InteractiveFly: GeneBrief

grim : Biological Overview | Regulation | Developmental Biology | Effects of Mutation or Overexpression | References


Gene name - grim

Synonyms -

Cytological map position - 75C1--2

Function - activator of apoptosis

Keywords - apoptosis, programmed cell death

Symbol - grim

FlyBase ID: FBgn0015946

Genetic map position - 3-

Classification - Grim-Reaper family of cell death activators

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
Recent literature
Sarkissian, T., Arya, R., Gyonjyan, S., Taylor, B. and White, K. (2016). Cell death regulates muscle fiber number. Dev Biol [Epub ahead of print]. PubMed ID: 27131625
Summary:
Cell death can have both cell autonomous and non-autonomous roles in normal development. Previous studies have shown that the central cell death regulators grim and reaper are required for the developmentally important elimination of stem cells and neurons in the developing central nervous system (CNS). This study shows that cell death in the nervous system is also required for normal muscle development. In the absence of grim and reaper, there is an increase in the number of fibers in the ventral abdominal muscles in the Drosophila adult. This phenotype can be partially recapitulated by inhibition of cell death specifically in the CNS, indicating a non-autonomous role for neuronal death in limiting muscle fiber number. It was also shown that FGFs produced in the cell death defective nervous system are required for the increase in muscle fiber number. Cell death in the muscle lineage during pupal stages also plays a role in specifying fiber number. Altogetger, data suggests that FGFs from the CNS act as a survival signal for muscle FCs. Thus, proper muscle fiber specification requires cell death in both the nervous system and in the developing muscle itself.
BIOLOGICAL OVERVIEW

Grim encodes a protein required for programmed cell death in Drosophila. grim, maps between two previously identified cell death genes in this region: reaper (rpr) and head involution defective (hid). Expression of Grim RNA coincides with the onset of programmed cell death at all stages of embryonic development, whereas ectopic induction of grim triggers extensive apoptosis in both transgenic animals and in cell culture. Cell killing by Grim is blocked by coexpression of p35, a viral product that inactivates ICE-like proteases, and does not require the functions of rpr or hid. The predicted Grim protein shares an amino-terminal motif in common with Rpr. However, Grim is sufficient to elicit apoptosis in at least one context, where Rpr is not. The grim gene product might thus function in a parallel circuit of cell death signaling that ultimately activates a common set of downstream apoptotic effectors (Chen, 1996).

The Grim N-terminus induces apoptosis by disrupting IAP blockage of caspases; however, N-terminally-deleted Grim retains pro apoptotic activity. This study describes GH3, a 15 amino acid internal Grim domain absolutely required for its proapoptotic activity and sufficient to induce cell death when fused to heterologous carrier proteins. A GH3 homology region is present in the Drosophila proapoptotic proteins Reaper and Sickle. The GH3 domain and the homologous regions in Reaper and Sickle are predicted to be structured as amphipathic alpha-helixes. During apoptosis induction, Grim colocalizes with mitochondria and cytochrome c in a GH3-dependent but N-terminal- and caspase activity-independent manner. When Grim is overexpressed in vivo, both the N-terminal and the GH3 domains are equally necessary, and cooperate for apoptosis induction. The N-terminal and GH3 Grim domains thus activate independent apoptotic pathways that synergize to efficiently induce programmed cell death (Clavería, 2002).

Secondary structure prediction of the Grim protein has identified three regions with a very high probability of conforming to an a-helical structure. These regions have been termed GH1, GH2 and GH3, for Grim Helix 1, 2 and 3. The GH3 domain shows similarity to a region in Reaper and Sickle, both also predicted to conform as an a-helix. The homology region spans the 15 amino acids predicted to conform as an a-helix in Grim, with two 5 amino acid regions of high similarity flanking a 5 amino acid central region with lesser homology. Representation in a helical wheel projection of GH3 residues and of those in the Reaper and Sickle homology regions reveals the amphipathic nature of the predicted a-helices (Clavería, 2002).

Tests were performed to see whether GH3 is important for Grim proapoptotic function by assaying the cell killing ability of several Grim mutants altered in the GH3 domain in Drosophila SL2 cells. Wild-type (WT) Grim induces cell death when overexpressed in this assay. In contrast, a Grim mutant form with a 13 amino acid deletion that removes the GH3 residues most reliably predicted to form an a-helix only marginally induces apoptosis. A 5'-shifted 11 amino acid deletion, such that the 3' part of GH3 was respected, was less effective in eliminating the proapoptotic activity than the complete GH3 deletion. An internal deletion removing four amino acids commonly deleted in the two larger deletions (Delta89-92) also resulted in strong impairment of Grim killing ability, but to a lesser extent than the complete GH3 deletion. The relevance was tested of L89, a conserved residue within positions 89-92, whose hydrophobicity could be relevant for the amphipathic nature of the GH3 domain. A non-conservative replacement of L89 by glutamic acid (L89E) impaired GH3 killing ability nearly to the same level as the Delta89-92 mutant. Semi-conservative replacement of L89 by alanine (L89A) had a mild effect on Grim proapoptotic function. These results show that the GH3 domain is required for Grim proapoptotic function and that L89 is a functionally relevant residue in the domain (Clavería, 2002).

Since N-terminally deleted Grim can still bind IAPs, GH3 domain function could be related to Grim's ability to bind IAPs and inhibit their protective function. Deletion of the Grim N-terminal domain was found to lead to a slight reduction in its ability to bind DIAP2, however, deletion of the GH3 domain, either alone or in combination with the N-terminal deletion, does not impair Grim's ability to bind DIAP2. These results suggest that GH3 activity is unrelated to the IAP inhibitory Grim activity (Clavería, 2002).

To determine whether the GH3 domain could function as a proapoptotic motif itself, a Grim fragment containing the GH3 domain was fused to green fluorescent protein (GFP) as a carrier protein (GH3-GFP) and the ability of this fusion protein to induce apoptosis in Drosophila SL2 cells was tested. GH3-GFP induces cell death with the same efficiency as does the complete Grim-GFP fusion protein. The cell death observed is specific to GH3 domain activity, since it is largely abolished by an L-to-E mutation in the residue equivalent to Grim L89. In all cases, cell death was rescued by coexpression with the baculoviral caspase inhibitor p35. The GH3 domain is therefore sufficient to trigger a specific proapoptotic route in SL2 cells (Clavería, 2002).

Immunocytochemistry was used to identify Grim subcellular localization in Drosophila SL2 cells. In phases previous to any obvious apoptotic phenotype, Grim generally shows a diffuse distribution in the cytoplasm, but also displays rings of stronger Grim staining. Grim rings are mitochondria-associated, as indicated by the presence of a mitochondrial matrix marker, mainly inside the rings, but also in colocalization with strong Grim staining. Grim colocalization with cytochrome c is similar to that observed with the mitochondrial matrix marker but, in addition, Grim and cytochrome c show extensive colocalization in larger dots. These larger cytochrome c dots are not observed in untransfected cells: they increase in size and abundance as apoptosis progresses, and do not colocalize with a mitochondrial marker. No substantial cytochrome c release to cytosol was found. Instead, Grim promotes redistribution of cytochrome c signal in large dots in colocalization with Grim itself (Clavería, 2002).

Grim subcellular localization is dependent on the presence of an intact GH3 domain. GH3-deficient Grim shows no obvious organization in rings associated with mitochondria and does not colocalize with either the mitochondrial marker or cytochrome c. In contrast, deletion of Grim 2-14 amino acids does not alter subcellular distribution of the Grim protein, nor the changes induced in cytochrome c display (Clavería, 2002).

The relevance of the GH3 and N-terminal Grim domains was examined by overexpressing the Grim mutants in transgenic flies using the Gal4-UAS system. To drive Grim expression, the GMR-Gal4 line, which targets expression to the eye disc, and the MS1096-Gal4 line, which directs expression to the wing imaginal disc, were used. Overexpression of WT Grim with either driver causes total or partial lethality in all transgenic lines. These results suggested that leaky expression from both promoters in vital tissues produces sufficient cell death to block development. Surviving adult flies overexpressing WT Grim display a considerable reduction in eye size with the GMR driver, and wing agenesis plus notum reduction and elimination of macro- and microchaetae with the MS1096 driver (Clavería, 2002).

In contrast to these results, overexpression of a GH3-deleted Grim (Delta86-98) results in very low lethality, as well as rescue of the eye, notum and wing phenotypes. Elimination of amino acids 89-92 results in a lesser impairment of Grim killing ability, showing that the 3' part of the GH3 domain is important for proapoptotic function. In correlation with these results, the Delta89-92 mutant rescues the eye phenotype induced by WT Grim, but only partially rescues the more sensitive wing and notum phenotypes. Elimination of amino acids 83-93, which extends the deletion N-terminal to the putative helical domain, does not increase the rescue observed with the 89-92 deletion, suggesting that residues 5' of leucine 89 might be less important for proapoptotic function. The relevance of leucine 89 was again shown by the significant rescue of viability and of eye, notum and wing phenotypes in flies overexpressing the non-conservative L89E substitution. In contrast, the semi-conservative substitution L89A results in mild, but significant, impairment of Grim death induction and targeted tissue deletion (Clavería, 2002).

In accordance with the results observed in cultured cells, mutations of the GH3 domain impairs Grim proapoptotic activity in transgenic flies. Deletion of the N-terminal domain, in contrast to the results observed in cultured cells, results in highly significant elimination of Grim proapoptotic function in vivo. Both viability and appearance of the tissues targeted by the GMR and MS1096 drivers were rescued by the 2-14 deletion to a level similar to that observed for the GH3 deletion. Simultaneous deletion of the N-terminus and amino acids 89-92 of the GH3 domain results in even lower lethality and fewer alterations in the targeted tissues than those induced by each deletion in isolation (Clavería, 2002).

To determine whether the N-terminal and GH3 Grim domains can function independently of each other, tests were performed to see whether the simultaneous expression of independent 2-14- and GH3-deleted Grim proteins could induce apoptosis. Flies were generated carrying independent transgenes for 2-14 and 86-98 Grim deletion mutants driven by MS1096 expression. Whereas males carrying either protein alone showed little or no lethality and no alterations in wing development, double transgenic males simultaneously expressing Delta2-14 and Delta86-98 Grim mutants display severe lethality and reduced wings. Females did not display lethality in any situation, but frequently showed reduced wings in the double transgenics, although not in single transgenics. Functions of both the N-terminal and the GH3 domains are therefore essential for Grim activity in vivo and they independently activate specific death mechanisms that synergize to trigger apoptosis (Clavería, 2002).

Several lines of evidence point to the mitochondrial-cytochrome c pathway as the target of GH3 action. Grim associates with mitochondria in colocalization with cytochrome c and this activity resides in the GH3 domain. In vertebrate cells, Grim targets the mitochondria and induces cytochrome c release in a GH3-dependent and N-terminal-, caspase- and IAP-independent manner, suggesting functional conservation of the pathway. Interestingly, during apoptosis induction by Grim and Reaper in flies, changes in cytochrome c display are observed, rather than its free release to the cytosol as in vertebrates. Grim-expressing cells specifically show large cytoplasmic deposits of cytochrome c at sites where Grim itself is present, but other mitochondrial markers are not. The changes observed in the distribution of cytochrome c may result from its relocation from mitochondria to hypothetical specialized cytoplasmic structures involved in apoptosis induction. Alternatively, the apoptosome might be formed in the vicinity of the mitochondria, and cytochrome c deposits may constitute the remnants of damaged mitochondria, which have lost some of their constitutive components, but retain cytochrome c and Grim. The relevance of the cytochrome c proapoptotic pathway in Drosophila PCD is supported as well by the observation that elimination of Dark, a Drosophila homolog of Apaf-1 that mediates cytochrome c-primed apoptosis, impairs Reaper, Hid and Grim killing in flies. Even though no cytochrome c free release appears to take place in Drosophila cells, it is possible that a mechanism homologous to that of vertebrate cells is activated, but from different subcellular compartments (Clavería, 2002).

The involvement of the GH3 domain in a mitochondrial pathway and its predicted structure, an amphipathic a-helix, resemble the characteristics of the widespread proapoptotic BH3 domain. These similarities could be interpreted as functional homology between the two pathways; however, no association has been detected between Grim and either mammalian (Bcl-2 and Bcl-xL) or insect (Debcl) Bcl-2-family members, as would be expected for a BH3-containing protein. Rather than representing homologous proapoptotic pathways, BH3 and GH3 domains may have converged during evolution to a similar proapoptotic mechanism. Since BH3-containing proteins coexist in Drosophila with GH3-containing proteins, the two pathways may operate in alternative routes, or even cooperate in apoptosis induction, not only in Drosophila, but perhaps also in other species (Clavería, 2002).

Two independent pathways may thus be triggered by Grim; an IAP inhibitory pathway activated by the N-terminal domain, and a mitochondrial-cytochrome c route activated by the GH3 domain. Either pathway could be alternatively or simultaneously promoted by Grim, and the relevance of each may depend on cellular context. The presence of a GH3 homology region in Reaper and Sickle suggests functional conservation of this domain in at least these other two Drosophila proapoptotic proteins. In this context, it is important to consider that Reaper promotes cytochrome c release in a cell-free Xenopus egg extract and does not require the N-terminal domain for this function (Clavería, 2002).

Although Reaper, Hid, Sickle and Grim induce specific apoptotic pathways in vertebrate cells, and in the fly participate in highly conserved routes, such as the p53 and Ras-MAPK pathways, no homolog for these proteins has been yet identified in any other organism. The vertebrate Smac/Diablo protein may, however, represent a functional homolog of the IAP inhibitory pathway. Smac/Diablo can bind to and block the protective effect of IAPs. However, it is unlikely that Smac/Diablo represent homologs of the mitochondrial-cytochrome c pathway. Database searches have failed to identify any protein with sequence similarity to the GH3 domain but, given the restricted sequence conservation among, for example, BH3 family members, this does not exclude conservation of this pathway. Whether vertebrate proapoptotic proteins exist that represent direct or functional homologs of the GH3 proapoptotic activity thus remains to be determined (Clavería, 2002).


REGULATION

Promoter

Coordinated expression of cell death genes regulates neuroblast apoptosis

Properly regulated apoptosis in the developing central nervous system is crucial for normal morphogenesis and homeostasis. In Drosophila, a subset of neural stem cells, or neuroblasts, undergo apoptosis during embryogenesis. Of the 30 neuroblasts initially present in each abdominal hemisegment of the embryonic ventral nerve cord, only three survive into larval life, and these undergo apoptosis in the larvae. This study used loss-of-function analysis to demonstrate that neuroblast apoptosis during embryogenesis requires the coordinated expression of the cell death genes grim and reaper, and possibly sickle. These genes are clustered in a 140 kb region of the third chromosome and show overlapping patterns of expression. Expression of grim, reaper and sickle in embryonic neuroblasts is controlled by a common regulatory region located between reaper and grim. In the absence of grim and reaper, many neuroblasts survive the embryonic period of cell death and the ventral nerve cord becomes massively hypertrophic. Deletion of grim alone blocks the death of neuroblasts in the larvae. The overlapping activity of these multiple cell death genes suggests that the coordinated regulation of their expression provides flexibility in this crucial developmental process (Tan, 2011).

The patterns of developmental cell death are extremely complex and dynamic, and the fate of an individual cell probably represents the integration of multiple signaling pathways. Intrinsic pathways that define the identity and health of a cell, as well as extrinsic growth and survival pathways, are all likely to contribute to the life or death decision. In Drosophila, the transcriptional regulation of the RHG genes is an important output of the pathways that regulate cell death. The clustering of these genes to a single large genomic locus suggests that cell death signals can be integrated by regulatory sequences that control the coordinated expression of these genes (Tan, 2011).

This work shows that rpr, grim and skl are co-regulated to eliminate NBs during embryonic development. A genomic region important for this regulation was identified, located between rpr and grim. Identified genes or transcripts are surprisingly lacking in the 93 kb region between these genes, which is highly conserved between Drosophila species. This suggests that crucial cis-regulatory elements might be localized to this region (Tan, 2011).

The genomic region important for NB apoptosis was narrowed down to 22 kb. The number of NBs expressing grim and skl is significantly decreased in stage 14/15 embryos homozygous for a deletion of this region. The number of NBs expressing rpr is also affected, but to a smaller extent. Interestingly, there is still expression of grim, rpr and skl in NBs from stage 10 through stage 16. However, in the wild type, the number of NBs expressing all three genes increases at stage 14, presaging the loss of these cells by several hours. This increase in NBs expressing rpr, grim and skl is not seen in MM3 homozygous embryos. As overall grim, rpr and skl expression levels are not significantly altered in the mutants, the deleted regulatory sequences must control grim, rpr and skl expression in both a temporal- and tissue-specific manner, and baseline NB expression must be regulated by sequences outside of this region (Tan, 2011).

It is also interesting to note that the MM3 deletion has a more significant effect on grim and skl expression than on rpr expression. grim and skl are co-expressed in most cells of the stage 15 embryonic CNS, whereas rpr expression is less overlapping. As grim and skl lie 134 kb apart, on either side of rpr, long range enhancer interactions might regulate subsets of genes in this cell death locus. This type of long-range interaction has been implicated previously in the response to irradiation. Here again, a single regulatory region regulates multiple cell death genes at a great distance. The radiation responsive element activates hid and rpr, which are 200 kb apart, without having a major effect on the intervening gene grim. It will be important to examine higher order chromatin interactions to understand these long-range enhancer interactions (Tan, 2011).

The structure of the RHG cell death locus is conserved in other Drosophila species, but not obviously in more distantly related insect species, even though IAP inhibitory proteins are found in other species. This suggests that a coordinately regulated RHG gene cluster might be particularly important for Drosophila development (Tan, 2011).

As demonstrated by these studies, the individual roles for the cell death genes in developmental apoptosis are not well understood. Strong genetic and molecular data support a role for hid in regulating cell death in the embryonic head and in the developing eye. A role for grim in the death of microchaete glial cells has been described recently (Wu, 2010). Shown here is a specific role for grim in the elimination of the normal abdominal neuroblasts in the third instar larvae. As described in this work, rpr alone is not required for abdominal NB death in the embryo. However, this double mutant analysis demonstrates that rpr and grim must act together to eliminate these cells (Tan, 2011).

A possible role for hid in NB death cannot be eliminated, although no hid mRNA is detected in the VNC outside of the midline. A recent study suggests that rpr requires hid for efficient induction of apoptosis (Sandu, 2010). However, loss of hid does not result in increased NB survival, as would be expected if hid is required for rpr activity. Furthermore, loss of one copy of hid along with rpr and grim does not increase NB survival. Knockdown of rpr, grim and hid in NBs with an RHG miRNA also does not increase the number of surviving NBs over loss of rpr and grim. Further studies are needed to examine how hid and grim, rpr and skl differentially contribute to developmental apoptosis (Tan, 2011).

Although skl has similar pro-apoptotic activity to hid, grim and rpr, a role for skl in developmental apoptosis has not previously been demonstrated, owing to the lack of a skl mutation. This study shows that skl deletion alone does not alter NB death. In addition, it was found that deletion of one copy of skl along with grim and rpr slightly increase NB survival over deletion of grim and rpr alone. Deletion of skl and rpr, along with the NBRR in XR38/X20 also results in a larger VNC than deletion of rpr and the NBRR in XR38/H88, again suggesting a role for skl in cell death in this tissue. Additional double mutant analysis will be needed to test whether deletion of skl along with grim or rpr supports a role for skl in developmental apoptosis. Given the strongly overlapping expression of grim and skl in the embryonic VNC, it might be found that skl is redundant with grim in the CNS (Tan, 2011).

What are the developmental advantages of multiple cell death regulators? This work demonstrates that multiple RHG genes are activated within a single NB to activate apoptosis. In the salivary gland, hid and rpr, but not grim, are transcribed in the dying tissue, whereas the radiation response appears to involve hid, rpr and skl. These data suggest that the complex upstream regulation of cell death is likely to impact on different subsets of the RHG genes. If each gene is controlled by different combinations of enhancers this could provide greater flexibility in controlling the activation of cell death. In addition, a requirement for multiple cell death activators could ensure that cells are not killed inappropriately by the accidental activation of a single RHG gene (Tan, 2011).

The requirement for multiple RHG genes to kill cells might also reflect differences in the pro-apoptotic activities of these genes. For example, the RHG proteins show differential binding preferences to specific domains of the DIAP1 protein, and inhibit DIAP1 anti-apoptotic functions through different mechanisms. Additional activities, such as mitochondrial permeabilization, translational repression or interaction with other proteins such as Bruce or Scythe, also differ between RHG genes. Furthermore, post-translational regulation of particular RHG proteins might regulate their activity. This has been demonstrated for Hid; phosphorylation by MAPK downregulates the killing activity of Hid. It remains to be demonstrated whether different cell types have differential sensitivities to particular combinations of cell death genes when expressed at normal levels. This might provide yet another layer of control of the cell death process (Tan, 2011).

In sum, this work demonstrates that the regulation of NB apoptosis during development involves the coordinated temporal and spatial regulation of multiple cell death genes, controlled by a common regulatory region. By dissecting the RHG gene locus, specific genomic elements that regulate these genes in other developmentally important cell deaths are likely to be identified (Tan, 2011).

Transcriptional Regulation

An important issue in Metazoan development is to understand the mechanisms that lead to stereotyped patterns of programmed cell death. In particular, cells programmed to die may arise from asymmetric cell divisions. The mechanisms underlying such binary cell death decisions are unknown. A Drosophila sensory organ lineage is described that generates a single multidentritic neuron in the embryo. This lineage involves two asymmetric divisions. Following each division, one of the two daughter cells expresses the pro-apoptotic genes reaper and grim and subsequently dies. The protein Numb appears to be specifically inherited by the daughter cell that does not die. Numb is necessary and sufficient to prevent apoptosis in this lineage. Conversely, activated Notch is sufficient to trigger death in this lineage. These results show that binary cell death decision can be regulated by the unequal segregation of Numb at mitosis. This study also indicates that regulation of programmed cell death modulates the final pattern of sensory organs in a segment-specific manner (Orgogozo, 2002).

