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INTRODUCTION
Cell death is a ubiquitous feature of metazoan development
(see for example Wyllie et al., 1980; Bowen and Lockshin,
1981; Truman, 1984; Ellis et al., 1991; Tomei and Cope,
1991; Raff, 1992). Studies from a broad range of vertebrate
and invertebrate systems have established that such natu-
rally occurring cell death results from an active develop-
mental program often triggered by systemic hormones,
withdrawal of trophic factors and local cell interactions
(Saunders, 1966; Truman, 1984; Oppenheim, 1985). In
many animal systems, this deliberate removal of cells pro-
ceeds through a series of distinct morphological stages
known as apoptosis (Kerr et al., 1972; Wyllie et al., 1980).
During apoptotic death, the cytoplasm and nucleus of the
dying cell condense while the morphology of cellular
organelles remains rather well preserved. In many cases,
the condensing cell breaks up into fragments (apoptotic
bodies) and is eventually engulfed by phagocytic cells. The
regional incidence of cell death during development is often
predictable (Whitten, 1969; Hinchcliffe, 1981; Hurle, 1988)
and, at least in some cases, depends on protein synthesis
(Tata, 1966; Lockshin, 1969; Farbach and Truman, 1988;
Martin et al., 1988; Oppenheim et al., 1990). Genetic
studies of cell death in C. elegans have identified several
loci that are required for this process during development
(Ellis and Horvitz 1986; Yuan and Horvitz, 1990; Ellis et
al., 1991; Hengartner et al., 1992). These and other obser-
vations have led to the concept of ‘programmed cell death’,
which views cell loss as the consequence of an active phys-
iological process analogous, in some ways, to differen-
tiation (Saunders, 1966; Lockshin and Zakeri, 1991; Raff,
1992). Although the extrinsic signals that elicit programmed
cell death have been characterized in some cases, the exact
molecular mechanisms underlying programmed cell death
remain unknown.
We are interested in a molecular genetic analysis of cell
death in Drosophila. Several examples of naturally occur-
ring cell death during postembryonic development have
been documented in this organism. In these cases, the deci-
sion to die can require hormone induction (Truman, 1984;
Kimura and Truman, 1990) and may be influenced by cell
interactions (Fischbach and Technau, 1984; Wolff and
Ready, 1991; Campos et al., 1992). In contrast, little is
known about programmed cell death in the Drosophila
embryo (Campos-Ortega and Hartenstein, 1985).
29
Development 117, 29-43 (1993)
Printed in Great Britain © The Company of Biologists Limited 1993
The deliberate and orderly removal of cells by pro-
grammed cell death is a common phenomenon during
the development of metazoan animals. We have exam-
ined the distribution and ultrastructural appearance of
cell deaths that occur during embryogenesis in
Drosophila melanogaster. A large number of cells die
during embryonic development in Drosophila. These
cells display ultrastructural features that resemble
apoptosis observed in vertebrate systems, including
nuclear condensation, fragmentation and engulfment by
macrophages. Programmed cell deaths can be rapidly
and reliably visualized in living wild-type and mutant
Drosophila embryos using the vital dyes acridine orange
or nile blue. Acridine orange appears to selectively stain
apoptotic forms of death in these preparations, since
cells undergoing necrotic deaths were not significantly
labelled. Likewise, toluidine blue staining of fixed tissues
resulted in highly specific labelling of apoptotic cells,
indicating that apoptosis leads to specific biochemical
changes responsible for the selective affinity to these
dyes. Cell death begins at stage 11 (~7 hours) of embryo-
genesis and thereafter becomes widespread, affecting
many different tissues and regions of the embryo.
Although the distribution of dying cells changes drasti-
cally over time, the overall pattern of cell death is highly
reproducible for any given developmental stage.
Detailed analysis of cell death in the central nervous
system of stage 16 embryos (13-16 hours) revealed asym-
metries in the exact number and position of dying cells
on either side of the midline, suggesting that the deci-
sion to die may not be strictly predetermined at this
stage. This work provides the basis for further molecu-
lar genetic studies on the control and execution of pro-
grammed cell death in Drosophila.
Key words: apoptosis, Drosophila; embryogenesis, programmed
cell death, vital dyes, nervous system development, macrophages
SUMMARY
Programmed cell death during Drosophila embryogenesis
John M. Abrams
1
, Kristin White
1
, Liselotte I. Fessler
2
and Hermann Steller
1
1
Howard Hughes Medical Institute, Department of Brain and Cognitive Sciences and Department of Biology, Massachusetts
Institute of Technology, Cambridge, Massachusetts 02139, USA
2
Molecular Biology Institute and Biology Department, University of California, Los Angeles, CA 90024, USA
30
Since we felt that the Drosophila embryo would be par-
ticularly amenable to future genetic and molecular analy-
ses of programmed cell death, we have investigated the
morphology and distribution of cell deaths that normally
occur during embryonic development. We find that a large
number of cells undergo programmed death, and that these
cells display many of the characteristic ultrastructural fea-
tures described for apoptosis in other organisms. Apoptotic
cells can be rapidly and reliably visualized in live embryos
with vital dyes. We demonstrate that at least one of these
stains, acridine orange, is specific for apoptotic forms of
cell death and does not significantly label cells undergoing
necrotic death provoked by injury. Although the number
and location of apoptotic cells changes dramatically over
the course of embryogenesis, we find that the overall pat-
tern of cell death is reproducible for any given develop-
mental stage. However, close examination of certain
regions, particularly in the developing central nervous
system, indicates that the exact number and position of
apoptotic cells can vary. These observations suggest that
the decision to die is not strictly stereotyped for all cells in
the embryo and may be influenced by local intercellular
interactions. Finally, this work has provided the basis for
the isolation of cell death defective mutations in Drosophila
(White, K., Abrams, J. M., Grether, M., Young, L. and
Steller, H. unpublished data).
MATERIALS AND METHODS
Egg collection and embryo staging
Wild-type (Canton S) eggs were collected on molasses/agar plates,
either at 25°C or at 18°C, and staged according to Campos-Ortega
and Hartenstein (1985). Tightly staged populations of embryos
were prepared by sorting blastoderms on the basis of their char-
acteristic morphology. Where appropriate, age is stated as time
after egg laying (AEL). Embryos from stocks of polyhomeotic
505
(Dura et al., 1987) and crumbs
11A22
(Tepass et al., 1990) were
also analyzed.
Staining with vital dyes
Embryos were dechorionated with 50% bleach, rinsed with water
and placed in an equal volume of heptane and either 5 µg/ml of
acridine orange (Sigma) or 100 µg/ml nile blue A (Sigma) in 0.1
M phosphate buffer, pH ~ 7.2. After 5 minutes of shaking,
embryos were removed from the interface and placed under series
700 Halocarbon oil, (Halocarbon products corp., Hackensack, NJ).
Samples were viewed either with a conventional fluorescence
microscope or with an MRC 600 confocal scanning laser micro-
scope (Bio-Rad) using a BHS color cube filter to detect green flu-
orescence or a YHS color cube filter to detect red fluorescence.
