Programmed cell death first becomes apparent during Drosophila development at ~7 h after egg laying (AEL). However, it is possible to induce apoptosis at earlier times by DNA damaging agents (X-irradiation) or by overexpression of apoptotic activators such as rpr or grim. Since all these stimuli activate a caspase-dependent apoptotic program, the caspases required for cell death must be present at all times at which death can be induced. A developmental Northern blot of Ice expression shows clearly that Ice expression is highest at 2-6 h AEL. Intriguingly, this is not a period in which any detectable cell death is observed in the embryo: however, Ice is synthesized as an inactive pro enzyme, so mRNA expression gives no indication of enzyme activity. Thereafter, Ice expression remains detectable throughout Drosophila development, consistent with the notion that caspase machinery is present at all stages at which PCD can occur (Fraser, 1997a).
Regulated cell death and survival play important roles in neural development. Extracellular signals are presumed to regulate seven apparent caspases to determine the final structure of the nervous system. In the eye, the EGF receptor, Notch, and intact primary pigment and cone cells have been implicated in survival or death signals. An antibody (CM1) raised against a peptide from human caspase 3 was used to investigate how extracellular signals control spatial patterning of cell death. The antibody crossreacts specifically with dying Drosophila cells and labels the activated effector caspase Ice. The initiator caspase Dronc and the proapoptotic gene head involution defective are important for activation in vivo. Dronc may play roles in dying cells in addition to activating downstream effector caspases. Epistasis experiments ordered the EGF receptor, Notch, and primary pigment and cone cells into a single pathway that affects caspase activity in pupal retina through hid and Inhibitor of Apoptosis Proteins. None of these extracellular signals appear to act by initiating caspase activation independently of hid. Taken together, these findings indicate that in eye development spatial regulation of cell death and survival is integrated through a single intracellular pathway (Yu, 2002).
A particularly useful feature of the CM1 antiserum is the detection of cells that would otherwise be marked for death but which are protected by baculovirus p35 expression. The morphological protection provided by p35 may permit better investigation of the location and autonomy of death and survival signals. On Western blots the CM1 antibody detects activated Ice but not the Ice zymogen; Ice is the Drosophila sequence most similar to the immunizing peptide. Definitive evidence that CM1-stained cells are apoptotic comes from the dependence of embryonic CM1-staining on the 75C1,2 chromosome region, and from the p35-sensitivity of larval and pupal CM1-stained cells. The morphology of all CM1-stained cells is altered by p35 expression. In the presence of p35, CM1-stained cells become indistinguishable from normal cells by morphological criteria, and are not distinguishable except by CM1 staining. Since baculovirus p35 blocks cell death by inhibiting caspase activity, p35-dependent morphology of CM1-stained cells shows that such morphology depends on caspase activity in the cells, which are therefore apoptotic. These results show that only apoptotic cells are labelled by the CM1 antiserum. Apoptotic cells that are unlabelled by CM1 might also exist, although none have been noticed (Yu, 2002).
The results indicate that effector caspases such as Ice can be processed in the presence of baculovirus p35. The initiator caspase Dronc is responsible for effector caspase processing in p35 expressing cells. p35-insensitive caspases are also thought to initiate cell death in other insect cells. In Drosophila eye development, Dronc always functions redundantly with other, p35-sensitive initiator caspases. Such redundancy explains why Dronc-DN had no effect in earlier studies of eye development. Dronc has been implicated in embryonic cell death by RNA interference studies (Yu, 2002).
If feedback of effector caspases on their own activation is essential for cell death, it would be expected that Dronc alone is insufficient for CM1 labelling in p35-expressing cells. By contrast, CM1 labelling persists in most cells, indicating that feedback of effector caspases is dispensable for the activation of at least one effector caspase. However, the results do not exclude the possibility that feedback makes a quantitative contribution to the pace of death or is required for a subset of effector caspases. Results are different for neuronal photoreceptor cells. R8 photoreceptor survival is rescued in the absence of EGFR by p35 expression, but such rescued R8 cells are not labelled strongly by CM1. It is possible that amplification of the apoptotic cascade might be more important for apoptosis of R8 precursors than for unspecified cells. It is also possible that R8 cell apoptosis involves different effector caspases or is initiated independently of Dronc (Yu, 2002).
Self-digestion of cytoplasmic components is the hallmark of autophagic programmed cell death. This auto-degradation appears to be distinct from what occurs in apoptotic cells that are engulfed and digested by phagocytes. Although much is known about apoptosis, far less is known about the mechanisms that regulate autophagic cell death. Autophagic cell death is regulated by steroid activation of caspases in Drosophila salivary glands. Salivary glands exhibit some morphological changes that are similar to apoptotic cells, including fragmentation of the cytoplasm, but do not appear to use phagocytes in their degradation. Changes in the levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin and nuclear Lamins precede salivary gland destruction, and coincide with increased levels of active Caspase 3 and a cleaved form of nuclear Lamin. Mutations in the steroid-regulated genes ßFTZ-F1, E93, BR-C and E74A that prevent salivary gland cell death possess altered levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin, nuclear Lamins and active Caspase 3. Inhibition of caspases, by expression of either the caspase inhibitor p35 or a dominant-negative form of the initiator caspase Dronc, is sufficient to inhibit salivary gland cell death, and prevent changes in nuclear Lamins and alpha-Tubulin, but not to prevent the reorganization of filamentous Actin. These studies suggest that aspects of the cytoskeleton may be required for changes in dying salivary glands. Furthermore, caspases are not only used during apoptosis, but also function in the regulation of autophagic cell death (Martin, 2004).
Studies of dying salivary glands indicate that autophagic cell death has
both similarities to and differences from apoptosis. Several markers of
apoptosis, including nuclear staining by Acridine Orange and DNA
fragmentation, appear 2 hours following the rise in steroid hormone that
triggers salivary gland cell death. The cytoplasm and membranes of salivary gland cells bleb and fragment as these cells degrade, and these are characteristics that are common to apoptotic cells. These common morphologies raise the questions of what distinguishes apoptosis from autophagic cell death, and what common and different regulatory mechanisms mediate these forms of cell killing (Martin, 2004).
The mechanisms of cell degradation and removal appear to provide the
clearest distinction between apoptosis and autophagic cell death. In salivary
glands, dynamic changes in vacuole structure immediately precede their demise,
and such changes have not been reported in apoptotic cells. Within one hour of salivary gland DNA degradation, large vacuoles appear to break into smaller vacuoles, and this occurs within 2 hours of complete tissue destruction. As
these large vacuoles fragment, smaller but distinct vacuoles accumulate near
the plasma membrane, and autophagic vacuoles containing components of the
cytoplasm, including mitochondria, are formed. Salivary gland cells then begin
to fragment, and nuclei and components of the cytoplasm then disperse within
the haemocoel.
Although a role for phagocytes in autophagic cell death cannot be excluded,
salivary gland cells proceed to late stages of degradation without the
assistance of phagocytes. It has been suggested that phagocytes may play a
secondary role in the removal of cellular debris toward the end of autophagic
cell death, and the results are consistent with this possible
conclusion. The presence of autophagic vacuoles in degrading salivary glands
further indicates that autophagic cells use their own lysosomes for
degradation, whereas apoptotic cells depend on phagocytes for the bulk
degradation of long-lived cellular proteins (Martin, 2004).