The vmd1a neuron is located within a cluster of five multidendritic (md) neurons in the ventral region of abdominal segments A1-A7. The vmd1a neuron can be distinguished from the other ventral md neurons (vmd1-4) using the B6-2-25 enhancer-trap marker. The origin of this vmd1a neuron is not known. vmd1-4 neurons are generated by the four vp1-4 external sensory (es) organ primary precursor (pI) cells. Each vp1-4 pI cell follows a lineage called the md-es lineage. This lineage is composed of four successive asymmetric cell divisions that generate five distinct cells, the four cells of the es organ at the position where the pI cell has formed and one md neuron that will then migrate to the ventral md cluster. In the md-es lineage, the membrane-associated protein Numb is segregated into one of the two daughter cells at each cell division. Numb establishes a difference in cell fate by antagonizing Notch in the Numb-receiving cell. Because no es organ is found in the vicinity of the vmd1a neuron, this neuron is probably not generated by a md-es lineage (Orgogozo, 2002).

rpr and grim, but not hid, are expressed specifically in the pIIa and pIIIb cells of the vmd1a lineage. By contrast, these genes are not expressed in cells of the vp1-4 lineages. In embryos in which a pIIb cell divides at the vp1 position in at least one abdominal segment, most segments contain a vmd1a pIIa-pIIb pair with one cell expressing rpr or grim. This cell is the pIIa cell fated to die. In some other segments, neither of these two cells accumulates rpr (25%) or grim (8%). Since the development of segments is not perfectly synchronous, it is assumed that this represents a situation preceding the onset of rpr and grim expression in the pIIa cell. In the remaining segments, a single Cut-positive cell is detected indicating that the pIIa cell has died. In those segments, expression of rpr and grim is never detected in the remaining pIIb cell (Orgogozo, 2002).

During the pIIb division, Numb was shown to segregate into the dorsal pIIb daughter cell. This cell is not fated to die and differentiates as a vmd1a neuron. By contrast, it could not be directly determined which one of the two pI daughter cells inherits Numb. Indeed, since the orientation of the vmd1a pI cell division is random, the pIIa and pIIb cells could not be identified from their relative positions. Nevertheless the vmd1a pIIa and pIIIb cells appear to generate ectopic shaft/socket and neuron/sheath cell pairs when cell death is prevented. In the md-es lineage, these cell pairs are the progeny of the cells that do not inherit Numb. This suggests that both the vmd1a pIIIb cell and the pIIa cell do not inherit Numb. Thus, Numb appears to segregate in the cells that do not die in the vmd1a lineage (Orgogozo, 2002).

The role of Numb was tested in regulating rpr and grim expression as well as cell death in the vmd1a lineage. In numb mutant embryos in which a secondary precursor cell divides at the vp1 position in at least one abdominal segment, it was observed that the two Cut-positive vmd1a pI daughter cells accumulate rpr or grim transcripts (54% of the segments for rpr, 52% for grim). In other segments a single Cut-positive pI daughter cell was found accumulating rpr or grim. In these segments one pI daughter cell has already died and the other one is undergoing apoptosis. These two phenotypes are not seen in wild-type embryos. Thus, in the absence of numb, both pI daughter cells undergo programmed cell death. Consistently, no Cut-positive cell is observed at the vmd1a position in numb mutant embryos in most segments. It is concluded that numb is required to inhibit the expression of rpr and grim and to prevent cell death in the pIIb cell (Orgogozo, 2002).

To test whether numb is sufficient to prevent cell death, the progeny of the vmd1a pI cell was analyzed in arm-Gal4 UAS-numb embryos that express high levels of Numb. In wild-type embryos in which a vp1 pIIIb cell is dividing in at least one segment, one or two Cut-positive cells are observed at the vmd1a position. In contrast, four Cut-positive cells are observed in 50% of the segments in arm-Gal4 UAS-numb embryos at the same stage. In 8 out of the 9 segments with four cells, two cells accumulating high levels of Pros and two cells accumulating low levels of Pros are seen, suggesting that these cells are two vmd1a neurons and two pIIIb cells. These data indicate that the pIIa cell death is inhibited and that the pIIa cell is transformed into a pIIb-like cell (Orgogozo, 2002).

Numb is known to function by antagonizing Notch activity. This therefore suggests that Notch promotes cell death in the vmd1a lineage and that Numb blocks this activity of Notch. Unfortunately, the strong effect of Notch loss-of-function alleles on the selection of the vmd1a pI cell means that it was not possible to test directly whether Notch is required for cell death in the vmd1a lineage. Therefore the conditional Notchts1 allele was used. However, when Notchts1 embryos are shifted to a restrictive temperature (31°C) soon after the specification of the vmd1a pI cell (i.e., at 13-14.5 hours after egg laying at 19°C), no significant reduction was seen in the number of rpr- or grim-expressing pIIa cells. A stronger Notchts1/Notch55e11 combination causes the appearance of additional vmd1a pI cells even at the permissive temperature (19°C). It is therefore not possible to determine whether an increase in the number of rpr- or grim-negative cells results from a lack of Notch-dependent apoptosis or from an excess of vmd1a pI cells due to reduced Notch signaling during lateral inhibition (Orgogozo, 2002).

Therefore a test was performed to see whether an activated form of Notch, Nintra, can promote the death of the pIIb cell when expressed around the time of the vmd1a pI cell division. In 6% of the segments from embryos in which at least one segment shows a dividing vp1 pIIb cell, rpr or grim transcripts accumulate in both vmd1a pI daughter cells. In other segments, a single Cut-positive cell remains at the vmd1a position and accumulates rpr or grim. These expression patterns are not seen in heat-shocked control embryos. Importantly, these observations are similar to those made in numb mutant embryos. Thus, both loss of numb activity and ectopic Notch signaling lead to transcriptional activation of pro-apoptotic genes in the pIIb cell. Finally, a similar effect of Nintra on rpr and grim expression is seen in the vmd1a pIIb daughter cells when Nintra expression was induced at a later stage, i.e., when the vmd1a pIIb cell is dividing. Together, these results indicate that Notch signaling is sufficient to promote cell death in the vmd1a lineage (Orgogozo, 2002).

In summary, the lineage generating the vmd1a neuron has been described. This lineage is composed of two asymmetric divisions following which one daughter cell undergoes apoptosis. These two binary cell death decisions are regulated by the unequal segregation of Numb at mitosis. Therefore, the data provide the first experimental evidence that alternative cell death decision can be regulated by the unequal segregation of a cell fate determinant. The conserved role of Numb and Notch in neuronal specification in flies and vertebrates suggests that Numb-mediated inhibition of Notch may play a similar role in regulating cell death decisions in vertebrates (Orgogozo, 2002).

Segment-specific prevention of pioneer neuron apoptosis by cell-autonomous, postmitotic Hox gene activity

In vertebrates, neurons often undergo apoptosis after differentiating and extending their axons. By contrast, in the developing nervous system of invertebrate embryos apoptosis typically occurs soon after cells are generated. The Drosophila dMP2 and MP1 pioneer neurons undergo segment-specific apoptosis at late embryonic stages, long after they have extended their axons and have performed their pioneering role in guiding follower axons. This segmental specificity is achieved by differential expression of the Hox gene Abdominal B, which in posterior segments prevents pioneer neuron death postmitotically and cell-autonomously by repressing the RHG-motif cell death activators reaper and grim. These results identify the first clear case of a cell-autonomous and anti-apoptotic role for a Hox gene in vivo. In addition, they provide a novel mechanism linking Hox positional information to differences in neuronal architecture along the anteroposterior axis by the selective elimination of mature neurons (Miguel-Aliaga, 2004).

How does Abd-B prevent the function of RHG-motif genes? It is likely that Abd-B prevents pioneer neuron apoptosis by repressing the transcription of, at least, rpr and grim. This idea is supported by four facts: (1) the H99 deletion is epistatic to (functions downstream of) Abd-B; (2) Abd-B is a transcription factor; (3) rprGAL4 is activated posteriorly in Abd-Bm mutants; (4) when misexpressed postmitotically, Abd-B can fully rescue both types of pioneer neurons. Given that loss of rpr is critical for anterior dMP2 survival, whereas loss of grim is critical for anterior MP1s, Abd-B must prevent the expression of at least these two cell death activators (Miguel-Aliaga, 2004).

The combined activity of RHG-motif genes is critical to the initiation of all cell death in the Drosophila embryo. These genes act in an additive manner. However, not all cell death activators are simultaneously expressed in every cell fated to die, and their specific expression patterns do not always overlap. Therefore, it is likely that they are differentially regulated by specific developmental signals. While Abd-B acts to repress rpr and grim function in posterior pioneer neurons, the developmental stimulus activating their expression in these neurons throughout the cord is currently unknown. Three developmental signals are known to regulate the function of RHG-motif genes in the Drosophila nervous system. The insect hormone ecdysone appears to be important for blocking cell death of certain peptidergic neurons during metamorphosis. However, the ecdysone-receptor complex has also been shown to promote cell death by activating rpr transcription in other tissues during Drosophila metamorphosis. While an embryonic ecdysone pulse occurs around the time when pioneer neurons die, preliminary experiments have failed to lend any support to an ecdysone-dependent activation of apoptosis in these neurons. The EGF-receptor/Ras/MAPK pathway has been shown to phosphorylate Hid protein, thereby preventing apoptosis of midline glial cells. However, neither Rpr nor Grim appear to be regulated in this fashion, and this model would not address the specific transcriptional activation of these genes in pioneer neurons. Lastly, Notch signaling has been described as resulting in both activation and inhibition of apoptosis. In Drosophila, recent studies have revealed that Notch can act cell-autonomously to induce apoptosis during final mitotic divisions both in the central and peripheral nervous systems. Although this Notch-induced developmental apoptosis is prevented in H99 mutant embryos, the molecular mechanisms by which activated Notch signaling results in the activation of IAP inhibitors are still unknown. Nevertheless, Notch signaling is unlikely to be relevant to dMP2 death, since it is not active in dMP2 neurons. It is, therefore, likely that an as yet unidentified factor is responsible for the activation of the apoptotic machinery in pioneer neurons. This factor could be Odd-skipped, given its specific expression in dMP2 and MP1 neurons. Because of the early role of odd in embryonic patterning, its possible postmitotic function in these neurons cannot be addressed using the currently available odd mutants (Miguel-Aliaga, 2004).

Developmental apoptosis in invertebrate embryos typically occurs shortly after cells are generated. In Drosophila, this has often precluded the identification of dying cells until apoptosis has been genetically prevented. Consequently, progress in identification of the mechanisms controlling apoptosis has been relatively slow, and little is known about the upstream pathways that initiate cell death in specific tissues or lineages. Furthermore, in the Drosophila VNC, studies have shown that apoptotic corpses are engulfed by glia, transported to the dorsal surface of the VNC and transferred to macrophages for final destruction. The molecular genetic mechanisms underlying this intriguing series of events are only just beginning to be unraveled. The identification of a late apoptotic event in two of the best-studied and least complex lineages in the Drosophila CNS, as well as the characterization of the dMP2-GAL4 line, should contribute to the elucidation of the mechanisms involved in both the developmental initiation and execution of apoptosis (Miguel-Aliaga, 2004).

Post-transcriptional Regulation of Grim

MicroRNAs (miRNAs) are small regulatory RNAs that are between 21 and 25 nucleotides in length and repress gene function through interactions with target mRNAs. The genomes of metazoans encode on the order of several hundred miRNAs, but the processes they regulate have been defined for only a few cases. New inhibitors of apoptotic cell death were sought by testing existing collections of P element insertion lines for their ability to enhance a small-eye phenotype associated with eye-specific expression of the Drosophila cell death activator Reaper. The Drosophila miRNA mir-14 has been identified as a cell death suppressor. Loss of mir-14 enhances Reaper-dependent cell death, whereas ectopic expression suppresses cell death induced by multiple stimuli. Animals lacking mir-14 are viable. However, they are stress sensitive and have a reduced lifespan. mir-14 mutants have elevated levels of the apoptotic effector caspase Ice, suggesting one potential site of action. Mir-14 also regulates fat metabolism. Deletion of mir-14 results in animals with increased levels of triacylglycerol and diacylglycerol, whereas increases in mir-14 copy number have the converse effect (Xu, 2003).

The two C. elegans miRNAs with known functions, lin-4 and let-7, are thought to regulate development by binding to the 3'untranslated region of target transcripts and thereby repressing the translation of their products. In these examples, the analysis of genetic interactions provides important clues as to the identity of targets. In the absence of this sort of information, it is difficult to predict miRNA targets in animals. This is because base pairing between the mature miRNA and its target is imperfect and the rules that govern which base pair interactions are important are unknown. Potential Mir-14 binding sites were sought in a number of apoptotic regulators, including Dronc, Rpr, Hid, and Grim. Potential target sites were identified in the transcripts of several genes, including Ice, Dcp-1, Scythe, SkpA, and Grim (however, the Grim target is present in the 3'UTR, which was absent in the GMR-Grim transgene). Of these, Ice, an apoptotic effector caspase, is of particular interest. Ice is required for at least some cell deaths and is activated by Dronc, which promotes cell death induced by Rpr, Hid, and Grim. Ice levels in adults were measured by using an anti-Ice antibody. Ice is elevated in mir-14Δ1 flies as compared to the wild-type, and this increase is suppressed in the presence of two copies of the mir-14-containing 3.4 kb genomic DNA fragment. Whereas these observations alone do not prove that Ice is a direct target of Mir-14, they do suggest that Ice is regulated, either directly or indirectly, by Mir-14 levels (Xu, 2003).

Principles of microRNA-target recognition: Targeting of bagpipe 3'UTR by microRNAs

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression in plants and animals. Although their biological importance has become clear, how they recognize and regulate target genes remains less well understood. This study systematically evaluates the minimal requirements for functional miRNA-target duplexes in vivo and classes of target sites with different functional properties are distinguished. Target sites can be grouped into two broad categories. 5' dominant sites have sufficient complementarity to the miRNA 5' end to function with little or no support from pairing to the miRNA 3' end. Indeed, sites with 3' pairing below the random noise level are functional given a strong 5' end. In contrast, 3' compensatory sites have insufficient 5' pairing and require strong 3' pairing for function. Examples and genome-wide statistical support is presented to show that both classes of sites are used in biologically relevant genes. Evidence is provided that an average miRNA has approximately 100 target sites, indicating that miRNAs regulate a large fraction of protein-coding genes and that miRNA 3' ends are key determinants of target specificity within miRNA families (Brennecke, 2005).

To improve understanding of the minimal requirements for a functional miRNA target site, use was made of a simple in vivo assay in the Drosophila wing imaginal disc. A miRNA was expressed in a stripe of cells in the central region of the disc and its ability to repress the expression of a ubiquitously transcribed enhanced green fluorescent protein (EGFP) transgene containing a single target site in its 3' UTR was assessed. The degree of repression was evaluated by comparing EGFP levels in miRNA-expressing and adjacent non-expressing cells. Expression of the miRNA strongly reduced EGFP expression from transgenes containing a single functional target site (Brennecke, 2005).

In a first series of experiments it was asked which part of the RNA duplex is most important for target regulation. A set of transgenic flies was prepared, each of which contained a different target site for miR-7 in the 3' UTR of the EGFP reporter construct. The starting site resembled the strongest bantam miRNA site in its biological target hid and conferred strong regulation when present in a single copy in the 3' UTR of the reporter gene. The effects were tested of introducing single nucleotide changes in the target site to produce mismatches at different positions in the duplex with the miRNA (note that the target site mismatches were the only variable in these experiments). The efficient repression mediated by the starting site was not affected by a mismatch at positions 1, 9, or 10, but any mismatch in positions 2 to 8 strongly reduced the magnitude of target regulation. Two simultaneous mismatches introduced into the 3' region had only a small effect on target repression, increasing reporter activity from 10% to 30%. To exclude the possibility that these findings were specific for the tested miRNA sequence or duplex structure, the experiment was repeated with miR-278 and a different duplex structure. The results were similar, except that pairing of position 8 was not important for regulation in this case. Moreover, some of the mismatches in positions 2-7 still allowed repression of EGFP expression up to 50%. Taken together, these observations support previous suggestions that extensive base-pairing to the 5' end of the miRNA is important for target site function (Brennecke, 2005).

Next the minimal 5' sequence complementarity necessary to confer target regulation was determined. The core of 5' sequence complementarity essential for target site recognition is referred to as the 'seed'. All possible 6mer, 5mer, and 4mer seeds complementary to the first eight nucleotides of the miRNA were tested in the context of a site that allowed strong base-pairing to the 3' end of the miRNA. The seed was separated from a region of complete 3' end pairing by a constant central bulge. 5mer and 6mer seeds beginning at positions 1 or 2 are functional. Surprisingly, as few as four base-pairs in positions 2-5 confers efficient target regulation under these conditions, whereas bases 1-4 are completely ineffective. 4mer, 5mer, or 6mer seeds beginning at position 3 are less effective. These results suggest that a functional seed requires a continuous helix of at least 4 or 5 nucleotides and that there is some position dependence to the pairing, since sites that produce comparable pairing energies differ in their ability to function. These experiments also indicate that extensive 3' pairing of up to 17 nucleotides in the absence of the minimal 5' element is not sufficient to confer regulation. Consequently, target searches based primarily on optimizing the extent of base-pairing or the total, and ranking miRNA target sites according to overall complementarity or free energy of duplex formation might not reflect their biological activity (Brennecke, 2005).

To determine the minimal lengths of 5' seed matches that are sufficient to confer regulation alone, single sites were tested that pair with eight, seven, or six consecutive bases to the miRNA's 5' end, but that do not pair to its 3' end. Surprisingly, a single 8mer seed (miRNA positions 1-8) is sufficient to confer strong regulation by the miRNA. A single 7mer seed (positions 2-8) is also functional, although less effective. The magnitude of regulation for 8mer and 7mer seeds is strongly increased when two copies of the site are introduced in the UTR. In contrast, 6mer seeds show no regulation, even when present in two copies. Comparable results have been reported for two copies of an 8mer site with limited 3' pairing capacity in a cell-based assay. These results do not support a requirement for a central bulge (Brennecke, 2005).

From these experiments it is concluded that (1) complementarity of seven or more bases to the 5' end miRNA is sufficient to confer regulation, even if the target 3' UTR contains only a single site; (2) sites with weaker 5' complementarity require compensatory pairing to the 3' end of the miRNA in order to confer regulation, and (3) extensive pairing to the 3' end of the miRNA is not sufficient to confer regulation on its own without a minimal element of 5' complementarity (Brennecke, 2005).

While recognizing that there is a continuum of base-pairing quality between miRNAs and target sites, the experiments presented here suggest that sites that depend critically on pairing to the miRNA 5' end (5' dominant sites) can be distinguished from those that cannot function without strong pairing to the miRNA 3' end (3' compensatory sites). The 3' compensatory group includes seed matches of four to six base-pairs and seeds of seven or eight bases that contain G:U base-pairs, single nucleotide bulges, or mismatches (Brennecke, 2005).

It is useful to distinguish two subgroups of 5' dominant sites: those with good pairing to both 5' and 3' ends of the miRNA (canonical sites) and those with good 5' pairing but with little or no 3' pairing (seed sites). Seed sites are considered to be those where there is no evidence for pairing of the miRNA 3' end to nearby sequences that is better than would be expected at random. The possibility cannot be excluded that some sites identified as seed sites might be supported by additional long-range 3' pairing. Computationally, this is always possible if long enough loops in the UTR sequence are allowed. Whether long loops are functional in vivo remains to be determined (Brennecke, 2005).

Canonical sites have strong seed matches supported by strong base-pairing to the 3' end of the miRNA. Canonical sites can thus be seen as an extension of the seed type (with enhanced 3' pairing in addition to a sufficient 5' seed) or as an extension of the 3' compensatory type (with improved 5' seed quality in addition to sufficient 3' pairing). Individually, canonical sites are likely to be more effective than other site types because of their higher pairing energy, and may function in one copy. Due to their lower pairing energies, seed sites are expected to be more effective when present in more than one copy (Brennecke, 2005).

Most currently identified miRNA target sites are canonical. For example, the hairy 3' UTR contains a single site for miR-7, with a 9mer seed and a stretch of 3' complementarity. This site has been shown to be functional in vivo , and it is strikingly conserved in the seed match and in the extent of complementarity to the 3' end of miR-7 in all six orthologous 3' UTRs (Brennecke, 2005).

Although seed sites have not been previously identified as functional miRNA target sites, there is some evidence that they exist in vivo. For example, the Bearded (Brd) 3' UTR contains three sequence elements, known as Brd boxes, that are complementary to the 5' region of miR-4 and miR-79. Brd boxes have been shown to repress expression of a reporter gene in vivo, presumably via miRNAs; expression of a Brd 3' UTR reporter is elevated in dicer-1 mutant cells, which are unable to produce any miRNAs. All three Brd box target sites consist of 7mer seeds with little or no base-pairing to the 3' end of either miR-4 or miR-79. The alignment of Brd 3' UTRs shows that there is little conservation in the miR-4 or miR-79 target sites outside the seed sequence, nor is there conservation of pairing to either miRNA 3' end. This suggests that the sequences that could pair to the 3' end of the miRNAs are not important for regulation as they do not appear to be under selective pressure. This makes it unlikely that a yet unidentified Brd box miRNA could form a canonical site complex (Brennecke, 2005).

The 3' UTR of the HOX gene Sex combs reduced (Scr) provides a good example of a 3' compensatory site. Scr contains a single site for miR-10 with a 5mer seed and a continuous 11-base-pair complementarity to the miRNA 3' end. The miR-10 transcript is encoded within the same HOX cluster downstream of Scr, a situation that resembles the relationship between miR-iab-5p and Ultrabithorax in flies and miR-196/HoxB8 in mice. The predicted pairing between miR-10 and Scr is perfectly conserved in all six drosophilid genomes, with the only sequence differences occurring in the unpaired loop region. The site is also conserved in the 3' UTR of the Scr genes in the mosquito, Anopheles gambiae, the flour beetle, Tribolium castaneum, and the silk moth, Bombyx mori. Conservation of such a high degree of 3' complementarity over hundreds of millions of years of evolution suggests that this is likely to be a functional miR-10 target site. Extensive 5' and 3' sequence conservation is also seen for other 3' compensatory sites, e.g., the two let-7 sites in lin-41 or the miR-2 sites in grim and sickle (Brennecke, 2005).