Confocal image processing was performed either with software
provided by the manufacturer or with the Voxel View (Vital
Images, Iowa) program on a Silicon Graphics computer. Acridine-
stained embryos can be viewed with filters for either green or red
fluorescence and, in general, these patterns are similar. Photo-
graphic representations shown here have been viewed using the
green filter unless otherwise noted. For time-lapse studies, acri-
dine-stained embryos were placed under Voltalef oil (3 s or 10 s)
on Petri-perm dishes (Bachofer, Reutlingen, Germany).
Fixed tissue spreads
Embryos were stained with acridine orange as described above,
washed in phosphate buffer to remove heptane, and individually
placed on slides coated with 0.5% gelatin and 0.05% chrom alum.
A siliconized cover slip placed over the embryo was used to gently
spread the tissue into a monolayer. After photographic recordings
of representative fields, the slides were rapidly frozen at −70°C.
To fix the tissue, the cover slip was quickly removed and slides
were immediately submerged in 2.5% glutaraldehyde for 20 min-
utes. For toluidine blue staining, glutaraldehyde-fixed tissue was
placed in 1% osmium tetroxide for 5 minutes, washed with water,
air dried and then stained with a solution of 0.1% toluidine/0.1%
sodium borate for 5 minutes at 55°C.
Electron microscopy
Dechorionated embryos were shaken in equal volumes of heptane
and a fixative solution of 1.5% acrolein, 1% paraformaldehyde,
2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.0, for 20 min-
utes. Embryos were then freed of the surrounding vitelline mem-
branes by hand dissection in 0.1 M phosphate buffer, pH 7.0, (PB)
and refixed for another 30 minutes in the above fixative solution.
After several washes in PB, the embryos were treated with a 2%
solution of osmium tetroxide in PB for 1 hour. Following several
washes in PB, the embryos were dehydrated through an ethanol
series, washed in several changes of propylene oxide and then
embedded in Spurr’s media (Polysciences, Warrington, PA; Spurr,
1969). Thin sections were stained with uranyl aceytate and lead
citrate according to Osborne (1980). Alternatively, after osmium
fixation, some embryos were washed in water and incubated in
1% uranyl acetate (in water) at 50°C for 12-16 hours. Thin sec-
tions from these samples were viewed directly by electron
microscopy without any subsequent staining. For light
microscopy, sections were stained with 0.01% toluidine/0.05%
sodium borate with or without 0.05% methylene blue at 55°C for
approximately 5 minutes.
Irradiation of embryos and cycloheximide
treatment
3-4 hours after egg laying (AEL), Canton S embryos were exposed
to 600 or 4000 rads of X-irradiation using a Torrex 120D X-ray
inspection system (Astrophysics Research Corp., CA). Compared
to a hatching frequency >90% for untreated embryos, these pro-
tocols of X-irradiation reduced the hatching frequency to ~ 5%
for a 600 rad exposure or 0% for a 4000 rad exposure. Some
embryos irradiated at 4000 rads were treated with cycloheximide
immediately afterward, by shaking them in heptane and 10 µg/ml
cycloheximide (in 0.1 M phosphate buffer). These embryos, along
with mock-treated samples, were aged for various times under
Voltalef oil.
Hypoxia treatments
10-12 hours AEL embryos were dechorinonated and placed under
heptane for 4 hours at 25°C. Survival after this treatment is 0%
(no hatched embryos observed out of 400 scored). Embryos sub-
jected to this treatment were either stained with vital dyes or pre-
pared for electron microscopy.
Antibody staining
Phagocytic hemocytes are detected with a rat polyclonal antibody
to protein X. Protein X is one of the abundant secreted proteins
found in the medium of Kc cell cultures (Echalier, 1976). This
protein has been purified by previously described techniques
(Olson et al., 1990). Anti-X polyclonal antibodies were raised in
a rat using pure, nonreduced protein X as immunogen. For some
whole-mount preparations, a 1:500 dilution of this antiserum was
used and immunostaining with HRP-conjugated secondary anti-
body was performed essentially as described in Steller et al. (1987)
except that post-fixation washes and blocking steps were done as
J. M. Abrams and others
31Cell death in the Drosophila embryo
described in Lee et al. (1991). Embryos prepared for subsequent
histology were immunostained with anti-X, biotinylated anti-rat-
IgG and avidin-peroxidase using the ABC Vectastain kit (Vector
Labs, Burlingame, CA). After color development, the embryos
were dehydrated, embedded in Epon (Polysciences, Warrington,
PA) and sectioned. The sections were then stained with 0.01%
toluidine Blue, 0.025% methylene blue in 0.025% sodium tetrab-
orate at 70-77°C.
RESULTS
Cell death in the Drosophila embryo occurs by
apoptosis
Comparative studies from a wide variety of organisms have
defined a set of strikingly conserved morphological features
associated with the regulated death of cells during devel-
opment (Kerr et al., 1972; Wyllie et al., 1980). This type
of death, conventionally referred to as apoptosis or pro-
grammed cell death, involves a generalized condensation of
the cytoplasm and nucleus, separation of the dying cell from
its neighbors, fragmentation into discrete membrane-bound
bodies and eventual engulfment of cellular debris and
corpses by phagocytes (Kerr et al., 1972; Wyllie et al.,
1980). A characteristic feature of apoptotic death is that, in
spite of these cytologic changes, organelles remain intact
and are often identifiable even after engulfment (Kerr and
Harmon, 1991). In contrast, a distinctly different set of
ultrastructural features is typically observed under con-
ditions that induce cellular injury or necrosis (Wyllie, 1981;
Kerr and Harmon, 1991). This form of death includes a
general swelling of the cell and its organelles, loss of mem-
brane integrity, lysosomal rupture and ultimate disintegra-
tion of organelles (for example see Trump et al., 1981;
Wyllie, 1981; see also Fig. 7). We wanted to determine
whether the ultrastructural morphology of embryonic cell
death in Drosophila resembles the apoptotic cell deaths
previously observed in other systems. Fig. 1 is a compila-
tion of various stages of cellular degeneration from elec-
tron microscopic images of a stage 13 embryo. At this point
during embryogenesis, cell death is prominent and wide-
spread. The resemblance of these degenerating bodies to
the apoptotic figures that have been described in other
Fig. 1. Electron micrographs of cell death in a stage 13 wild-type embryo. (A) Early stage of cell death in the epidermal layer. The dying
cell has begun to separate from its neighbors and masses of compacted nuclear material are apparent (white arrow) yet mitochondria
appear normal. A macrophage, already containing one apoptotic corpse (open white arrow), is extending a phagocytic pseudopodium
(black arrow) around the dying cell which is already an apparent target for engulfment; scale bar, 2.4 µm. (B) A fragmenting cell corpse is
fully separated from neighboring cells. Cytoplasmic bridges (black arrow) still join the separating fragments; scale bar, 1 µm. (C)
Fragmented cell corpse. Cytoplasm is condensed yet mitochondria (arrow) are still recognizable; scale bar, 1 µm. (D) Macrophage
engorged with cell corpses, one of which (arrow) contains readily identifiable mitochondria; scale bar, 2 µm.