Dying cells exhibit dynamic changes in cell shape.
The changes in cell organization that occur during salivary gland cell death
are likely to be controlled by the modification of structural protein
organization. Indeed, dynamic changes in the abundance and localization of
filamentous Actin, alpha-Tubulin, alpha-Spectrin and nuclear Lamins
precede the death of salivary glands. At least two possible explanations exist for how such changes
in protein expression are regulated in dying cells. One possibility is that
proteases cleave structural proteins, and that this results in the changes in cell shape. Alternatively, changes in cell shape could be regulated by the assembly of cytoskeletal proteins, such as filamentous Actin, through signaling that is mediated by small GTPases (Martin, 2004).
Proteolysis and changes in the assembly of the cytoskeleton both appear to
be involved in the regulation of changes that occur during autophagic cell
death of salivary glands. Although caspases play an important role in the cell
death of salivary glands, several lines of evidence suggest that some changes
in the structure of the cytoskeleton may occur in a caspase-independent
manner. First, whereas changes in filamentous Actin localization occur in
synchrony with changes in proteins such as nuclear Lamins that are cleaved by
caspases, changes in Actin protein levels are delayed by 4 hours. Second, mutations in steroid-signaling genes, such as ßFTZ-F1, that prevent
expression of active caspase-3 and cleavage of nuclear Lamins do not prevent
changes in filamentous Actin localization. Third, although
inhibition of caspases by expression of either p35 or a dominant-negative form
of Dronc is sufficient to prevent changes in nuclear Lamins and
alpha-Tubulin, these inhibitors are not sufficient to block changes in
filamentous Actin. These data are further supported by the observation that numerous small GTPases increase their expression immediately prior to salivary gland cell death. Although previous studies have suggested that changes in the Actin cytoskeleton are required for autophagic cell death, the
failure to distinguish between cytoskeleton proteolysis and rearrangement has
made it difficult to interpret the potential significance of maintenance of
the cytoskeleton during cell death (Martin, 2004).
Several possibilities exist to explain why the cytoskeleton is maintained
during cell death. The cytoskeleton could be used to restrict the subcellular
location and activity of pro-apoptotic regulators of the Bcl-2 family, and
activation of apoptosis. This mechanism seems unlikely during salivary gland cell
death because the Actin cytoskeleton is maintained after caspase-dependent
cleavage of substrates, including nuclear Lamins. Alternatively, the Actin
cytoskeleton could be maintained as a substrate to localize proteins,
membranes and vacuoles within the cell. Intracellular trafficking plays an
important role in autophagy, as evidenced by the observation that membrane-bound cytoplasmic components
(autophagic vacuoles) are transported to the lysosome for degradation. Since
autophagic vacuoles are observed at stages after caspase activation and cleavage of
substrates such as nuclear Lamins, it is possible that the Actin cytoskeleton is maintained to enable transport of vacuoles to lysosomes (Martin, 2004).
Studies of salivary glands indicate that caspases play an important role in
their autophagic cell death. The caspase-encoding genes dronc and
drice show an increase in their transcription following the rise in
steroid that triggers salivary gland autophagic cell death. This
increase in caspase transcription corresponds to the increase in active
caspase protein levels and in the cleavage of substrates such as nuclear
Lamins in dying salivary glands. Mutations in the
steroid-regulated ßFTZ-F1, E93 and BR-C genes, which
prevent salivary gland cell death, exhibit little or no active Caspase-3/Drice
expression, and have altered alpha-Tubulin, alpha-Spectrin and nuclear
Lamin expression in salivary glands. Although E74A
mutants prevent salivary gland cell death, they have elevated Caspase-3/Drice
levels and degraded nuclear Lamins. Although these data are consistent with the partially degraded morphology of E74A mutant salivary glands, it
remains unclear what factor(s) E74A may regulate that are required
for normal cell death. However, the data indicate that ßFTZ-F1,
E93 and BR-C play a crucial role in determining caspase levels
in dying salivary gland cells, and this is supported by the impact of these
genes on the transcription of dronc.
Significantly, inhibition of caspases by expression of either p35 or
dominant-negative Dronc is sufficient to prevent DNA fragmentation, changes in
nuclear Lamins and alpha-Tubulin, and death of salivary glands (Martin, 2004).
The morphologies of dying cells indicate that apoptosis and autophagy are
distinct. However, the difference between these cells becomes less apparent
when one considers characteristics that were previously considered to be
specific to apoptosis. Clearly, markers such as DNA fragmentation, expression
and function of caspases, and cleavage of caspase substrates can exist in
cells that possess the morphology of autophagic cell death. As expression of
dominant-negative Dronc is sufficient to block caspase-dependent changes in
salivary glands, these studies also indicate that the mechanism for caspase
activation during autophagic cell death is similar to apoptosis during
Drosophila development; the initiator caspase Dronc regulates the
activation of the executioner caspase Drice and cleavage of cell substrates. It is
surprising how little is known about the activity of caspases in developing
animals; these proteases have been a subject of substantial investigation.
Recent studies indicate that caspases do not only function during autophagic
and apoptotic cell death, but that they are also used to degrade proteins
during the differentiation of sperm in Drosophila. Studies
of salivary glands indicate that the distinction between apoptosis and
autophagic cell death may be more subtle than their morphology suggests, and
raise the question of what makes these cells look so different. Restriction of
caspase activity within compartments of the dying cell may provide one
possible explanation, but it is also possible that other mechanisms of
proteolysis occur during autophagic cell death. This possibility is supported
by the large increase in transcription of non-caspase proteases just before
cell death of salivary glands, and by the fact that, unlike apoptotic cells that require phagocyte lysosomes, salivary gland cells appear to degrade themselves through autophagy. Future studies should provide important insights into the
similarities and differences in the mechanisms that regulate apoptosis and
autophagic programmed cell death (Martin, 2004).
In both vertebrates and invertebrates, developing organs and tissues must be precisely patterned. One patterning mechanism is Notch/Delta-mediated lateral inhibition. Through the process of lateral inhibition, Drosophila sensory organ precursors (SOPs) are selected and sensory bristles form into a regular pattern. SOP cell fate is determined by high Delta expression and following expression of neurogenic genes like neuralized. SOP selection is spatially and temporally regulated; however, the dynamic process of precise pattern formation is not clearly understood. In this study, using live-imaging analysis, it was shown that the appearance of neuralized-positive cells is random in both timing and position. Excess neuralized-positive cells are produced by developmental errors at several steps preceding and accompanying lateral inhibition. About 20% of the neuralized-positive cells show aberrant cell characteristics and high Notch activation, which not only suppress neural differentiation but also induce caspase-dependent cell death. These cells never develop into sensory organs, nor do they disturb bristle patterning. This study reveals the incidence of developmental errors that produce excess neuralized-positive cells during sensory organ development. Notch activation in neuralized-positive cells determines aberrant cell fate and typically induces caspase-dependent cell death, as detected using SCAT3, a fluorescence resonance energy transfer (FRET) indicator for effector caspase activity. Apoptosis is utilized as a mechanism to remove cells that start neural differentiation at aberrant positions and timing and to ensure robust spacing pattern formation (Koto, 2011).