Several families of miRNAs have been identified whose members have common 5' sequences but differ in their 3' ends. In view of the evidence that 5' ends of miRNA are functionally important, and in some cases sufficient, it can be expected that members of miRNA families may have redundant or partially redundant functions. According to this model, 5' dominant canonical and seed sites should respond to all members of a given miRNA family, whereas 3' compensatory sites should differ in their sensitivity to different miRNA family members depending on the degree of 3' complementarity. This is being tested using the wing disc assay with 3' UTR reporter transgenes and overexpression constructs for various miRNA family members (Brennecke, 2005).

miR-4 and miR-79 share a common 5' sequence that is complementary to a single 8mer seed site in the bagpipe 3' UTR. The 3' ends of the miRNAs differ. miR-4 is predicted to have 3' pairing at approximately 50% of the maximally possible level (~10.8 kcal/mol), whereas the level of 3' pairing for miR-79 is approximately 25% maximum (~6.1 kcal/mol), which is below the average level expected for random matches. Both miRNAs repressed expression of the bagpipe 3' UTR reporter, regardless of the 3' complementarity. This indicates that both types of site are functional in vivo and suggests that bagpipe is a target for both miRNAs in this family (Brennecke, 2005).

To test whether miRNA family members can also have non-overlapping targets, 3' UTR reporters were used of the pro-apoptotic genes grim and sickle, two recently identified miRNA targets. Both genes contain K boxes in their 3' UTRs that are complementary to the 5' ends of the miR-2, miR-6, and miR-11 miRNA family. These miRNAs share residues 2-8 but differ considerably in their 3' regions. The site in the grim 3' UTR is predicted to form a 6mer seed match with all three miRNAs, but only miR-2 shows the extensive 3' complementarity that would be needed for a 3' compensatory site with a 6mer seed to function (~19.1 kcal/mol, 63% maximum 3' pairing, versus ~10.9 kcal/mol, 46% maximum, for miR-11 and ~8.7 kcal/mol, 37% maximum, for miR-6). Indeed, only miR-2 is able to regulate the grim 3' UTR reporter, whereas miR-6 and miR-11 are non-functional (Brennecke, 2005).

The sickle 3' UTR contains two K boxes and provides an opportunity to test whether weak sites can function synergistically. The first site is similar to the grim 3' UTR in that it contains a 6mer seed for all three miRNAs but extensive 3' complementarity only to miR-2. The second site contains a 7mer seed for miR-2 and miR-6 but only a 6mer seed for miR-11. miR-2 strongly downregulates the sickle reporter, miR-6 has moderate activity (presumably via the 7mer seed site), and miR-11 has nearly no activity, even though the miRNAs were overexpressed. The fact that a site is targeted by at least one miRNA argues that it is accessible (e.g., miR-2 is able to regulate both UTR reporters), and that the absence of regulation for other family members is due to the duplex structure. These results are in line with what would be expected based on the predicted functionality of the individual sites, and indicate that the model of target site functionality can be extended to UTRs with multiple sites. Weak sites that do not function alone also do not function when they are combined (Brennecke, 2005).

To show that endogenous miRNA levels regulate all three 3' UTR reporters, EGFP expression was compared in wild-type cells and dicer-1 mutant cells, which are unable to produce miRNAs. dicer-1 clones did not affect a control reporter lacking miRNA binding sites, but showed elevated expression of a reporter containing the 3' UTR of the previously identified bantam miRNA target hid. Similarly, all 3' UTR reporters above were upregulated in dicer-1 mutant cells, indicating that bagpipe, sickle, and grim are subject to repression by miRNAs expressed in the wing disc. Taken together, these experiments indicate that transcripts with 5' dominant canonical and seed sites are likely to be regulated by all members of a miRNA family. However, transcripts with 3' compensatory sites can discriminate between miRNA family members (Brennecke, 2005).

Experimental tests such as those presented in this study and the observed evolutionary conservation suggest that all three types of target sites are likely to be used in vivo. To gain additional evidence the occurrence of each site type was examined in all Drosophila 3' UTRs. Use was made of the D. pseudoobscura genome, the second assembled drosophilid genome, to determine the degree of site conservation for the three different site classes in an alignment of orthologous 3' UTRs. From the 78 known Drosophila miRNAs, a set of 49 miRNAs with non-redundant 5' sequences was chosen. Whether sequences complementary to the miRNA 5' ends are better conserved than would be expected for random sequences was tested. For each miRNA, a cohort of ten randomly shuffled variants was constructed. To avoid a bias for the number of possible target matches, the shuffled variants were required to produce a number of sequence matches comparable (±15%) to the original miRNAs for D. melanogaster 3' UTRs. 7mer and 8mer seeds complementary to real miRNA 5' ends were significantly better conserved than those complementary to the shuffled variants. Conserved 8mer seeds for real miRNAs occur on average 2.8 times as often as seeds complementary to the shuffled miRNAs. For 7mer seeds this signal was 2:1, whereas 6mer, 5mer, and 4mer seeds did not show better conservation than expected for random sequences. To assess the validity of these signals and to control for the random shuffling of miRNAs, this procedure was repeated with 'mutant' miRNAs in which two residues in the 5' region were changed. There was no difference between the mutant test miRNAs and their shuffled variants. This indicates that a substantial fraction of the conserved 7mer and 8mer seeds complementary to real miRNAs identify biologically relevant target sites (Brennecke, 2005).

Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development: Differential posttranscriptional repression of the proapoptotic factors hid, grim, reaper, and sickle

MicroRNAs are small noncoding RNAs that control gene function posttranscriptionally through mRNA degradation or translational inhibition. Much has been learned about the processing and mechanism of action of microRNAs, but little is known about their biological function. Injection of 2′O-methyl antisense oligoribonucleotides (2'OM-ORNs) into early Drosophila embryos leads to specific and efficient depletion of microRNAs and thus permits systematic loss-of-function analysis in vivo. Twenty-five of the forty-six embryonically expressed microRNAs show readily discernible defects; pleiotropy is moderate and family members display similar yet distinct phenotypes. Processes under microRNA regulation include cellularization and patterning in the blastoderm, morphogenesis, and cell survival. The largest microRNA family in Drosophila (miR-2/6/11/13/308) is required for suppressing embryonic apoptosis; this is achieved by differential posttranscriptional repression of the proapoptotic factors hid, grim, reaper, and sickle. These findings demonstrate that microRNAs act as specific and essential regulators in a wide range of developmental processes (Leaman, 2005).

miR-2/13 and miR-6 depletion results in catastrophic apoptosis: Embryos injected with miR-2/13 and miR-6 antisense 2′OM-ORNs fail to differentiate normal internal and external structures. At the end of embryogenesis, the embryos fall apart on touch, and no cuticle is recovered. To determine the onset of these problems, blastoderm embryos were examined, and it was found that cellularization and early pattern formation along the anteroposterior axis occur normally for both miRNAs, indicating that early fating and morphogenesis are intact. Interestingly, in miR-6, but not miR-2/13 depleted embryos, pole cell formation at the posterior end is disrupted (Leaman, 2005).

One possible cause of the catastrophic defects observed in miR-2/13 and miR-6 depleted embryos is excessive and widespread apoptosis. In both miR-2/13 and miR-6 antisense injected embryos, the number of apoptotic cells is greatly increased compared to wild-type by stage 13. Notably, the overall morphology of miR-6 depleted embryos is much more affected than that of miR-2/13 depleted embryos. miR-6 depleted embryos are generally smaller in size and have fewer and abnormally large (para-) segments, suggesting greater excess or earlier onset of apoptosis (Leaman, 2005).

To determine the specificity of the effects of miR-6 and miR-2/13 antisense injections, genomic rescue experiments were carried out. Embryos ubiquitously overexpressing mir-6 or mir-2 (Actin-Gal4;UAS-mir6-3/2b-2) show normal cell-death patterns. When injected with miR-6 or miR-2/13 antisense, they show significant rescue of miR-6 antisense by mir-6, with respect to both cell death and morphology, and of miR-2/13 antisense by mir-2. Interestingly, crossrescue of miR-6 antisense by mir-2 overexpression and of miR-2/13 antisense by mir-6 is weak (Leaman, 2005).

The miRNA sequence family miR-6 and miR-2/13 belong to has two additional members, miR-11 and miR-308. Depletion of miR-11 results in a moderate and of miR-308 in a mild increase in apoptosis in midembryogenesis. Thus, for all members of the miR-2 family, antisense-induced depletion results in excess embryonic cell death, but with marked differences in phenotypic strength. This differential could be due to differences in expression level or to sequence divergence and thus differential interaction with target mRNAs (Leaman, 2005).

The miR-2 family regulates cell survival by translational repression of proapoptotic factors: In Drosophila, three pathways are known to control caspase activity. The main control is thought to come from the proapoptotic factors Hid, Grim, and Reaper (Rpr), which are transcriptionally activated in response to a range of natural and toxic conditions; they promote caspase activation through inhibition of the caspase inhibitor Diap1. The three factors appear to act independently, with each being sufficient to drive apoptosis. When miR-2/13 and miR-6 antisense 2′OM-ORNs are injected into embryos deficient for the hid, grim, and rpr genes (H99 deficiency), they are unable to trigger apoptosis, indicating that these miRNAs act through hid, grim, and/or rpr (Leaman, 2005).

To determine whether the regulation of the three proapoptotic factors occurs at the transcriptional or at the posttranscriptional level, their RNA expression was examined in miR-2/13 and miR-6 depleted embryos using in situ hybridization and quantitative PCR. No significant increase was found in the expression level or broadening of the pattern compared to control embryos for any of three transcripts, either at embryonic stage 13 or 1 hr earlier at embryonic stage 12. By contrast, the protein expression of Hid is dramatically increased in miR-6 depleted embryos and modestly in miR-2/13 depleted embryos. These results strongly argue against a transcriptional and in favor of a posttranscriptional regulation of the proapoptotic factors by miR-2/13 and miR-6 (Leaman, 2005).

To test this directly, two existing translation control assays were adapted to the embryonic paradigm. In the first assay, full-length 3′UTRs are fused to a ubiquitously transcribed sensor (tub-GFP); transgenic embryos are injected with sense or antisense 2′OM-ORNs, and GFP fluorescence is measured. The 3′UTRs of hid, grim, rpr, and sickle (skl, a structurally related but less potent proapoptotic factor display marked differences in sensor expression, with rpr showing no expression, hid and skl low uniform expression, and grim strong and spatially modulated expression, indicating that these proapoptotic factors experience quite different levels of translation control. To gauge the efficacy of the assay, hid GFP sensor embryos were injected with bantam antisense 2′OM-ORNs, and mild but statistically significant derepression of GFP expression was found as compared to control, consistent with the weak cell-death phenotype of bantam depleted embryos. Antisense injection of miR-2 family members reveals strong derepression of the hid GFP sensor by miR-6 antisense, but not by miR-2/13, 11, or 308 antisense. Conversely, the grim GFP sensor shows significant derepression as a result of miR-2/13, 11, and 308, but not miR-6 depletion. Finally, the skl GFP sensor shows significant derepression for all four family members (Leaman, 2005).

To assess effects on rpr, a second, more sensitive assay was developed that employs transient expression of a dual-luciferase vector in injected embryos. For initial comparison with the GFP assay, a hid luciferase sensor was tested against the entire miR-2 family and the same profile was found. The rpr luciferase sensor shows strong derepression in miR-6 and 2/13, moderate derepression in miR-11, and no significant effect in miR-308 depleted embryos. Thus, the 3′UTRs of all four proapoptotic factors are subject to translational repression by the miR-2 family, but each miRNA displays a distinct interaction profile. The interaction preferences correlate well with the observed differences in phenotype: miR-6 has the most severe death phenotype and is the only family member to regulate hid, the factor with the broadest expression and the strongest proapoptotic effect. mir-2/13 and miR-11 have the same overall profile, but they differ in the strength of their interaction with rpr and show a corresponding differential in phenotypic strength. Finally, miR-308, which has the mildest death phenotype, interacts only with the weakly proapoptotic skl and with grim (Leaman, 2005).

The differences in target interaction profile between the miR-2 family members are pronounced and do not merely reproduce differences in the strength or onset of miRNA expression. This suggests that differential pairing outside the 5′ core sequence shared by all members has an important role in target selection. Computational predictions indicate that miR-2 family binding sites are present in the 3′UTRs of all four proapoptotic factors: rpr and grim have one, hid and skl two predicted sites. All six miRNA target sites lie in sequence blocks that are conserved between the six sequenced Drosophilid species, spanning an evolutionary distance of 40 Myr. Interestingly, for all sites, absolute conservation extends well beyond the bases complementary to the 5′ core of the miRNA and includes adjacent stretches suitable for pairing with the 3′ end. All but one of the sites show Watson-Crick pairing with miRNA positions 2-7 and variable pairing at the 3′ end. One of the hid sites (hid468) has a mismatch in the core but shows strong pairing with miR-6 at the 3′ end. The rules for 3′ pairing between miRNAs and their targets are not yet well understood, but it is clear that the miR-2 family members differ considerably in their ability to form 3′ matches with the six target sites. Further experimentation will be required to better understand how the observed differences in regulatory effect relate to differences in sequence pairing (Leaman, 2005).

Protein Interactions

Assay of Grim function in mammalian cells: Grim can be processed as a consequence of caspase proteolytic activity

Genetic studies have shown that grim is a central genetic switch of programmed cell death in Drosophila; however, homologous genes have not been described in other species, nor has its mechanism of action been defined. grim expression is shown to induce apoptosis in mouse fibroblasts. Cell death induced by grim in mammalian cells involves membrane blebbing, cytoplasmic loss and nuclear DNA fragmentation. The conserved N-terminal domain is not required for either the initiation or the execution of apoptosis by Grim. Grim-induced apoptosis is blocked by both natural and synthetic caspase inhibitors. Grim itself shows caspase-dependent proteolytic processing of its C-terminus in vitro. Three clustered aspartate residues near the Grim C-terminus (positions 126, 128 and 129) could then be used by a caspase as a target sequence. The downshift predicted by digestion at these sites is between 1 and 1.3 kDa; however, the observed size for the short form is ~4 kDa less than that observed for the intact protein. Nevertheless, C-terminal deletions of the protein provoke downshifts greater than expected, which are in the range of the observed downshift for the proteolysed form of the protein. These results show that besides activating caspases, Grim itself can be processed as a consequence of caspase proteolytic activity. Even though inhibitors of apoptosis OpIAP and DEVD.cho do not block Grim-induced apoptosis, they are both functionally active in repressing endogenous caspases in either treated or transfected cells, since they prevented Grim cleavage. Therefore, it is likely that caspases insensitive to OpIAP and/or DEVD.cho are sufficient to achieve Grim-induced apoptosis (Clavería, 1998).

Grim-induced death is antagonized by bcl-2 in a dose-dependent manner; neither Fas signaling nor p53 are required for Grim pro-apoptotic activity. The sensitivity of the grim-induced death to bcl-2 levels suggests that Grim acts by activating a mitochondrial pathway. To determine the site of Grim action, its subcellular localization was explored in transfected cells prior to the time they show morphological symptoms of apoptosis. Pre-apoptotic fibroblasts simultaneously show Grim in two different cytoplasmic localizations: diffuse cytosolic and a punctate pattern. However, while Grim-transfected cells show an almost exclusive cytosolic localization, cells treated with cell-permeable broad specificity caspase inhibitor zVAD.fmk show predominantly the punctate pattern. The same pattern is observed in Grim-expressing p35-rescued cells. Co-localization of Grim protein with a mitochondrial-specific antibody shows that the punctate pattern corresponds to mitochondria. It is concluded that Grim localizes initially in the cytoplasm but accumulates progressively in the mitochondria, correlating with apoptosis progression. It is possible that Grim translocation to mitochondria is the event that triggers the apoptotic pathway. In that case, the increased mitochondrial localization of Grim in zVAD-treated cells would be the result of the apoptosis blockade by zVAD at a point downstream of Grim incorporation in the mitochondria. These results show that Drosophila Grim induces death in mammalian cells by specifically acting on mitochondrial apoptotic pathways executed by endogenous caspases (Clavería, 1998).

It is possible that mitochondrial components of the mammalian cellular apoptotic machinery recognize the Drosophila Grim protein and respond by activating the apoptotic programme, as they would after the proper endogenous stimulus. In good agreement with this view, Grim action is counteracted by the overexpression of the anti-apoptotic factor bcl-2, which generally inhibits all mitochondrial death pathways. bcl-2 inhibits Grim-induced death in a dose-dependent manner, suggesting that they mutually antagonize to either promote or inhibit caspase activation. A bcl-2-like molecule has not yet been isolated in Drosophila, but mammalian and nematode members of this family have been shown to rescue ced-3- and Reaper-induced apoptosis in cultured fly cells. One possibility is that Grim promotes cytochrome c release from the mitochondria, as has been shown for Reaper in an in vitro amphibian system. However, Reaper does not induce apoptosis in mouse fibroblasts, arguing for a mechanism of action or regulation different from that of Grim. The activity of caspases insensitive to OpIAP, DEVD.cho and YVAD.cho is sufficient to execute the apoptosis induced by Grim. Caspase 9, which has been shown to transduce apoptotic mitochondrial signals, may be one of those activated by mitochondrial Grim. Nevertheless, group II caspases are activated as well, since Grim cleavage is inhibited by caspase inhibitors specific for this group and it cannot be excluded that activation of group II caspases may also serve to execute Grim-induced apoptosis. Inhibitor of apoptosis proteins (IAPs), the natural inhibitors of group II caspases, have been shown to inhibit apoptosis by directly binding to Grim, Reaper and Hid in a lepidopteran cell line. In contrast, neither OpIAP nor DEVD block Grim-induced death. These results, nevertheless, show that group II caspases are activated, suggesting that some of the Grim-induced caspase activation cascades are similar between mouse fibroblasts and cultured lepidopteran cells, whereas others may differ. It is possible that the particular caspase activation cascade triggered by grim depends more on the cell type in which it is expressed rather than on any intrinsic specificity of its action (Claveria, 1998).

Mutations that remove Nedd2-like caspase (DRONC) are not available. Therefore, to examine a possible role for DRONC as a cell death effector a form of DRONC, DRONCC318S, was generated in which the active site cysteine was altered to serine. Expression of similar forms of other caspases results in a suppression of caspase activity and caspase-dependent cell death. This may occur as a result of interaction of DRONCC318S with the Drosophila homolog of the caspase-activating protein Apaf-1, thus preventing the Drosophila Apaf-1 from binding to wild type DRONC and promoting its activation in a manner similar to that described for mammalian Apaf-1 and caspase-9. Transgenic Drosophila were generated in which DRONCC318S was expressed under the control of a promoter, known as GMR, that drives transgene expression specifically in the developing fly eye. The eyes of these flies, known as GMR-DRONCC318S flies, appear similar to those of wild type flies. To assay the ability of DRONCC318S to block cell death, GMR-DRONCC318S flies were crossed to flies overexpressing rpr (GMR-rpr), hid (GMR-hid), or grim (GMR-grim) under the control of the same promoter. GMR-driven expression of rpr, hid, or grim results in a small eye phenotype due to activation of caspase-dependent cell death. However, flies coexpressing GMR-DRONCC318S and one of the cell death activators showed a dramatic suppression of the small eye phenotype, indicating that cell death had been suppressed. The possibility cannot be ruled out that this suppression is a result of DRONCC318S forming nonproductive interactions with the Drosophila Apaf-1 that block its ability to activate other long prodomain caspases such as DCP-2/DREDD. However, these possibilities notwithstanding, these results suggest that DRONC activity is important for bringing about rpr-, hid-, and grim-dependent cell death (Hawkins, 2000).

Grim promotes cell death by binding to DIAP1, thereby inhibiting its function as a caspase inhibitor

Ectopic expression of Reaper or Grim induces substantial apoptosis in mammalian cells. Reaper- or Grim-induced apoptosis is inhibited by a broad range of caspase inhibitors and by human inhibitor of apoptosis proteins cIAP1 and cIAP2. Additionally, in vivo binding studies demonstrate that both Reaper and Grim physically interact with human IAPs through a homologous 15-amino acid N-terminal segment. Deletion of this segment from either Reaper or Grim abolishes binding to cIAPs. In vitro binding experiments indicate that Reaper and Grim bind specifically to the BIR domain-containing region of cIAPs, since deletion of this region results in loss of binding. The physical interaction has been further confirmed by immunolocalization. When co-expressed, Reaper or Grim co-localize with cIAP1. However, deletion of the N-terminal 15 amino acids of Reaper or Grim abolishes co-localization with cIAP1, suggesting that this homologous region can serve as a protein-protein interacting domain in regulating cell death. Moreover, by virtue of this interaction, it has been demonstrated that cIAPs can regulate Reaper and Grim by abrogating their ability to activate caspases and thereby inhibit apoptosis. This is the first function attributed to this 15-amino acid N-terminal domain, which is the only region having significant homology between these two Drosophila death inducers (McCarthy, 1998).

Reaper (Rpr), Hid, and Grim activate apoptosis in cells programmed to die during Drosophila development. Transient overexpression of Rpr in the lepidopteran SF-21 cell line induces apoptosis. Members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit Rpr-induced apoptosis and physically interact with Rpr through IAP family members' BIR (baculovirus IAP repeat) motifs. Transient overexpression of HID and GRIM also induces apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs (see Drosophila Thread) block HID- and GRIM-induced apoptosis and also physically interact with them through the BIR motifs of the IAPs. The region of sequence similarity shared by Rpr, Hid, and Grim (the N-terminal 14 amino acids of each protein) is required for the induction of apoptosis by Hid and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of Hid and Grim are sufficient to induce apoptosis and confer IAP binding activity. However, Grim is more complex than HID since the C-terminal 124 amino acids of Grim retain apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within Grim. Coexpression of IAPs with Hid stabilizes Hid levels and results in the accumulation of Hid in punctate perinuclear locations that coincide with IAP localization. The physical interaction of IAPs with Rpr, Hid, and Grim provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins (Vucic, 1998).

Induction of apoptosis in Drosophila requires the activity of three closely linked genes: reaper, hid and grim. The proteins encoded by reaper, hid and grim activate cell death by inhibiting the anti-apoptotic activity of the Drosophila IAP1 (Diap1, also known as Thread) protein. In a genetic modifier screen, both loss-of-function and gain-of-function alleles in the endogenous diap1 gene were obtained, and the mutant proteins were functionally and biochemically characterized. Gain-of-function mutations in diap1 strongly suppress reaper-, hid- and grim-induced apoptosis. Sequence analysis of these diap1 alleles reveals that they are caused by single amino acid changes in the baculovirus IAP repeat domains of Diap1, a domain implicated in binding Reaper, Hid and Grim. Significantly, the corresponding mutant Diap1 proteins display greatly reduced binding of Reaper, Hid and Grim, indicating that Reaper, Hid and Grim kill by forming a complex with Diap1. Collectively, these data provide strong support for the idea that Reaper, Hid and Grim kill by inhibiting DIAP1's ability to antagonize caspase function (Goyal, 2000).

