Key outstanding questions that need to be addressed are how autophagy and apoptosis pathways interact with each other, and whether common regulatory mechanisms exist between these two processes. We have shown here that six known cell death genes and the Ras–Raf–MAPK signaling pathway not only function in apoptosis but also act to regulate autophagy in D. melanogaster l(2)mbn cells. We cannot rule out the possibility that additional cell death genes that we screened may also function in autophagy but were not detected in our assay because of insufficient knockdown by RNAi, a long half-life of the corresponding proteins, and/or functional redundancy.
Consistent with our in vitro data, the involvement of Hid in autophagy regulation has been demonstrated in D. melanogaster.
Overexpression of Hid induced autophagy in the fat body, larval epidermis, midgut, salivary gland, Malpighian tubules, and trachea epithelium (Juhasz and Sass, 2005
). Further, expression of the constitutively active Ras form (RasV12
), which has been shown to inhibit Hid activity in apoptosis (Bergmann et al., 1998
), can also block Hid-induced autophagy (Juhasz and Sass, 2005
). In D. melanogaster
salivary glands, the Ras signaling pathway has also been shown to inhibit the autophagy process (Berry and Baehrecke, 2007
). Based on our loss-of-function findings and these previous gain-of-function studies, we speculate that the Ras–Raf–MAPK pathway acts upstream to inhibit Hid activity in autophagy.
Poor nutrition has a dramatic effect on egg production in D. melanogaster
. Flies fed on a protein-deprived diet showed an increase in cell death in germaria and midstage egg chambers (Drummond-Barbosa and Spradling, 2001
). These two stages have been proposed to serve as nutrient status checkpoints where defective egg chambers are removed before the investment of energy into them. The molecular mechanisms of germarium cell death are still largely unknown, and Daughterless, a helix-loop-helix transcription factor, was the only known regulator involved in cell death of germaria (Smith et al., 2002
). Nurse cell death during mid-oogenesis is also different from most developmental cell death in other D. melanogaster
tissues because apoptotic regulators such as rpr
, or grim
are not required for cell death in these cells (Peterson et al., 2007
). However, the activity of caspases, particularly Dcp-1, was shown to be required for mid-oogenesis cell death (Laundrie et al., 2003
; Baum et al., 2007
). Our findings implicate several additional genes, Dcp-1
, and Atg1
, in nutrient deprivation–induced cell death in the germarium, as well as during mid-oogenesis.
Other forms of cell death, such as autophagic cell death, have been proposed previously to be involved in the elimination of defective egg chambers during mid-oogenesis. Known signaling pathways, including the insulin and ecdysone pathways, have been shown to be required not only for the survival of nurse cells in mid-oogenesis; they are also known to regulate the autophagy process, supporting the notion that autophagy plays a role in mid-oogenesis cell death (Drummond-Barbosa and Spradling, 2001
; McCall, 2004
). Features of autophagy were observed during D. virilis
mid-oogenesis cell death as shown by monodansylcadaverine staining and transmission electron microscopy (Velentzas et al., 2007
). Our results using GFP-LC3 and LTG demonstrate that autophagy occurs in degenerating midstage egg chambers and also in germaria of nutrient deprived D. melanogaster
. We found that mutation of Atg7
results in a significant decrease of autophagy in dying mid-stage egg chambers and in germaria of starved flies, further supporting the presence of autophagy during these stages.
The role of autophagy in cell survival or cell death is still not well resolved and is likely to be context dependent. Our results show that autophagy contributes to the cell death process in the ovary. Loss of Atg7 or Atg1 activity in both dying midstage egg chambers and germaria leads to decreased TUNEL staining, which indicates a reduction in DNA fragmentation. Consistent results were observed previously in the larval midguts of Atg7
mutants, which also showed an inhibition of DNA fragmentation (Juhasz et al., 2007
). Interestingly, lack of autophagy function does not appear to affect nuclear DNA condensation in nurse cells. Nurse cells in degenerating stage 8 egg chambers of starved Atg7
mutants or Atg1
GLCs appeared to still have condensed nuclei, as shown by DAPI staining (). Thus, based on Atg7
mutant analyses, autophagy contributes to DNA fragmentation but not all aspects of nurse cell death. Future studies are required to determine how autophagy is connected to known pathways leading to DNA fragmentation and chromatin condensation during cell death.
The IAP family member Bruce was shown previously to repress cell death in the D. melanogaster
eye (Vernooy et al., 2002
). Bruce was also shown to protect against excessive nuclear condensation and degeneration, perhaps by limiting excessive caspase activity, during sperm differentiation (Arama et al., 2003
). Other IAP family members have been shown to bind caspases via a BIR domain and inhibit apoptosis (Riedl and Shi, 2004
). The presence of a BIR domain in Bruce suggests that it may also have caspase-binding activity. We found that lack of Bruce function resulted in an increase in both LTR and TUNEL staining in germaria and degenerating midstage egg chambers. Thus, the Bruce
mutant-degenerating phenotype in ovaries suggests that Bruce might function normally to restrain or limit caspase activity in this tissue. Because we found that Dcp-1 and Bruce are both required for the regulation of autophagy and DNA fragmentation in germaria and dying midstage egg chambers, it is possible that Bruce acts to bind and degrade Dcp-1 in nurse cells under nutrient-rich conditions. Future studies using epistasis and protein interaction analyses will be required to test this prediction. We cannot rule out the possibility that other IAP proteins, such as DIAP1, and other caspases also play a role during these stages. However, at least in response to starvation signals, Bruce and Dcp-1 play a nonredundant dual role in the regulation of autophagy and cell death in the ovary.
Numerous studies have linked caspase function to apoptosis, but recent findings indicate that caspases are also required for nonapoptotic processes including immunity and cell fate determination (for reviews see Kumar, 2004
; Kuranaga and Miura, 2007
). We have shown here that Dcp-1 is also required for starvation-induced autophagy. In the ovary, it appears that both apoptotic and autophagic events occur in the germaria and midstage egg chambers after nutrient deprivation. It is possible that Dcp-1 coordinates autophagy and apoptosis at these two nutrient status checkpoints to ensure elimination of defective egg chambers in the most efficient manner possible. Dcp-1
mutants exhibit intact nuclei in stage 8 defective egg chambers, which indicates a block in both DNA fragmentation and nuclear condensation, and further supports a dual regulatory role for Dcp-1 in mid-oogenesis cell death. Dcp-1 might function to induce autophagosome formation while coordinately acting upon alternate proteolytic targets to complete execution of apoptosis. Future studies to elucidate upstream regulators and downstream substrates of Dcp-1 in cells undergoing autophagy or apoptosis will help to establish the regulatory mechanisms governing the crosstalk between these two cellular processes. Given the multiple cellular effects associated with autophagy, our results also have important therapeutic implications for the use of modulators of caspase or IAP activity in the treatment of cancer and other diseases.