32
animal systems is striking. Early signs of programmed cell
death are depicted in Fig. 1A, where compaction of chro-
matin into electron-dense masses and cellular shrinkage
have become apparent. The mitochondria are clearly iden-
tifiable and apparently unaffected at this stage of cell death.
As degeneration progresses, dying cells separate from their
neighbors, becoming further condensed and osmiophilic. At
this stage, fragmentation into membrane-bound bodies often
ensues (see Fig. 1B). While nuclear components become
highly compacted, the condensing cytoplasm frequently
contains intact mitochondria (Fig. 1C). Dying cells and
corpses can exist as free, unengulfed material or, more
often, they are observed inside large phagocytes that may
contain several degenerate bodies (see Fig. 1D and below).
From our analyses, it appears that the engulfment of cell
corpses during embryogenesis occurs mostly, if not exclu-
sively, by circulating macrophage-like cells (see below).
The stage at which a dying cell becomes a target for
engulfment is apparently quite variable. Late-stage apop-
totic bodies, which have deteriorated to the extent that
organelles are no longer identifiable, are frequently being
engulfed by macrophages (not shown). Some dying cells
that have been entirely phagocytosed, however, contain
identifiable mitochondria and other organelles characteris-
tic of earlier stages of degeneration (see Fig. 1D). We have
also observed macrophages approaching cells showing only
very early signs of death. For example, in Fig. 1A, a cell
in a very early stage of apoptosis (its nucleus is only par-
tially osmophilic) is in close contact with an engulfing
macrophage. Thus, while many dying cells are engulfed at
a fairly late stage of degeneration, when the entire cell is
fully condensed, there are also instances of early engulf-
ment prior to complete nuclear condensation.
From our ultrastructural analyses, it is clear that the vast
majority of natural cell deaths in the Drosophila embryo
occur by apoptosis. These structural features contrast
starkly with the cytologic changes observed when embry-
onic cells undergo necrosis (see Fig. 7).
Degenerating cells in fixed tissue preparations are read-
ily identified with histological stains (Bowen, 1981, Fis-
chbach and Technau, 1984), including toluidine blue
(Fristrom, 1969; Murphy, 1974; Giorgi and Deri, 1976).
However, these studies made no clear distinction between
apoptotic and necrotic forms of death. To examine the cor-
respondence between the above ultrastructural observations
with light microscopy, we stained semithin plastic sections
with toluidine blue, and examined adjacent ultrathin sec-
tions in the electron microscope (Fig. 2). In this way, we
were able to compare the ultrastructure and staining prop-
erties of the same cell. In the light microscope, dying cells
are distinguished as condensed figures which become heav-
ily stained with toluidine blue (Fig. 2C,D). Ultrastructural
examination of the same region revealed that the intensely
stained (chromophilic) cells displayed obvious apoptotic
features (Fig. 2, compare A,C with B,D). Note that only
the apoptotic corpses, but not the cytoplasm of the engulf-
J. M. Abrams and others
Fig. 2. Apoptotic cells are stained by toluidine blue. (A) Electron micrograph of phagocytic macrophages containing apoptotic cell
corpses and (C) the same region of tissue in a neighboring toluidine-blue-stained semithin section seen by Nomarski optics. (B) Electron
micrograph of unengulfed apoptotic bodies and (D) the same region of tissue in a neighboring toluidine-blue-stained semithin section
seen by Nomarski optics. Images are from a stage 13 embryo. Scale bars, 5 µm.
33Cell death in the Drosophila embryo
ing phagocytes, are stained by toluidine blue (for example
see Fig. 2C). These results show that toluidine blue is a
reliable stain for apoptotic cells in the Drosophila embryo.
Furthermore, it generally appears that ultrastructural
changes associated with cell death precede the changes that
cause dying cells to become characteristically chromophilic
(data not shown). Also, as we show below, this increased
chromophilic property appears to be specific for apoptotic
forms of cell death, since necrotic cells remain unstained.
Identification of apoptotic cells in live embryos
In order to investigate the pattern of programmed cell death
throughout embryonic development, and as a prerequisite
for genetic screens, we sought rapid and reliable methods
to visualize apoptotic cells in the Drosophila embryo. Vital
dyes have been previously used to investigate cell death in
a variety of developmental preparations (Saunders et al.,
1962; Robbins and Marcus, 1963; Spreij, 1971; Starre-van
der Molen and Otten, 1974; Wolff and Ready, 1991; Bonini
et al., 1990). Based on these methods, we developed a
simple protocol for staining apoptotic cells in live
Drosophila embryos (see Materials and Methods). Using
heptane permeabilization, live embryos were stained with
the vital dyes acridine orange (AO) or nile blue (NB) and
then viewed by conventional or confocal microscopy. With
confocal microscopy and time-lapse imaging, we can
usually follow these AO-positive cells for at least 30 min-
utes (not shown). The patterns of staining obtained in this
fashion were found to closely resemble the distribution of
pyknotic figures in toluidine-blue-stained plastic sections.
Fig. 3 compares optical sections obtained by confocal
microscopy from the head region of an AO-stained embryo
(stage 13) with toluidine-blue-stained plastic sections from
equivalent sagittal planes of a comparably staged embryo.
There is an excellent correspondence between the position
of brightly fluorescent cells in whole-mount embryos and
the distribution of darkly stained degenerating figures in
plastic sections. Embryos stained with another vital dye,
nile blue (NB), display patterns which are identical to those
seen with AO (not shown). Furthermore, simultaneous co-
staining with these two dyes shows a precise correspon-
dence between AO-positive figures and NB-positive figures
(not shown).
Previous studies (Saunders, 1962; Spreij, 1971) did not
resolve whether vital dyes label dying cells per se, or
whether they merely provide an indicator of cellular activity
associated with degeneration, such as phagocytosis (for
example see Savill et al., 1990 and below). Therefore, to
achieve higher resolution of the structures that stain with
vital dyes and compare them to those that stain with tolu-
idine blue after fixation, we analyzed tissue spreads of
embryonic material. Because specific staining with these
vital dyes is compromised after fixation, our analyses were
performed in sequential fashion. Embryos were first stained
in vivo with AO and then spread onto coated glass slides
to dissociate the tissue (see Methods). After photographi-
cally recording representative fields of tissue under fluo-
rescence microscopy, these preparations were then fixed
and stained with toluidine blue using standard histological
procedures (see methods). In this fashion, the cytology of
previously identified AO-positive cells could then be
reassessed by returning to the exact same field of tissue (see
Methods). The vast majority of toluidine-blue-positive cells
(Fig. 4B) were clearly positive for AO fluorescence (Fig.