It is thought that the previously proposed function of IAPs upstream of reaper, hid and grim is simply an artifact of unphysiologically high levels of protein expression in heterologous systems. When IAP expression constructs are introduced into cultured cells under the control of strong promoters and at high copy numbers, the levels of proteins expressed far exceed those of the endogenous cellular IAP proteins. Under these unphysiological conditions, cellular IAPs can display properties that do not reflect their normal mechanism of action. In particular, the current results demonstrate that mutant proteins that completely lack anti-apoptotic activity in vivo can still inhibit cell death in vitro as long as they can bind to Reaper, Hid and Grim. Conversely, gain-of-function diap1 alleles that display reduced binding to Reaper, Hid and Grim have strongly increased anti-apoptotic function in vivo, but show reduced protection in heterologous cell transfection assays. These results clearly reveal the limitations of overexpression studies in cultured cells for determining the normal mechanism of action of these proteins in the cell death pathway (Goyal, 2000).

The proapoptotic genes reaper (rpr), grim, and head involution defective (hid) are required for virtually all embryonic apoptosis. The proteins encoded by these genes share a short region of homology at their amino termini. The Drosophila IAP homolog Thread/Diap1 (Th/Diap1) negatively regulates apoptosis during development. It has been proposed that Rpr, Grim, and Hid induce apoptosis by binding and inactivating TH/Diap1. The region of homology between the three proapoptotic proteins has been proposed to bind to the conserved BIR2 domain of TH/Diap1. An analysis of loss-of-function and gain-of-function alleles of th indicates that additional domains of Th/Diap1 are necessary to allow th to inhibit death induced by Rpr, Grim, and Hid. In addition, analysis of loss-of-function mutations demonstrates that th is necessary to block apoptosis very early in embryonic development. This may reflect a requirement to block maternally provided Rpr and Hid, or it may indicate another function of the Th/Diap1 protein (Lisi, 2000).

Several mechanisms of action have been suggested for the antiapoptotic properties of the IAP family of proteins. Among these are the binding of the Drosophila IAPs to the proapoptotic proteins Rpr, Grim, and Hid. This interaction has been demonstrated in overexpression systems, and has been proposed to involve the homologous amino-terminal 14 amino acid sequences of the apoptosis initiators with the second BIR domain of the IAPs. The data presented here suggest that this is an oversimplification. Another mechanism that has been proposed for IAP antiapoptotic activity is the direct binding and inhibition of caspases. Th/Diap1 binds to the Drosophila caspases drICE and DCP-1 and to inhibit their ability to induce apoptosis. Here again, this binding activity appears to rest within BIR2 (Lisi, 2000).

These physical interactions support a simple model of IAP action. In this model, IAPs act within viable cells to inhibit caspase function. The action of Rpr, Hid, and Grim interferes with the ability of IAPs to inhibit caspases, thus inducing apoptosis. On the basis of the model, the LOF mutations identified in this study would be predicted to interfere with the ability of the Th/Diap1 protein to inhibit caspase function. This is likely to be true for th109.07, which lacks most of the protein, as well as for th5 and th4, which affect conserved residues in BIR2. BIR2 is sufficient to inhibit apoptosis induced by the active form of the Drosophila caspase drICE. The th9 mutation in BIR1 suggests that this BIR is also important for the full function in caspase inhibition. Alternatively, this change in BIR1 might have long-range effects on BIR2 structure or on protein stability (Lisi, 2000).

It is interesting to note that th7, which acts as a very strong LOF mutation and seems to show some dominant-negative properties, has only the BIR1 attached to the spacer and ring domains. Thus, despite the extensive homologies between the two BIR domains of the protein, a single BIR is not sufficient for Th/Diap1 function, at least in the presence of an attached ring domain. BIR2 of Th/Diap1 and Op-IAP, as well as the single BIR of survivin, are able to inhibit apoptosis (Lisi, 2000).

Again, on the basis of the model above, the GOF mutations identified would be predicted to bind to caspases, but not to the inducers. The thSL mutation maps to a weakly conserved residue in BIR1 and does not result in increased th protein levels. This suggests that BIR1 is important for Rpr and Grim binding, but not for Hid binding, as Hid activity is unaffected in this mutation. Even in the context of overexpression in the eyes of transgenic flies, this mutant IAP retains some specificity for Rpr and Grim killing. This implies that the simple model of BIR2 binding to the conserved NH2-terminal sequences of Rpr, Grim, and Hid is not accurate, and that other residues in the protein are differentially important for Rpr and Grim, as opposed to Hid binding (Lisi, 2000).

The importance of regions outside of BIR2 for Diap1 activity is supported by the analysis of the GOF1 class of mutations, th6B and th81.03. Both of these mutations suppress Hid killing and would be predicted to inhibit Hid binding. These mutations change conserved cysteines in the ring domain to tyrosines. This suggests that the ring is important for Hid/Diap1 interaction. However, the region of Hid binding to Diap1 and Op-IAP has been mapped to BIR2, while the ring does not show any ability to bind to Hid. In addition, mutations in the ring, including those in conserved cysteines, have little effect on the ability of Op-IAP to protect against Hid killing. These data, together with the finding that both GOF1 mutations are cysteine-to-tyrosine changes, suggest that these mutations might have a novel ability to interfere with binding of Hid to BIR2. In addition, the observation that the GOF1 mutations slightly enhance Rpr and Grim killing suggests that these mutants are less potent inhibitors of caspases. This might result from weaker binding to caspases or from proteins that are slightly less stable. This second attribute would be predicted to enhance killing by any inducer that binds the IAP, but not to have an effect on Hid, which is unable to bind (Lisi, 2000 and references therein).

In conclusion, the data support a model where Rpr, Grim, and Hid interact with Th/Diap1 to induce apoptosis. Mutations that affect killing by Rpr and Grim or by Hid can be isolated, indicating that these inducers interact with Th/Diap1 in different ways. The GOF mutations that have been identified also provide useful tools to examine the roles of IAPs, rpr, grim, and hid during Drosophila development. The other Drosophila IAP homolog, DIAP2, has been shown to selectively inhibit Rpr- and Hid-induced but not Grim-induced death (Lisi, 2000).

In LOF th alleles, a developmental arrest occurs at the blastoderm stage and, subsequently, a synchronous apoptosis of all the nuclei. Earlier reports that homozygous th embryos show no ectopic apoptosis probably reflects the very early stage at which this apoptosis occurs. At this time, a direct requirement for th to block apoptosis cannot be distinguised from a requirement for th in another developmental process. This developmental defect could then result in secondary apoptosis. The latter possibility is reasonable, as many failures in development result in ectopic apoptosis. A BIR containing protein from Caenorhabditis elegans is required for cytokinesis in embryos. However, it is also possible that developmental arrest occurs as a result of the initiation of apoptosis, which is manifest only as DNA damage several hours later (Lisi, 2000).

Does this early requirement for th reflect a need to inhibit apoptosis induced by rpr, grim, and hid? Double mutants of th and Df(3L)H99, the deletion that removes rpr, grim, and hid, show a phenotype similar to th alone. This indicates that Th/Diap1 is not required to suppress zygotic Rpr, Grim, and Hid activity. However, hid and rpr mRNA can be seen in a subset of cells in the blastoderm embryo, as judged by in situ analysis. This may indicate that these gene products are supplied maternally. Th/Diap1 may be required to suppress maternally supplied Rpr, Grim, or Hid. Allelic differences in the stage at which apoptosis begins in the th mutants parallel the general ability of the alleles to inhibit apoptosis induced by Rpr, Hid, and Grim. The strong LOF alleles arrest at the blastoderm stage; the GOF1 alleles arrest much later, and the GOF2 allele is completely viable (Lisi, 2000).

Many members of the inhibitor of apoptosis (IAP) family of proteins suppress programmed cell death, at least in part, by physically interacting with and inhibiting the catalytic activity of caspases. An important functional unit in all death-inhibiting IAP proteins is the so-called baculoviral IAP repeat (BIR), which contains approximately 80 amino acids folded around a zinc atom. The Drosophila genome contains four genes that encode proteins with BIR domains. The overexpression of two of these, DIAP1 and DIAP2, inhibit both normal developmental cell death and apoptosis induced by expression of proapoptotic genes. In addition, DIAP1 is required for cell survival in the embryo and in a number of adult tissues. These observations, in conjunction with others showing that DIAP1 binds and inactivates several Drosophila caspases and that loss of DIAP1 results in an increase in caspase activity in vivo, argue that DIAP1's function as a caspase inhibitor is required for cell survival. DIAP1 contains two N-terminal BIR repeats and a C-terminal RING domain. DIAP1 fragments containing the BIR2 domain are sufficient to prevent cell death in a number of contexts. Interestingly, fragments consisting of the BIR2 and surrounding linker sequences also bind multiple proapoptotic proteins, including the apical caspase DRONC, and Hid, Grim, and Reaper (Wu, 2001 and references therein).

One mechanism by which Hid, Grim, and Reaper promote cell death is by binding to DIAP1, thereby inhibiting its function as a caspase inhibitor. Although Hid, Grim, and Reaper perform a similar function in promoting cell death, they only share homology in the N-terminal 14 residues of their primary sequences. These N-terminal sequences are sufficient to mediate interactions with DIAP1 and with several mammalian IAPs. In the case of Hid in insects, and Hid and Reaper in mammalian cells, these N-terminal sequences are essential for proapoptotic function (Wu, 2001 and references therein).

In mammalian cells, caspase inhibition by IAPs is negatively regulated by a mitochondrial protein Smac/DIABLO, which is released from the mitochondrial intermembrane space into the cytosol upon apoptotic stimuli. Smac/DIABLO physically interacts with multiple IAPs and relieves their inhibitory effect on both initiator and effector caspases. Thus, Smac/DIABLO represents the mammalian functional homolog of the Drosophila Hid, Grim, and Reaper proteins. Recent structural studies reveal that the N-terminal tetrapeptide of Smac/DIABLO binds a surface groove on XIAP-BIR3, thus competitively removing the inhibition of caspase-9 by XIAP. Smac/DIABLO shares sequence homology with Hid, Grim, and Reaper only in the N-terminal 4 residues, prompting the hypothesis that Hid, Grim, and Reaper interact with DIAP1 using similar tetrapeptides and binding to a similar surface groove on DIAP1 (Wu, 2001 and references therein).

There is currently no structural information on DIAP1 or Hid, Grim, or Reaper. To investigate the structural mechanisms of DIAP1 recognition by the Drosophila Hid, Grim, and Reaper proteins, the DIAP1-BIR2 domain was crystalized by itself and in complex with the N-terminal peptides from both Hid and Grim (these structures were determined at 2.7, 2.7, and 1.9 Angstrom resolution, respectively). By analogy to the Smac-XIAP interactions, the first four amino acids of Hid and Grim bind an evolutionarily conserved surface groove on DIAP1-BIR2. The next 3 conserved residues of Hid and Grim also contribute to the interactions with DIAP1 through extensive van der Waals contacts. Interestingly, peptide binding to DIAP1-BIR2 appears to induce the formation of an additional alpha helix, which appears to stabilize peptide binding. In conjunction with biochemical analysis, this structural study reveals a molecular basis for the conservation and diversity necessary for the recognition of IAPs by the Drosophila Hid/Grim/Reaper and the mammalian Smac proteins. These results have important ramifications for the design of IAP inhibitors toward therapeutic applications (Wu, 2001).

Inhibitor of apoptosis (IAP) proteins suppress apoptosis and inhibit caspases. Several IAPs also function as ubiquitin-protein ligases. Regulators of IAP auto-ubiquitination, and thus IAP levels, have yet to be identified. Head involution defective (Hid), Reaper (Rpr) and Grim downregulate Drosophila melanogaster IAP1 (DIAP) protein levels. Hid stimulates DIAP1 polyubiquitination and degradation. In contrast to Hid, Rpr and Grim can downregulate DIAP1 through mechanisms that do not require DIAP1 function as a ubiquitin-protein ligase. Observations with Grim suggest that one mechanism by which these proteins produce a relative decrease in DIAP1 levels is to promote a general suppression of protein translation. These observations define two mechanisms through which DIAP1 ubiquitination controls cell death: first, increased ubiquitination promotes degradation directly; second, a decrease in global protein synthesis results in a differential loss of short-lived proteins such as DIAP1. Because loss of DIAP1 is sufficient to promote caspase activation, these mechanisms should promote apoptosis (Yoo, 2002).

Grim binds Sythe thereby inducing apoptosis

Reaper is a potent apoptotic inducer critical for programmed cell death in the fly Drosophila melanogaster. While Reaper homologs from other species have not yet been reported, ectopic expression of Reaper in cells of vertebrate origin can also trigger apoptosis, suggesting that Reaper-responsive pathways are likely to be conserved. Reaper-induced mitochondrial cytochrome c release and caspase activation in a cell-free extract of Xenopus eggs requires the presence of a 150 kDa Reaper-binding protein, Scythe. Reaper binding to Scythe causes Scythe to release a sequestered apoptotic inducer. Upon release, the Scythe-sequestered factor(s) is sufficient to induce cytochrome c release from purified mitochondria. Moreover, addition of excess Scythe to egg extracts impedes Reaper-induced apoptosis, most likely through rebinding of the released factors. In addition to Reaper, Scythe binds two other Drosophila apoptotic regulators: Grim and Hid. Surprisingly, however, the region of Reaper that is detectably homologous to Grim and Hid is dispensable for Scythe binding (Thress, 1999).

Bruce inhibits Grim mediated cell death

Mammalian Bruce is a large protein (530 kDa) that contains an N-terminal baculovirus IAP repeat (BIR) and a C-terminal ubiquitin conjugation domain. Bruce upregulation occurs in some cancers and contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs. However, it is still unknown whether Bruce inhibits apoptosis directly or instead plays some other more indirect role in mediating chemoresistance, perhaps by promoting drug export, decreasing the efficacy of DNA damage-dependent cell death signaling, or by promoting DNA repair. Using gain-of-function and deletion alleles, it has been demonstrated that Drosophila Bruce can potently inhibit cell death induced by the essential Drosophila cell death activators Reaper (Rpr) and Grim but not Head involution defective (Hid). The Bruce BIR domain is not sufficient for this activity, and the E2 domain is likely required. Drosophila Bruce does not promote Rpr or Grim degradation directly, but its antiapoptotic actions do require that their N termini, required for interaction with DIAP1/Thread BIR2, be intact. Bruce does not block the activity of the apical cell death caspase Dronc or the proapoptotic Bcl-2 family member Debcl/Drob-1/dBorg-1/Dbok. Together, these results argue that Bruce can regulate cell death at a novel point (Vernooy, 2002).

In Drosophila, the products of the reaper (rpr), head involution defective (hid), and grim genes are essential activators of caspase-dependent cell death. A genetic screen was carried out for suppressors of Rpr-, Hid-, and Grim-dependent cell death to identify regulators of their activity. Approximately 7000 new insertion lines of the GMREP P element transposon were generated. GMREP contains an engineered eye-specific enhancer sequence (GMR). This sequence is sufficient to drive the expression of linked genes in and posterior to the morphogenetic furrow during eye development. Thus, insertion of GMREP within a region can lead to the eye-specific expression of nearby genes. Each insertion line was crossed to flies that had small eyes due to the eye-specific expression of Rpr (GMR-Rpr flies), Hid (GMR-Hid flies), or Grim (GMR-Grim flies), and the progeny were scored for enhancement or suppression. A number of suppressors were identified. Five lines (GMREP-86A-1–5) mapped to the 86A region, and each strongly suppressed cell death induced by eye-specific expression of Rpr or Grim but not Hid. These lines mapped within a 6-kb interval. A number of other lines were obtained with P-element insertions located in the nearby region. Four of these, EP(3)0359, EP(3)0739, l(3)j8B6, and l(3)06142, mapped within six base pairs of the GMREP-86A-3–5 insertion sites. None of these, nor a fifth nearby line, l(3)06439, acted as a suppressor of GMR-Rpr-, GMR-Grim-, or GMR-Hid-dependent cell death. These results argue that the cell death suppression seen with the GMREP-86A lines was not due to a transposon-induced loss of function, but rather to the GMREP-dependent expression of a nearby gene. All of the GMREP-86A insertions were located 5' to a gene encoding the Drosophila homolog, Bruce, of murine Bruce (also known as Apollon in humans, suggesting this as an obvious candidate. The results of tissue in situ hybridizations with a Drosophila Bruce probe and immunocytochemistry with a Bruce-specific antibody support this possibility. Bruce transcript and protein are expressed at uniform low levels in wild-type eye discs. However, in the GMREP86A lines, they are expressed at high levels in and posterior to the morphogenetic furrow of the eye disc, which is where the GMR element drives expression (Vernooy, 2002).

To demonstrate that Bruce is responsible for the GMREP-86A-dependent suppression of Rpr- and Grim-dependent cell death, levels of the Bruce transcript were specifically downregulated in the eyes of flies carrying a GMR-Rpr transgene as well as a GMREP-86A element. Analysis focussed on one line, GMREP-86A-1, since all five lines behaved similarly with respect to cell death suppression and Bruce overexpression. Flies were generated that carried a Bruce RNA interference (RNAi) construct driven under GMR control (GMR-Bruce-RNAi flies). The eyes of GMR-Bruce-RNAi flies were normal. These animals were crossed to flies in which GMR-Rpr-dependent cell death was suppressed by the presence of the GMREP-86A-1 transposon and progeny from this cross were identified that carried all three transgenes, GMR-Bruce-RNAi, GMR-Rpr, and GMREP-86A-1. It was reasoned that if ectopic expression of Bruce in the eye, driven by the GMREP-86A-1 insertion, was responsible for the suppression of Rpr-dependent cell death, then expression of Bruce-RNAi should downregulate levels of the Bruce sense transcript. This should lead to an attenuation of the GMR-EP-86A-1-dependent suppression of Rpr-dependent cell death, causing a decrease in eye size. Such an attenuation was in fact observed. These observations, in conjunction with those obtained from studies with Bruce deletion mutants, argue that Bruce can suppress Rpr- and Grim-dependent cell death (Vernooy, 2002).

cDNAs encompasing the Bruce coding region were sequenced. This allowed an accurate map to be assembled of the Bruce exon-intron structure, which differs in some respects from that of the BDGP predicted gene. Overall, Bruce is 30% identical to murine Bruce. However, the Bruce N-terminal BIR domain and the C-terminal E2 domain show much higher degrees of homology, 83% and 86% identity, respectively. C. elegans homologs of Bruce were not apparent. Mutations in the Bruce gene were generated by carrying out imprecise excision of a P element, EP3731, located 3' to the Bruce transcript. Two deletions were generated that extended only in one direction, into the 3' end of the Bruce coding region. E12 deleted a relatively small region of the C terminus that includes the E2 domain, while E16 deleted approximately the C-terminal half of the Bruce coding region. Both lines were homozygous viable but male sterile. The possibility that E12 and E16 represent neomorphic mutations in Bruce cannot be excluded. However, the hypothesis that they represent hypomorphs or null mutations is favored, since they had the opposite phenotype of the GMREP-86A Bruce expression lines when in combination with GMR-Rpr, acting as enhancers rather than suppressors of Rpr-dependent cell death in the eye. E12 and E16 also enhanced GMR-Grim, but this effect is much more modest. E12 and E16 have no clear effect on cell death due to expression of Hid (Vernooy, 2002).

These results argue that endogenous Bruce levels, at least in the eye, are sufficient to act as a brake on Rpr-, and to some extent, Grim-dependent cell death. How does Bruce suppress apoptosis? A number of observations argue that Rpr- and Grim-dependent killing proceeds through distinct mechanisms and/or is regulated differently from those activities that is due to Hid. These differences are manifest at multiple points. At the level of DIAP1, point mutations of DIAP1 have effects on Rpr- and Grim-dependent cell death that are the opposite of those due to Hid. In addition, in a Drosophila extract, Hid, but not Rpr and Grim, promotes DIAP1 polyubiquitination. In contrast, in a different set of assays, Rpr and Grim, but not Hid, act as general inhibitors of protein translation. Finally, Rpr and Grim, but not Hid, show strong synergism with the effector caspase DCP-1 in terms of their ability to induce cell death in the eye. Each of these points defines a possible target for Bruce antiapoptotic action (Vernooy, 2002 and references therein).

Because Bruce strongly suppresses cell death induced by Rpr and Grim but not by Hid, one obvious possibility was that Bruce promotes Rpr and Grim ubiquitination and degradation. This hypothesis was tested by generating mutant versions of Grim and Rpr that lacked all lysines, the amino acid to which ubiquitin is added. These genes were introduced into flies under GMR control. GMR-Rpr-lys- and GMR-Grim-lys- flies have small eyes, indicating that these mutant proteins are effective cell death inducers. GMREP-86A-1-dependent Bruce expression suppresses this death very effectively, indicating that Bruce cannot be promoting ubiquitin-dependent degradation of Rpr or Grim. Interestingly, however, Bruce expression does not suppress cell death induced by expression of versions of Rpr (GMR-RprC) or Grim (GMR-GrimC) lacking their N termini, which are required for their IAP-caspase-disrupting interactions with the DIAP1 BIR2. This result is important because it argues that Bruce does not act to regulate this relatively uncharacterized death pathway (Vernooy, 2002).

The N-terminal Bruce BIR lacks a number of residues thought to be important for binding of Rpr, Hid, and Grim to DIAP1 BIR2. Thus, it seems unlikely that GMR-driven expression of Bruce inhibits cell death by simply titrating Rpr and Grim away from interactions with DIAP1 BIR2 as a result of similar interactions with the Bruce BIR. Nonetheless, the high degree of conservation between Bruce and mammalian Bruce in the BIR suggests that it is functionally important. To explore this role further, a fragment of Bruce that contained residues 1–531, including the BIR domain, was expressed under GMR control. Flies carrying this construct, GMR-Bruce-BIR flies, had normal appearing eyes, and in crosses to flies expressing GMR-Rpr, -Hid, or -Grim, GMR-Bruce-BIR did not enhance or suppress these eye phenotypes. These results do not rule out a role for the Bruce BIR in suppressing Rpr- and Grim-dependent cell death. However, they do suggest that the BIR alone is unlikely to mediate this inhibition (Vernooy, 2002).