The Drosophila sensory organ is a typical model for the study of Notch/Delta-mediated lateral inhibition. Tracking the process of cell fate determination in each cell lineage is presumed to be effective in revealing the mechanisms behind precise pattern formation (Koto, 2011).
The first finding in this study is that bristle patterning starts in a random fashion: about 20% of neuralized-positive cells are fated to become aberrant SOP-like cells, and Notch signaling is involved in determining the fate of SOP-like cells. However, the mechanisms proposed for the production of SOP-like cells in previous reports do not coincide perfectly with the current observations. Previous studies showed that the conventional model of Notch/Delta-mediated lateral inhibition is not sufficient to produce the precise bristle pattern but that cell-autonomous interaction or filopodia-mediated intermittent Notch/Delta signaling makes lateral inhibition robust enough to suppress the neural differentiation of surrounding cells. In contrast, the current results suggest that lateral inhibition from adjacent SOPs is not the sole source of Notch activation in SOP-like cells, because a portion of SOP-like cells preceded the nearby SOPs. Also, SOP-like cells showed ongoing Notch activity even in the absence of adjacent SOPs. One possible explanation for these SOP-like cells failing to develop into sensory organs may be that they appear too early in the developmental time course and cannot complete the developmental program to become sensory organs in a cell-autonomous manner. In any case, the decrease in SOP-like cells in the N55e11 heterozygous mutant reliably suggests that Notch activation in cells that start neural differentiation contributes to the determination of their cell fate as aberrant SOP-like cells (Koto, 2011).
Dynamic oscillation of the Notch effector gene Hes1 has been observed in neural progenitors of the developing mouse brain with the aid of a short-half-life indicator using ubiquitinated firefly luciferase. In Drosophila sensory organ development, the technical limitations of the GFP reporter make it difficult to confirm this type of oscillation pattern in Notch signaling. However, given that Notch oscillation occurs in cells in the proneural stripe regions at the beginning of SOP selection, it is conceivable that SOP-like cells might be the product of fluctuating Notch signaling at inappropriate times, developmentally speaking (Koto, 2011).
The second finding in this study is that a program of caspase-dependent cell death specifically eliminates SOP-like cells. Ablating an incipient SOP removes Notch/Delta-mediated lateral inhibition and allows a nearby epithelial cell to become the SOP, as has also been observed in the embryonic central nervous system of grasshoppers. However, the adjacent SOP-like cells never develop into sensory organs, suggesting that the fate of SOP-like cells is irreversible. By observing the nuclear morphology along with an indicator for caspase activation, it was noted that in the process of sensory organ development, only SOP-like cells showed the typical features of programmed cell death. These results indicate that programmed cell death ensures robust pattern formation by eliminating aberrantly differentiated cells (Koto, 2011).
The significance of programmed cell death in pattern formation has been well studied, especially in the development of the fly eye. Each ommatidium is composed of eight photoreceptor neurons and six support cells, consisting of four cone cells and two primary pigment cells. Between each ommatidium, remaining cells form the interommatidial lattice. Excess pigment cells are eliminated through programmed cell death. Notch functions within the interommatidial lattice to induce cell death, and the primary pigment cells send a survival signal to adjacent cells. The life-and-death fate of interommatidial cells is decided by their position and the cells to which they are attached. In the case of sensory organ formation, Notch signaling is crucial in determining the aberrant cell fate of SOP-like cells. However, Notch activation alone seems insufficient to induce programmed cell death, because the surrounding epithelial cells do not disappear, even though they exhibit high levels of Notch activation during sensory organ development. Therefore, some factor that marks neural differentiation in SOP-like cells may be required to induce cell suicide. This study found that ectopic neuralized expression did not induce the aberrant cell fate or cell death in epithelial cells, suggesting that neuralized itself is not essential in determining the aberrant cell fate of SOP-like cells. Therefore, to determine how apoptosis is induced in SOP-like cells, the effect of Notch activation in neuralized-positive cells was examined at the one-cell stage using the temporal and regional gene expression targeting (TARGET) system with tub-GAL80ts. As reported previously, activated Notch induced the multiple-sockets phenotype. At the same time, about 50% of neuralized-positive cell lineages died, accompanied by nuclear fragmentation, causing a dramatic bald phenotype that was observed in the adult flies. These findings suggest that the combination of neural differentiation in the SOP lineage and Notch activation switches on cell death signaling. One possible future approach to searching for the killing factor expressed in SOP-like cells would be gene profiling using laser microdissection (Koto, 2011).
When the apoptotic pathway is blocked, the inhibition of cell death results in cell fate transformation. In C. elegans, cell death survivors in ced-3 mutants exhibit an ambiguous cell fate. The most disruptive alternative cell fate occurs when the remaining cells differentiate into tumor-like proliferating cells, as shown in the development of the Drosophila serotonin lineage. Under apoptosis-deficient conditions, other types of cell death occur, such as necrosis or autophagic cell death. These alternate reactions could mask the incidence of programmed cell death; therefore, it is possible that the role of the apoptotic pathway has been missed in the case of sensory organ development. This study has shown that SOP-like cells differentiate into epithelial cells when the cell death pathway is blocked. Time-lapse imaging made it possible to trace the transient fate of dying SOP-like cells, revealing the contribution of programmed cell death in the SOP selection process. Although the function of apoptosis has been emphasized in various developmental processes, the principle message is that several pathways exist to overcome the appearance of excess or aberrant cells and to make the developmental process more robust. This study reveals that programmed cell death plays an important role in overcoming innately induced developmental errors and contributing to robust neural cell selection (Koto, 2011).
In Drosophila oogenesis, the programmed cell death of germline cells occurs predominantly at three distinct stages: stage 2a/2b (germarium), stage 8 (midoogenesis), and stages 10-13 (late oogenesis). These cell deaths are subject to distinct regulatory controls, since cell death during early and midoogenesis is stress-induced, whereas the cell death of nurse cells in late oogenesis is developmentally regulated. This report shows that the effector caspase Drice is activated during cell death in both mid- and late-oogenesis, but that the level and localization of activity differ depending on the stage. Active Drice forms localized aggregates during nurse cell death in late oogenesis; however, active Drice is found more ubiquitously and at a higher level during germline cell death in midoogenesis. Because Drice activity is limited in late oogenesis, an examination was performed to see whether another effector caspase, Dcp-1, could drive the unique morphological events that occur normally in late oogenesis. Premature activation of the effector caspase, Dcp-1, results in a disappearance of filamentous actin, rather than the formation of actin bundles, suggesting that Dcp-1 activity must also be restrained in late oogenesis. Overexpression of the caspase inhibitor DIAP1 suppresses cell death induced by Dcp-1 but has no effect on cell death during late oogenesis. This limited caspase activation in dying nurse cells may prevent destruction of the nurse cell cytoskeleton and the connected oocyte (Peterson, 2003).
