Department of Biological Sciences and Science and Technology Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA
* Author of correspondence (e-mail: minden{at}cmu.edu)
Accepted 20 August 2003
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SUMMARY |
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Key words: Drosophila, Embryo, Engulfment, Apoptosis, H99, p35, Phagocytosis
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Introduction |
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A number of Drosophila genes necessary for cell death have been
identified (Abrams, 1999;
Lee and Baehrecke, 2000
;
Richardson and Kumar, 2002
).
Drosophila cells carry a full complement of caspases that are
maintained in an inactive state by Drosophila inhibitor of apoptosis
1, DIAP1 (Hay, 2000
). A trio
of well-characterized cell death genes are known to inhibit DIAP1 and allow
for caspase activation and cell death
(White et al., 1994
). These
genes, reaper (rpr), head involution defective
(hid) and grim lie in close proximity to one another on the
third chromosome in a region spanned by the H99 deletion. Embryos
that are homozygous for the H99 deletion lack cell death and exhibit
several developmental abnormalities, including hyperplasia of the central
nervous system, defects in head involution and delayed germband retraction
(White et al., 1994
;
Pazdera et al., 1998
;
Vucic et al., 1998
;
Wang et al., 1999
;
Goyal et al., 2000
;
Holley et al., 2002
;
Yoo et al., 2002
). Ectopic
expression of these genes, alone or in combination, causes apoptosis in a
tissue dependent fashion (Chen et al.,
1998
; Quinn et al.,
2000
).
Several other regulators of cell death are known in Drosophila
that also have homologues in C. elegans and mammalian cells. DARK,
which is homologous to CED-4 and APAF-1 in C. elegans and mammalian
cells, respectively, is an apoptosis adaptor protein that functions downstream
of RPR, HID and GRIM (Rodriguez et al.,
1999). DARK initiates caspase function by binding to the class I
caspases, DRONC and DREDD (Rodriguez et
al., 1999
; Kanuka et al.,
1999
). dark mutant embryos show reduced levels of cell
death and hyperplasia in the central nervous system
(Rodriguez et al., 1999
;
Kanuka et al., 1999
).
debcl, the Drosophila homologue of ced-9 and a
member of the Bcl2/Bax family, causes increased apoptosis when
overexpressed and reduced cell death when inhibited by RNA interference
(Colussi et al., 2000
;
Igaki et al., 2000
;
Zhang et al., 2000
;
Chen and Abrams, 2000
).
One of the most challenging aspects of studying apoptosis is developing
assays to measure the various cellular events that occur during cell death.
The majority of our knowledge about apoptosis is derived from genetic analysis
of C. elegans and from mammalian tissue culture systems. Time-lapse
analysis of C. elegans mutants has uncovered a long list of genes
required for cell death and phagocytosis. This time-lapse analysis relies on
transmitted light microscopy to observe cellular changes. Tissue culture
systems have revealed many of the biochemical details about the signals
leading to caspase activation and the caspase activation cascade itself. In
addition, the tissue culture methods use transmitted-light microscopy and
assays for nuclear decay, such as TUNEL and Acridine Orange (AO) staining. A
very useful marker for plasma membrane changes during apoptosis is the
flipping of phosphatidylserine from the inner leaflet to the outer surface of
the membrane (Martin et al.,
1995). Unfortunately, most of these methods are not suitable for
studying apoptosis in living insects or mammals.
Cell death is a highly dynamic and patterned process. To monitor cell death
in living Drosophila embryos, we have used time-lapse microscopy of
AO-injected embryos (Pazdera et al.,
1998). This revealed that apoptosis in the abdominal epidermis is
patterned with a majority of cells dying adjacent to the segment boundaries.
Removal of apoptotic cells by macrophages and neighboring cells occurs in
approximately 40 minutes of the first appearance of an AO signal.