Bruce overexpression in the eye also does not suppress cell death resulting from GMR-driven expression of the caspase Dronc, which is required for many apoptotic cell deaths in the fly, including those induced by expression of Rpr, Grim, and Hid. Dronc most resembles mammalian caspase-9, and its activation is likely to involve interactions with the Drosophila Apaf-1 homolog Ark. Thus, this result strongly suggests that Bruce does not block Ark-dependent Dronc activation or Dronc activity. This result is also suggested by the observation that decreasing Ark or Dronc in the eye strongly suppressed Hid-dependent cell death, which Bruce does not. A similar lack of cell death suppression is seen in the progeny of crosses between GMR-Bruce flies and flies expressing a second long prodomain caspase, Strica, whose mechanism of activation and normal functions are unknown. Finally, GMREP-86A-1 also fails to suppress the cell death due to GMR-dependent expression of the Drosophila proapoptotic Bcl-2 family member known variously as Debcl, Drob-1, dBorg-1, or Dbok (Vernooy, 2002).

Thus, the Bruce gene is found in mammals and flies, but not in the worm C. elegans. In humans, it is upregulated in some cell lines derived from gliomas and an ovarian carcinoma, and the results of antisense inhibition of Bruce suggest that it contributes to the resistance of these cells to DNA-damaging chemotherapeutic drugs. The Drosophila homolog of Bruce, can potently inhibit cell death induced by Rpr and Grim but not Hid. In addition, flies with C-terminal deletions that removed the Bruce ubiquitin conjugation domain, or much larger regions of the coding region, acted as dominant enhancers of Rpr- and Grim-dependent, but not Hid-dependent, cell death. Together, these observations clearly demonstrate that Bruce can function as a cell death suppressor. The results with the deletion mutants suggest, but do not prove, that Bruce's death-inhibiting activity requires its function as a ubiquitin-conjugating enzyme. Based on the general conservation of cell death regulatory mechanisms, these results argue that mammalian Bruce is likely to facilitate oncogenesis by directly promoting cell survival in the face of specific death signals. One mechanism by which Rpr, Grim, and Hid promote apoptosis is by binding to DIAP1, thereby blocking its ability to inhibit caspase activity. It will be interesting to determine if mammalian Bruce also inhibits cell death induced by the expression of specific IAP binding proteins (Vernooy, 2002).

How does Bruce inhibit cell death? It does not promote the ubiquitination and degradation of Rpr and Grim directly. However, the possibility cannot be ruled out that Bruce somehow sequesters these proteins from their proapoptotic targets. The fact that it does not inhibit cell death due to Hid or Dronc expression argues that it is unlikely to be acting on core apoptotic regulators such as Ark, Dronc, or DIAP1, which are important for Hid-, Rpr-, and Grim-dependent cell death. An attractive hypothesis is that Bruce, perhaps in conjunction with apoptosis-inhibiting ubiquitin-protein ligases such as DIAP1 or DIAP2, promotes the ubiquitination and degradation of a component specific to Rpr- and Grim-dependent death signaling pathways. What might such a target be? Little is known about how Rpr- and Grim-dependent death signals differ from those due to Hid. However, one possibility is suggested by the recent observation that Rpr and Grim, but not Hid, can inhibit global protein translation. This creates an imbalance between levels of short-lived IAPs and the caspases they inhibit, thereby sensitizing cells to other death signals. Perhaps Bruce targets a protein(s) required for this activity (Vernooy, 2002).

Finally, Bruce is a very large protein, and thus its coding region might be expected to be subject to a relatively high frequency of mutation. Truncation of Bruce through the introduction of a stop codon or a frame shift is thus likely to be a relatively common form of Bruce mutation. The results of the deletion analysis show that C-terminal Bruce truncations act to enhance cell death in response to several different signals. Given this, it will be interesting to determine if human Bruce mutations are associated with a predisposition to pathologies that involve an inappropriate increase in cell death (Vernooy, 2002).

Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination

The inhibitor of apoptosis protein DIAP1 inhibits Dronc-dependent cell death by ubiquitinating Dronc. The pro-death proteins Reaper, Hid and Grim (RHG) promote apoptosis by antagonizing DIAP1 function. This study reports the structural basis of Dronc recognition by DIAP1 as well as a novel mechanism by which the RHG proteins remove DIAP1-mediated downregulation of Dronc. Biochemical and structural analyses revealed that the second BIR (BIR2) domain of DIAP1 recognizes a 12-residue sequence in Dronc. This recognition is essential for DIAP1 binding to Dronc, and for targeting Dronc for ubiquitination. Notably, the Dronc-binding surface on BIR2 coincides with that required for binding to the N termini of the RHG proteins, which competitively eliminate DIAP1-mediated ubiquitination of Dronc. These observations reveal the molecular mechanisms of how DIAP1 recognizes Dronc, and more importantly, how the RHG proteins remove DIAP1-mediated ubiquitination of Dronc (Chai, 2003).

Reaper and Grim induce stable inactivation in voltage-gated K+ channels

Drosophila genes reaper, grim, and head-involution-defective (hid) induce apoptosis in several cellular contexts. Voltage-dependent Shaker (Sh) K+ channels open in response to depolarization and subsequently undergo N-type inactivation by a "ball and chain" mechanism. The 20 N-terminal residues of the ShB channel form the inactivation "ball," which is tethered to membrane-spanning channel domains by the following ~200-residue "chain." Inactivation occurs when the N-terminal inactivation ball physically occludes the inner pore of the channel from the cytoplasmic side. Stability of the inactivated state is enhanced by the hydrophobicity of approximately the first 10 residues of the inactivation ball, whereas positively charged amino acids within the following 10 residues promote entry into the inactivated state via electrostatic interactions. Deletions in the distal N terminus of the channel disrupt inactivation, which can be reversed by application of a 20-residue synthetic peptide corresponding to the initial N-terminal sequence of the channel. Ancillary beta subunits in some K+ channel complexes serve to produce N-type inactivation by a similar mechanism. The conserved N-terminal sequences of Reaper, Grim, and Hid resemble those N-terminal Sh K+ channel domains that are involved in inactivation. This sequence similarity led to the hypothesis that Reaper, Grim, and Hid facilitate initiation of apoptosis by inducing inactivation of K+ channels. Sustained inactivation of K+ channels will result in chronic membrane depolarization that may lead to the initiation of the caspase-dependent apoptotic program, perhaps by increasing the level of cytosolic free Ca2+. Synthetic Reaper and Grim N terminus peptides are shown to induce fast inactivation of Shaker-type K+ channels when applied to the cytoplasmic side of the channel that is qualitatively similar to the inactivation produced by other K+ channel inactivation particles. Mutations that reduce the apoptotic activity of Reaper also reduced the synthetic peptide's ability to induce channel inactivation, indicating that K+ channel inactivation correlates with apoptotic activity. Coexpression of Reaper RNA or direct injection of full length Reaper protein causes near irreversible block of the K+ channels. These results suggest that Reaper and Grim may participate in initiating apoptosis by stably blocking K+ channels (Avdonin, 1998).

Grim stimulates Diap1 poly-ubiquitination by binding to UbcD1

Diap1 is an essential Drosophila cell death regulator that binds to caspases and inhibits their activity. Reaper, Grim and Hid each antagonize Diap1 by binding to its BIR domain, activating the caspases and eventually causing cell death. Reaper and Hid induce cell death in a Ring-dependent manner by stimulating Diap1 auto-ubiquitination and degradation. It has not been clear how Grim causes the ubiquitination and degradation of Diap1 in Grim-dependent cell death. This study found that Grim stimulates poly-ubiquitination of Diap1 in the presence of UbcD1 and that it binds to UbcD1 in a GST pull-down assay, so presumably promoting Diap1 degradation. The possibility that dBruce is another E2 interacting with Diap1 was examined. The UBC domain of dBruce slightly stimulated poly-ubiquitination of Diap1 in Drosophila extracts but not in a reconstitution assay. However Grim does not stimulate Diap1 poly-ubiquitination in the presence of the UBC domain of dBruce. Taken together, these results suggest that Grim stimulates the poly-ubiquitination and presumably degradation of Diap1 in a novel way by binding to UbcD1 but not to the UBC domain of dBruce as an E2 (Yoo, 2005).

Drosophila IAP antagonists form multimeric complexes to promote cell death

Apoptosis is a specific form of cell death that is important for normal development and tissue homeostasis. Caspases are critical executioners of apoptosis, and living cells prevent their inappropriate activation through inhibitor of apoptosis proteins (IAPs). In Drosophila, caspase activation depends on the IAP antagonists, Reaper (Rpr), Head involution defective (Hid), and Grim. These proteins share a common motif to bind Drosophila IAP1 (DIAP1) and have partially redundant functions. This study shows that IAP antagonists physically interact with each other. Rpr is able to self-associate and also binds to Hid and Grim. The domain involved in self-association has been defined and it was demonstrated to be critical for cell-killing activity in vivo. In addition, Rpr requires Hid for recruitment to the mitochondrial membrane and for efficient induction of cell death in vivo. Both targeting of Rpr to mitochondria and forced dimerization strongly promotes apoptosis. These results reveal the functional importance of a previously unrecognized multimeric IAP antagonist complex for the induction of apoptosis (Sandu, 2010).

This study shows that IAP antagonists undergo self-association and hetero-association that is essential for their full killing activity. Specifically, the physical association between Rpr, Hid, and Grim involves the central helical domain of Rpr. Disrupting this protein-protein interface leads to a significant loss of Rpr’s ability to induce cell death in vivo. The importance of Rpr self-association was revealed by generating enforced Rpr dimers in which the central helical domain of this protein is replaced by defined dimerization motifs. These experiments revealed that enforced parallel, but not anti-parallel dimerization of Rpr (RprLZ) can induce cell death very efficiently in transgenic Drosophila. The resulting cell death occurred by apoptosis and was rescued by the overexpression of the caspase inhibitor p35, or through Rpr-insensitive diap1 alleles. Furthermore, mutants that inhibit the self-association of Rpr have reduced pro-apoptotic activity, providing independent support for the importance of Rpr multimerization. Because an anti-parallel Rpr dimer (RprProP) was not efficiently inducing cell death in transgenic animals, it appears that the IBM motifs of multimeric Rpr have to be in a specific conformation, or at least in close proximity for efficient DIAP1 inactivation. This may occur, for example, by engaging both BIR domains of one DIAP1 molecule in a similar fashion to how SMAC can engage XIAP (Sandu, 2010).

The association of Rpr with the other IAP antagonists Grim and Hid is also reported. Hid is the only IAP antagonist that has a defined mitochondrial targeting sequence at its C terminus and is targeted to the mitochondria by itself; therefore, focus was placed particularly on the interaction between Rpr and Hid. Consistent with previous reports, it was found that Hid consistently localizes to the mitochondria in both human and Drosophila cells. Although it has been previously reported that Rpr localizes to the mitochondria through the GH3-lipid interaction, the current results support an alternative view that Rpr’s ability to translocate to the mitochondria is an indirect consequence of associating with Hid. Specifically, in support of this model, it was shown that Rpr is uniformly distributed in cells when transfected alone in heterologous cells, translocating to the mitochondria only when cotransfected with Hid. It was further shown that the GH3 mutant F34AL35A, unlike wild-type Rpr, does not coimmunoprecipitate with Hid. This is in agreement with previous observations that a GH3 mutant failed to localize to the mitochondria in Drosophila S2 cells (Sandu, 2010).

Rpr induces ubiquitination of DIAP1 in vitro and in HEK293 cells. Unlike Rpr, Hid is not able to perform this function. Thus, the significance of Rpr-Hid interaction might be to bring Rpr at the mitochondrial surface to degrade DIAP1. Although both Rpr and Hid belong to the IAP antagonists family, share a conserved IBM motif, bind DIAP1, and induce cell death, their role in induction of cell death seems to be distinct. In many paradigms Hid appears to be a more potent inducer of cell death than Rpr. It is possible that the primary role of Hid is to assemble a complex at the mitochondrial membrane that recruits Rpr as one the players. The role of Rpr in this complex is to induce DIAP1 ubiquitination. Inability of Hid itself to induce DIAP1 degradation might be related to its larger size (410 amino acids) as compared with Rpr (64 amino acids) or even Grim (138 amino acids). Potentially, the bulkier Hid might interfere with conformational changes in DIAP1 or with the ubiquitin-related transfer process (Sandu, 2010).

In addition, evidence is provided that Rpr is more potent at inducing apoptosis when present at the mitochondrial membrane. When Rpr was fused to the mitochondrial targeting sequence from Hid and expressed in Drosophila eyes, strong cell killing and pupal lethality were observed. Flies dissected from the pupal cases show severely ablated eyes that are reduced to black spots. Even the inactive GH3 mutant F34AL35A, when artificially targeted to the mitochondria using the Hid MTS, induces significant eye ablation. Therefore, Rpr is more potent when present at the mitochondrial membrane. Two possible explanations are considered for this enhanced pro-apoptotic activity: First, Rpr may be more active at the mitochondrial surface because of increased protein stability. Consistent with this idea, cytoplasmic Rpr is not very stable and it was found that Rpr accumulates to higher protein levels when the presence of Hid permits mitochondrial localization. The resulting high local concentration of Rpr may be critical for DIAP1 ubiquitination. As predicted by this model, it was found that Rpr-induced cell death is less efficient when Hid is depleted by RNA knockdown. The model is also in agreement with several previous observations. For example, it has been reported that Rpr and Hid localize to mitochondria and can induce changes of the mitochondrial ultrastructure. This study also showed that inhibition of Rpr localization to mitochondria significantly inhibits cell killing, and that Rpr and Hid act in concert with caspases to promote mitochondrial disruption and Cyt C release. In addition, overexpression of both rpr and hid is required to induce cell death in midline cells of the nervous system, and neither of them kills well individually. This is consistent with the observation that more than one IAP antagonist is expressed and they act synergistically in the dying midline glia cells. Finally, Drosophila salivary gland cell death is preceded by the expression of both rpr and hid, and RNAi knockdown of hid alone is sufficient to block the death of these cells. The second, and not mutually exclusive explanation is that Rpr may be more active at the mitochondria because of local concentration of apoptosis regulators that operate at this surface. It has been previously shown that Dronc and active Drice are present at the mitochondrial membrane, and more recently that mammalian XIAP can translocate to the mitochondrial surface in response to apoptotic stimuli. In addition, mitochondrial proteins involved in energy metabolism have been recently described to modulate caspase activity and cell death in Drosophila cells. Recently, it was shown by coimmunoprecipitation experiments in fly cell culture that Grim interacts with the Bcl-2 family proteins Debcl and Buffy. Thus, Rpr may be part of a higher-order complex at the mitochondria to locally regulate IAP turnover and caspase activity (Sandu, 2010).

Taken together, this study uncovered the role of the Rpr helical domain in self-association and interaction with Hid and Grim. The mechanism of Rpr recruitment to the mitochondria by interaction with Hid was revealed. Most importantly, this study has provided a new concept with respect to IAP antagonist activity in fly, which acts cooperatively by physical interaction rather than by additive cell death output (Sandu, 2010).


DEVELOPMENTAL BIOLOGY

Embryonic

To understand the role apoptosis plays in nervous system development and to gain insight into the mechanisms by which steroid hormones regulate neuronal apoptosis, the death of a set of peptidergic neurons was investigated in the CNS of Drosophila. Typically, apoptosis in Drosophila is induced by the expression of the genes reaper, grim, or head involution defective (hid). Genetic evidence is provided that the death of these neurons requires reaper and grim gene function. Consistent with this genetic analysis, these doomed neurons accumulate reaper and grim transcripts prior to the onset of apoptosis. These neurons also accumulate low levels of hid, although the genetic analysis suggests that hid may not play a major role in the induction of apoptosis in these neurons. The death of these neurons is dependent on the fall in the titer of the steroid hormone 20-hydroxyecdysone that occurs at the end of metamorphosis: the accumulation of both reaper and grim transcripts is inhibited by this steroid hormone. These observations support the notion that 20E controls apoptosis by regulating the expression of genes that induce apoptosis (Draizen, 1999).

Larval and Pupal

Hormones and trophic factors provide cues that control neuronal death during development. These developmental cues in some way regulate activation of apoptosis, the mechanism by which most, if not all, developmentally programmed cell deaths occur. In Drosophila, apoptosis can be induced by the expression of the genes reaper, grim, or head involution defective. Prior to the death of a set of identifiable doomed neurons, these neurons accumulate transcripts of the reaper and grim genes, but do not accumulate transcripts of the head involution defective gene. Death of these doomed neurons can be suppressed by two manipulations: either by increasing the levels of the steroid hormone 20-hydroxyecdysone (see Ecdysone receptor) or by decapitation. The impact that these two manipulations have on reaper expression has been investigated. Steroid treatment prevents the accumulation of reaper transcripts, whereas decapitation results in the accumulation of lower levels of reaper transcripts that are not sufficient to activate apoptosis. These data demonstrate that in vivo, reaper, and grim transcripts accumulate coordinately in a set of identified doomed neurons prior to the onset of apoptosis. These observations raise the possibility that products of the reaper and grim genes act in concert in postembryonic neurons to induce apoptosis. That reaper transcript accumulation is regulated by the steroid hormone titer and by the presence of the head is evidence that developmental factors control programmed cell death by regulating the expression of genes that induce apoptosis (Robinow, 1997).

The Drosophila compound eye is formed by selective recruitment of undifferentiated cells into clusters called ommatidia during late larval and early pupal development. Ommatidia at the edge of the eye often lack the full complement of photoreceptors and support cells, and undergo apoptosis during mid-pupation. This cell death is triggered by the secreted glycoprotein Wingless, which activates its own expression in peripheral ommatidia via a positive feedback loop. Wingless signaling elevates the expression of the pro-apoptotic factors head involution defective, grim and reaper, which are required for ommatidial elimination. It is estimated that approximately 6%-8% of the total photoreceptor pool in each eye is removed by this mechanism. In addition, the retinal apoptosis previously reported in apc1 mutants occurs at the same time as the peripheral ommatidial cell death and also depends on head involution defective, grim and reaper. The implications of these findings for eye development and function in Drosophila and other organisms is considered (Lin, 2004).


EFFECTS OF MUTATION

To discover whether expression of apoptosis activators reaper, grim and hid triggers the accumulation of Death related ced-3/Nedd2-like protein (DREDD) mRNA, the three apoptosis activators were ectopically expressed in mesoderm, and the expression of DREDD mRNA examined. Expression of the apoptosis activators triggers excessive apoptosis in mesoderm. During stage 13 and beyond, DREDD mRNA is not widely expressed in the developing musculature in wild-type flies. However, when misexpression of each of the death activators is directed to these tissues, prominent levels of ectopic DREDD mRNA are detected. Expression of grim in the ectoderm also results in DREDD mRNA accumulation. DREDD mRNA accumulation has also been examined in embryos homozygous for crumbs (crb). In crb mutants, reaper is ectopically expressed in the disorganized epidermis. As anticipated, ectopic accumulation of DREDD mRNA is found scattered throughout the ectoderm in crb embryos, coincident with widespread patterns of rpr expression. Perhaps the most compelling evidence for a direct role for Dredd in apoptosis comes from an examination of accumulation of DREDD mRNA in embryos carrying a homozygous deletion of the entire reaper region (mutated for rpr, hid, and grim). No apoptosis occurs in these deletion mutants. The selective accumulation of DREDD mRNA fails to occur in these mutants. This is the first report of a molecular activity that is completely blocked by the absence of H99-associated signaling (Chen, 1998).

In Drosophila, the induction of apoptosis requires three closely linked genes, reaper, head involution defective, and grim. The products of these genes induce apoptosis by activating a caspase pathway. Two very similar Drosophila caspases, DCP-1 and drICE, have been previously identified. DCP-1 has a substrate specificity that is remarkably similar to that of human caspase 3 and Caenorhabditis elegans CED-3, suggesting that DCP-1 is a death effector caspase. drICE and DCP-1 have similar yet different enzymatic specificities. Although expression of either in cultured cells induces apoptosis, neither protein is able to induce DNA fragmentation in Drosophila SL2 cells. Ectopic expression of a truncated form of dcp-1 (DeltaN-dcp-1) in the developing Drosophila retina under an eye-specific promoter results in a small and rough eye phenotype, whereas expression of the full-length dcp-1 (fl-dcp-1) has little effect. However, expression of either full-length drICE (fl-drICE) or truncated drICE (DeltaN-drICE) in the retina shows no obvious eye phenotype. Although active DCP-1 protein cleaves full-length DCP-1 and full-length drICE in vitro, GMR-DeltaN-dcp-1 does not enhance the eye phenotype of GMR-fl-dcp-1 or GMR-fl-drICE flies. Significantly, GMR-rpr and GMR-grim, but not GMR-hid, dramatically enhance the eye phenotype of GMR-fl-dcp-1 flies. These results indicate that Reaper and Grim, but not HID, can activate DCP-1 in vivo (Song, 2000).

The proapoptotic proteins encoded by rpr, hid, and grim all require caspase activity to kill cells. Whether coexpression of caspases and these proapoptotic genes could lead to significantly enhanced cell killing was investigated. For this purpose, flies carrying GMR-rpr, GMR-hid, and GMR-grim were crossed to GMR-fl-dcp-1 and GMR-fl-drICE flies. Two different GMR-fl-dcp-1 transgenic fly lines were crossed to GMR-rpr46, GMR-hid1M, and GMR-grim flies, with identical results. Likewise, two GMR-fl-drICE transgenic fly lines were crossed to GMR-rpr46, GMR-hid1M, and GMR-grim flies, again with identical results. Flies carrying one copy of GMR-fl-dcp-1 or GMR-fl-drICE have almost normal eye morphology. Flies transgenic for GMR-hid1M, GMR-grim, or GMR-rpr46 have a mild but easily detectable eye phenotype. The coexpression of hid and full-length drICE produces no obvious enhancement of the eye phenotype, but rather an additive effect of the two transgenes. Also, the expression of hid together with full-length dcp-1 enhanced the eye phenotype only weakly, comparable to what is seen for coexpression of many other proapoptotic gene combinations. In stark contrast, expression of either rpr or grim together with GMR-fl-dcp-1 yields a dramatically enhanced eye phenotype that cannot be simply explained by additive effects. rpr produces a stronger effect than grim. The expression of rpr also enhances the eye phenotype of GMR-fl-drICE flies, whereas grim is not very effective. This finding is consistent with the observation that drICE is activated in rpr-transfected S2 cells. Among the different cell types of the Drosophila retina, the pigment cells appear to be particularly sensitive to DCP-1. Both the truncated and the full-length DCP-1 cause pigment cell death. Judging by the complete loss of eye color, all pigment cells are eliminated in flies that coexpress DCP-1 with either rpr and grim. In order to further investigate the specificity of this interaction, GMR-fl-dcp-1 flies were also crossed to a transgenic line with strong hid expression: GMR-hid 10 flies. Again, the eye phenotype observed for this combination is not significantly enhanced. Overall, rpr and grim were found to interact with dcp-1 much more strongly than hid and interact more effectively with dcp-1 than with drICE. Taken together, these observations suggest that dcp-1 is rate limiting for cell killing by rpr and grim, but not hid. Therefore, it is proposed that rpr and grim function upstream of dcp-1 in vivo (Song, 2000).