The cytoskeletal events induced by expression of activated Dcp-1 (tdcp-1) are significantly different from those normally seen in nurse cells during late oogenesis. Normally during stage 10B of oogenesis, actin bundles form in the cytoplasm of nurse cells, connecting the plasma membrane with the nucleus. After actin bundle formation, actin-myosin-based contraction occurs and drives nurse cell cytoplasm dumping. Expression of tdcp-1 does not lead to actin bundles or nurse cell dumping. Instead, actin forms clumps and disappears as the egg chambers degenerate. Similar cytoskeletal events have been reported for egg chambers that degenerate during stage 8 in response to other stimuli. It is possible that factors required for actin bundle formation are not expressed during midoogenesis. However, egg chambers from midway mutants that degenerate prematurely during stages 8-9 do show actin bundles. Thus, midway, which encodes an acyl coenzyme (A:diacylglycerol acyltransferase) may normally act to control the timing of actin bundle formation and nurse cell death. It has been reported that dcp-1 germline clone mutants are defective in actin bundle formation and nurse cell dumping; however, recent findings suggest that these effects are due to a neighboring gene affected by the P-element alleles and that dcp-1 activity is not required for these cytoskeletal events. Nonetheless, because nurse cell nuclear breakdown has already begun prior to dumping, it has been suggested that the cytoskeletal events are part of the nurse cell death process (Peterson, 2003).
The formation of actin bundles in the cytoplasm of dying late-stage nurse cells differs from the actin rearrangements that have been reported in apoptotic cells and shows similarity to the actin structures in autophagic MCF-7 cells. In cultured mammalian cells undergoing apoptosis, filamentous actin relocalizes to form a dense network in the cytoplasm at the base of the membrane blebs, and later disappears. However, in MCF-7 cells undergoing autophagy, actin forms fibers stretching from the nucleus to the plasma membrane, resembling the actin bundles seen in nurse cells. Autophagic cell death has been observed in groups of cells that die, such as cells of the Drosophila salivary gland during metamorphosis. Caspase activity is required for Drosophila salivary gland autophagy, but it is not known if caspase activity is regulated differently in autophagy and apoptosis. There are several modes of cell death, including deaths that show properties of both autophagy and apoptosis; nurse cell death may fall into this class. Indeed, autophagic vacuoles have been reported in nurse cells, although follicle cells appear to engulf nurse cell material as well (Peterson, 2003).
Egg chambers degenerating during midoogenesis in response to nutrient deprivation or tdcp-1 expression do not show expression of the altered form of cytochrome c, suggesting that this may be a stage-specific event. One explanation is that the factors required to alter cytochrome c may not be expressed in midoogenesis. Alternatively, a mitochondria-independent cell death pathway may be utilized in midoogenesis. The lack of cytochrome c alteration suggests that cytochrome c involvement is not necessary for Drosophila apoptosis when a high level of caspase activity is present. However, it is possible that cytochrome c plays no direct role in apoptotic signaling in Drosophila, and this antibody simply recognizes cytochrome c in dividing or otherwise altered mitochondria (Peterson, 2003).
Interestingly, egg chambers prior to stage 8 were largely resistant to any apoptotic effects of activated Dcp-1. Stage 8 has been shown to be a checkpoint stage for a number of signals, including reduced food availability, ecdysone signaling, treatment with chemicals, ectopic death of follicle cells, or abnormal egg chamber development. Because vitellogenesis begins during stage 8, it has been suggested that the state of the egg chambers is monitored before making the investment of vitellogenesis. However, it is interesting that the egg chambers prior to stage 8 are well-protected from strong death-inducing stimuli, including expression of a truncated caspase. This stage-specific protection may be the result of a high level of caspase inhibitors like IAPs early in oogenesis. Indeed, transcriptional downregulation of Drosophila IAPs during stage 8 has been reported. Overexpression of DIAP1 can indeed block cell death induced by expression of truncated Dcp-1 in midoogenesis (Peterson, 2003).
The limited activation of Drice and the unusual cytoskeletal events that normally occur in late oogenesis suggest that caspase activity is carefully controlled during nurse cell death in late oogenesis. One model to explain the observed cytoskeletal differences is that only a subset of the usual cytoskeletal targets of caspases are cleaved in late oogenesis. This limited cleavage would allow for the formation and persistence of cytoplasmic actin bundles, which are necessary for proper nurse cell cytoplasm transfer. Alternatively, the cytoskeletal events may be controlled by Damm or Decay or may be caspase-independent. However, these models would still require that the activity of Drice and Dcp-1 be curtailed to prevent disassembly of actin cytoskeleton (Peterson, 2003).
Overexpression of the caspase inhibitor DIAP1 does not affect normal nurse cell death. This suggests that nurse cell death may be caspase-independent, or utilize caspases that are not readily inhibitable by DIAP1. Alternatively, mechanisms may exist to compartmentalize caspase activation or to degrade DIAP1, even when it is overexpressed. Support for this idea comes from the observations that full-length DIAP1 does not inhibit naturally occurring cell death in the eye as well as a version of DIAP1 lacking the RING finger. The RING finger has been shown to be critical for rapid turnover of DIAP1 protein (Peterson, 2003).
The controlled caspase activation that occurs in nurse cells during late oogenesis may explain why this form of cell death is not regulated by the cell death activators: Reaper, Hid, and Grim. Reaper, Hid, and Grim induce apoptosis in many cell types by triggering the degradation of DIAP1. Embryos homozygous for the H99 chromosomal deletion, which removes reaper, hid, and grim, are completely lacking in normal programmed cell death (White, 1994). However, flies carrying H99 germline clones undergo normal nurse cell death, indicating that nurse cell death is regulated differently from the vast majority of cell deaths in Drosophila. Perhaps the Reaper, Hid, Grim/DIAP1 mechanism of apoptosis induction would not permit such localized and limited caspase activation. This regulation of caspase activity may be necessary for the systematic destruction of nurse cells while the oocyte is protected from active caspases and other dangerously cleaved proteins (Peterson, 2003).
In the Drosophila antenna, sensory lineages selected by the basic helix-loop-helix transcription factor Atonal are gliogenic while those specified by the related protein Amos are not. What are the mechanisms that cause the two lineages to act differentially? Ectopic expression of the Baculovirus inhibitor of apoptosis protein (p35) rescues glial cells from the Amos-derived lineages, suggesting that precursors are removed by programmed cell death. In the wildtype, glial precursors express the extracellular-signal regulated kinase (phosphoERK) transiently, and antagonism of Epidermal growth factor pathway signaling compromises their development. It is suggested that all sensory lineages on the antenna are competent to produce glia but only those specified by Atonal respond to EGF signaling and survive. These results underscore the importance of developmental context of cell lineages in their responses to non-autonomous signaling in the choice between survival and death (Sen, 2004).
Several lines of investigation have ascertained that the first cells to divide in the sensory lineages are the secondary progenitors: PIIa, PIIb and PIIc. The numbers of sensory cells undergoing division at different times in the developing antenna were estimated by staining mitotic nuclei with antibodies against phosphorylated histone. A peak of cell division was observed between 16 and 24 h after puparium formation (APF). It has been considered that only in those sensory lineages specified by Ato, PIIb produces a glial cell and a tertiary progenitor, PIIIb, which in turn divides to form the sheath cell and a neuron. In Amos dependent lineages, PIIb is believed to directly give rise to a neuron and a sheath cell. The difference between the two lineages could be entirely dependent on the nature of the proneural genes activated; Amos, for example, could direct a non-gliogenic lineage. Alternatively, the two proneural genes could specify similar division patterns but the glial cell precursor in Amos-lineages could be removed by PCD, resulting in non-gliogenic lineages (Sen, 2004).