In C. elegans, the pattern of cell death is invariant. In organisms that employ regulative development, such as Drosophila and mammals, cell death is a more stochastic process. In these organisms, most tissues are formed from an excess of cells. Apoptosis is required to remove these excess cells after pattern formation. Thus, it is likely that there will be additional factors involved in cell death in regulative organisms. One obvious distinction is that in C. elegans dying cells are engulfed by their immediate neighbors; whereas in Drosophila and mammals, apoptotic cells are engulfed by macrophages, as well as their neighbors. Thus, there are probably different engulfment signals.
Genetic analysis of cell death in C. elegans has revealed seven
genes that are required for cell engulfment
(Ellis et al., 1991). These
seven genes can be grouped into two pathways: ced-1, ced-6, ced-7 and
ced-2, ced-5, ced-10, ced-12
(Ellis et al., 1991
;
Gumienny et al., 2001
). Six of
these genes are required exclusively in the engulfing cells. Only
ced-7, which is homologous to human ABC1 (ABCA1
Human Gene Nomenclature Database) is required in both the dying cells
and the engulfing cells. There are no known genes required for engulfment of a
dying cell that are exclusively expressed in the dying cell.
Very little is known about engulfment in Drosophila. Drosophila
homologues of the C. elegans engulfment genes have been identified:
draper ced-1, dcrk
ced-2, myoblast city
ced-5 (Wu and Horvitz,
1998
; Nolan et al.,
1998
) and rac1
ced-10
(Galletta et al., 1999
;
Reddien and Horvitz, 2000
;
Hakeda-Suzuki et al., 2002
).
Because an in vivo assay for phagocytosis in Drosophila embryos has
not been established, the role of these genes in cell engulfment is not known.
croquemort (crq), which is homologous to mammalian
CD36, encodes a macrophage receptor for dying cells
(Franc et al., 1996
;
Franc et al., 1999
). Ectopic
expression of crq is sufficient to confer phagocytic ability on
non-phagocytic cells (Franc et al.,
1996
). The ligand for crq has not been identified.
To begin investigating phagocytosis in living Drosophila embryos, we developed a novel engulfment assay based on a fluorogenic ß-galactosidase substrate, which we call VGAL. VGAL is non-fluorescent in the cytoplasm of living cells, but becomes fluorescent when a cell is phagocytosed by a neighbor or macrophage. The pattern of engulfment closely paralleled the cell death pattern as indicated by Acridine Orange fluorescence. Thus, we have developed a reliable in vivo assay for studying phagocytosis in living embryos. Surprisingly, the pattern of cell engulfment persisted in embryos that were deficient for hid, rpr, and grim. Likewise, the engulfment pattern persisted in embryos that globally expressed the pan-caspase inhibitor, p35. These results indicate that there is a significant caspase-independent, cell engulfment pathway operating in Drosophila embryos.
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Materials and methods |
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Embryo preparation
Embryos were collected at stage 3 and prepared for time-lapse imaging and
photoactivation as previously described
(Minden et al., 2000).
Briefly, embryos were collected on apple juice agar plates, dechorionated in
50% bleach for 90 seconds, then rinsed in egg-wash solution (0.12 M NaCl and
0.04% Triton X-100). Selected embryos were aligned on an agar block, placed
onto a glue-coated coverslip and oriented with watchmaker's forceps so that
the region to be imaged or photoactivated was flush with the coverslip.
Embryos were staged by morphology according to Campus-Ortega and Hartenstein
(Campus-Ortega and Hartenstein,
1985
).
Time-lapse microscopy
Time-lapse microscopy was performed using a Delta Vision microscope system
controlled by softWoRx software (Applied Precision, Issaquah, WA) configured
around an Olympus IX70 inverted microscope. Dying cells were visualized by
injection of Acridine Orange (0.25-0.5 mg/ml) into syncytial embryos and
imaged with a fluorescein filter set. Cellular engulfment was visualized by
injection of an in vivo ß-galactosidase substrate,
resorufin-ß-galactoside-polyethylene glycol1,900 (referred to
as VGAL), at 1.8 mg/ml and imaged with a rhodamine filter set
(Minden, 1996). Segment
boundaries were observed with transmitted light.