These results indicate that Reaper and Grim, but not Hid, can lead to DCP-1 activation. Several other observations also indicate that rpr and grim have cell killing properties that are distinct from those of hid. For example, the Ras/MAPK pathway inhibits hid-induced cell death but has no effect on rpr- or grim-induced death (Bergmann, 1998). In addition, mutations in the diap1 gene of Drosophila have been isolated that enhance rpr- and grim-induced cell killing but suppress hid-induced cell killing (J. Agapite, K. McCall, and H. Steller, unpublished data cited in Song, 2000). The easiest interpretation of all these observations is that rpr and grim kill cells by activating the same (set of) caspases and that hid activates a distinct caspase. Since it has been recently shown that rpr, hid, and grim induce cell death by inhibiting the antiapoptotic activity of diap1, diap1 must control at least two distinct caspase pathways. According to this model, Reaper and Grim and HID would interact selectively with specific DIAP1-(pro)caspase complexes. The binding of Reaper, Grim, or HID to the relevant DIAP1-(pro)caspase complex is thought to result in caspase activation. This model is consistent with a variety of findings from both invertebrate and vertebrate systems. However, the possibility that rpr and grim may also activate DCP-1 through a DIAP1-independent pathway cannot be ruled out. Although several Drosophila caspases have been described, these results indicate that additional caspases, in particular ones activated by HID, remain to be identified (Song, 2000).

Grim-specific functions as revealed by studies of Grim-induced apoptosis in the CNS midline

The Drosophila reaper, head involution defective (hid), and grim genes play key roles in regulating the activation of programmed cell death. Two useful systems for studying the functions of these genes are the embryonic CNS midline and adult eye. In this study the Gal4/UAS targeted gene expression system was used to demonstrate that unlike reaper or hid, expression of grim alone is sufficient to induce ectopic CNS midline cell death. In both the midline and eye, grim-induced cell death is not blocked by the Drosophila anti-apoptosis protein Diap2, which does block both reaper- and hid-induced cell death. grim can also function synergistically with either reaper or hid to induce higher levels of midline cell death than those observed for any of the genes individually. Analysis was made of the function of a truncated Reaper-C protein, which lacks the NH2-terminal 14 amino acids that are conserved between Reaper, Hid, and Grim. Ectopic expression of Reaper-C reveals cell killing activities distinct from full length Reaper, and indicates that the conserved NH2-terminal domain acts in part to modulate Reaper activity (Wing, 1998).

Reaper, Hid, and Grim are three Drosophila cell death activators that each contain a conserved NH2 -terminal Reaper-Hid-Grim (RHG) motif. The importance of the RHG motifs in Reaper and Grim have been examined for their different abilities to activate cell death during development. Analysis of chimeric R/Grim and G/Reaper proteins indicates that the Reaper and Grim RHG motifs are functionally distinct and help to determine specific cell death activation properties. A truncated GrimC protein lacking the RHG motif retains an ability to induce cell death, and unlike Grim, R/Grim, or G/Reaper, its actions are not efficiently blocked by the cell death inhibitors Diap1, Diap2, p35, or a dominant/negative Dronc caspase. Finally, a second region of sequence similarity was identified in Reaper, Hid, and Grim, that may be important for shared RHG motif-independent activities (Wing, 2001a).

Analyses of R/Grim and G/Reaper chimeras have indicated that the closely related RHG motifs of Reaper and Grim are not functionally interchangeable. Instead, the four amino acid substitutions between their RHG motifs help determine the unique cell killing abilities of Reaper and Grim. For example, unlike Grim, R/Grim resembles Reaper and is unable to induce cell death in the CNS midline. In contrast, one P[UAS-g/reaper] strain induces significant midline cell death, implying that the presence of the Grim RHG motif can confer Grim-like cell killing abilities on Reaper. It is important to note, however, that the identity of the RHG motif does not completely transform the cell killing properties of the chimeras, indicating that other regions of Reaper and Grim proteins are also crucial for their distinct actions. In this regard, like Grim, both R/Grim and G/Reaper are able to act synergistically with Reaper to induce CNS midline cell death (Wing, 2001a).

While the Grim-Reaper proteins do not contain defined structural domains, they each share sequence similarity in the 14 amino acids at their NH2-termini. This RHG (Reaper, Hid, Grim) motif is most similar between Reaper and Grim (71.4% identity), and least similar between Hid and Grim (21.4% identity). The RHG motif plays a key role in interactions between Grim-Reaper proteins and members of the Inhibitor-of-Apoptosis-Protein (IAP) family, including Drosophila Diap1 and Diap2. Like other IAPs, Diap1 and Diap2 both contain related baculovirus IAP repeat (BIR) motifs, as well as a Really Interesting New Gene (RING) finger. Diap1 is an essential cell death regulator and diap1 mutants exhibit early embryonic lethality due to massive ectopic cell death. The functions of Diap2 in regulating cell death are less clear; however, it does share a number of functional properties with Diap1. Diap1 can directly bind caspases and repress their proteolytic activities. Significantly, caspase inhibition by Diap1 is antagonized by Hid, suggesting a double-repression model where the Grim-Reaper proteins promote cell death by binding to Diaps, suppressing their ability to inhibit caspases. Recent studies have indicated that the vertebrate Diablo/SMAC protein also promotes cell death activation by binding to IAPs and suppressing their death inhibitory activities. Thus, IAP suppression may be an evolutionarily conserved cell death regulatory mechanism. In this regard, while Grim-Reaper orthologs have not been identified, the expression of each protein can induce vertebrate cells to die, implying that they may suppress vertebrate IAPs (Wing, 2001a).

As with Reaper or Grim, P[GMR-gal4]-targeted expression of R/Grim or G/Reaper is very effective at inducing cell death. However, the actions of the chimeras are distinct from those of Reaper or Grim. In particular, the cell death phenotypes resulting from R/Grim or G/Reaper expression are completely blocked by Diap1 and partially blocked by Diap2. In contrast, both Diap1 and Diap2 completely block the effects of Reaper expression, but do not affect cell death induced by Grim. Thus, as a result of the presence of the Reaper RHG motif, R/Grim exhibits an increased sensitivity to repression by the Diaps compared with Grim. Similarly, the presence of the Grim RHG motif in G/Reaper results in decreased sensitivity to repression by Diaps compared with Reaper. These results indicated that the sequence differences in the RHG motifs of Reaper and Grim may strongly influence functional interactions with the Diaps (Wing, 2001a).

Diap1, like Diap2, exhibits distinct abilities to repress cell death induced by Reaper, Hid, or Grim. In the CNS midline, Diap1 more effectively blocks Grim-induced cell death than cell death induced by Reaper and Hid. In contrast, when examined in the adult eye, Diap1 is most effective at blocking Reaper-induced cell death, moderately effective against Hid, and ineffective against Grim. Similar results were obtained using the thsl gain-of-function diap1 mutant allele, which represses Reaper-induced eye cell death more effectively than death induced by Hid or Grim. Importantly, these data indicate that Diap1 has distinct, tissue-specific effects on cell death induced by Grim-Reaper proteins, and that these effects differ from those of Diap2. The basis for these functional distinctions are not yet clear. One possibility is that the associations between each Diap and Grim-Reaper protein may differ in strength, or be influenced by specific ancillary factors. Differences have been noted between Diap1 and Diap2 in their ability to bind and repress the actions of certain caspases, and Reaper, Hid, and Grim can act through different downstream caspases. Taken together, these findings suggest potentially complex functional interactions between Grim-Reaper proteins, Diaps, and caspases. It is likely that distinct activities of individual Grim-Reaper and Diap proteins provide enhanced capabilities for regulating cell death processes in different developmental and physiological contexts (Wing, 2001a).

Do Reaper, Hid, and Grim share RHG-independent functions? Both truncated ReaperC and GrimC proteins induce cell death in developing tissues, indicating that regions outside the RHG motif also have death-inducing activities. Surprisingly, it was found that cell death induced by GrimC or ReaperC is only partially repressed by p35, suggesting a distinct mode of action compared with native Reaper or Grim. Similar to Reaper, Hid and Grim, GrimC does apparently act through the p35-insensitive caspase, Dronc, as GrimC-induced death is partially suppressed by a dominant/negative DroncC318S protein. However, the persistence of some eye cell death in the presence of DroncC318S indicates that GrimC and ReaperC also act through alternate pathways. Perhaps GrimC acts through pro-apoptotic Drosophila Bcl-2 orthologs that may induce cell death which is not blocked by p35. Another interesting possibilty is that GrimC might act via a Drosophila ortholog of Scythe, a Xenopus cell death regulator that binds Reaper, Hid, and Grim independently of the RHG motif (Wing, 2001a and references therein).

A second region of sequence similarity, the 30 amino acid Trp-block, has been identified that is present once in Reaper and Grim, and four times in Hid. The Trp-blocks may be important for the cell death activation capabilities of GrimC and ReaperC, as well as for potentially shared RHG motif-independent activities of native Grim-Reaper proteins. This additional sequence similarity also suggests a modular organization of the Grim-Reaper proteins, where distinct functions may be afforded by the RHG motif and Trp-block. Taken together, the sequence similarities of the Grim-Reaper proteins, as well as the organization and chromosomal location of the corresponding genes, imply that the grim-reaper genes arose from duplication of a common ancestor and have diverged to assume overlapping yet distinct cell death activation functions. It will be of interest to determine the representation of grim-reaper orthologs in other species, information that could provide important insights into the evolution of cell death control mechanisms. This is of particular relevance given that inhibition of IAP activity is likely to constitute a conserved mechanism to regulate cell death activation (Wing, 2001a).

The Drosophila reaper, head involution defective, and grim genes play key roles in regulating the activation of programmed cell death. Two useful systems for studying the functions of these genes are the embryonic CNS midline and adult eye. The Gal4/UAS targeted gene expression system has been used to demonstrate that unlike reaper or hid, expression of grim alone is sufficient to induce ectopic CNS midline cell death. In both the midline and eye, grim-induced cell death is not blocked by the Drosophila anti-apoptosis protein Diap2, which does block both reaper- and hid-induced cell death. grim can also function synergistically with reaper or hid to induce higher levels of midline cell death than observed for any of the genes individually. Finally the function was analyzed of a truncated Reaper-C protein that lacks the NH2-terminal 14 amino acids that are conserved between Reaper, Hid, and Grim. Ectopic expression of Reaper-C reveals cell killing activities distinct from full length Reaper, and indicates that the conserved NH2-terminal domain acts in part to modulate Reaper activity (Wing, 2001b).

Conditional expression of grim provokes apoptogenic cytochrome c display

An overt alteration in cytochrome c anticipates programmed cell death (PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs without release of the protein into the cytosol. Conditional expression of the Drosophila death activators, reaper or grim, provoke apoptogenic cytochrome c display and, surprisingly, caspase activity is necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase activation is triggered by mitochondria from apoptotic cells but identical preparations from healthy cells are inactive. These observations provide compelling validation of an early role for altered cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal, feed-forward amplification involving cytochrome c and caspases (Varkey, 1999).

Previous studies on Drosophila SL2 cells have shown that conditional expression of rpr or grim triggers apoptosis in cultured cells and in transgenic animals. Transiently transfected SL2 cells were induced for rpr or grim and, at various time intervals after induction, the preparations were examined for cytochrome c immunoreactivity with mAb 2G8. Apoptotic cultures exhibit profound staining with the antibody. To test the possibility that cytochrome c might be released into the cytosol during apoptosis, healthy SL2 cells and apoptotic rpr- or grim-expressing cells were fractionated, and assayed for cytochrome c in the mitochondrial and cytosolic fractions. Surprisingly, these cells showed no difference in cytochrome c distribution and no evidence was found for the transit of cytochrome c to the cytosol as a correlate to apoptosis. Biochemical data indicating retention of cytochrome c in mitochondria during apoptosis is consistent with cytological studies. These observations indicate that appreciable efflux of cytochrome c from mitochondria does not occur during apoptosis in Drosophila cells (Varkey, 1999).

Mitochondria isolated from apoptotic cells trigger caspase activation in vitro. Caspase activation was measured in L2 cell cytosol that had been coincubated with mitochondria isolated from parental L2 cells or from pre-apoptotic cells (induced either for rpr or grim). Caspase activation was detected, as measured by signature cleavage of a bovine substrate, PARP. Cleavage of PARP in this assay is indistinguishable from the signature activity reported in many mammalian systems and is readily detected in the cytosol of pre-apoptotic cells but not in cytosol from parental L2. These observations emphasize the importance of one or more mitochondrial factors in the activation of caspase function triggered by rpr or grim (Varkey, 1999).

The Drosophila death activators, rpr and grim, activate one or more caspases to elicit apoptosis. To study the temporal relation of cytochrome c display with respect to caspase activity, SL2 cells were cotransfected with rpr and p35 plasmids. Six hours after induction, cells induced for rpr alone show pronounced labeling with mAb 2G8 whereas cells expressing rpr together with p35 are prevented from apoptosis and do not bind the mAb. These observations suggest that apoptogenic cytochrome c display requires caspase activity, a presumption that is further substantiated when rpr-expressing cells are treated with the peptide caspase inhibitors zDEVD-fmk and zVAD-fmk. As seen for p35-blocked cells, these inhibitors similarly prevent mAb 2G8 labeling and subsequent apoptosis. Parallel results are observed in grim-expressing cells (Varkey, 1999).

These data demonstrate that caspase activity is required for apoptogenic cytochrome c display. To determine if caspase function is sufficient to trigger this change, apoptosis was induced in SL2 cells by conditional expression of an activated version of the Drosophila caspase, dcp-1. If deleted for its prodomain, this caspase provokes considerable apoptosis in mammalian cells and SL2 cells. When labeled with mAb 2G8, cells transfected and induced for dcp-1 expression exhibit profound punctate cytochrome c staining with features indistinguishable from those associated with expression of the death activators (Varkey, 1999).

Two potential explanations reconcile the in vivo observations reported here on apoptogenic cytochrome c with reports from mammalian cell-free systems that cytochrome c can trigger caspase activation. One possibility is that the order and/or nature of cytochrome c apoptotic function is not conserved between mammals and insects and thus, relative to caspase action, cytochrome c is upstream in the former case and downstream in the latter case. This scenario, however, seems unlikely given the widespread conservation of apoptotic components, the fact that display of fly cytochrome c in the animal significantly precedes all signs of programmed cell death, and reports from mammalian systems that upstream caspases can trigger cytochrome c release. Therefore, a more likely interpretation of the results reported here is that cytochrome c propagates apoptotic physiology by functioning together with caspases in a feed-forward amplification loop. In this scenario, altered cytochrome c and caspase activity exert positive and reciprocal feedback on one another, similar to observations recently reported for caspase 8. Thus, agents that restrain caspase action (p35) are also predicted to suppress pro-apoptotic display of cytochrome c, which behaves as an amplifier of caspase function. This interpretation is also consistent with recent studies on Fas signaling in type II cells, where molecular ordering studies found that activation of an initiator caspase (caspase 8/Flice) occurs upstream of changes associated with cytochrome c (Varkey, 1999).

sickle strongly enhances the eye cell death induced by expression of either an reaper/grim chimera or reaper

A novel grim-reaper gene, termed sickle, has been identified that resides adjacent to reaper. The sickle gene, like reaper and grim, encodes a small protein which contains an RHG motif and a Trp-block. In wild-type embryos, sickle expression is detected in cells of the developing central nervous system. Unlike reaper, hid, and grim, the sickle gene is not removed by Df(3L)H99, and strong ectopic sickle expression is detected in the nervous system of this cell death mutant. sickle very effectively induced cell death in cultured Spodoptera Sf-9 cells, and this death is antagonized by the caspase inhibitors p35 or DIAP1. Strikingly, unlike the other grim-reaper genes, targeted sickle expression does not induce cell death in the Drosophila eye. However, sickle strongly enhanced the eye cell death induced by expression of either an reaper/grim chimera or reaper (Wing, 2002).

To test sickle's ability to induce cell death, transient transfection assays were performed using cultured Spodoptera Sf-9 cells. Survival of the transfected cells was monitored using a LacZ reporter construct. Compared to the empty vector, transfection with the sickle expression construct results in a dramatic increase in cell death levels, as evidenced by an 18-fold reduction in LacZ expression. Transfection of a reaper expression construct also results in significant cell loss, although not to the same extent as is observed with sickle. The cell death induced by either sickle or reaper is suppressed ~3- to 6-fold by coexpression of the genes encoding the caspase inhibitory proteins p35 or DIAP1. These data imply that like other Grim-Reaper proteins, Sickle acts upstream of caspases and induces apoptosis via a mechanism involving inhibition of IAP function. The cell death-inducing capabilities of sickle were also investigated in Drosophila. Surprisingly, P[GMR-gal4]-targeted sickle expression using P[UAS-sickle] strains is ineffective at inducing ectopic cell death in the adult eye. Thus, P[GMR-gal4]/P[UAS-sickle] animals survive to adulthood, and nearly all exhibit normal eye morphology. (A few of these flies did have slightly roughened eyes, suggesting weak cell killing effects of sickle expression.) This result is in stark contrast to the lethality and complete loss of eye tissue seen for P[GMR-gal4]-targeted expression of reaper, hid, or grim. The use of several additional P[gal4] strains also failed to yield any evidence for sickle-induced ectopic cell death. While the basis for the distinct effects of sickle expression in cultured cells and Drosophila tissue is not yet clear, cell-specific effects of ectopic grim-reaper expression have been previously noted (Wing, 2002).

Because of the synergistic activities of reaper, hid, and grim in embryonic CNS midline, attempts were made to determine if sickle might enhance the actions of other grim-reaper genes. Since P[GMR-gal4]-targeted sickle expression failed to induce ectopic cell death, this issue was addressed by coexpression of either sickle and an reaper/grim chimera or reaper in the adult eye. P[GMR-gal4]-targeted expression of reaper/grim results in viable adults that exhibit a temperature-sensitive loss of eye tissue and pigmentation. In contrast, at either 25°C or 21°C P[GMR-gal4]-targeted coexpression of sickle and reaper/grim results in complete lethality. At 18°C, where the reaper/grim effects are reduced, a few flies coexpressing sickle and reaper/grim did emerge, and these exhibited a much greater loss of eye tissue than flies expressing either sickle or reaper/grim alone. Thus, sickle exhibits strong synergistic actions with reaper/grim. As expected, the effects of reaper/grim, as well as reaper/grim and sickle, are repressed by coexpression of p35; these animals are viable when raised at 25°C and exhibit essentially normal eye size and pigmentation. To demonstrate that sickle-dependent synergism is not restricted to the reaper/grim chimera, whether P[GMR-gal4]-targeted sickle expression would enhance cell death induced by P[GMR-reaper] was also examined. Flies bearing a single copy of P[GMR-reaper] exhibit a moderate loss of eye tissue. In contrast, flies bearing one copy each of P[GMR-reaper], P[GMR-gal4], and P[UAS-sickle], exhibit much more severe eye cell death, with greatly reduced eye size and pigmentation. This ectopic cell death is repressed by expression of p35. Synergistic eye cell death effects are also observed for coexpression of sickle and a g/reaper chimera, as well as sickle and grim. These results indicate that sickle can potentiate the cell killing effects of grim-reaper genes, and they provide the first examples of synergistic action for grim-reaper genes outside of the embryonic CNS midline (Wing, 2002).

In summary, these data demonstrate that sickle is a novel member of the grim-reaper family of cell death activators and suggest that functional interactions may be a general mechanism underlying the actions of Grim-Reaper proteins. The sequence of the Sickle protein strongly suggests that it has unique RHG motif-dependent and RHG motif-independent functions. Overall, the identification of sickle reveals further complexity in the regulation of cell death activation in Drosophila and provides additional evidence that these linked genes at 75C be considered a genetic complex (Wing, 2002).

Programmed cell death in the embryonic central nervous system of Drosophila

Although programmed cell death (PCD) plays a crucial role throughout Drosophila CNS development, its pattern and incidence remain largely uninvestigated. This study provides a detailed analysis of the occurrence of PCD in the embryonic ventral nerve cord (VNC). The spatio-temporal pattern of PCD was traced and the appearance of, and total cell numbers in, thoracic and abdominal neuromeres of wild-type and PCD-deficient H99 mutant embryos were compared. Furthermore, the clonal origin and fate of superfluous cells in H99 mutants was examined by DiI labeling almost all neuroblasts, with special attention to segment-specific differences within the individually identified neuroblast lineages. These data reveal that although PCD-deficient mutants appear morphologically well-structured, there is significant hyperplasia in the VNC. The majority of neuroblast lineages comprise superfluous cells, and a specific set of these lineages shows segment-specific characteristics. The superfluous cells can be specified as neurons with extended wild-type-like or abnormal axonal projections, but not as glia. The lineage data also provide indications towards the identities of neuroblasts that normally die in the late embryo and of those that become postembryonic and resume proliferation in the larva. Using cell-specific markers it was possible to precisely identify some of the progeny cells, including the GW neuron, the U motoneurons and one of the RP motoneurons, all of which undergo segment-specific cell death. The data obtained in this analysis form the basis for further investigations into the mechanisms involved in the regulation of PCD and its role in segmental patterning in the embryonic CNS (Rogulja-Ortmann. 2007).

