by cell processes and eventually phagocytosed by the neighbouring mesenchymal cells, which appear to transform into macrophages. Our ultrastructural
and enzymic analysis agrees with previous accounts of this process in limb
mesenchyme cell death (Ballard, 1965; Ballard & Holt, 1968; Dawd & Hinchliffe, 1971; Pautou & Kieny, 1971).
Scanning electron microscopy
Figure 9 represents a general survey of the PNZ under the SEM. Within the
area, healthy mesenchymal cells, rounded dead cells and cell fragments and
large macrophages can be easily identified, but identification of actively fragmenting cells or cells in the initial stages of degeneration is usually more
difficult. Dead or degenerating cells and macrophages are absent from regions
of mesenchyme adjacent to the PNZ (Fig. 10). Healthy mesenchymal cells have
a typical stellate appearance, with numerous cell processes (Figs 9-11). In the
PNZ they form a loose mesh-work within which are located the different types
of degenerative cells.
Cells in the initial stages of degeneration tend to lose their stellate profile,
FIGURES
5-8
Fig. 5. Mesenchymal cell of PNZ, showing extensive vacuolization of the cytoplasm.
Arrows show two levels where confluence of vacuoles will produce fragmentation.
Note the detachment of the nucleus and adjacent cytoplasm from the vacuolized
cytoplasm.
Fig. 6. Mesenchymal cell of PNZ undergoing fragmentation. Note large AP positive
autophagic vacuoles in the cytoplasm. The nucleus shows early stages of chromatin
condensation.
Fig. 7. Cytoplasmic fragment of a dead cell showing AP activity within the vacuoles
and also free in the cytoplasm (arrow).
Fig. 8. Nuclear fragment of a dead cell without AP activity. Note the presence of
AP activity in Golgi cisternae of surrounding mesenchymal cells.
130
J. H U R L E AND J. R. H I N C H L I F F E
£•:#"
'TUB'
Fig. 9. SEM micrograph showing a panoramic view of PNZ. Stellate mesenchymal
cells, dead cells (DC) and fragments (F), and macrophages (M) are clearly distinguishable. Ectoderm (E).
Fig. 10. SEM micrograph, for comparison with Fig. 9, showing viable mesenchyme
adjacent to the PNZ. Note stellate cell profile and filopodia.
Cell death in the chick wing-bud
131
becoming rounded and showing constrictions which suggest that they are in
initial stages of fragmentation (Fig. 11). Cells in a more advanced stage of
degeneration are clearly recognized as fragmenting cells and appear to consist
of four to five (Fig. 12) or more (Fig. 13) rounded bodies detaching from each
other. One fragment is usually relatively large and probably corresponds to the
nuclear fragment described using the TEM. In addition to these changes in
cell surface structure, the surface is frequently (Fig. 12) but not always (Fig. 13)
pitted with small holes.
Cell fragments appear as rounded bodies 3-6 (im in diameter, their surface
usually rough with numerous small holes. They are usually isolated between the
mesenchymal cells (Fig. 14), but some clusters of small fragments can be found.
Macrophages may be clearly differentiated from mesenchymal cells by their
round shape, lack of cell processes and large size, 14-18 /on in diameter (Fig. 15)
They are usually surrounded by mesenchymal cells. Macrophages have 5-6 ^m
diameter ovoid protrusions, pitted with small holes and probably representing
recently ingested dead cells. Small cell fragments are often in contact with the
macrophage surface and they eventually appear to sink below the cell surface
in a manner similar to the in vitro latex particle ingestion by macrophages
described by Kaplan, Gaudernack & Seljelid (1975).
DISCUSSION
Our results suggest the following sequence in the process of cell death in the
PNZ: (1) appearance in the cytoplasm of small AP-positive autophagic vacuoles
enclosing organelles such as mitochondria. (2) An increase in size of the autophagic vacuoles, which may now become autolytic. Under the SEM, cells of
this stage are rounded with the cell surface often containing small holes, presumably representing superficial vacuoles (Kieny, Sengel & Pautou, 1973). (3)
Cell fragmentation, visible with both TEM and SEM, frequently beginning
with separation of a large nucleus-containing fragment from the cytoplasmic
portion. Later, chromatin condensation is marked in the nuclear fragment,
while the cytoplasmic portion becomes more and more fragmented. (4) Phagocytosis of the cell fragments by macrophages.