Photoactivated gene expression
Photoactivated gene expression was done as previously described
(Cambridge et al., 1997;
Minden et al., 2000
). Briefly,
GAL4VP16 protein was purified and caged with nitroveratryl chloroformate.
Caged GAL4VP16, at 115 µg/ml, was injected into syncytial embryos.
Photoactivation was performed on an IX70 Olympus microscope using a DAPI
bandpass filter to generate a beam of 365 nm light. The beam diameter was
adjusted using different sized pinholes inserted into the conjugate image
plane on one side of a dual-beam, epi-fluorescence illuminator. After
photoactivation, the embryos were imaged by time-lapse microscopy.
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Results |
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The final step in apoptosis is the engulfment of the cell corpse. Although
there is a great deal known about apoptotic cell engulfment in C.
elegans, there is very little known about this process in
Drosophila. Our goal was to establish an in vivo engulfment assay
using a fluorogenic, membrane-impermeant ß-galactosidase substrate,
resorufin-ß-galactoside-polyethylene glycol1,900 (referred to
as VGAL) (Minden, 1996). Two
key features of VGAL are: the ß-galactoside-quenched resorufin
fluorophore, which develops a red fluorescence when cleaved by
ß-galactosidase, and the long hydrophilic, polyethylene glycol tail that
prevents the compound from crossing lipid membranes or gap junctions.
Drosophila embryos have an endogenous, lysosomal ß-galactosidase
activity (MacIntyre, 1974
;
Fuerst et al., 1987
).
Injecting VGAL into the cytoplasm of syncytial-stage embryos loads it into all
cells of the embryo where it is maintained in a non-fluorescent state. We
reasoned that when a cell is engulfed, the cytoplasmic VGAL of the engulfed
cell would be mixed with the ß-galactosidase-containing, lysosomal
compartment of the engulfing cell. This would lead to the cleavage of VGAL and
cause the lysosomes of the engulfing cell to fluoresce red. To demonstrate
this effect, syncytial-stage embryos were co-injected with AO and VGAL. Prior
to the start of germband retraction, at late stage 11, there was no
fluorescence from either fluorophore, aside from background yolk
autofluorescence. Cell death first appears after the start of germband
retraction as evidenced by the green fluorescence of AO-positive nuclei in the
head and abdominal epidermis. Soon after the first AO-positive nuclei appear,
the dying cells are engulfed by migrating macrophages. This is particularly
evident in the head and tip of the retracting germband. After a lag of about
15 minutes, one can see red fluorescent bodies within the migrating
macrophages (Fig. 1A-C). These
VGAL-positive bodies ranged in size from 1-3 µm. The AO-positive and
VGAL-positive vacuoles were mostly non-overlapping, indicating that the
macrophages engulf cells in a piecemeal fashion. This has been observed in
time-lapse recordings where several macrophages appeared to participate in
engulfing a single dying cell (data not shown).
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To demonstrate that the large, motile cells that contained the AO- and
VGAL-positive vacuoles were indeed macrophages, we used the photoactivated
gene expression system (Cambridge et al.,
1997) to activate UAS-nGFP expression selectively in
macrophage progenitors. The photoactivated gene expression system relies on
the injection of a caged form of the transcriptional activator, GAL4VP16 into
syncytial embryos. Expression of a UAS-transgene is activated by briefly
irradiating the cells of interest with a long-wavelength, UV microbeam,
causing the uncaging of the GAL4VP16 protein.
Macrophages originate from a small patch of ventral mesoderm cells just
anterior to the cephalic furrow (Tepass et
al., 1994). Syncytial UAS-nGFP embryos were co-injected
with caged-GAL4VP16 and VGAL and a patch of five to eight cells in this region
was photoactivated at the start of gastrulation, causing them to express
nuclear-localized GFP. The resulting nGFP-positive macrophages migrated
throughout the embryo along their characteristic pathways. Some of these
marked macrophages contained two GFP bodies
(Fig. 1G). One of these is
mostly likely the functional nucleus, while the other object is an engulfed
nucleus derived from another photoactivated cell. The size of the
photoactivation beam did not limit the irradiated area to macrophage
precursors only. Over time, these marked macrophages accumulated
red-fluorescent, VGAL-positive vacuoles. All photoactivated embryos with
nGFP-positive macrophages contained VGAL-positive vacuoles of the same size
range as observed in the AO-VGAL-injected embryos (9/9 embryos,
Fig. 1G-I).