In this analysis of PCD distribution it was found that, macroscopically, the CNS of wt and PCD-deficient (H99) embryos do not show large differences. These observations indicate that the supernumerary cells do not disturb developmental events in the CNS of H99 embryos, such as cell migration and axonal pathfinding. The glial cells mostly find their appropriate positions accurately. The DiI-labeled NB lineages were, in the majority of cases, easily identifiable based on their shape, position and axonal pattern, despite the supernumerary cells. The FasII pattern showed that the axonal projections form and extend along their usual paths. In fact, the supernumerary cells themselves are capable of differentiating i.e. expressing marker genes and extending axons, as shown by clones of several NBs and by cell marker expression analysis in H99 (e.g. NB7-3) (Rogulja-Ortmann. 2007).

It has been shown that a large number of CNS cells undergo PCD during embryonic development. The distribution of activated Caspase-3-positive cells in wt embryos suggests that the death of some cells is under tight spatial and temporal control, as revealed by their regular, segmentally repeated occurrence. Other dying cells were rather randomly distributed, suggesting a certain amount of developmental plasticity. The overall counts of Caspase-3-positive cells give an estimate of the numbers of dying cells at a given time. They indicate that PCD becomes evident in the CNS at stage 11 and is most abundant in the late embryo (from stage 14). It is however difficult to estimate the total number of apoptotic cells throughout CNS development by anti-Caspase-3 labeling, because the cell corpses are removed fairly quickly. Therefore the total number of cells were counted per thoracic and abdominal hemineuromere in the late embryo. Comparison between stage 16 and stage 17 wt embryos indicates that 25-30 % of all cells are removed in both tagmata after stage 16, which in turn suggests that the total percentage of removed cells must be high, since PCD occurs at high levels already from stage 14 on. In comparison to the developing nervous system of C. elegans, where PCD removes about 10% of cells, and of mammals, where this number can be as high as 50-90%, PCD in the fly CNS appears to show an intermediate prevalence. This lends support to the hypothesis of an increasing contribution of PCD in shaping more advanced nervous systems during evolution (Rogulja-Ortmann. 2007).

Comparisons between wt and H99 reveal, as expected, a greater number of cells in both tagmata of H99 embryos (151% increase in the thorax and 162% in the abdomen at stage 17). These additional cells in H99 may reflect the total number of cells normally undergoing cell death until stage 17. However, there is a large variability in the total number of cells, especially within the H99 strain. In wt embryos, it seems to be more pronounced in the thorax and at stage 17, which might be a consequence of variable amounts of PCD occurring until this stage. The even higher variability within the H99 strain (both in thorax and abdomen) is likely to reflect variable numbers of additional cell divisions. The great majority of abdominal NBs are normally removed by PCD after they have generated their embryonic progeny, whereas in the thoracic neuromeres most of the NBs enter quiescence at the end of embryogenesis and continue dividing as postembryonic NBs in larval stages. Thus, there are few mitoses occurring in the wt CNS from stage 16 onwards. BrdU labeling experiments revealed a high number of BrdU-positive cells in some H99 embryos injected at early stage 17. It is assumed that these are progeny of mitotic NBs and/or GMCs that survive and continue dividing, generating cells that do not exist in wt. Clones obtained by DiI labeling in H99 confirm this conclusion. The finding that surviving cells divide already in the embryo complement results that showed that, in reaper mutants, NBs in the abdominal neuromeres survive and generate progeny in larval stages (Rogulja-Ortmann. 2007).

Among the DiI-labeled clones in H99 embryos, very few NB lineages were obtained which did not differ from their wt counterparts. The majority contained, as expected, supernumerary cells. In some cases axons projected by these cells could be identified, showing that they are specified as neurons. In fact, in three cases (NB4-2, NB5-3 and NB7-3), these additional cells were found to be specified as motoneurons. As additional axons within a fascicle were generally difficult to identify, it is possible that these are not the only lineages which make additional motoneurons in H99. Whether these cells are normally born and apoptose, or originate from additional divisions of surviving NBs or GMCs, cannot be determined from these experiments, but similar observations have been made for both cases. It is interesting that none of these cells, regardless of their origin, are specified as glia. No additional glia were observed in the NB clones in H99 embryos, and equal numbers of Repo-expressing glial cells were found in wt and H99. It is concluded that PCD occurs almost exclusively in neurons and/or undifferentiated cells, and that lateral glia are not produced in excess numbers in the embryo. Furthermore, because it is likely that NBs, which normally die, stay in a late temporal window in H99, one could speculate that NBs in this window normally do not give rise to glia. These results are not in agreement with the notion that LG are overproduced, and their numbers adjusted through axon contact. Occasional apoptotic LG have been observed and it is possible that the current method of counting does not allow a resolution fine enough to account for an occasional additional Repo-positive cell in H99 embryos. However, if LG were consistently overproduced, a higher number of glia in would be expected H99 embryos. It is assumed that LG cell death may reflect a small variability in the number of cells needed, and not a general mechanism for adjusting glial cell numbers (Rogulja-Ortmann. 2007).

Generally, no difference was found between Repo-expressing glia numbers in wt and H99. However, a small difference does become apparent when one separates the total cell counts into those in the CNS and those in the periphery: 25.67±0.45 cells/hs and 28.42±0.64 cells/hs for wt and H99, respectively, were counted in the CNS, whereas 8.50±0.28 cells/hs and 6.35±0.82 cells/hs for wt and H99, respectively, were found in the periphery. The reasons for this difference might be the greater width of the CNS in H99 embryos, and that the cues required for proper migration of the peripheral glia are disturbed by additional cells. Alternatively, the difference might be due to differentiation defects in these cells (Rogulja-Ortmann. 2007).

In addition to NB clones with too many cells and wild-type-like axon projections in H99, some lineages were obtained whose clones exhibited atypical projection patterns. These projections were found to belong both to motoneurons (e.g. in NB4-2) and interneurons (e.g. NB5-3, NB7-2 and NB-7-4). NB4-2 normally produces two motoneurons (RP2 and 4-2Mar) and 8-14 interneurons. In two out of three NB4-2 clones in H99 two additional motoneurons that project anteriorly were found, similar to RP2. One of the two clones was found in the thorax and had a normal cell number (16), whereas the other was abdominal and had too many cells (25). Thus, the two additional motoneurons are likely to be the progeny of divisions occurring in the wt, and not of an additional NB or GMC mitosis. The fact that the third NB4-2 clone (found in the abdomen and comprising 17 cells) did not show the same motoneuronal projections could be due to these cells not being differentiated at the time of fixation (clones of different ages were occasionally observed in the same embryo), or they may not have differentiated at all. It would be interesting to determine the target(s) of these additional motoneurons and thereby perhaps gain insight into physiological reasons for their death. However, such an experiment has to await tools that allow specifically labeling of the NB4-2 lineage, or these motoneurons, in the H99 mutant background (Rogulja-Ortmann. 2007).

The other three lineages (NB5-3, NB7-2 and NB7-4) all have atypical interneuronal projections. The cells which these atypical axons belong to may represent evolutionary remnants that are not needed in the Drosophila CNS. Alternatively, they might have a function earlier in development and be removed when this function is fulfilled. Such a role has been shown for the dMP2 and MP1 neurons, which are born in all segments and pioneer the longitudinal axon tracts. At the end of embryogenesis these neurons undergo PCD in all segments except A6 to A8, where their axons innervate the hindgut. It is known that some cells of the NB5-3 lineage express the transcription factor Lbe, and that H99 mutants show about three additional Lbe-positive neurons per hemisegment, which mostly likely belong to NB5-3. The DiI-labeling results complement this finding in that four or more additional neurons were also found in H99 clones. The supernumerary Lbe-positive neurons in H99 could possibly be the ones producing the atypical axonal projections (Rogulja-Ortmann. 2007).

In the wt embryo, only eight NB lineages show obvious tagma-specific differences in cell number and composition. Tagma-specific differences among serially homologous CNS lineages have been shown to be controlled by homeotic genes. Therefore, these lineages provide useful models for studying homeotic gene function on segment-specific PCD. In H99 embryos, further lineages were observed that were differently affected in the thorax and abdomen. How these tagma-specific differences arise in a PCD-deficient background is an interesting question. For example, NB4-3 shows a wild-type cell number in the thorax (8 and 12-13), but has too many cells in the abdomen (15, 15 and 22). There are a couple of plausible scenarios to explain this observation. (1) The development of the NB4-3 lineage, including the involvement of PCD, could actually differ in the thorax and abdomen of wt embryos, with the final cell number being similar by chance. The DiI-labeled clones allow determination of the final cell number, but do not reveal how this number is achieved. The difference would become obvious in an H99 mutant background, at least regarding the involvement of PCD. (2) This possibility does not exclude the first one, the thoracic NB4-3 could become a postembryonic NB (pNB) and the abdominal NB4-3 might undergo PCD after generating the embryonic lineage. In H99, the abdominal NB would be capable of undergoing a variable number of additional divisions to generate a variable number of progeny. This would easily explain larger discrepancies in cell number between individual clones in H99 (e.g. the abdominal NB4-3 clone with 22 cells), and is in agreement with occasional observations of H99 embryos with a very high CNS cell number per segment, and with the two observed classes of H99 embryos with high and low numbers of BrdU-positive cells (Rogulja-Ortmann. 2007).

NB6-2 is another lineage whose clones differ in the two tagmata of H99 embryos. In this case, the abdominal clones showed no difference to their wt counterparts, whereas the thoracic clones did (18 and 19 cells). Although no difference in cell number between thoracic and abdominal clones was reported for this lineage, a rather large count range (8-16 cells) was given, which would allow for a thorax-specific PCD of two to three postmitotic progeny. Alternatively, the thoracic NB6-2 might undergo cell death upon generating its progeny, which would make it the first identified apoptotic NB in the thorax. When PCD is prevented, this NB may undergo a few additional rounds of division. The data obtained in these experiments do not counter this notion, but the number of clones obtained in the thorax was not sufficient to draw a definite conclusion. As the abdominal NB6-2 lineage in H99 did not differ from the one in wt, its NB may be one of the few abdominal postembryonic NBs (Rogulja-Ortmann. 2007).

A specific set of NBs undergoes PCD in the late embryo, whereas surviving NBs resume proliferation in the larva as pNBs, after a period of mitotic quiescence. The identities of the individual NBs undergoing PCD versus those surviving as pNBs are still unknown. The sizes of NB lineages obtained in H99 embryos may provide hints for identifying candidate pNBs in the abdomen [12 NBs/hs in A1, four in A2 and three in A3 to A7, and NBs that undergo PCD in the thorax at the end of embryogenesis [seven NBs/hs in T1 to T3. In the abdomen, NB1-1a and NB6-2 are obvious candidates for pNBs, as they remained consistently unchanged in H99 embryos. Two other NBs, NB1-2 and NB3-2, are also potential abdominal pNBs as they mostly did not differ from their wt counterparts, and only occasionally contained one additional cell. On the other hand, clones which showed more than twice the cell number in H99 (NB2-1, NB5-4a and NB7-3) than in wt, strongly suggest that these NBs normally undergo PCD in the abdomen (but perform additional divisions in H99), because, even if one daughter cell of each GMC undergoes PCD, they still cannot account for all cells found in H99 clones (Rogulja-Ortmann. 2007).

Regarding thoracic NBs, it can only be speculated on account of low sample numbers. NBs which seem to become pNBs in the thorax, as they showed no difference between wt and H99 clones, are NB3-2, NB4-3 and NB4-4. Potential candidates for NBs which do not become pNBs, but undergo PCD in the thorax, are expected to consistently have a significant increase in cell number in H99. These are NB5-1 and NB5-5. In addition, lineages for which one clone was obtained in H99 but which also showed many more cells in the thorax than normal are NB2-2t, NB5-4t and NB7-3 (Rogulja-Ortmann. 2007).

In order to investigate the developmental signals and mechanisms involved in the regulation of PCD in the embryonic CNS, some of the apoptotic cells were identified which will be used as single-cell PCD models. These are the dHb9-positive RP neuron from NB3-1, Lbe-positive neurons from NB5-3, the Eg-positive GW neuron from NB7-3 and the Eve-positive U neurons from NB7-1. As not much is known about the dying RP motoneuron or the Lbe-positive neurons, the first goal will be to characterize each of these cells more closely, based on the combination of expressed molecular markers (Rogulja-Ortmann. 2007).

Some of the dying NB7-3 cells are already known to be undifferentiated daughter cells of the second and third GMC, which undergo PCD shortly after birth. Notch has been identified as the signal initiating PCD. The surviving daughters receive the asymmetrically distributed protein Numb, which counteracts the PCD-inducing Notch signal. The same had been shown in a sensory organ lineage of the embryonic peripheral nervous system, where cells produced in two subsequent divisions undergo Notch-dependent PCD. Both the PCD in the NB7-3 lineage and in the sensory organ lineage require the Hid, rpr and grim genes. It will be interesting to see whether the Notch-Numb interaction also plays a role in the segment-specific PCD of the differentiated GW motoneuron, or if another signal is used for the removal of this, and possibly other, differentiated cells (Rogulja-Ortmann. 2007).

The U motoneurons also show a segment-specific cell death pattern (they apoptose in A6 to A8), thus somewhat resembling the MP1 and dMP2 neurons. However, in contrast to MP1 and dMP2, the U neurons survive in the anterior segments and undergo PCD in the posterior ones. Whether homeotic genes play any role in the survival or death of these cells remains to be investigated (Rogulja-Ortmann. 2007).

In summary, this study has presented descriptions of PCD in the developing CNS of the wt Drosophila embryo, and of the CNS of PCD-deficient embryos. The pattern of Caspase-dependent PCD is partly very orderly, suggesting tight spatio-temporal control of cell death, and partly random, which suggests a certain amount of plasticity already in the embryo. The CNS of PCD-deficient embryos is nevertheless well organized, despite the presence of too many cells. These superfluous cells come from both a block in PCD and from additional divisions that surviving NBs go through. It was possible to link the occurence of cell death to identified NB lineages by clonal analysis in PCD-deficient embryos, to uncover segment-specific differences, and to establish single-cell PCD models that will be used in further studies to investigate mechanisms responsible for controlling PCD in the embryonic CNS (Rogulja-Ortmann. 2007).

Lineage-specific cell death in postembryonic brain development of Drosophila

The Drosophila central brain is composed of thousands of neurons that derive from approximately 100 neuroblasts per hemisphere. Functional circuits in the brain require precise neuronal wiring and tight control of neuronal numbers. How this accurate control of neuronal numbers is achieved during neural development is largely unclear. Specifically, the role of programmed cell death in control of cell numbers has not been studied in the central brain neuroblast lineages. This study focusses on four postembryonic neuroblast lineages in the central brain identified on the basis that they express the homeobox gene engrailed (en). For each lineage, the total number of adult-specific neurons generated was determined, as well as number and pattern of en-expressing cells. Programmed cell death has a pronounced effect on the number of cells in the four lineages; approximately half of the immature adult-specific neurons in three of the four lineages are eliminated by cell death during postembryonic development. Moreover, programmed cell death selectively affects en-positive versus en-negative cells in a lineage-specific manner and, thus, controls the relative number of en-expressing neurons in each lineage. Furthermore, evidence is provided that Notch signaling is involved in the regulation of en expression. Based on these findings, it is concluded that lineage-specific programmed cell death plays a prominent role in the generation of neuronal number and lineage diversity in the Drosophila brain (Kumar, 2009).

In postembryonic CNS development of holometabolous insects such as flies, a combination of programmed cell death and neuronal process re-innervation allows the larval nervous system to reorganize and innervate new body structures. During metamorphosis many adult-specific neurons in the ventral ganglia are targeted by programmed cell death, particularly in abdominal segments. Furthermore, extensive cell death occurs during postembryonic development in the insect visual system, where cells are overproduced and those that do not make the appropriate targets are eliminated by apoptosis. By contrast, very little is currently known about the prevalence and functional roles of programmed cell death in development of the insect adult central brain (Kumar, 2009).

This report identifies four neuroblast lineages in the postembryonic central brain and finds that programmed cell death occurs in all four lineages, albeit to different extents. Whereas cell death plays only a minor role in the medial cluster MC1 lineage, it has dramatic effects in anterior cluster (AC), posterior cluster (PC) and medial cluster MC2 lineages, in which nearly half of the adult-specific neuronal progeny are programmed to die during larval development. It is noteworthy that the adult-specific neurons targeted by cell death are generated during larval development and are eliminated before their respective neuroblasts stop proliferating (12-24 hours after pupal formation). Because the cell death reported here occurs before neuronal differentiation, it is probably not involved in events of brain reorganization that take place during metamorphosis (Kumar, 2009).

Another central feature of the cell death events demonstrated here is that none of the four lineages is completely eliminated by cell death; all four neuroblasts and a significant number of their neuronal progeny survive at the end of larval development, and these neuronal progeny are largely present in the adult. In this sense, the programmed cell death reported here is likely to be functionally different from the cell death observed in the ventral ganglia, where the neuroblast itself undergoes apoptosis, regulating neuronal numbers in the abdominal segments (Kumar, 2009)

These experiments indicate that programmed cell death plays a prominent role in determining lineage-specific features; if cell death is blocked the total neuronal number increases in all four lineages and the number of en-expressing neurons increases in AC, PC and MC2. Furthermore, the axonal projection pattern of H99 mutant (deleting rpr, hid and grim) and Notch mutant en-expressing lineages was examined, comparing them to wild type. Both cell death defective H99 and Notch mutant PC lineages showed an additional projection that was not present in the wild type, whereas the other three H99 lineages did not appear to change drastically in their projection patterns. In conclusion, programmed cell death appears to contribute to the cellular diversity of neuronal lineages in the central brain (Kumar, 2009).

Studies on neuroblast lineages in the developing ventral ganglia indicate that proliferating neuroblasts generate a largely invariant clone of neural cells. In general, each neuroblast division produces a distinctly fated GMC, and each GMC division produces two sibling progeny of different fates. There is some evidence that the fate of these progeny is controlled by the parental GMC; the two siblings are restricted to a pair of different cell fates, with one sibling adopting an 'A' fate and the other adopting a 'B' fate. This, in turn, has led to a model in which a neuroblast lineage can be thought of as composed of two hemilineages, with one hemilineage comprising 'A'-fate cells and the other hemilineage comprising 'B'-fate cells. It is thought that an interaction between Notch and Numb is responsible for generating distinct neural fates of the two GMC daughter cells, with a loss of Notch or Numb resulting in reciprocal cell-fate duplication. However, Notch signaling does not appear to confer a particular fate; rather, it acts generically as a mechanism to enable two siblings to acquire different fates, and other developmental control genes that are inherited from the specific parental GMC are thought to be instrumental in determining the final identity of each progeny (Kumar, 2009).

Findings on lineage-specific cell death support a comparable model in which all four brain neuroblasts can generate one en-positive hemilineage and one en-negative hemilineage. In this model, programmed cell death is then targeted in a lineage-specific manner to either the en-negative hemilineage (AC, PC), or the en-positive hemilineage (MC2), or neither hemilineage (MC1). Alternatively, en-positive and en-negative neurons in the lineages could be generated in a temporal fashion and subsequently en-positive or en-negative neurons could be eliminated in a lineage-specific manner. However, the results suggest that this is unlikely. In particular, in the PC lineage, more than 80% of the two-cell clones examined were composed of one en-positive and one en-negative cell. If the above did occur, a significant number of two-cell en-positive clones should have been obtained along with two-cell clones comprising one en-positive and one en-negative neuron. Similar analysis of single and two cell clones in the other three en lineages is further required to confirm the occurrence of hemilineage-specific programmed cell death (Kumar, 2009).

Based on these experimental results, it was postulate that Notch signaling is an important generic mechanism underlying generation of the two different hemilineages, as in the absence of Notch signaling, cell-fate duplication of GMC siblings occurs. Indeed, analysis of Notch loss-of-function neuroblast clones suggests that in the absence of Notch signaling most of the neurons in the four lineages acquire an en-positive cell fate. Alternatively, in the four lineages examined, being positive for en may be the default state of the cells, and Notch induces secondary fate by repressing en in subsets of cells in each lineage. These en-positive neurons then appear to survive or undergo programmed cell death depending on the lineage-specific context. However, it remains to be seen whether Notch itself acts on the apoptotic machinery, independent of en (Kumar, 2009).

This study, used en as a molecular marker to identify four lineages in the postembryonic central brain. Might en itself be functionally involved in regulating programmed cell death in these lineages? For the PC lineage, there is some indication that the total clone size is reduced by approximately half in en loss-of-function mutants, compared with wild type. Although this suggests that en may be involved in promoting survival of en-positive neurons in PC (and probably AC), it does not explain the role of en in the MC2 lineage, where it would have to play an opposing role, as en-positive neurons die in this lineage. Thus, en could act either as a pro-apoptotic or an anti-apoptotic factor, depending on the lineage-specific context. Moreover, a direct genetic interaction between en and the apoptotic machinery remains to be investigated. As en is known to have multiple interactions with other proteins, a complex regulatory network involving target proteins of en may be responsible for regulating apoptosis in a lineage-specific manner. Further analysis of interactions with such target proteins is necessary to reveal the full regulatory network in more detail (Kumar, 2009).

The lineage-specific effects of cell death and of Notch signaling in AC and PC are distinctly different from those observed in MC1 or MC2 lineages. However, when compared with each other, many aspects of AC and PC lineages are similar. In wild type, both lineages consist of similar numbers of adult-specific neurons, and the majority (approximately 80%) of these neurons are positive for en, whereas neuroblasts and GMCs are negative for en in both lineages. Blocking cell death results in a substantial (approximately double) increase in total cell number in both lineages, and this increase is almost exclusively due to an increase in the number of surviving en-negative neurons in both lineages. Moreover, loss of Notch function causes a marked increase in the number of surviving en-positive neurons without affecting the number of en-negative neurons in both lineages. The only significant difference between AC and PC lineages observed in this study is that the AC lineage is located in the protocerebrum, whereas the PC lineage is located in the deutocerebrum (Kumar, 2009).

What might be responsible for these similarities in the AC and PC neuroblast lineages? There is some evidence for the existence of serially homologous neuroblasts in the fly brain and VNC. In the VNC, serially homologous neuroblasts, defined by comparable time of formation, similar positions in the neuromeric progenitor array and similar expression of developmental control genes, such as segment polarity genes, dorsoventral patterning genes and other molecular markers, can give rise to almost identical cell lineages. This suggests that similar regulatory interactions take place during development of serially homologous neuroblasts and their neural lineages. A comparison of molecular expression patterns in neuroblasts from different neuromeres of the brain and ventral ganglia suggests that several of them might be serial homologs of each other. For example, neuroblasts NB5-6 in the abdominal, thoracic and subesophageal ganglia have been proposed to be homologous to NBDd7 in the deutocerebrum and NBTd4 in the tritocerebrum (Kumar, 2009).