4A). We also observed a few cells that stained for either
AO or toluidine blue but failed to stain for both. Reasons
Fig. 3. AO-stained cells in vivo
correspond to the position of
pyknotic cells in fixed tissue. (A,C)
Confocal optical sections from head
region of AO-stained embryos and
(B,D) toluidine-stained plastic
sections from the head region of
stage 13/14 embryos. At this stage
of head development, intensely
stained cells are observed in the
clypeolabrum and in the
subepidermal spaces associated
with the the maxillary bud, the
dorsal ridge and the brain. A and B
are comparable sections as are C
and D. Anterior is to the left, dorsal
is up. Note that the in vivo
distribution of brightly stained AO-
positive cells is similar to the
distribution of dark, pyknotic
figures in plastic sections. Scale bar,
20 µm.
34
for the occasional discrepancy between these staining pro-
cedures may include different dye affinity properties for
cells at different stages of degeneration and/or losses of
cells during the procedure. Nevertheless, these sequential
comparisons established an excellent overall correspon-
dence between cells that stained with AO in vivo and
pyknotic cells that stained intensely with toluidine blue after
fixation. Because toluidine-blue-stained figures are clearly
apoptotic when examined by electron microscopy (see Fig.
2), these experiments establish that apoptotic cells in live
Drosophila embryo are stained by the vital dyes AO and
NB.
Vital dyes stain unengulfed and engulfed cell
corpses
Our analyses of plastic sections and electron micrographs
showed that many apoptotic cells were engulfed by
macrophage-like cells. These phagocytes display an activity
that is exceptionally similar to the scavenger-receptor medi-
ated endocytosis found in mammalian macrophages
(Abrams et al., 1992). To compare patterns of vital dye
staining with the distribution of these engulfing cells, we
sought additional markers to visualize these phagocytes. For
this purpose, we used an antibody against protein X, which
labels vesicles and membranes of hemocytes in the
Drosophila embryo. Immunolocalization studies and com-
parisons of cDNA sequences encoding protein X indicate
that this protein is a product of hemocytes with functions
characteristic of macrophages (Nelson, R. E., Fessler, L. I.
and Fessler, J. H., unpublished data). When the distribution
of this immunogen is compared to the AO-staining pattern
of stage 13/14 embryos, a pattern of staining remarkably
similar to the distribution of dying cells can be observed
(Fig. 5, compare A with B). The coincidence between these
staining patterns is particularly well illustrated at the lead-
ing edge of the dorsal epidermis during dorsal closure. This
similarity persists during most embryonic stages with the
notable exception of late stages in central nervous system
development where prominent AO staining occurs in the
absence of co-localizing hemocytes (not shown). Double-
labelling experiments with anti-X antibody and toluidine
blue demonstrate the presence of toluidine-blue-stained
apoptotic bodies in many of the anti-X-positive cells (Fig.
5C-E). Note that only a portion of phagocytic cells that cor-
responds to the apoptotic corpses (see Fig. 2A,B) is stained
by toluidine blue.
It was evident from our studies that AO and NB detect
both free and phagocytosed cell corpses. Close examina-
tion of whole-mount and dissociated embryos stained in
vivo shows small, individual, uniformly fluorescent cells
representing unengulfed apoptotic bodies (small arrow in
Fig. 6B). Free apoptotic corpses are the predominant, if not
exclusive, form of staining in the maturing ventral nerve
cord, as no phagocytosis of dead cells was observed in this
tissue at this stage. Cell corpses that have already become
engulfed can also be observed as discrete, vesicular stain-
ing inside phagocytes (Fig. 6A,C). Time-lapse studies show
that AO staining of dead cell corpses can persist after
engulfment for over 2 hours (data not shown). However,
even in these advanced stages of cell death, the staining of
these vital dyes is restricted to the engulfed cell corpses
inside phagocytes. These experiments demonstrate that AO
and NB, like toluidine blue, do not label macrophages
directly. Phagocytes are only labelled when they contain
one or more engulfed apoptotic corpses which are them-
selves selectively stained by these dyes. Finally, labelled
corpses within macrophages are detected immediately upon
staining of live embryos. Since the staining procedure takes
only a few minutes, the majority, if not all, of the apop-
totic corpses must have been engulfed prior to the AO treat-
ment of embryos. We conclude that AO and NB are capable
of staining apoptotic bodies after their engulfment and
therefore must be able to readily enter and penetrate live
phagocytes. These observations are relevant for the mech-
anism by which these vital dyes stain (see below and dis-
cussion).
Acridine orange staining is specific For apoptotic
cell death
The mechanisms by which AO and NB stain degenerating
cells are not known. In particular, it was not clear whether
these vital dyes are general stains for dead cells, or whether
they are selective for apoptosis. We explored this question
J. M. Abrams and others
Fig. 4. Colocalization of acridine orange and toluidine blue in
dissociated tissue. In order to assess the histogenetic properties of
AO-positive cells, we examined preparations of tissue spreads
before and after fixation (see methods). (A) A field of dissociated
tissue from an AO-stained embryo prior to fixation. (B) The same
field of tissue after fixation and subsequent toluidine blue staining
(B). Arrows indicate several examples of in vivo AO-stained cells
(white arrows) that also stain darkly with toluidine blue after
fixation (black arrows). Scale bar, 75 µm.
35Cell death in the Drosophila embryo
by inducing an alternative form of cell death referred to as
necrosis. Necrosis results from exposure to various exter-
nal injuries, such as oxygen deprivation (hypoxia), abnor-
mal temperature (hypo- and hyperthermia), or certain toxins
(reviewed in Wyllie, 1981; Kerr and Harmon, 1991).
Necrotic deaths are characterized by a general swelling of
the cell, mitochondrial dilation, loss of membrane integrity
and eventual plasma membrane rupture. This mode of death
Fig. 5. Comparison of AO staining with the distribution of phagocytic hemocytes. (A) Dorsal view of a stage 14 embryo immunostained
with anti-X antibody; scale bar, 50 µm. (B) Similarly aged embryo stained with AO; scale as in A, anterior is left. (C-E) High
magnification views of toluidine-blue-stained plastic sections from anti-X-immunostained embryos. Note the presence of pyknotic, darkly
stained material inside large, anti-X-positive cells. Scale bars in C and E, 10 µm.