There is controversy concerning the role of AP-positive lysosymes in cell
death, since in some cases lysosomes appear to play a primary role, involving
first autophagy and then autolysis, while in other cases no early involvement of
lysosomes has been detected (Lockshin & Beaulaton, 1974Ö). However, associated with PNZ mesenchymal deterioration, we found that cell fragmentation
frequently results in a nuclear part with a thin rim of almost AP-negative cytoplasm becoming detached from other cytoplasmic fragments that contained AP
activity both diffuse and localized in autophagic vacuoles. This result makes it
necessary to reinterpret the previous studies on limb-bud mesenchymal cell
death in other regions, which concluded there was no AP participation in the
132
J. HURLE AND J. R. H I N C H L I F F E
Cell death in the chick wing-bud
133
initial stages of cell death (Ballard, 1966; Ballard & Holt, 1968; Dawd &
Hinchliffe, 1971). These analyses paid more attention to the more prominent
but mainly AP-negative nuclear fragment than to the AP-positive cytoplasmic
fragments. In the 'opaque patch' Dawd & Hinchliffe described AP-positive
'large lysosomes' or autophagic vacuoles in apparently viable cells (similar
AP-positive bodies may be seen in Ballard & Holt, 1968, e.g. figs. 7 and 8)
interpreted as undergoing non-lethal autophagy, but they did not consider
such cells as predecessors of the AP-negative nuclear fragments also described.
With the improved technique of the present study, however, it is clear that
autophagy accompanied by AP participation occurs at an early stage of
deterioration of the PNZ mesenchyme. However, it is not possible to define
precisely the lysosomal role in PNZ cell death. Autolysis caused by lysosomes
may be the cause of cell death, as originally proposed by de Duve (1959);
alternatively lysosomal autophagy may be a defence mechanism eliminating
organelles already caused to deteriorate by some other unknown necrotic
process.
Thus the PNZ mesenchyme undergoes firstly autolytic digestion, followed by
digestion within macrophages. The origin of the increased AP in the deteriorating mesenchyme cells could be either from new synthesis, or by activation of
previously synthesised AP (Lockshin & Beaulaton, 1974«). The decreasing
synthesis of DNA, RNA and protein reported as taking place in the prospective
PNZ in the stages preceding cytological deterioration (Pollak & Fallon, 1974,
1976) suggests activation. However, the hypothesis that the remaining protein
synthesis is of acid hydrolases cannot be ruled out. Previously it has been
assumed that the intense AP and other acid hydrolase activity within the macrophage (e.g. Ballard & Holt, 1968; Dawd & Hinchliffe, 1971) is due to active
synthesis by the macrophage. Perhaps, however, the phagocytosed dead cells
FIGURES
11-15
Fig. 11. SEM micrograph of cell in early stages of degeneration (D). Note the
rounded shape, the presence of a constriction (arrows) in the middle, and small
holes in the surface, and compare with the cell shown in Fig. 5. Note the much
larger macrophage (M), and normal mesenchymal cell (C).
Fig. 12. SEM micrograph of fragmenting mesenchymal cell. Note the presence of a
large rounded protrusion, probably the nuclear fragment, and some small pieces
showing holes in the surface.
Fig. 13. SEM micrograph of a mesenchymal cell undergoing active fragmentation
into numerous small pieces.
Fig. 14. SEM micrograph of isolated dead cell fragments lying between normal
mesenchymal cells. Note the constant presence of small holes in the surface.
Fig. 15. SEM micrograph of a macrophage located under the basal lamina of the
ectoderm. Note the presence of round protrusions and small holes in the surface.
Note also the group of cell fragments (F), possibly from several cells and no longer
actively fragmenting, awaiting phagocytosis.
134
J. HURLE AND J. R. H I N C H L I F F E
are themselves contributing much of the AP activity within the macrophage
vacuoles.
Cell fragmentation is another constant feature in the degeneration of PNZ
cells. The SEM allows us to follow clearly all the stages of fragmentation,
which begin with rounding and pitting of the cell surface and end in the detachment of fragments. However, in order to follow this fragmentation process, it
is important to conserve cell surface structure using the critical-point drying
method omitted in previous SEM surveys of interdigital cell death (Kieny et al.