To test the latter possibility, cell death profiles were examined in developing pupal antennae using the terminal transferase assay (TUNEL) and attempts were made to correlate the timing of PCD with cell division profiles discussed above. The appearance of TUNEL-positive cells peaked between 22 and 24 h APF consistent with the occurrence of PCD immediately after division of secondary progenitors (Sen, 2004).
TUNEL reactions were performed on 22-24 h APF antennae from lz-Gal4; UAS-lacZnls and ato-Gal4; UAS-lacZnls animals. Double labeling with antibodies against ß-galactosidase marked sensory cells arising from the Lz and Ato lineages. Lz::lacZ overlaps the regions of the antennal disc where amos expression occurs and labels all the basiconic and trichoid sensilla in the mature (36 h APF) antenna. Hence for the purpose of this study, all cells in which lz-Gal4 expresses will be considered to belong to the Amos-dependent lineages. ato-Gal4 drives reporter activity in proneural domains of the disc and subsequently in all cells of the coeloconic sense organs (Sen, 2004).
Most of the apoptotic nuclei observed during olfactory sense organ development co-localized with Lz::LacZ suggesting that death occurred mainly within the 'Amos-dependent' sensory clusters. Only very few TUNEL-positive cells were detected in regions where ato-lacZ expressed and these did not co-localize with the reporter expression. If PCD is the mechanism used to remove glial precursors from Amos lineages, then their rescue would be expected to result in additional peripheral glia in the antenna (Sen, 2004).
The GAL4/UAS system was used to target ectopic expression of baculovirus inhibitor of apoptosis protein (p35) to different cell types within the developing antennal disc. distalless981-Gal4 (henceforth called dll-Gal4), which drives expression in all cells of the antennal disc, resulted in the formation of >300 glial cells as compared to ~100 in the wildtype. Other sensory cells--neurons, sheath, socket and shaft cells--within sense organs were unaffected. Ectopic expression of p35 specifically in Ato lineages (ato::p35) did not alter glial number. This means that the `additional' glial cells rescued in dll::p35 must arise from lineages other than Ato. Mis-expression of p35 in Amos-dependent lineages using lz-Gal4, on the other hand, resulted in a significant increase in glial number. While other explanations are possible, it is believed that the somewhat lower number of glia obtained in lz::p35 as compared to dll::p35 could be accounted for by the strength of the P(Gal4) driver (Sen, 2004) (Sen, 2004).
In order to identify the cell within the Amos lineage that is fated to die, the cellular events during development of sense organs were re-examined. At approximately 12-14 h APF, most sensory cells are associated in clusters of secondary progenitors. Two cells in each cluster -- PIIb and PIIc -- express the homeodomain protein Prospero (Pros). pros-Gal4;UAS-GFP recapitulates Pros expression at this stage and marks PIIb and PIIc and their progeny in all olfactory lineages. In the wildtype, a Repo-positive cell was associated with only a few of the total sensory clusters, these were all located within the coeloconic domain of the antenna. Targeted expression of p35 using pros-Gal4 increased glial number indicating that cells which are the progeny of either PIIb or PIIc could be rescued from apoptosis. In the pros-Gal UAS-2XEGFP/UAS-p35 genotype, a glial cell was associated with most clusters at 18 h APF rather than in Ato lineages alone (Sen, 2004).
In order to directly visualize the cell undergoing apoptosis, 22-24 h APF antenna from the neuA101 strain were stained with antibodies against ß-galactosidase to mark the sensory cells and with TUNEL. Sensory clusters located in basiconic and trichoid domains of the pupal antenna each had a single associated TUNEL positive cell. Since TUNEL reactivity data does not reflect the initiation of the death program, developing antennae were also stained at different time points with an antibody that recognized the activated caspase -- Drice. At 20 h APF, a single Drice-positive cell was found within each sensory cluster within the basiconic and trichoid domains of the pupal antenna. This cell also expressed low levels of Pros suggesting that it could arise from either PIIb or PIIc. This means that the PIIb/c in Amos lineages, like that in Ato, divides to give rise to a PIIIb and its sibling. The sibling in the former lineage was not previously detected because it expresses only low levels of Pros and soon dies. Since this cell is capable of expressing the glial-identity gene repo when rescued from death, it is denoted as a glial precursor (Sen, 2004).
How is apoptosis of a specific cell within the lineage regulated? In Drosophila three genes [reaper (rpr), grim and head involution defective (hid)] which all map under the Df(3L)H99 are necessary for the initiation of the death program. Heterozygotes of Df(3L)H99 show a small but significant increase in glial number over that of normal controls. hid-lacZ was used to follow expression during antennal development; reporter activity occurs at low levels ubiquitously including in glial cells. Levels of reporter expression indicate somewhat higher hid transcription in glia rescued by p35 mis-expression. The presence of Hid in the 'normal' glial precursors suggests a mechanism dependent on possible trophic factors to keep cells alive. In several other systems signaling, mainly through the EGFR pathway, results in an antagonism of Hid action and transcription. The sustained levels of hid transcription in the rescued glia, is not unexpected since inhibitors of apoptosis act by antagonizing a downstream event of caspase activation, rather than on Hid itself (Sen, 2004).
Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. This study demonstrates that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. These results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process (Huh, 2004; full text of article).
In Drosophila, the APAF-1 homolog ARK is required for the activation of the initiator caspase DRONC, which in turn cleaves the effector caspases DRICE and DCP-1. While the function of ARK is important in stress-induced apoptosis in Drosophila S2 cells, since its removal completely suppresses cell death, the decision to undergo apoptosis appears to be regulated at the level of caspase activation, which is controlled by the IAP proteins, particularly DIAP1. This study further dissects the apoptotic pathways induced in Drosophila S2 cells in response to stressors and in response to knock-down of DIAP1. The induction of apoptosis is dependent in each case on expression of ARK and DRONC and surviving cells continue to proliferate. A difference was noted in the effects of silencing the executioner caspases DCP-1 and DRICE; knock-down of either or both of these have dramatic effects to sustain cell survival following depletion of DIAP1, but have only minor effects following cellular stress. These results suggest that the executioner caspases are essential for death following DIAP1 knock-down, indicating that the initiator caspase DRONC may lack executioner functions. The apparent absence of mitochondrial outer membrane permeabilization (MOMP) in Drosophila apoptosis may permit the cell to thrive when caspase activation is disrupted (Kiessling, 2006).
While a requirement for ARK and DRONC was observed under all of the pro-apoptotic conditions in S2 cells, a difference was noted in the effects of knock-down of the executioner caspases DCP-1 and DRICE. Knock-down of either or both of these has dramatic effects to sustain cell survival following knock-down of DIAP1, but has only minor effects following cellular stress (Kiessling, 2006).