Fig. 6. Vital dyes stain cell corpses prior to and after engulfment. (A) Confocal image of a macrophage containing multiple AO-positive
corpses in an AO-stained embryo; scale bar is 5 µm. (B) Nomarski optics of dissociated tissue from an AO-stained embryo. At this
magnification, AO-positive material shows orange coloration without UV illumination. Note AO-positive unengulfed cell corpse (small
arrow) and engulfed corpses (large arrow) which are also AO positive. Scale bar is 10 µm. (C) Nomarski optics of dissociated tissue from
a nile-blue-stained embryo showing macrophage with engulfed nile-blue-positive corpses; scale bar, 10 µm.
36
is dramatically distinct from apoptosis. To induce necrosis,
Drosophila embryos were deprived of oxygen for a period
of 4 hours (see methods) and then examined by electron
microscopy. Cells from these embryos exhibit the charac-
teristic features of necrosis including dilated mitochondria
(Fig. 7C). When these embryos were stained with AO and
observed in the green fluorescence channel (see Material
and Methods), stage-specific staining of apoptotic cells was
generally preserved, yet an enhanced level of background
nuclear staining tended to obscure these images. Observa-
tions of AO staining in the red (rhodamine) channel showed
no ectopic AO staining even though essentially every cell
in these embryos suffered necrosis (Fig. 7A). Futhermore,
embryos treated in this fashion retained the AO-staining
pattern reminiscent of the staining observed at the devel-
opmental stage during which necrosis was induced (com-
pare Fig. 7A to Fig. 9E). Similar results were obtained by
staining plastic sections of these embryos with toluidine
blue (Fig. 7B). Necrotic cells showed no significant increase
in their affinity for toluidine blue, but the staining of apop-
totic bodies was preserved.
AO staining in vivo and toluidine staining of plastic sec-
tions thus show parallel properties with respect to necrotic
tissue. Necrotic cells are apparently not recognized by these
dyes under the described conditions. We conclude that pref-
erential staining with these dyes requires biochemical
changes that are specific for apoptotic forms of death.
Analysis of ectopic cell death in embryonic
mutants
We assessed the ability of vital dyes to detect ectopically
induced cell death in mutants that perturb embryonic devel-
opment. Mutations at polyhomeotic cause extensive degen-
eration in the ventral epidermis (Dura et al., 1987; Smouse
and Perrimon, 1990). Embryos mutant for polyhomeotic
exhibited excessive AO staining in this region and else-
where (Fig. 8B). Mutations at crumbs, which cause mas-
sive degeneration of epithelial tissue (Tepass et al., 1990),
also display ectopic AO staining that is widespread through-
out the epidermis (Fig. 8C). Similar observations were
obtained with NB (not shown).
We also induced cell death using X-irradiation which, in
other systems, has been shown to cause protein synthesis-
dependant apoptosis (see, for example, Umansky, 1991;
Tomei, 1991). When 3-4 hour old wild-type embryos were
exposed to a low dose of X-rays (600 rads), no staining
was noted immediately after irradiation. However, after
aging these embryos for 7 hours of physiological time, an
excessive number of AO-positive cells was observed (Fig.
8D). Higher doses of X-irradiation (4000 rads) gave a more
rapid and very noticeable increase of AO-labelled dying
cells that displayed the ultrastructural features of apoptosis.
In fact, AO-stained cells could be induced prior to the
developmental stage at which the onset of cell death first
appears. When 3-4 hour old embryos were irradiated at
4000 rads, aged for various times and then examined for
AO staining, we found that dying cells were readily
observed at least 2 hours earlier than would otherwise nor-
mally occur (see Fig. 8F). The appearance of precocious
dying cells could be suppressed by cycloheximide treat-
ments that immediately followed exposure to X-rays, sug-
J. M. Abrams and others
Fig. 7. Acridine orange does not detect necrotic cell death. (A)
Embryo 10-12.5 hour AEL deprived of oxygen for 4 hours (see
methods) and then stained with AO. This image is a
superimposition of confocal optical sections collected in the red
channel summed over a depth of ~30 µm. Note scattered AO-
positive cells along the midline region; scale bar, 50 µm. (B)
Toluidine-blue-stained plastic section of embryo (10-12 hour
AEL) treated as in A. Arrow indicates macrophage containing at
least one corpse; bar is 10 µm. (C) Electron micrograph from
embryo (10-12 hour AEL) treated as in A. Arrow indicates
swollen mitochondria indicative of necrotic death. Note extensive
blebbing on the plasma membrane of the cell which is left of
center. Several engulfed apoptotic cells are also visible. Inset
shows typical mitochondrial morpholgy in these embryos. Scale
bar, 2 µm, for inset the scale bar, 19 µm.
37Cell death in the Drosophila embryo
gesting that some aspect of irradiation-induced cell death
is apparently dependent upon protein synthesis (Fig. 8E).
These experiments serve to demonstrate the utility of the
vital dyes AO and NB as tools to investigate the effect of
mutations, environmental factors or chemical agents on pro-
grammed cell death in the Drosophila embryo.
Patterns of cell death during embryogenesis
Although the pattern of cell death during embryogenesis in
Drosophila is quite dynamic, the overall distribution of
dying cells is fairly reproducible for any given develop-
mental stage. Prior to and during their ingestion by
macrophages, dying cells are often extruded from a devel-
oping organ or cell layer (for example see Fig. 3). Because
detection of cell death with vital dyes includes these late
stages of degeneration, patterns observed with AO or NB
reflect the position of dying cells as well as the accumula-
tion of corpses in circulating phagocytes. Therefore, our
observations do not necessarily reveal the precise original
position of a dying cell. However, since the pattern of
cell death is very reproducible for a particular develop-
mental stage, it appears that the original position of a
dying cell typically approximates the final position of its
corpse.
Since the following descriptive accounts of cell death are
based on observations of embryos stained with AO and NB,
it is important to keep in mind that these stains detect cell
corpses that may have already fragmented into two or more
apoptotic bodies. It is furthermore evident that one phago-
cyte may contain several stained corpses which might
appear as a single stained structure (see Fig. 6). Finally, the
continual disappearance of AO-stained cells with time
allows only dynamic snapshots of cell death patterns, poten-
tially leading to a significant underestimate of the total
number of cell deaths. For these reasons, it is difficult to
derive an accurate numerical estimate by this method. Nev-
ertheless, to provide an impression of the scope of this
process during embryogenesis, we occasionally cite num-
Fig. 8. Visualization of ectopic cell death by acridine orange and suppression of induced cell death by cycloheximide. Acridine-stained
embryos in A, B and C are of comparable age. (A) Wild-type embryo, stage 13 (~10 hour AEL), (B) polyhomeotic mutant embryo (from
an 8-11 hour AEL collection), (C) crumbs mutant embryo (from an 8-13 hour AEL collection), (D) X-irradiated embryo exposed to 600
rads at 3-4 hours AEL and then aged 14 hours at 18°C (see Methods). Embryos in E and F were exposed to 4000 rads X-irradiation and
then aged 1.5 hours. Embryos in E were treated with cycloheximide immediately after irradiation whereas those in F received no such
treatment (see Materials and Methods). Note that cycloheximide suppresses the appearance of acridine-positive cells. The brightly stained
object in the lower right corner in E is an unfertilized egg which has considerable background fluorescence due to its high yolk content.