To demonstrate that dying cells elicit both AO and VGAL responses, the photoactivated gene expression system was used to induce a patch of apoptotic cells. UAS-rpr, UAS-hid/TM3 embryos were co-injected with caged GAL4VP16, AO and VGAL. A patch of five to eight cells in the abdominal epidermis was photoactivated at the start of gastrulation (4 hours prior to the first apoptotic events) and analyzed by time-lapse microscopy (Fig. 1J-O). The first AO-positive, ectopic cell deaths appeared near the end of full germband elongation just prior to the start of germband retraction (Fig. 1J). Normally, endogenous cell death begins at the start of germband retraction. Ectopic VGAL-positive vacuoles were observed within 15 minutes of the AO-positive nuclei (8/8 embryos containing ectopic AO-positive nuclei, Fig. 1K). This time-lapse series shows a number of AO- and VGAL-positive spots persisting over the following 80 minutes (Fig. 1L-O). The duration of the AO signal was longer in these experiments than previously reported because the experiments were performed at 18°C, rather than 22°C, which slows development by about half. This characterization of VGAL indicates that it is a reliable and sensitive in vivo marker for apoptotic cell engulfment.
In vivo mapping of engulfment in wild-type embryos
Previous studies showed that cell death in the abdominal epidermis is
patterned (Pazdera et al.,
1998). Cell death begins at late stage 11, during the initiation
of germband retraction, and continues through development to hatching.
Time-lapse recordings of AO-injected embryos revealed that cell death occurs
adjacent to the segment boundaries in three clusters along the dorsoventral
axis. These initial studies also showed that dying epidermal cells in the
abdomen are preferentially engulfed by their epithelial neighbors, not
macrophages.
To explore the relationship between dying cells and their engulfment, we used time-lapse microscopy of embryos injected with AO and VGAL to map cell death and engulfment in the lateral abdominal epidermis. As was the case with macrophages, AO-positive nuclei generally appeared in the abdominal epidermis slightly before VGAL-positive vacuoles, with a lag of about 15 minutes. In addition, like the macrophages, the VGAL spots did not exactly overlap with the AO spots. However, the overall segmentally repeated, striped distribution of AO- and VGAL-spots was very similar. Fig. 2 shows selected images from a time-lapse recording of a wild-type embryo injected with AO and VGAL at 1/2, 3/4 and full germband retraction. These results confirm that dead epidermal cells are engulfed by their neighbors and that the VGAL pattern of engulfment mimics the AO pattern of apoptosis.
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The median number of VGAL spots per segment was 35±11. This is more
than twice the number of dying cells, which is 12-16 cells per segment between
stage 12-14 (Pazdera et al.,
1998). Following the fate of AO-positive nuclei showed that 72%
were fragmented into 2-7 pieces. This further demonstrates that dying cells
are engulfed in multiple pieces.
Cell engulfment persists in cell-death-deficient embryos
To explore the relationship between AO-positive cell death and engulfment,
we examined VGAL signaling in cell-death-deficient embryos. In
Drosophila, apoptosis is controlled by a set of three closely linked
genes: hid, rpr and grim
(White et al., 1994). Embryos
that are homozygous deficient for these three genes, which is covered by the
H99 deficiency, have very few AO-positive nuclei or engulfing
macrophages (White et al.,
1994
). Preliminary experiments showed that homozygous H99
embryos, that lacked AO-positive nuclei and macrophages, also lacked
VGAL-positive macrophages. Time-lapse recordings of H99 embryos
injected with VGAL alone were made. Embryos that lacked VGAL-positive
macrophages were scored as homozygous for the H99 deficiency, which
accounted for the expected one quarter of all embryos examined. These embryos
also displayed head involution and germband retraction defects. Surprisingly,
all H99 homozygous embryos developed the usual striped pattern of
VGAL-positive spots in the epidermis (Fig.