Several possible explanations were envisioned for this difference. One possibility is that knock-down of DIAP1 leads to caspase activation uniquely through permitting DRONC function to activate DCP-1 and DRICE, while stressors somehow engage other caspase activation pathways (and other caspases). This, however, is inconsistent with the observation that stress-induced apoptosis is clearly dependent on ARK and DRONC. Alternatively, it may be that stress-induced death also involves inhibition of other IAPs, such as DIAP-2 and dBRUCE, which may have a wider spectrum of effects to engage additional caspases not affected by DIAP1 alone. Previously, however, it was noted that knock-down of DIAP-2 does not trigger apoptosis, but greatly enhances susceptibility to death induced by stressors such as were used in this study. This argues that DIAP-2 function, at least, continues following such stress (such that its knock-down has an effect) and thus it is less likely to be an important explanation for the current effects (Kiessling, 2006).
The final possibility is perhaps the most interesting. The knock-down of DIAP1 leads to death, presumably through permitting low levels of ongoing (and otherwise repressed) caspase activation to function and any subsequent effect may depend on amplification, as active executioner caspases cleave and activate others. Therefore, knock-down of even one caspase in the cell may dampen this amplification so that cells survive. In contrast, the induction of apoptosis by stress may involve not only blockade of DIAP1 function (through the N-termini of Reaper, Hid, Grim and Sickle) but also another signal that amplifies caspase activation upstream of ARK. Such an upstream effect has been suggested by studies of the so-called 'GH3' region in these proteins, that appears to be required for death in Drosophila cells and can function to promote the mitochondrial pathway in vertebrate systems. Reaper, Hid, Grim, and probably Sickle are necessary for stress-induced apoptosis in Drosophila, and therefore their effects are likely to depend on ARK and ARK-DRONC interactions. Nevertheless, this line of reasoning suggests that they function not only to de-repress caspases (though blocking DIAP1), but also to do something else to bypass full dependence on DCP-1 and DRICE, perhaps by amplifying caspase activation at the level of the ARK-DRONC interaction. While speculative, this possibility is intriguing, and suggests that the induction of apoptosis in Drosophila may prove to be more complex than simple models indicate (Kiessling, 2006).
Caspase family proteases play important roles in the regulation of
apoptotic cell death. Initiator caspases are activated in response to death
stimuli, and they transduce and amplify these signals by cleaving and thereby
activating effector caspases. In Drosophila, the initiator caspase Nedd2-like caspase (Nc; previously called Dronc) cleaves and activates two short-prodomain caspases, Dcp-1
and Ice (previously Drice), suggesting these as candidate effectors of Nc
killing activity. dcp-1-null mutants are healthy and possess few
defects in normally occurring cell death. To explore roles for Ice in cell
death, an Ice null mutant was generated and characterized. Animals
lacking Ice show a number of defects in cell death, including those
that occur during embryonic development, as well as during formation of adult
eyes, arista and wings. Ice mutants exhibit subtle defects in the
destruction of larval tissues, and do not prevent destruction of salivary
glands during metamorphosis. Cells from Ice animals are also markedly
resistant to several stresses, including X-irradiation and inhibition of
protein synthesis. Mutations in Ice also suppress cell death that is
induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These
observations demonstrate that Ice plays an important non-redundant role as a
cell death effector. Finally, Ice participates in, but is
not absolutely required for, the non-apoptotic process of spermatid
differentiation (Muro, 2006; full text of article).
Post-eclosion elimination of the Drosophila wing epithelium was examined in vivo where collective 'suicide waves' promote sudden, coordinated death of epithelial sheets without a final engulfment step. Like apoptosis in earlier developmental stages, this unique communal form of cell death is controlled through the apoptosome proteins, Dronc and Dark, together with the IAP antagonists, Reaper, Grim, and Hid. Genetic lesions in these pathways caused intervein epithelial cells to persist, prompting a characteristic late-onset blemishing phenotype throughout the wing blade. This phenotype wase leveraged in mosaic animals to discover relevant genes. homeodomain interacting protein kinase (HIPK) was shown to be required for collective death of the wing epithelium. Extra cells also persisted in other tissues, establishing a more generalized requirement for HIPK in the regulation of cell death and cell numbers (Link, 2007).
Elimination of cells by programmed cell death (PCD) is a universal feature of development and aging. In both vertebrates and invertebrates, dying cells often progress through a stereotyped set of transformations referred to as apoptosis. In this form of PCD the nucleus condenses, and the collapsing cell corpse fragments into 'apoptotic bodies' that are engulfed by specialized phagocytes or neighboring cells. Apoptosis requires autonomous genetic functions within the dying cell, and extrinsic cues that elicit apoptosis have been investigated in numerous experimental models. Other forms of death are also thought to contribute during development and differ from apoptosis with respect to cellular morphology, mechanism, or mode of activation. These may include necrosis, characterized by swelling of the plasma membrane, or autophagic cell death, which is linked to extensive vacuolization in the cytoplasm. These forms of cell death can be caspase dependent or independent and may or may not be under deliberate genetic control (Link, 2007).
Two conserved protein families comprise central elements of the apoptotic machinery. Orthologous proteins represented by Ced4 in the nematode, Apaf1 in mammals, and Drosophila Ark (Dark) function as activating adaptors for CARD-containing apical caspases. During apoptosis, Ced4/Apaf1/Dark adaptors associate with pro-caspase partners (Ced3, Caspase 9, and Dronc) in a multimeric complex referred to as the 'apoptosome'. This complex is regulated by Bcl2 proteins, but apparently through a diverse group of mechanisms (Link, 2007).
Components of the Drosophila apoptosome have been genetically examined. dark and dronc are recessive, lethal genes. Both exert global functions during PCD and in stress-induced apoptosis. However, their roles in apoptosis are not absolute because rare cell deaths occurred in embryos lacking maternal and zygotic product of either gene. Elimination of dronc in the wing causes a unique, age-dependent phenotype associated with late-onset blemishing throughout the wing blade (Chew, 2004). This study shows that this progressive phenotype is characteristic for wing epithelia that lack apoptogenic functions and is caused by defects in a communal form of PCD where epithelial cells are collectively and rapidly eliminated. These findings were leveraged to discover additional genes required for PCD, and a limited set of loci, many of which were previously unknown to function in cell death, were recovered. This study establish that homeodomain interacting protein kinase (HIPK) is essential for coordinated death in the wing epithelium and, consistent with PCD functions in earlier developmental stages, regulates proper cell number in diverse tissue types (Link, 2007).
Wings mosaic for dronc- tissue exhibit normal morphology at eclosion but develop progressive, melanized blemishes with age (Chew, 2004). Similar methods were applied to determine whether lesions in other apoptogenic genes present a similar phenotype. After eclosion, wings mosaic for Df(H99), a deletion removing the apoptotic activators reaper (rpr), grim, and hid, were morphologically normal at eclosion, but over 3-7 d, melanized blemishes appeared at random throughout the wing (Link, 2007).
Likewise, homozygous driceDelta1 adult 'escapers' deficient for the effector caspase Drice also presented normal wings at eclosion but developed blemishes with age. Wings mosaic for dark82, a null allele of dark, were indistinguishable from wild-type (WT) at eclosion, but within 4 d developed wing blemishes. These late-onset blemishes became markedly more severe as animals aged. Similar yet less severe wing blemishes occurred in adults homozygous for darkCD4, a hypomorphic allele of dark. Together, these observations establish that late-onset progressive blemishing in mosaic wings is a characteristic phenotype shared among mutants in canonical PCD pathways (Link, 2007).