Anterior is left, dorsal is up. Scale bars, 50 µm.
38
bers of AO-stained figures in the following descriptions.
Because phagocytic cells do not circulate within the cen-
tral nervous system, our quantitation of AO-stained figures
in the ventral nerve cord are likely to represent a fairly
accurate, stage-specific estimate of the number of cell
deaths in this tissue.
Stage 11
We were unable to detect any signs of cell death until ~ 7
hours after egg laying (AEL) corresponding to the later part
of the fully extended germ band stage. The first dying cells
are invariably observed in the dorsal region of the head just
anterior to the extended tip of the germ band. Apoptotic
cells are also observed inside the epidermal cell layer of
the gnathal segments and near the caudal tip of the extended
germ band.
Stage 12
As the germ band retracts, stage 12, cell death becomes
more widespread and prominent. During early germ band
retraction, dying cells accumulate just beneath the devel-
oping epithelium of the gnathal segments, throughout the
procephalic lobe region and within the interstitial space of
the clypeolabrum (Fig. 9A). As the retracting germ band
reaches 50% egg length, cell death becomes more promi-
nent within the most posterior abdominal segments (Fig.
9A) and early signs of degeneration along the ventral mid-
line can be observed within the most anterior thoracic seg-
ments. Cell death in the dorsal cephalic region, just beneath
the dorsal ridge, also becomes very prominent (as in Fig.
9C). Scattered cell deaths also begin to appear in a seg-
mentally reiterated pattern within the lateral portions of the
ventral region (Fig. 9B). Toward the completion of germ
band retraction, cell death is very conspicuous in the ven-
J. M. Abrams and others
Fig. 9. Patterns of cell death during embryogenesis. AO-stained embryos viewed by graphic superimpositions of confocal optical sections
(A-C) or fluorescence microscopy (D, double exposure of Nomarski and red channel, E, and F, green channel). Anterior is left. Note that
yolk material throughout the center of the embryos gives diffuse, non-specific staining which is particularly prominent during earlier
developmental stages. (A) Stage 12, lateral view. At this early stage, dying cells are found in the gnathal segments, the clypeolabrum and
near the caudal tip of the retracting germ band. (B) Late stage 12, ventral view. Cell death spreads from both cephalic and caudal
extremeties along the ventral midline axis. Prominent cell death in the ventral cephalic region is also found. (C) Stage 13, lateral view.
Dying cells accumulate around the cephalic ganglia and beneath the dorsal ridge. Scattered deaths occur throughout the lateral epidermis.
(D) Stage 13, ventral view. Segmentally reiterated deaths are found in the ventrolateral epidermis. (E) Stage 13/14, ventral view. One
central and two lateral columns of cell deaths are apparent in the ventral portions of the embryo. (F) Late stage 16, lateral view. Note that
prominent numbers of cells die in the central nervous system. Scale bar, 50 µm.
39Cell death in the Drosophila embryo
tral neurogenic region. It is interesting to note that the onset
of cell death in this area does not occur simultaneously in
all segments. Prominent numbers of dying cells first appear
along the ventral midline in the thoracic and posterior
abdominal segments yet they are nearly absent from mid-
abdominal segments (Fig. 9B). Cell death within the mid-
abdominal segments occurs on a slightly later schedule and,
as germ band retraction proceeds, the waves of cell death
along the midline eventually converge to form one contin-
uous line (as in Fig. 9D).
Stage 13
With the exception of the central nervous system, all major
zones of degeneration have been fully established by the
completion of germ band retraction. By this stage (~ 9.5-
10.5 hours AEL), cell death in the dorsal portion of the
head becomes very prominent, as marked numbers of
corpses accumulate around the supraoesophageal ganglia
and beneath the dorsal ridge (Fig. 9C). Noticeable accu-
mulation of corpses has also occurred within the clypeo-
labrum and just anterior of the salivary duct.
As this stage progresses, scattered and variable numbers
of cell deaths are evident throughout the epidermis of the
embryo (Fig. 9C). Within the dorsolateral portion of each
hemisegment, for example, 15 to 50 AO-stained figures are
typically observed at any given point during this stage. Seg-
mentally reiterated AO staining in the ventrolateral portions
of the epidermis is also very prominent (Fig. 9D). Clusters
of AO-positive cells accumulate along the midline (Fig.
9B,D), which are clearly associated with phagocytic
macrophages (not shown). Lateral to the midline, up to 30
AO-positive figures appear at a slightly later point in stage
13 and are scattered throughout the most ventral portion of
each hemisegment (compare Fig. 9D with B). The vast
majority of dying cells in this region accumulate in the
interstitial spaces between the ventral epidermis and the
nerve cord. We have thus far been unable to determine
whether these corpses originated from cells that were com-
mitted to neural or epidermal fates. By the end of this stage,
the pattern of cell death in the ventrolateral region gradu-
ally evolves into one central and two lateral columns of
macrophage associated staining along this portion of the
embryo (Fig. 9E). The position of cells in the central, mid-
line column is fairly consistent among the segments
whereas the lateral columns, although also segmented in
character, tend to show more variably positioned cell death
figures.
Stage 14
The AO- and NB-staining cells generally tend to persist
from the previous stages, especially along the ventral mid-
line and in the head region. As this stage progresses (~10.5-
11.5 hours AEL), a new and continuous ring of dying cells
becomes evident at the leading edge of the dorsally clos-
ing tissue during gut closure (see Fig. 5B). At this stage,
degenerating cells are rapidly phagocytosed by neighbor-
ing macrophages (see Fig. 5).
Stage 15
Once dorsal closure is complete (~13 hours AEL), the gen-
eral domains of vital dye staining from earlier stages fade
and sporadic cell deaths occur throughout the body cavity
(not shown). Many of these dying cells are localized just
inside of the body wall or around the mid-gut. The appear-
ance and position of this staining pattern suggests an asso-
ciation with phagocytic macrophages. Toward the end of
this stage, cell death begins to occur in the condensing cen-
tral nervous system.