1973). Such fragmentation or 'apoptosis' has been emphasized by Kerr as a
general feature of cell death (Kerr et al. 1972), but the mechanism by which
fragmentation is produced has not been explained. Most studies regard it as a
consequence of previous degenerative events (Dawd & Hinchliffe, 1971;
Lockshin & Beaulaton, 19746; Ojeda & Hurle, 1975). In our study, it sometimes seems to be a consequence of previous vacuolization, but in other instances, one or more constrictions are clearly detectable by SEM and TEM
(Fig. 6) without vacuolization. An active mechanism involving microtubules
and microfilaments could be involved, since Mullinger & Johnson (1976) have
described a similar process of fragmentation resulting from altered cell division
in chilled mitotic HeLa cells. The theory that cell death is in fact an aberrant
form of mitosis has been advanced (Forsberg & Källen, 1968; Gondos & Hobel,
1973).
One striking feature of the PNZ (Saunders & Fallon, 1967) is the substantial
24 h time interval between the irreversible programming at stage 21 of the PNZ
cells to die, and the first appearance of deterioration at the cytological level at
stage 24. During this gap DNA and RNA synthesis decreases by stage 22 and
protein synthesis by stage 23 (Pollak & Fallon, 1974, 1976). The cytochemical
changes preceding the unequivocal cytological deterioration typical of cell
death occur no earlier than stage 23/4, so events during the gap between determination and expression of cell death cannot yet be explained in ultrastructural
terms.
One of us (J.H.) wishes to acknowledge receipt of a Postdoctoral grant of the Spanish
Ministry of Education and Science.
REFERENCES
BALLARD, K. J. (1965). Studies on the participation of hydrolytic enzymes in physiological autolysis in degenerating regions of the rat foetus. Ph.D. thesis, University of London.
BALLARD, K. J. & HOLT, S. J. (1968). Cytological and cytochemical studies on cell death and
digestion in foetal rat foot; the role of macrophages and hydrolytic enzymes. /. Cell Sei.
3, 245-262.
BARKA, T. & ANDERSON, P. J. (1963). Histochemistry. New York, Evanston and London:
Hoeber.
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. J. Embryol. exp. Morph. 26, 401-424.
Cell death in the chick wing-bud
135
D E DUVE, C. (1959). Lysosomes, a new group of cytoplasmic particles. In Subcellular Particles
(ed. T. Hayashi), pp. 128-59. Washington: The Ronald Press.
EL-SHERSHABY, A. M. & HINCHLIFFE, J. R. (1975). Epithelial autolysis during implantation of
the mouse blastocyst: an ultrastructural study. J. Embryol. exp. Morph. 33, 1067-1080.
ERICCSON, J. L. E. (1969). Mechanism of cellular autophagy. In Lysosomes in Biology and
Pathology, vol. II (ed. J. T. Dingle & H. B. Fell), pp. 354-394. Amsterdam: North Holland
Publishing Company.
FORSBERG, J. G. & KALLEN, B. (1968). Cell death during embryogenesis. Revue Roumaine
Embryol. Cytol. 5, 91-102.
GONDOS, B. & HOBEL, C. J. (1973). Germ cell degeneration and phagocytosis in the human
foetal ovary. In The Development and Maturation of the Ovary and its Functions (H.
Peters), pp. 77-83. Amsterdam: Excerpta Medica.
HAFFEN, K. & SALZEGEBER, B. (1977). Les modalités de la régression du mésonephros chez les
amniotes. In Mécanismes de la Rudimentation des Organes chez les Embryons de Vertébrés, (ed. A. Raynaud). Colloques Internationaux du CNRS 266, 251-262.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages of development of the
chick embryo. / . Morph. 88, 49-92.
HINCHLIFFE, J. R. (1974). The patterns of cell death in chick limb morphogenesis. Lib. J. Sei.
4, 23-32.
HINCHLIFFE, J. R. & EDE, D. A. (1973). Cell death and the development of limb form and
skeletal pattern in normal and wingless (ws) chick embryos. / . Embryol. exp. Morph. 30,
753-772.
HINCHLIFFE, J. R. & THOROGOOD, P. V. (1974). Genetic inhibition of cell death and the
development of form and skeletal pattern in the limb of talpid3 (ta3) mutant chick embryos
/ . Embryol. exp. Morph. 31, 747-760.
JURAND, A. & PAVAN, C. (1975). Ultrastructural aspects of histolytic processes in the salivary
gland cells during metamorphic stages in Rhynchosciara hollaenderi (Diptera, Sciaridae).
Cell Differentiation 4, 219-236.