3D-F). The temporal appearance of the VGAL signal was the same as
wild-type embryos. There was no significant difference between the VGAL signal
in wild-type and H99 embryos with respect to the number or size of
the VGAL spots.
To test if the engulfment pattern persisted in other cell-death-deficient
paradigms, the photoactivated gene expression system was used to globally
express the pan-caspase inhibitor, p35
(Hay et al., 1994).
UAS-p35 embryos were injected with caged GAL4VP16, AO and VGAL.
Ninety percent of the irradiated embryos had fewer than 12 AO-positive nuclei
per embryo, indicating that whole embryo irradiation globally activated p35
gene expression. To ensure that only segments expressing p35 were analyzed,
UAS-p35; UAS-cGFP embryos were injected with caged GAL4VP16 and VGAL,
then photoactivated over the entire embryo. The number of VGAL spots was
counted in segments expressing GFP. All of these embryos continued to show
segmentally repeated stripes of VGAL fluorescence
(Fig. 3G-I). These
photoactivated UAS-p35 embryos had more VGAL-positive macrophages
than did homozygous H99 embryos, but the number of macrophages was
significantly less than wild type. Most of these macrophages were restricted
to the head region where the concentration of p35 was reduced due to limited
diffusion of the caged GAL4VP16. Similar to homozygous H99 embryos,
global expression of p35 caused germband retraction defects in about half of
the embryos.
Quantitation of the distribution and number of VGAL spots showed very similar values for wild-type, homozygous H99 and p35-expressing embryos (the number for H99 embryos was 29 segments in five embryos; the number for photoactivated UAS-p35 embryos was 37 segments in 13 embryos). In all three cases, about 80% of the VGAL spots were located in the anterior or posterior third of the segment (Fig. 3K). Wild-type embryos appeared to have a bias toward the anterior compartment, while H99 and p35-expressing embryos had a more even distribution that was still slightly biased to the anterior of the segment.
The average number of VGAL spots per segment was 30±10 in H99 embryos. This was not significantly different from wild-type, which was 35±11. For p35-expressing embryos, the average was 18±6. This difference in spot number per segment can be attributed to the genetic background of the UAS-p35 embryos, which had an average of 21±10 VGAL-positive spots per segment in the absence of p35 expression (35 segments in six embryos). These data indicate that the pattern of cell engulfment is unchanged in the absence of AO-positive cell death.
Engulfment of neuronal progenitors in H99 and p35-expressing
embryos
Inhibition of AO-positive cell death does not appear to affect the
engulfment of cells expected to die in the epidermis. Dying epidermal cells
are generally engulfed by neighboring cells. What is the fate of cells that
are usually cleared by macrophage engulfment in caspase-inhibited embryos?
Dying neuronal cells are normally removed by macrophages. To demonstrate this, UAS-nGFP embryos were injected with caged GAL4VP16 and VGAL and a patch of five to eight cells was photoactivated in the lateral procephalic ectoderm of the early gastrula to mark neuronal progenitor and epidermal cells (Fig. 4A-C). The development and engulfment of GFP-positive cells was followed by time-lapse microscopy. Photoactivated cells that were engulfed gave rise to GFP-positive vacuoles within macrophages that also carried VGAL-positive vacuoles (n=14 embryos, Fig. 4A-C). This indicated the some of the cells emerging from the proneural region of the embryo had died and were engulfed by macrophages.
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To determine if the entire cell, including the nucleus, was engulfed, UAS-p35/UAS-nGFP embryos were injected with caged GAL4VP16 and VGAL. As these embryos were heterozygous for UAS-p35 and UAS-nGFP, the appropriate controls were performed to ensure that a sufficient amount of p35 was induced to block AO-positive cell death and that a sufficient amount of nGFP was express to be visible (data not shown). Photoactivation of the neurogenic region generated GFP-positive brain neurons and over time gave rise to macrophages that harbored both VGAL- and GFP-containing vacuoles in all embryos examined (n=18, Fig. 4G-I). Thus, the presence of cGFP- and nGFP-vacuoles within macrophages indicates that caspase-inhibited cells are engulfed in entirety.