In the wing of newly eclosed adults, PCD removes the epithelium that forms the dorsal and ventral cuticles. To determine whether the cause of the blemish phenotype might trace to defective death in the wing epithelium, this tissue was examined in dark mutants. For these studies, wings of darkCD4 adults were prepared for light and electron microscopy. Histological analyses at the light level showed that on the first day of eclosion, the dorsal and ventral cuticles of WT animals became tightly merged with no intervening tissue evident between these layers. However, even 14 d after eclosion, cells and cell remnants remained situated between the dorsal and ventral cuticles in dark mutants. This 'undead' tissue was most easily visualized in lateral sections through melanized blemishes. Further examination of the persisting epithelium at the EM level showed evidence of intact cells soon after eclosion and ectopic cellular material 24 h after eclosion (Link, 2007).
To directly examine the death of wing epithelial cells in vivo, a transgenic nuclear DsRed reporter was used that driven by vestigial-Gal4 (vg:DsRed), allowing visualization of the fate of these cells soon after eclosion. Observations with this pan-epithelial marker in the wing confirmed earlier studies. Within 1 h of eclosion, intact epithelial cells are clearly present and regularly patterned throughout the wing. 1-2 h later (2-3 h after eclosion), the entire intervein epithelium disappears, manifested here by the abrupt loss of DsRed throughout the wing blade. Live, real-time imaging of the wing in newly eclosed adults revealed unexpected features associated with elimination of the intervein epithelium. Epithelial cells, labeled by nuclear fluorescence, were arranged in a regular, predictable pattern throughout the wing. Then, consistent with nuclear breakdown, fluorescence became redistributed throughout the cell followed by indications of blebbing and the appearance of fragmenting cells. Occasionally, weak fluorescence enclosed in cell corpses condensed to bright punctate bodies. This series of apoptogenic changes spread extremely rapidly throughout the epithelium, appearing here as a collective wave initiating from the peripheral edge and moving across the wing blade (Link, 2007).
Within just 4 min, virtually all nuclei (~450 cells) within a space of ~114 mm2 converted from viable to apoptotic morphology. The process involved tight coordination at the group level because the likelihood of a single cell apoptosing was clearly linked to similar behaviors by nearest neighboring cells over short time frames. Also, the direction and size of the cell death wave may not be fixed in every region of the wing, but centrally located cell groups were generally eliminated earlier (Link, 2007).
Unlike conventional examples of PCD in development, no indication was found that overt engulfment of apoptotic corpses occurred at the site of death. Instead, DsRed-labeled cell remnants were passively swept en masse toward the nearest wing vein where, apparently under hydrostatic pressure, cell debris streamed proximally toward the body through the wing or along the wing vein. Together, these observations describe a communal form of PCD that rapidly eliminates the wing epithelium through coordinated group behavior (Link, 2007).
The vg:DsRed reporter was used to track the fate of mosaic wing epithelia where mutant clones were induced. In sharp contrast to WT wings, abnormally persisting cells could be readily detected as patches of DsRed in the nuclei of epithelial cells in mosaic tissues. For example, wings mosaic for dronc- clones retained extensive patches of persisting DsRed-labeled cells. Here, cells and nuclei were readily detected 4 d after eclosion, and even at 11 d post-eclosion, extensive evidence of cell debris was seen (not depicted). Wings mosaic for the H99 deletion gave identical results. Likewise, adults mutated for dark exhibited persisting cells throughout the wing blade. Consistent with this, rare driceDelta1 escapers also showed evidence of persisting cells after eclosion. These observations link failures in PCD to progressive melanized wing blemishes, raising the possibility that other apoptogenic mutants might also produce this phenotype (Link, 2007).
Unlike previously described wing defects, which are congenital and evident at eclosion, the age-dependent phenotype described in in this study is characteristic of mutations in genes that function in canonical PCD pathways. Moreover, when the dosage of dronc was reduced by half in darkCD4 adults or if WT Dmp53 was removed from these same animals, melanized blemishes became far more severe. These genetic interactions are highly specific because wing defects were never observed in Dmp53- homozygotes or in dronc51 heterozygotes. Numerous other mutants showed no such effects in combination with a dark hypomorph. It was reasoned that, if genetically eliminated, additional regulators and effectors in PCD pathways should phenocopy wings mosaic for dark- or dronc- tissue. A collection of preexisting transposon mutants was screened to capture insertions that exhibit normal wings at eclosion but develop melanized blemishes with age. This strategy exploits the FLP/FRT system together with wing-specific drivers to interrogate animals bearing wing genotypes mosaic for clones of P element-derived lethal mutations. Progeny with mosaic wings were examined for late-onset wing blemishes at 1, 7, and 14 d post-eclosion. Over 1,000 lethal insertions were screened, representing 356 2nd chromosome mutations and 707 3rd chromosome mutations (Link, 2007).
The majority of insertions (87%) produced no visible defects as wing mosaics. 13% of insertions tested produced abnormalities, and these were scored for the phenotypic categories. Congenital defects including notched, blistered, or wrinkled wings occurred alone or occasionally as compound phenotypes. The candidate strains that developed wing blemishing were further subdivided based on phenotypic severity. Insertions in class A developed pronounced blemishes within a week of eclosion, whereas those in class B developed relatively light-colored patches between 1 and 2 wk after eclosion. Mutant lines exhibiting class A phenotypes were rare (~2%). All members of this class lacked blemishes at eclosion and displayed progressive blemishing occasionally associated with fragile and sometimes broken wings. A new allele of dark (l(2)SH0173) was recovered in this class, providing reassuring validation for the screening strategy. Some members among these classes exhibited congenital notches or blisters, but congenital blemishes present at eclosion were not found (Link, 2007).
Inverse PCR was applied to map or confirm insertion sites of many class A and B strains. In addition to darkl(3)SH0173, several mutations associated with genes previously implicated in PCD were isolated. For example, l(2)SH2275 contains an insertion 2 kb upstream of mir-14, a microRNA capable of modulating Rpr-induced cell death. Likewise, l(3)S048915 maps to the first intron of DIAP1 and may represent a hypermorphic allele at this locus. l(3)S055409 maps near misshapen, a gene implicated in cell killing triggered by Rpr or Eiger, the fly counterpart of TNF. Several insertions map in or near transcriptional or translational regulators that might alter the expression of cell death genes. For example, grunge (l(3)S146907), an Atrophin-like protein, functions as a transcriptional repressor, while belle (l(3)S097074) belongs to the DEAD-box family of proteins often implicated in translational regulation and RNA processing. A portion of the class A and B hits were also directly examined for defective PCD by applying the vg:DsRed reporter in mosaic wings. Of the 29 strains tested, 14 showed obvious evidence for persisting cells in the wing epithelium (Link, 2007).
Mutants identified that exhibit both blemishing and persisting cells are likely candidates for PCD genes. One strain, l(3)S134313, produced severe late-onset blemishing and a persisting cell phenotype. After mapping this insertion to the first intron of the HIPK, null alleles at this locus were produced (Link, 2007).