Given the remarkable similarities in AC and PC neuroblast lineages, it is possible that the protocerebral AC lineage and the deutocerebral PC lineages represent serial homologs. If this is the case, then investigations of the cellular and molecular mechanisms that control their lineage-specific development should be useful for understanding of how regionalized neural diversity in the brain evolves from a basic metameric ground state. However, as neither the combination of developmental control genes expressed in AC and PC neuroblasts nor the position of the two brain neuroblasts in their neuromeres of origin are currently known in sufficient detail, further experiments are needed before the issue of serial homology can be resolved for these brain neuroblast lineages (Kumar, 2009).

The role of apoptosis in shaping the tracheal system in the Drosophila embryo

The tubular network of the tracheal system in the Drosophila embryo is created from a set of epithelial placodes by cell migration, rearrangements, fusions and shape changes. A designated number of cells is initially allocated to each branch of the system. The final cell number in the dorsal branches is not only determined by early patterning events and subsequent cell rearrangements but also by elimination of cells from the developing branch. Extruded cells die and are engulfed by macrophages. These results suggest that the pattern of cell extrusion and death is not hard-wired, but is determined by environmental cues (Baer, 2010).

In live studies of the tracheal system using embryos expressing GFP under the control of the breathless (btl) promoter GFP-expressing motile cells were observed that were not attached to the tracheal system. The btl gene, which encodes an FGF receptor homolog, is mainly expressed in the tracheal system, but also in glial cells and a few other cell type. However, it had not previously been described as being expressed in individual cells that were dispersed in the embryo. Since it was not clear what the individual cells outside the tracheal system were, other tracheal markers were used to determine whether they indeed derived from the tracheal system, or rather represented an as yet undiscovered cell type in which the btl promoter is active. When the tracheal system was marked with lacZ expressed under the control of the promoter of another tracheal gene, trachealess (trh-lacZ), it was observed that lacZ was also expressed not only in the tubular tracheal epithelia but also in single cells detached from the tracheal system (Baer, 2010).

In the live observations using GFP it was noticed that these cells appeared to be moving around in the embryo, preferentially along the dorsal trunk of the trachea. Previously hemocytes had been described to move along the tracheal system. Thus it was asked whether these cells might be a subpopulation of hemocytes that shared expression of some genes with the tracheal system. To assess their cell type, the cells were analyzed in embryos in which markers for the tracheal system and for hemocytes were simultaneously visualized. Hemocytes were visualized by using a croquemort-GAL4 transgene to control the expression of GFP. Croquemort is expressed in a subpopulation of hemocytes, the macrophages. For the tracheal system trh-lacZ was used (Baer, 2010).

It was found that the individual lacZ-expressing cells that are detached from the tracheal system also expressed the crq-controlled GFP. In addition, there were also many cells that showed only crq-GFP staining and no lacZ. There are two possible explanations for these findings. Either a subset of macrophages can activate transcription of the btl and trh promoters, or the doubly-stained objects are not single cells. Consistent with the latter explanation, it was noticed that the two fluorescent markers were often localized in non-overlapping parts of the cells, suggesting that lacZ-expressing cells or cell fragments might have been engulfed by macrophages. If this were the case, then the doubly-stained objects should contain two nuclei. Indeed, in triple stainings with the nuclear marker TOTO-3 two TOTO-3 signals were observed in the majority of the doubly-stained objects, indicating the presence of two nuclei. It is concluded therefore that the objects are hemocytes that have engulfed tracheal cells that have left the tracheal epithelium (Baer, 2010).

To test directly whether these single cells originate from the developing tracheal tree, more detailed live observations were performed. In previous experiments it was noticed that the disconnected cells can be preferentially found near the branches that undergo cellular rearrangements, for example, at the bases of dorsal branches. The areas around these sites were chosen for further studies. Live imaging of dorsal branches of the embryos expressing α-cat-GFP or sqh-GFP revealed that during the outgrowth of the dorsal branches individual tracheal cells detach from the tracheal system at, or near the site where the dorsal branches emerge from the dorsal trunk. The numbers of cells leaving the system from the base of branches and from the interbranch-regions of the dorsal trunk were compared in 12 videos of wildtype embryos. The 'base of the branch' is described as those cells that bordered at least on one side on a cell that was part of the dorsal branch. 42 branches were evaluated and cells were found leaving from the base in 20 cases, whereas in 40 interbranch-regions seven cells leaving the system were found. Thus, cells are almost threefold less likely to be extruded in the large interbranch area than from the small area at the base of the branch (Baer, 2010).

Since the detached cells appeared to be engulfed by macrophages and one function of macrophages is to clear up apoptotic cells, whether the trh-lacZ positive cells seen inside the hemocytes displayed markers for apoptosis was tested. It was found that some but not all of the trh-lacZ cells that were detached from the tracheal system gave a positive signal in a TUNEL reaction. Since the TUNEL assay only detects cells in a brief phase of apoptosis it was not surprising that only a subset of the detached cells were labelled. Thus this result is consistent with the notion that the engulfed cells are dying or dead cells. A further hallmark of apoptosis is the activation of caspase 3, which can be detected with an antibody that specifically recognizes the activated form. Trh-lacZ-expressing cells that were detached from the tracheal system also gave a positive signal when stained with antibodies against activated caspase3 (Baer, 2010).

Next, it was asked whether the detached cells undergo apoptosis because they have lost contact with the tracheal epithelium, or, conversely, whether they die within the epithelium and are subsequently extruded. This question was addressed by two approaches: tracing caspase activity in vivo and blocking apoptosis. To examine when the apoptotic program is induced in tracheal cells leaving the system an in vivo fluorescent sensor of caspase activity, the 'Apoliner' transgene (Bardet, 2008), was used. The Apoliner consists of two fluorophores, a membrane-anchored RFP and a GFP with nuclear localisation signal (NLS), which are linked by a caspase sensitive fragment of the DIAP1 protein. Upon caspase activation within the cell, the sensor is cleaved, resulting in translocation of GFP into the nucleus, whereas RFP stays at the membrane. The Apoliner was expressed using btl-GAL4 and the development of dorsal branches was followed in vivo. Nuclear GFP can be observed in individual cells in the tracheal system. Shortly after they begin to accumulate nuclear GFP these cells detach from the system. Thus apoptosis precedes cell removal from the tissue (Baer, 2010).

If death is a prerequisite for expulsion, then suppressing apoptosis should reduce the number of detached cells. To test this, the tracheal system was examined in embryos homozygous for a deficiency Df(3L)H99, which removes the three pro-apoptotic genes, grim, reaper and hid, and in which apoptosis therefore cannot occur. Since homozygous Df(3L)H99 embryos show strong overall developmental defects, the analysis was restricted to the dorsal branches in segments that do not show gross morphological abnormalities, i.e. segments 3-7. Whereas wild type stage 14/15 embryos show approximately 2-4 detached cells in segments 3-7 on each side, no detached cells were observed in embryos that were homozygous for Df(3L)H99. To exclude the possibility that this was due to a non-autonomous effect of the mutant phenotype of surrounding tissues in Df(3L)H99 mutant embryos, an alternative way of blocking apoptosis only in the tracheal system was used. The baculoviral inhibitor of apoptosis p35 was expressed in tracheal cells using the btl-GAL4 driver line. To confirm that apoptosis was blocked efficiently, the Apoliner was co-expressed together with p35. In these embryos no Apoliner signal was detected, indicating the absence of caspase activity, and no cells were seen to leave the tracheal system. This shows that the completion of the apoptotic program is necessary for detachment of the tracheal cells (Baer, 2010).

The fact that the detached cells were frequently found near the base of the dorsal branches of the tracheal system raises the question whether their leaving the epithelium might be associated with a specific developmental programme, such as the morphogenesis of the dorsal branches. Since each dorsal branch arises from six cells, but the final branch typically consists of only five cells, it has been suggested that the sixth cell migrates back to the dorsal trunk. The results point to an alternative possibility suggesting that apoptosis might be a mechanism that contributes to the determination of cell number in the dorsal branches. If the assumption that cell death is involved in determining the number of cells is correct, then dorsal branches should contain more cells if apoptosis cannot occur. To test this, cell numbers were counted in the dorsal branches of Df(3L)H99 mutant embryos and in embryos expressing p35 in the tracheal system. The embryos were stained with an antibody against Trachealess to mark specifically the nuclei of tracheal cells. The nuclei in dorsal branches were then counted under the microscope by focusing through the entire depth of the dorsal branch. Branches with an unclear course were not included in the analysis (Baer, 2010).

All tested genotypes showed variability in cell number in the dorsal branches, but there were clear differences between the mutant and the wild type embryos. 76.3% of the 93 dorsal branches evaluated in 10 wild type embryos consisted of 5 cells, 8.6% consisted of six cells and 15.1% consisted of 3 or 4 cells. This shows that branches with six cells are possible, but 5 cells are preferred. By contrast, embryos with blocked apoptosis had 51.6% (btl-Gal4, UAS-p35 embryos) or 56.6% (Df(3L)H99 mutant embryos) dorsal branches with six cells. These results indicate that removal of the cells by apoptosis indeed contributes to the determination of the final cell number in the dorsal branches (Baer, 2010).

It is unclear how the emigrating cell is determined, and why it is not determined in every one of the branches. The fact that this is so shows that, in contrast to many other situations in which apoptosis eliminates unwanted cells during development, cell death in this case is not the result of a hard-wired developmental programme. Instead it is more likely to be a response to unfavourable conditions in the cell's environment. Changes in the microenvironment have been shown to induce cell death in mammalian endothelial cells, with signals from the extracellular matrix influencing the balance between cell survival and apoptosis. Adhesion via integrins can protect cells from FAS mediated apoptosis, and integrins may act as mechano-transducers in this context. Thus it is possible that weakening of cell-cell junctions resulting from tissue remodelling in the tracheal system could trigger the apoptotic pathway. Junction rearrangement also affects cell death in the wing disc, where an imbalance in the junctional forces within the epithelium was found to be rebalanced by the elimination of cells. In this system, the initial irregularity arose through cell proliferation, but it is imaginable that in the tracheal system cell intercalation might affect local junctional forces, and cell extrusion can be triggered to obtain a geometrically optimal structure (Baer, 2010).

The fact that only half of the dorsal branches in the mutant embryos had six cells shows that apoptosis cannot be the only mechanism for the removal of supernumerary cells. There must be other ways of losing cells, perhaps by re-integration into the dorsal trunk, as previously suggested. Such cases were indeed observed. More stringently, even embryos in which a larger number of branches have six cells, namely btl-Gal4, UAS-p35 embryos, can develop normally and hatch. It is of course possible that subtle defects, for example a reduced efficiency in oxygen delivery, would not manifest themselves in easily measurable phenotypes in a laboratory setting (Baer, 2010).

In summary, the results are consistent with a scenario in which cells that find themselves in a sub-optimal epithelial context either move away, or die and leave the epithelium. If the apoptotic pathway is blocked, they may be forced to use the option of moving elsewhere, or induce neighbouring cells to rearrange further, or they remain in place, and the system is able to tolerate a sub-optimal structure (Baer, 2010).

grim promotes programmed cell death of Drosophila microchaete glial cells

The Inhibitor of apoptosis (IAP) antagonists Reaper (Rpr), Grim and Hid are central regulators of developmental apoptosis in Drosophila. Ectopic expression of each is sufficient to trigger apoptosis, and hid and rpr have been shown to be important for programmed cell death (PCD). To investigate the role for grim in PCD, a grim null mutant was generated. grim was not a key proapoptotic gene for embryonic PCD, confirming that grim cooperates with rpr and hid in embryogenesis. In contrast, PCD of glial cells in the microchaete lineage required grim, identifying a death process dependent upon endogenous grim. Grim associates with mitochondria and has been shown to activate a mitochondrial death pathway distinct from IAP antagonization; therefore, the Drosophila bcl-2 genes buffy and debcl were investigated for genetic interaction withgrim. Loss of buffy led to microchaete glial cell survival and suppressed death in the eye induced by ectopic Grim. This is the first example of a developmental PCD process influenced by buffy, and places buffy in a proapoptotic role. PCD of microchaete glial cells represents an exceptional opportunity to study the mitochondrial proapoptotic process induced by Grim (Wu, 2010).

Shaping of the Drosophila embryo involves a series of morphogenetic movements, all of which are accompanied by cell death. Embryos homozygous for the H99 deletion lack developmental apoptosis. The three known proapoptotic genes in the H99 region, rpr, hid and grim are expressed during embryogenesis. Data using overlapping deficiencies, mutants and ectopic expression studies suggest that the combined activities of rpr, hid and grim are required for initiation of PCD during embryogenesis. This study of the first grim null mutant confirms that grim works cooperatively with the other RHG genes for embryonic PCD (Wu, 2010).

In development of the mechanosensory bristles of the Drosophila notum, an SOP gives rise to the four cells of the sensory organ and a glial cell that is doomed to die. Evaluation of the grim null mutant, knockdown of grim, and a large deletion that removes genomic sequences upstream of grim all demonstrated that PCD of this glial cell is dependent upon grim. Additionally, a small deletion within the glutamine-rich domain of Grim was identified that reduced the proapoptotic activity of Grim. Future investigations that focus on the role of the glutamine-rich domain in Grim function will first require separation of the grimΔ52-57 mutation from the f04656 P-element insertion (Wu, 2010).

An increase was observed in glial cell survival in the absence of buffy. Death of microchaete glial cells is the first developmental PCD identified that is modified by loss of buffy. buffy played a proapoptotic role in this process as well as in grim-dependent cell death in the eye. Expression studies in the eye have led to the conclusion that over-expressed Debcl is a proapoptotic Bcl-2 protein, whereas over-expressed Buffy moderately blocks the killing activity of Debcl and other pro-death proteins such as Rpr, Grim and Hid. In stress-induced apoptosis this study also found that Buffy inhibits apoptosis. Why then, does Buffy appear to be proapoptotic in the current study? To begin, although over-expressed Buffy suppresses cell death due to ectopic Grim in the eye, this study found that a high level of Buffy was unable to block PCD of the microchaete glial cell. This suggests that there is an important difference in the killing function of over-expressed Grim relative to a physiological amount of Grim. It is not simply that over-expressed Grim induces Debcl-dependent apoptosis that would be enhanced by loss of Buffy and suppressed by ectopic Buffy. If this were the case, then Grim-induced death should have been suppressed by loss of debcl, which it was not. How ectopic Buffy inhibits over-expressed Grim-dependent cell death (as well as other proapoptotic proteins when over-expressed may instead relate to its ability to suppress mitochondrial dysfunction. But in cells that respond to endogenous levels of Grim, Buffy is not antiapoptotic. This establishes that physiological levels of Grim do not utilize a killing mechanism that can be suppressed by Buffy. Furthermore, ectopic Buffy is not generally inhibitory to PCD in the animal. Secondly, the Drosophila Bcl-2 proteins can function as both pro- and anti-death proteins: Debcl protects cells from CED-3 and serum-deprivation induced cell death and protects neurons from expanded polyglutamine-mediated neurodegeneration whereas Buffy can promote cell death. The finding that Bcl-2 proteins have dual functions is not novel. Certain mammalian antiapoptotic Bcl-2 proteins can be converted into proapoptotic proteins that induce Cytochrome c release from mitochondria. CED-9, the C. elegans Bcl-2 protein, also has both pro- and antiapoptotic activity. Conversely, Bax, Bak and Bad are mammalian proapoptotic Bcl-2 proteins that can promote cell survival. In Drosophila, as in mammals, the ability of Bcl-2 proteins to promote or inhibit cell death likely depends on the specific cellular context (Wu, 2010).

Grim can activate two pathways leading to cell death: the first is dependent upon the IBM to block DIAP1 and release active caspases, and the second requires the GH3 domain for mitochondrial targeting. In the case of Rpr, these two pathways are inter-dependent in that DIAP1 degradation promoted by Rpr is significantly more efficient at the mitochondria. However, this may not be the situation for Grim as there are numerous observations that Grim maintains some killing activity even when unable to bind and antagonize DIAP1 and that the GH3 domain alone targets mitochondria and induces death. These data are suggestive of the existence of a DIAP1-independent killing mechanism for Grim that is mitochondrial. Expression of mutants lacking the ability to engage one or the other of the two mechanisms demonstrated that the two pathways could cooperate. It is not known how these two activities contribute to Grim function in an endogenous PCD setting. This study defines PCD of microchaete glial cells as the first example of grim-dependent PCD, and a very recent report demonstrates that life and death of these cells does not rely upon DIAP1 antagonization. In this report, DIAP1 protein turnover was monitored in live animals. DIAP1 protein was not detectable in cells of the microchaete SOP lineage from the 2-cell stage (PIIa and PIIb) through the 5-cell stage (shaft, socket, neuron, sheath and glial cell). Thus, even from birth, there was no DIAP1 protein in the glial cell. The lack of DIAP1 protein was not because RHG proteins induced DIAP1 protein degradation since there was also no detectable DIAP1 in H99−/− clones. Lastly, Rpr was expressed in cells of the SOP lineage and this did not lead to precocious glial cell death. If IAP protein was present and responsible for maintaining glial cell viability, then Rpr expression should have caused premature cell death. These data demonstrate that Grim does not induce PCD of microchaete glial cells solely through DIAP1 antagonization, and suggest that another mechanism utilized by Grim contributes to glial cell death. The previously described mitochondrial pathway for Grim-dependent death is the most likely candidate and is supported by the genetic and physical interaction of Grim with Buffy. Verification of the dependence of microchaete glial cell death on the GH3 domain of Grim awaits mutational analysis of grim at its endogenous locus (Wu, 2010).

A mitochondrial pathway activated by Grim could involve mitochondrial permeabilization or alternatively mitochondrial fragmentation and dysfunction, but must ultimately lead to cell death by activating caspases (microchaete glial cells have active caspase activity prior to death and p35 expression forces their survival. When expressed in vertebrate cells, Grim induced release of Cytochrome c, through a process that required the GH3 domain but was independent of IAP antagonization or Bcl-2 proteins. Although Cytochrome c is not released, mitochondrial permeabilization may play a role in Drosophila cell death (mitochondria are clearly affected as Cytochrome c changes confirmation early in death). Mitochondrial fragmentation has been observed in Drosophila cells undergoing PCD and occurs prior to caspase activation, suggesting that fragmentation may be causative. A large body of work has demonstrated that mitochondrial fission in mammalian cells accompanies apoptosis (Wu, 2010).

In this study, although it is possible that Grim and Buffy promote cell death through separate mechanisms, it is proposed that the current results are most consistent with Buffy enhancing or amplifying a mitochondrial death process activated by Grim. The physical interaction between the two proteins supports this. Such a model would explain why loss of buffy has only a partial effect on ectopic Grim-dependent death (likely mostly DIAP1-dependent) and why microchaete cell death was affected less by loss of buffy than by loss of grim (because Grim is augmented by Buffy). Endogenous Buffy must be sufficient for grim-dependent cell death because excess Buffy did not further increase microchaete cell death. Since buffy does not modify ectopic Rpr or Hid in the fly eye, and since there are situations in which Grim induces cell death in which neither Rpr nor Hid can, the interaction between Grim and Buffy may be unique to Grim (Wu, 2010).

There is significant evidence that mammalian Bcl-2 family members can influence the dynamics of mitochondrial fission and fusion in both healthy and apoptotic cells, possibly through direct interaction with core components of the mitochondrial fission/fusion machinery. Although a role for Buffy in mitochondrial morphogenesis has not been carefully investigated, Buffy expression suppresses mitochondrial phenotypes of PINK1 and Prel mutant animals. In the simplest scenario, either Grim induces mitochondrial dysfunction more efficiently through interaction with Buffy (perhaps leading to more mitochondrial fission) or mitochondrial dysfunction is actively amplified by Buffy (perhaps through release of a proapoptotic factor) (Wu, 2010).

PCD of microchaete glial cells is the first example of an in vivo death that utilizes buffy and requires grim and provides an unparalleled opportunity to investigate a cell death mechanism that is likely to elucidate a role for mitochondria in Drosophila PCD (Wu, 2010).


REFERENCES

Search PubMed for articles about Drosophila grim

Avdonin, V., et al. (1998). Apoptotic proteins Reaper and Grim induce stable inactivation in voltage-gated K+ channels. Proc. Natl. Acad. Sci. 95(20): 11703-8. PubMed Citation: 9751729

Baer, M. M., Bilstein, A., Caussinus, E., Csiszar, A., Affolter, M. and Leptin, M. (2010). The role of apoptosis in shaping the tracheal system in the Drosophila embryo. Mech. Dev. 127(1-2): 28-35. PubMed Citation: 19995601

Bardet, P. L. et al. (2008). A fluorescent reporter of caspase activity for live imaging. Proc. Natl. Acad. Sci. 105: 13901-13905. PubMed Citation: 18779587

Bergmann, A., Agapite, J., McCall, K. and Steller, H. (1998). The Drosophila gene hid is a direct molecular target of ras-dependent survival signaling. Cell 95: 331-341. 9814704

Brennecke, J., Stark, A., Russell, R. B. and Cohen, S. M. (2005). Principles of microRNA-target recognition. PLoS Biol. 3(3):e85. 15723116

Chai, J., Yan, N., Huh, J. R., Wu, J. W., Li, W., Hay, B. A. and Shi, Y. (2003). Molecular mechanism of Reaper-Grim-Hid-mediated suppression of DIAP1-dependent Dronc ubiquitination. Nat Struct Biol 10: 892-898. PubMed ID: 14517550

Chen, P., et al. (1996). grim, a novel cell death gene in Drosophila. Genes Dev. 10: 1773-1782. 8698237

Chen, P., et al. (1998). Dredd, a novel effector of the apoptosis activators Reaper, Grim, and Hid in Drosophila. Dev. Biol. 201(2): 202-16. PubMed Citation: 9740659

Clavería, C., et al. (1998). Drosophila grim induces apoptosis in mammalian cells. EMBO J. 17: 7199-7208. PubMed Citation: 9857177

Clavería. C., et al. (2002). GH3, a novel proapoptotic domain in Drosophila Grim, promotes a mitochondrial death pathway. EMBO J. 21: 3327-3336. 12093734

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Biological Overview

date revised: 25 February 2014

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