Homozygous H99 embryos do not develop many engulfing macrophages. To determine the fate of neuronal precursors in homozygous H99 embryos, UAS-nGFP; H99/TM3 embryos were injected with caged GAL4VP16 and VGAL. The same population of neurogenic cells were photoactivated as stated above. Homozygous H99 embryos were those that showed few VGAL-positive macrophages. Time-lapse recordings showed that dying epidermal progeny from this region of the embryo continued to be engulfed by neighbors, but the neuronal progeny were neither engulfed by macrophages nor by neighbors (n=7 embryos, Fig. 4J-L). Thus, localized, cell-autonomous caspase inhibition does not affect the engulfment of dying cells by macrophages, while globally blocking caspase activation appears to inhibit the phagocytic ability of macrophages.
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Discussion |
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As VGAL is initially confined to the cytoplasm and there does not appear to be any cytoplasmic ß-galactosidase activity, the only way to convert VGAL to its fluorescent form is to transport it into the lysosomal space. The usual routes into the lysosome are by engulfment or fluid-phase endocytosis. For endocytosis to occur, the VGAL must first leak out of the cell and then be taken up from the extracellular fluid. We do not believe this is happening for two reasons. First, we know that there is ß-galactosidase activity in the intervitelline space. If VGAL was to leak out of cells into this space, one would observe an increase in intervitelline VGAL fluorescence, which was not seen. Second, if the VGAL was endocytosed from the extracellular fluid, one would expect to observe a broad distribution of VGAL, which was also not seen. The fact that the VGAL signal is distributed in a similar pattern both spatially and temporally as the AO-cell death pattern argues against a necrotic release of VGAL and subsequent endocytosis. One might argue that endocytosis of the released VGAL is so fast that there is little time for diffusion. If this were the case, one would not expect to observe VGAL in macrophages. The time it takes for a macrophage to migrate to a dying cell would be more than enough time for the released VGAL to be endocytosed by neighboring cells. The fact that macrophages carry VGAL-positive vesicles indicates engulfment of VGAL-containing cytoplasm.
We do not believe that secondary necrosis is contributing to the VGAL
signal because secondary necrosis is primarily observed in tissue culture
experiments where engulfment is not occurring and in vivo when massive amounts
of apoptosis are induced and there are insufficient macrophages to clear the
dying cells (Honda et al.,
2000; Scaffidi et al.,
2002
). In the experiments reported here, we are mostly observing
naturally occurring cell death. The appearance of the VGAL signal occurs
within 10 minutes of the AO signal, thus there is not a significant delay in
engulfment. Moreover, the timing of VGAL fluorescence is the same for both
wild-type and H99 embryos. Finally, intervitelline injection of the
necrotic-cell marker, propidium iodide, did not label epidermal cells at any
point during embryogenesis (J. Minden, personal communication).
The aforementioned arguments leave engulfment as the most likely cause for VGAL fluorescence. We cannot claim with certainty that the engulfment is solely by neighboring cells or macrophages and not by autophagy. Autophagy may play a role in eliciting a VGAL signal, but the distribution of the VGAL signal and the involvement of macrophages argues against significant amounts of autophagy. In autophagy, one would expect to see the VGAL signal to always appear immediately adjacent to apoptotic nuclei, which we do not generally observe. This evidence clearly indicates that VGAL is an authentic marker for cellular engulfment.
Surprisingly, engulfment occurred even in embryos known to be deficient for
apoptotic cell death. Despite having no AO-positive cell death, homozygous
H99 embryos continued to show engulfment in the epidermis. The
pattern and number of VGAL-positive vacuoles was the same as wild type. The
VGAL-positive vacuoles were the same size in death-deficient embryos as in
wild-type embryos. The persistence of the engulfment pattern in
death-deficient embryos suggests that the cells expected to die in wild-type
embryos are still experiencing the cellular changes required for their
phagocytosis and still eliciting a signal for their neighbors to engulf them.