Two FRT-containing P element insertions flanking the coding region of HIPK were used to generate a novel deletion. PCR verified recombination between P elements, and 8 deletion strains were recovered. These validated alleles eliminate exons 4-12, removing over 92% of coding sequence in the predicted HIPK open reading frame. Deletions at the HIPK locus were uniformly lethal before the 3rd instar stage. However, zygotic HIPK is not essential to complete embryogenesis because ~70% of HIPK homozygotes hatch to 1st instar larvae. HIPKD1 was recombined on the FRT79 chromosome to generate adult wings mosaic for this allele, and like the original insertion, these animals also developed robust progressive blemishes and a persisting cell phenotype. Both phenotypes were more severe than the original P insertion, suggesting that the l(3)S134313 allele is hypomorphic for HIPK. These findings link loss of HIPK function to the query phenotypes, establishing that the action of HIPK is essential for post-eclosion PCD in the wing epithelium (Link, 2007).
Using general stains (acridine orange) or TUNEL methods, embryonic PCD was not overtly disturbed in HIPK mutants. To investigate the possibility of more subtle or specific phenotypes, the nervous system was examined using antibodies that label specific populations of neurons affected by the H99 deletion. Using anti-Kruppel antibody, it was confirmed that stage 14-15 WT embryos contained 9-12 Kruppel-positive cells in the Bolwig's Organ. However, a portion of animals lacking maternal HIPK contained as many as 15 cells per organ at a penetrance comparable to H99 animals, which are completely cell death defective. Neurons expressing dHb9, a homeodomain protein marking a subset of cells that persist in cell death-defective H99 embryos, was examined (Rogulja-Ortmann, 2007). In germline clones, distinct classes of dHb9 staining patterns emerged. A subset of animals exhibited extreme patterning defects. Other animals displayed a striking increase in dHb9-positive cell numbers when compared with WT embryos of the parental strain. These data establish that HIPK fundamentally regulates cell numbers in the nervous system, and because the same subpopulation of cells are affected by the H99 mutation, they implicate HIPK as a more general regulator of PCD (Link, 2007).
The pupal eye undergoes reorganization involving cell death of interommatidial cells after pupation. To determine if HIPK regulates cell death in the retina, whole eye clones were generated and the anti-Dlg (discs large) antibody was used to outline cell borders in dissected pupal eyes after pupation. Extra interommatidial cells were frequently retained in whole eye HIPK- clones. This phenotype is overtly similar to animals lacking the apical caspase Dronc and consistent with an essential role in retinal PCD (Link, 2007).
Elimination of the wing epithelium in newly eclosed adults is predictable, easily visualized, and experimentally tractable. The major histomorphologic events involve cell death, delamination, and clearance of corpses and cell remnants. Recent studies established that post-eclosion PCD is under hormonal control and involves the cAMP/PKA pathway (Kimura, 2004). While dying cells in the adult wing present apoptotic features (e.g., sensitivity to p35 and TUNEL positive), elimination of the epithelium is distinct from classical apoptosis in several important respects. (1) Unlike most in vivo models, overt engulfment of cell corpses does not occur at the site of death. Instead, dead or dying cells and their remnants are washed into the thoracic cavity via streaming of material along and through wing veins. (2) Extensive vacuolization is seen in ultrastructural analyses, which could indicate elevated autophagic activity. (3) Widespread and near synchronous death that occurs in this context defines an abrupt group behavior. The process affects dramatic change at the tissue level, causing wholesale loss of intervein cells and coordinated elimination of the entire layer of epithelium. Rather than die independently, these cells die communally, as if responding to coordinated signals propagated throughout the entire epithelium, perhaps involving intercellular gap junctions. This group behavior contrasts with canonical in vivo models where a single cell, surrounded by viable neighbors, sporadically initiates apoptosis (Link, 2007).
It has been proposed that an epithelial-to-mesenchymal transition (EMT) accounts for the removal of epithelial cells after eclosion (Kiger, 2007). Although the results do not exclude EMT associated changes in the newly eclosed wing epithelium, compelling lines of evidence establish that post-eclosion loss of the wing epithelium occurs by PCD in situ -- before cells are removed from the wing. First, before elimination, wing epithelial cells label prominently with TUNEL. Second, every mutation in canonical PCD genes so far tested failed to effectively eliminate the wing epithelium, and at least two of these were recovered in the screen described in this paper. Third, elimination of the wing epithelium was reversed by induction of p35, a broad-spectrum caspase inhibitor. Fourth, using time-lapse microscopy, condensing or pycnotic nuclei, followed by the rapid removal of all cell debris in time frames was detected that was not consistent with active migration. Instead, removal of cell remnants occurred by a passive streaming process, involving perhaps hydrostatic flow of the hemolymph (Link, 2007).
This study sampled over one fifth of all lethal genes and nearly 10% of all genes in the fly genome for the progressive blemish phenotype, a reliable indicator of PCD failure in the wing epithelium. Nearly half of the mutants that produced melanized wing blemishing also displayed a cell death-defective phenotype when examined with the vg:DsRed reporter. The precise link between these defects is unclear, but a likely explanation suggests that as the surrounding cuticle fuses, persisting cells, now deprived for nutrients and oxygen, become necrotic and may initiate melanization. Mutants could arrest at upstream steps, involving the specification or execution of PCD, or they might affect proper clearance of cell corpses from the epithelium. New alleles were recovered of dark (l(2)SH0173) and a likely hypermorph of thread (l(3)S048915), which provides reassuring validation of this prediction (Link, 2007).
By leveraging this distinct phenotype, novel cell death genes were captured, including the Drosophila orthologue of HIPK. Though first identified as an NK homeodomain binding partner, this gene is an essential regulator of PCD and cell numbers in diverse tissue contexts. Of the four mammalian HIPK genes, HIPK2, the predicted ortholog of Drosophila HIPK, has been placed in the p53 stress-response apoptotic pathway (D'Orazi, 2002; Hofmann, 2002; Di Stefano, 2004; Di Stefano, 2005), but whether the Drosophila counterpart similarly impacts this network is not yet known (Link, 2007).
Understanding the mechanism(s) by which dopaminergic (DAergic) neurons are eroded in Parkinson's disease (PD) is critical for effective therapeutic strategies. By using the binary tyrosine hydroxylase (TH)-Gal4/UAS-X RNAi Drosophila melanogaster system, it is reported that p53, basket and ICE gene knockdown in dopaminergic neurons prolong life span and locomotor activity in D. melanogaster lines chronically exposed to (1 microM) paraquat [PQ, oxidative stress (OS) generator] compared to untreated transgenic fly lines. Likewise, knockdown flies displayed higher climbing performance than control flies. Amazingly, gallic acid (GA) significantly protected DAergic neurons, ameliorated life span, and climbing abilities in knockdown fly lines treated with PQ compared to flies treated with PQ only. Therefore, silencing specific gene(s) involved in neuronal death might constitute an excellent tool to study the response of DAergic neurons to OS stimuli. It is proposed that a therapy with antioxidants and selectively 'switching off' death genes in DAergic neurons could provide a means for pre-clinical PD individuals to significantly ameliorate their disease condition (Ortega-Arellano, 2013).
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date revised: 10 February 2015
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