Stage 16
As the nerve cord condenses (~14 hours AEL), large num-
bers of cell deaths can be observed with vital dyes through-
out the central nervous system (note staining within the
brain and the ventral nerve cord in Fig. 9F). At this stage,
neuromuscular development matures to the extent that
twitching movements can be observed. Phagocytic hemo-
cytes do not invade the tightly packed cell body layer of
the central nervous system (CNS) and, hence, unlike cell
deaths in other regions, no engulfment by circulating
macrophages occurs in this region. The pattern of vital dye
staining in the CNS should therefore most accurately reflect
the precise position and numbers of apoptotic cells. We ana-
lyzed AO staining within the ventral nerve cord by super-
imposing optical sections that extend through this portion
of the CNS. These analyses result in a graphic summation
of cell death through the entire depth of the ventral cord
(Fig. 10A). Using this technique in combination with time-
lapse preparations (see Methods), we have followed many
Fig. 10. Cell death in the embryonic central nervous system. Stage
16 embryos were stained with AO and the ventral nerve cord was
viewed by graphic summation of confocal optical sections (A) or
fluorescence microscopy (B). Close examination of AO staining
on either side of the midline axis reveals symmetric and
asymmetric patterns of cell death in this tissue. Examples of
symmetric deaths are denoted by full arrow pairs and several
examples of asymetric cell deaths are denoted by arrowheads.
Anterior is left. Scale bars, 50 µm.
40
AO-positive cells in the CNS for up to 45 minutes. Fig.
10A shows one such example of cell death in a condensed
ventral nerve cord where most of the AO-stained cells are
positioned at the anterior and posterior termini. Approxi-
mately 140 AO-positive cells were detected at this stage.
At a slightly earlier stage, cell death is more uniformly dis-
tributed over the length of the ventral cord and appears
segmentally reiterated (Fig. 10B). A similar pattern of
degeneration has been reported during late embryogenesis
in the CNS of Calliphora (Starre-van der Molen and Otten,
1974).
Comparative analyses of AO staining on either side of
the midline reveals an overall symmetry in the pattern of
cell deaths in the condensing ventral nerve cord (Fig.
10A,B). Although the precise number and position of AO-
stained cells may vary, cell death on one side of the ven-
tral midline is often accompanied by a similarly positioned
dying cell(s) on the opposite side of the midline. There are,
however, clear instances of asymmetric cell deaths in the
nervous system as well. These asymmetries are more read-
ily observed at later stages of ventral nerve cord matura-
tion when neural cell death is somewhat less prominent. As
shown in Fig. 10A, AO-positive cells are not always accom-
panied by a similarly positioned dying cell(s) on the oppo-
site side of the midline. Because AO staining can persist
for relatively long periods in these preparations (at least 45
minutes), sporadic variances of a temporal nature are
unlikely to be the entire cause for asymmetric staining. We
suggest that asymmetric cell deaths in the nerve cord may
reflect some degree of plasticity in the control of cell death
during embryonic CNS development (see Discussion).
Summary of cell death during Drosophila
embryogenesis
The earliest appearance of cell death is observed in the
dorsal cephalic region, within the gnathal segments and in
the clypeolabrum as the germ band begins to retract (stage
11). Thereafter, as germ band retraction proceeds (stages
12 and 13), cell death becomes widespread throughout the
embryo, particularly in the ventrolateral portions and
around the procephalic lobes. Large numbers of degenerat-
ing cells accumulate in the interstitial spaces beneath the
dorsal ridge, along the ventral midline and within the
gnathal segments. During dorsal closure, a zone of degen-
erating cells, organized in the shape of a ring, forms around
the closing dorsal tissue (stage 14). As head involution
becomes well advanced (stage 15), zones of vital dye stain-
ing from earlier stages subside and scattered subepidermal
staining appears throughout the embryo. Eventually, promi-
nent cell death appears throughout the CNS as the ventral
nerve cord condenses (stage 16). In contrast to earlier
stages, cell death in the cell body layer of the ventral cord
and brain hemispheres at this time is not associated with
phagocytic macrophages.
DISCUSSION
The deliberate and orderly removal of cells by naturally
occurring cell death is an integral part of animal develop-
ment. Despite the importance of this regressive process
during normal development and in pathological situations,
much remains to be learned about the underlying molecu-
lar mechanisms. We believe that dramatic progress can be
made in this area by taking advantage of the powerful
genetic and molecular techniques available in Drosophila.
In this paper, we examined the general features and distri-
bution of programmed cell death in wild-type embryos to
establish the foundation for further molecular genetic
studies.
The results from our ultrastructural analysis indicate that
cell death in the Drosophila embryo occurs by apoptosis.
We found that embryonic cell deaths involve separation
from neighboring cells, nuclear condensation, cytoplasmic
shrinkage, fragmentation and engulfment by circulating
macrophages. These cytological changes closely parallel the
features of apoptotic cell death described in other systems
(for examples see Wyllie, 1981; Kerr and Harmon, 1991).
Degenerate ovarian chambers in adult Drosophila females
also show ultrastructural features that resemble apoptotic
cell death (Giorgi and Deri, 1976). Similarly, the eye disc
of wild-type or mutant larvae contain cells that appear apop-
totic (Fristrom, 1969, Wolff and Ready, 1991). However,
Wolff and Ready (1991) noted an absence of marginal con-
densation from the nuclear membrane during cell death in
the eye disc and also reported that, subsequent to cellular
fragmentation, the nuclei within subcellular bodies can
assume necrotic-like features. Because our ultrastructural
studies show little indication of necrosis in embryonic
tissue, we suspect that variations in the final mode of struc-
tural collapse may arise from tissue-specific differences
(Clarke, 1990).
In order to visualize cell death rapidly and reliably in the
Drosophila embryo, we adopted vital dye staining tech-
niques that were originally developed for the analysis of
imaginal tissues (Spreij, 1971). We find that acridine orange
(AO) and nile blue (NB) stain individual apoptotic cells,
both prior to and after engulfment, but not macrophages per
se. Because of the ease and speed of these assays, it should
be possible to study the effect of rather large numbers of
mutations or chemical compounds on apoptosis. The mode
by which these dyes preferentially stain dead cells is not
known, although several possible mechanisms, including
increased membrane permeability, seem plausible. How-
ever, we find that apoptotic corpses inside macrophages are
clearly labelled. Therefore, these dyes must be able to enter
and penetrate live cells readily. Furthermore, cells under-
going necrotic death induced by hypoxia were not stained
by AO (as visualized in the red channel). These observa-
tions indicate that AO can be used to visualize selectively
apoptotic forms of death, and that selective staining by AO
is not merely a passive consequence resulting from com-
promised membrane permeability (for a review of the cyto-
chemical properties of AO, see Kasten, 1967). Likewise,
toluidine blue staining of fixed tissue sections resulted in
highly specific labelling of apoptotic forms of death, leav-
ing necrotic cells completely unstained. We conclude that
biochemical changes characteristic for apoptotic forms of
death are responsible for the selective affinity to AO and
toluidine blue.
Many corpses resulting from programmed cell death are
avidly engulfed by circulating macrophage-like cells in the
J. M. Abrams and others
41Cell death in the Drosophila embryo
Drosophila embryo (see also Campos-Ortega and Harten-
stein, 1985). The characteristic phagocytic activity associ-
ated with these cells is further emphasized by the observa-
tion that they express an endocytic receptor activity
(Abrams et al., 1992) which is remarkably similar to the
scavenger receptor activity found in mammalian
macrophages (Goldstein et al., 1979, Kodama et al., 1990).