The presence of phagocytosis in the absence of rpr, hid and
grim suggests that there is a caspase-independent cell engulfment
pathway and that the signal for engulfment occurs upstream of caspase
activation. White et al. (White et al.,
1994) observed a significant reduction of phagocytosis by
macrophage in H99 homozygous embryos using TEM analysis. We also
observed the same reduction using time-lapse microscopy. Thus, there may be
two types of engulfment signal: a local signal for neighboring cells to
phagocytose dying cells and a global signal that attracts macrophages to the
site of cell death.
p35 is a pan-caspase inhibitor that functions by binding directly to
caspases. This caspase inhibition is irreversible resulting from the cleavage
of p35 (Bump et al., 1995;
Zhou et al., 1998
;
LaCount et al., 2000
).
Previous studies have demonstrated that expression of p35 results in the loss
of AO-positive cell death and leads to hyperplasia
(Hay et al., 1994
). As in
death-deficient, homozygous H99 embryos, p35-expressing embryos
showed engulfment in a wild-type pattern in the lateral abdominal epidermis,
in spite of not having any AO-positive nuclei. Furthermore, tissue specific
expression of p35 in neuronal precursors showed that these marked cells were
engulfed by macrophages that did not express p35. Thus, p35-expressing cells
are capable of sending a global engulfment signal that attracts macrophages.
It is curious that one does not observe many phagocytic macrophages in
homozygous H99 embryos. There are at least two possible explanations
for this observation. (1) p35 does not inhibit all caspases, such as DRONC
(Hawkins et al., 2000
). DRONC
is an initiator caspase that acts upstream of executioner caspases, which are
p35 sensitive. One would expect that inhibiting the executioner caspases would
inhibit all caspase-dependent cell death
(Adrain and Martin, 2001
).
Perhaps these p35-resistant caspases are required for a dying cell to elicit a
global engulfment signal. (2) A certain amount of cell death is required for
macrophages to mature from non-phagocytic, hemocytes to mature, phagocytic
macrophages (Tepass et al.,
1994
). Perhaps the signal from the cells being engulfed in
death-deficient embryos is not sufficient to activate the macrophage
population. Extensive DNA damage by X-irradiation of H99 embryos
indeed causes macrophages to engulf damaged cells
(White et al., 1994
), which is
further evidence for a threshold for macrophage maturation. A third possible
explanation, that was disproved, is that macrophages require caspase
activation to be phagocytic. Photoactivating UAS-p35 in macrophage
precursors yielded macrophages that continued to be phagocytic (data not
shown). Additional experimentation is required to determine why macrophages in
homozygous H99 embryos are not phagocytic.
One candidate for the local signal for cell engulfment is
phosphatidylserine, which is flipped from the cytoplasmic face of the plasma
membrane to the outer membrane surface of dying cells
(Martin et al., 1995). The
exposure of phosphatidylserine during death is a highly conserved process
across multiple species from C. elegans to vertebrates
(van den Eijnde et al., 1998
).
In addition, a phosphatidylserine receptor is found on virtually all cells
capable of phagocytosis; activation of this receptor has been shown to lead to
the engulfment of the dying cell (Fadok et
al., 2001
; Henson et al.,
2001
). We would very much like to monitor exposed
phosphatidylserine in living Drosophila embryos. Unfortunately, this
is not yet possible.
The results reported here indicate that the signal for cell engulfment may
be independent of the caspase cascade. In addition to the vast literature on
the necessity of caspases for apoptosis
(Fraser and Evan, 1997;
Fraser et al., 1997
;
Song et al., 1997
;
Chen et al., 1998
;
Dorstyn et al., 1999
;
Kumar and Doumanis, 2000
;
Quinn et al., 2000
;
Adrain and Martin, 2001
;
Harvey et al., 2001
), there is
growing evidence for caspase-independent death pathways in tissue culture
paradigms (Borner and Monney,
1999
; Leist and Jaattela,
2001
; Lockshin and Zakeri,
2002
). There are very few reports on caspase-independent cell
death in vivo. In Drosophila, it has been shown that rpr,
hid and grim are not required for apoptosis of the nurse cells
during oogenesis (Foley and Cooley,
1998
). Zhang et al. (Zhang et
al., 2000
) showed that Dbok is capable of inducing cytochrome
c release in a caspase-independent fashion when overexpressed. These
cultured Drosophila cells died in a non-apoptotic, presumably
necrotic, fashion in the presence of zVAD-fmk, a broad range caspase
inhibitor. It is very unlikely that the VGAL signal is the result of necrotic
death for the reasons stated earlier.