KAPLAN, G., GAUDERNACK, G. & SELJELID, R. (1975). Localization of receptors and early
events of phagocytosis in the macrophage. Expl Cell Res. 95, 365-375.
KERR, J. F. R., WYLLIE, A. H. & CURRIE, A. R. (1972). Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br. J. Cancer. 26, 239-257.
KIENY, M., PAUTOU, M. P. & SENGEL, P. (1976). Limb morphogenesis as studied by Janus
green B and vinblastine-induced malformations. In Tests of Teratogenicity in Vitro, pp.
389-415. Amsterdam: North Holland.
KIENY, M., SENGEL, P. & PAUTOU, M-P. (1973). Nécrose morphogène et phagocytose
étudiées dans le pied de l'embryon de poulet par la microscopie électronique à balayage.
Cr. hebd. Séanc. Acad. Sel, Paris 277D, 581-584.
LGCKSHIN, R. A. & BEAULATON, J. (1974a). Programmed cell death. Life Sciences 15,
1549-1566.
LOCKSHIN, R. A. & BEAULATON, J. (19746). Programmed cell death. Cytochemical evidence
for lysosomes during the normal breakdown of the intersegmental muscles. J. Ultrastruct.
Res. 46, 43-62.
MILAIRE, J. (1977). Rudimentation digitale au cours du développement normale de l'autopode
chez les mammifères. In Mécanismes de la Rudimentation des Organes chez les Embryons de
Vertébrés (ed. A. Raynaud). Colloques Internationaux du CNRS 266, 221-233.
MULLINGER, A. M. & JOHNSON, R. T. (1976). Perturbation of mammalian cell division. III.
The topography and kinetics of extrusion subdivision. J. Cell Sei. 22, 243-285.
OJEDA, J. L. & HURLE, J. M. (1975). Cell death during the formation of tubular heart of the
chick embryo. / . Embryol. exp. Morph. 33, 523-534.
PAUTOU, M-P. (1975). Morphogenèse de l'autopode chez l'embryon de poulet. J. Embryol.
exp. Morph. 34, 511-529.
PAUTOU, M-P. (1976). La morphogenèse du pied de l'embryon de poulet étudiée a l'aide de
malformations provoquées par le vert Janus. / . Embryol. exp. Morph. 35, 649-665.
PAUTOU, M-P. & KIENY, M. (1971). Sur les mécanismes histologiques et cytologiques de la
nécrose morphogène interdigitale, chez l'embryon de poulet. Cr. hebd. Séanc. Acad. Sei.,
Paris 272D, 2025-2028.
136
J. H U R L E A N D J. R. H I N C H L I F F E
POLLAK, R. D . & FALLON, J. F. (1974). Autoradiographic analysis of macromolecular synthesis in prospectively necrotic cells of the chick limb bud. I. Protein synthesis. Expl Cell Res.
86, 9-14.
POLLAK, R. D. & FALLON, J. F. (1976). Autoradiographic analysis of macromolecular synthesis in prospectively necrotic cells of the chick limb bud. 11. Nucleic acids. Expl Cell
Res. 100, 15-22.
REYNOLDS, E. A. (1963). The use of lead citrate at high pH as an electron opaque stain in
electron microscopy. J. Cell Biol. 17, 208-212.
RYDER, T. A. & BOWEN, I. D. (1975). A method for the fine structural localization of acid
phosphatase using jp-nitrophenylphosphate as substrate. J. Histochem. Cytochem. 23,
235-237.
SALZEGEBER, B. & WEBER, R. (1966). Le régression du mésonéphros chez l'embryon de poulet.
Étude des activités de la phosphatase acide et des cathepsines. Analyse biochimique,
histochimique et observations au microscope électronique. / . Embryol. exp. Morph. 15,
397-419.
SAUNDERS, J. W. JR. (1966). Death in embryonic systems. Science, N.Y. 154, 604-612.
SAUNDERS, J. W. JR. & FALLON, J. F. (1967). Cell death in morphogenesis. In Major Problems
in Developmental Biology (ed. M. Locke), pp. 289-314. London: Academic Press.
SAUNDERS, J. W., JR., GASSELING, M. T. & SAUNDERS, L. C. (1962). Cellular death in morpho-
genesis of the avian wing. Devi Biol. 5, 147-178.
WEBER, R. (1964). Ultrastructural changes in regressing tail muscles of Xenopus larvae at
metamorphosis. / . Cell Biol. 22, 481-487.
{Received 22 March 1977, revised 4 July 1977)