In addition, circulating phagocytes synthesize a number of
basement membrane components during embryogenesis
(Fessler and Fessler, 1989). Macrophage-like cells are thus
involved with multiple histogenetic functions during this
stage of Drosophila development.
We used vital dye staining to catalogue the pattern of
cell death throughout embryogenesis in Drosophila.
Although the distribution of dying cells changes dramati-
cally during development, the pattern of cell death for any
given developmental stage was remarkably reproducible.
Therefore, the induction of these deaths appears to be
tightly controlled and must result from ‘natural causes’. It
is apparent from our studies that a large number of cells
die at many different times and in many different tissues
and regions of the embryo. Our methods of imaging cell
death, however, do not readily allow for a precise census
of the number of cell deaths in most contexts. There are
several limitations to the use of AO and NB as a means to
count cell deaths. First, dying cells can fragment into mul-
tiple AO-stained apoptotic bodies. Second, most of the
dying cells are rapidly engulfed by phagocytes. A single
phagocyte usually contains multiple cell corpses, which are
labelled by AO and NB, but may appear as a single stained
structure. Finally, the continual loss of cells that were
‘pulse-stained’ over time means that these methods provide
only a static snap-shot image of a very dynamic process.
Nevertheless, for reasons discussed below, we feel that a
reasonable numerical assessment of cell deaths can be made
in the ventral nerve cord. Assuming that there are approx-
imately 300 cells in each segment at this stage (Poulson,
1950; Truman and Bate, 1988), we estimate that at least
4% of this neural population undergoes programmed cell
death. Because our counts derive from static rather than
cumulative images, we feel that this represents a very con-
servative estimate.
Even though bilateral symmetry of cellular age and iden-
tity is generally well preserved throughout the Drosophila
central nervous system (for example Campos-Ortega and
Hartenstein, 1985; Doe et al., 1988; Klämbt et al., 1991),
clear asymmetries in the exact number and position of cell
deaths on either side of the ventral cord midline were found.
These differences are significant because they are not easily
explained either by subtle temporal variances, or by relo-
cation of dead cells upon phagocytosis in migratory cells.
First, time-lapse studies demonstrate that apoptotic cells in
the CNS retain AO staining for at least 45 minutes, argu-
ing strongly against small sporadic temporal differences as
a potential explanation for our observations. Second, based
on electron microscopy and Anti-X staining, we know that
circulating macrophages do not have access to degenerat-
ing cells within the CNS cell body layer. In this context, a
dying cell is unlikely to wander far from its original posi-
tion. Therefore, it appears that the onset of cell deaths in
the CNS is at least somewhat variable. Although we cannot
entirely rule out positional or temporal variances as expla-
nations for the asymmetries in the pattern of cell deaths, it
is possible that some apoptotic deaths are not strictly pre-
determined during the later stages of CNS development in
the Drosophila embryo. Plasticity in the control of cell
deaths has been well documented during postembryonic
development (e.g. Kimura and Truman, 1990, Fischbach
and Technau, 1984, Steller et al., 1987, Wolff and Ready,
1990; Campos et al., 1992). Similar interactive processes
may regulate cell survival during embryogenesis. Many
mutants affecting embryonic (see, for example Dura et al.,
1987; Magrassi and Lawrence,1988; Tepass et al., 1990) or
imaginal development (Fristrom, 1969; James and Bryant,
1981; Bonini et al., 1990) show ectopic death. Moreover,
cell death in ftz mutants is not restricted to cells that would
normally express this gene product (Magrassi and
Lawrence, 1988). Hence, as is the case for many organ-
isms, Drosophila also displays a capacity to eliminate cells
that do not successfully complete their developmental pro-
gram.
In a variety contexts, it has been shown that cell death
is an active process which can be blocked or delayed by
inhibitors of protein synthesis (Tata, 1966; Lockshin, 1969;
Farbach and Truman, 1988; Martin et al., 1988; Oppenheim
et al., 1990). We explored this issue in the Drosophila
embryo with the use of cycloheximide. Although very early
treatments with this agent prevented the appearance of AO-
stained cells, it was also clear that these treatments had
imposed such general and widespread effects upon devel-
opment that a meaningful assessment of the result was
impossible. In contrast, when cycloheximide was intro-
duced at a later stage, shortly before the first onset of cell
deaths, their occurrence was not affected. However, we did
find that X-irradiation-induced cell deaths could be sup-
pressed by cycloheximide treatment. These precocious cell
deaths apparently depend upon some aspect of de novo pro-
tein synthesis. More direct evidence for the active nature
of cell death in Drosophila comes from the identification
of mutations that block all embryonic cell deaths (White,
K., Abrams, J. M., Grether, M., Young, L. and Steller, H.,
unpublished data, see below).
The work described in this paper provides the basis for
the isolation of cell death defective mutants in Drosophila.
Genes required for distinct steps in a cell death pathway
(eg. specification, execution, engulfment and degradation)
have already been identified in the nematode C. elegans
(reviewed in Ellis et al., 1991). Comparative analyses of
these loci with genetic components identified in Drosophila
may elucidate some aspects of the cell death pathway that
have been conserved between these evolutionarily disparate
organisms. In contrast to the situation in C. elegans (Sul-
ston and Horvitz, 1977; Ellis et al., 1991), at least some
programmed cell in Drosophila are not strictly predeter-
mined by lineage. For example, some postembryonic cell
deaths require hormone induction (Kimura and Truman,
1990) and the survival of certain cells in the visual system
depends on cellular interactions (see for example Fischbach
and Technau, 1984; Wolff and Ready, 1990, Campos et al.,
1992). This plasticity offers a unique opportunity to study
how cell interactions may lead to the specification of the
cell death fate.
42
We have recently completed a screen for cell death defec-
tive mutations and have identified one locus that is required
for virtually all programmed cell deaths that occur during
Drosophila embryogenesis (White, K., Abrams, J. M.,
Grether, M., Young, L. and Steller, H., unpublished data).
We expect that the biochemical characterization of this
locus and further genetic studies in Drosophila will con-
tribute significantly to a better understanding of the mole-
cular mechanisms underlying programmed cell death.
We are grateful to Dave Smith for help with the confocal micro-
scope and we thank Megan Grether and Lynn Young for com-
ments on the manuscript and other valuable contributions. This
work was supported in part by a Pew Scholars Award to H. S.
J. M. A. is an American Cancer Society postdoctoral fellow. K.
W. is a postdoctoral associate, and H. S. is an Assistant Investi-
gator with the Howard Hughes Medical Institute. L. I. F. was sup-
ported by U. S. Public Health Service grant AG02128.
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(Accepted 23 September 1992)