A very important question about engulfment in cell-death-deficient embryos
is why is there no AO signal from cells that have been engulfed? There are two
possible explanations: (1) chromosomes within the lysosomes of engulfing cells
do not adopt a suitable conformation for AO intercalation if they have not
been previously exposed to caspase-activated endonucleases; or (2) the acidic
environment of the lysosome maintains the AO in an aggregated state that is
not amenable to DNA intercalation. Delic et al.
(Delic et al., 1991) showed
that chromosomes within living nuclei do not bind AO. A key feature of
apoptosis is nuclear condensation. This condensation, which can be triggered
by caspase activation, may be essential for AO binding. Further
experimentation in vivo and in vitro is required to determine why engulfed
cell nuclei do not elicit AO fluorescence in caspase-inhibited embryos.
If cell engulfed continues in caspase-inhibited embryos, then why do these
embryos die? There is evidence that caspases are also required for
morphogenesis. rpr is expressed in the embryo several hours before
cell death appears in a pattern that covers many more cells than the number
that actually die (Nassif et al.,
1998; Pazdera et al.,
1998
). These observations spawned the notion that there is a
threshold of rpr expression below which permits morphogenesis; above
which triggers cell death. To further understand the relationship between
morphogenesis and cell death, a detailed developmental analysis of embryos
lacking various caspases alone or in combination must be performed.
Why do caspase-inhibited embryos have extra cells? White et al.
(White et al., 1994) showed
that there are extra cells in the CNS of homozygous H99 embryos. Our
time-lapse analysis has shown that dying neurons are generally cleared by
macrophages. We and others have also shown that macrophages do not mature
properly in homozygous H99 embryos
(Tepass et al., 1994
).
Therefore, the extra CNS cells in homozygous H99 embryos may be due
to an engulfment failure in addition to the blockage of the cell death
program. We showed that localized inhibition of caspases by p35 does not block
the expected phagocytosis of neuronal cells. In C. elegans, it has
been shown that cells slated to die can be rescued in engulfment-deficient
animals (Hoeppner et al.,
2001
; Reddien et al.,
2001
).
Finally, if cells are being engulfed in homozygous H99 and global,
p35-expressing embryos, should these still be considered cell-death-deficient
embryos? Once a cell is engulfed, it should be considered dead. Therefore,
homozygous H99 and global, p35-expressing embryos should not be
referred to as cell-death-deficient; perhaps they should be referred to as
caspase-inactivated or -inhibited embryos. It is not clear if this
caspase-independent engulfment can still be considered apoptosis as the
hallmark features of apoptosis do not occur in these embryos. One very
important distinction must be made between the results reported here and much
of the existing research on apoptosis. Aside from studies using C.
elegans embryos, the vast majority of cell death research has focused on
tissue culture models. In these tissue culture systems there are generally no
engulfing cells present and it typically takes several hours for most cells to
die upon triggering apoptosis. In living Drosophila embryos,
AO-positive cells are engulfed within 15 minutes of the AO signal, and in some
cases engulfment precedes the AO signal. The precise time lag between the
decision for cell death and the AO- or VGAL-signals is not known. We estimate
the lag to be about 1 hour. This is based on previous studies where excess
cells resulting from an ectopic round of cell division driven by Cyclin E
overexpression begin to produce ectopic AO signals within 1 hour of the
ectopic division (Li et al.,
1999). Thus, there are significant differences between cell death
in situ and in culture. Exploring these differences will play an important
role in our understanding of programmed cell death.
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ACKNOWLEDGMENTS |
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