Programmed Cell Death in Insects Part 3

The Drosophila Model

Choice of Drosophila melanogaster as a Model System for the Study of PCD of Nerve and Muscle Cells

Like moths, fruit flies are holometabolous insects. Three larval (feeding) stages are followed by pupation. The pupa forms inside the pupal case (puparium), the hardened cuticle of the final larval stage. Drosophila development is rapid and the entire life cycle is complete within 10 days at 25°C. While Drosophila have provided a powerful genetic model system for a century, it arrived relatively late to the modern study of PCD. Thus, it was not until 1990 that Kimura and Truman (1990) extended classic observations of muscle death during Drosophila metamorphosis by systematically documenting neuronal death in the fused ventral ganglia following adult eclosion. Injection of living flies with a toluidine blue solution revealed numerous examples of dying neurons in the dorsal and lateral regions of the abdominal and metathoracic neuromeres. These investigators also used bi-refringence to map degeneration of head and abdominal musculature during metamorphosis and adult eclosion. Several studies described below have made use of these baseline observations to explore ecdysteroid regulation and molecular mechanisms of PCD in the nervous system of newly-eclosed adult flies (Robinow et al., 1993, 1997; Draizen et al., 1999).

Interestingly, the first insight into the genetic basis of PCD in flies did not come from studies of metamorphosis but, rather, of embryogenesis. The laboratory of Hermann Steller used a classic histological technique, acridine orange staining, to identify dying cells with an apoptotic phenotype in wild type embryos (Abrams et al., 1993). A subsequent genetic screen using a set of 129 chromosomal deficiency strains identified a small region at 75C1,2 on chromosome 3L that is essential for developmental and X-irradiation induced apoptosis (White et al., 1994). Subsequent studies led to the identification of four related pro-apoptotic genes that reside within a 300-kb interval in this region: reaper (rpr), head involution defective (hid), grim, and sickle (skl) (White et al., 1994; Grether et al., 1995; Chen et al., 1996; Christich et al., 2002; Srinivasula et al., 2002; Wing et al, 2002) (Figure 4). A genetic modifier screen resulted in the identification of Drosophila Inhibitor of Apoptosis Proteins, DIAP1 and DIAP2, which are crucial inhibitors of caspase activities (Hay et al., 1995). DIAP1 mutants exhibit embryonic lethality associated with massive ectopic PCD, indicating it is essential for survival of many cell types (Hay, 2000).


The Reaper, Grim, and Sickle proteins are small (65, 138, and 108 amino acids, respectively), while Hid is substantially larger (410 amino acids). Each possesses a related 14-aa region at the N-terminus, designated the RHG (Reaper, Hid, Grim) motif or IBM (inhibitor of apoptosis (IAP)-binding motif). This motif has potent pro-apoptotic activities, and can bind to and repress the caspase-inhibiting activities of IAPs (Vaux and Silke, 2005; Steller, 2008; Orme and Meier, 2009; also discussed below). The RHG/IBM motifs of Reaper and Grim share 71% identity, with three of the four amino-acid differences conservative substitutions. However, despite this strong similarity, in vivo studies have shown that these domains possess distinct death-inducing activities (Wing et al., 1998). More recently, two unlinked Drosophila genes, omi/htr2A (a homolog of the mammalian Htr2A serine protease) and jafrac2 (a thiodoxin peroxidase) , were identified that encode proteins with similar RHG domains (see, for example, Challa et al., 2007), but for this review we will focus on the four linked RHG genes, reaper, hid, grim, and sickle. Reaper, Grim, and Sickle also share a second region of sequence similarity, a 15-aa Grim helix 1, 2, and 3 (GH3) domain or Trp block (Figure 4), which has distinct pro-apoptotic functions.

The grim-reaper genes encode related pro-apoptotic proteins. (A) The hid, grim, reaper, and sickle genes all reside within a 300-kb interval in the 75C region of chromosome 3L of Drosophila melanogaster. The four genes are transcribed in the same direction (arrows above genes) and are expressed predominantly in doomed and dying cells. No genes are predicted to reside between grim and reaper, or reaper and sickle, while four unrelated genes (orange blocks) reside between hid and grim. (B) reaper, sickle, and grim encode small proteins that contain an N-terminal RHG motif/IBM as well as a C-terminal Trp block/ GH3 domain. Hid is a substantially larger protein that also contains an RHG motif but does not exhibit strong similarity to the Trp block/GH3 domain.

Figure 4 The grim-reaper genes encode related pro-apoptotic proteins. (A) The hid, grim, reaper, and sickle genes all reside within a 300-kb interval in the 75C region of chromosome 3L of Drosophila melanogaster. The four genes are transcribed in the same direction (arrows above genes) and are expressed predominantly in doomed and dying cells. No genes are predicted to reside between grim and reaper, or reaper and sickle, while four unrelated genes (orange blocks) reside between hid and grim. (B) reaper, sickle, and grim encode small proteins that contain an N-terminal RHG motif/IBM as well as a C-terminal Trp block/ GH3 domain. Hid is a substantially larger protein that also contains an RHG motif but does not exhibit strong similarity to the Trp block/GH3 domain.

This domain is necessary for mitochondrial association of Reaper and Grim during apoptosis, and activates PCD independently of the IAP antagonizing functions of the NH2 RHG domain/IBM (see, for example, Abdelwahid et al., 2007). Hid possesses four regions that resemble a Trp block/GH3 domain, although the level of similarity is much lower (Wing et al., 2001). In addition, Hid also contains a mitochondrial localization signal and can form a heterodimer with Reaper that translocates to mitochondria to promote cell death (Sandu et al., 2010). Reaper also forms homodimers and heterodimers with Grim but not Sickle. Formation of these dimers appears essential for strong pro-apoptotic activity of RHG proteins (Sandu et al., 2010). Finally, Reaper and Grim, but not Hid, can induce apoptosis via general translation inhibition (Holley et al., 2002; Yoo et al., 2002). The related RHG proteins are multifunctional apoptosis activators that have distinct and overlapping modes of action.

Insights into the molecular mechanisms of PCD gained by further studies of RHG genes during Dro-sophila embryonic development and metamorphosis are described in the following sections. Significantly, targeted ectopic expression of these genes in cells that are normally fated to live has provided an invaluable tool for producing cell-specific lesions of the fly CNS, particularly of peptidergic neurons (see, for example, McNabb et al., 1997;Zhou et al.,1997; Renn et al., 1999; Rulifson et al., 2002; Park et al., 2003; Zhao et al., 2010).

Hormonal Regulation of Metamorphosis in Drosophila melanogaster

Hormonal regulation of metamorphosis in Drosophila is similar to that described for other insects, with post-embryonic development wholly dependent upon exposure of tissues to coordinated pulses of 20E, which in turn produce a coordinated cascade of gene expression (Riddiford, 1993; Truman and Riddiford, 2002). Cloning of the Drosophila ecdysone receptor (EcR) gene led to studies of the expression of EcR in tissues including neurons and muscle (Koelle et al., 1991; Robinow et al., 1993; Talbot et al., 1993; Truman et al., 1994). These studies support the view, based on earlier studies of Manduca, that ecdyster-oid regulation of PCD in insects is a result of direct action of the steroid on the cells that are fated to die.

PCD of Neurons during Early Development and Metamorphosis

Four periods of PCD have been described in the nervous system of Drosophila: during mid-to-late embryogenesis, in late third instar larvae, in pupae during metamorphosis, and in the newly-eclosed adult. The PCD that occurs during these stages generates in turn the larval and adult nervous systems by eliminating cells that are produced in excess, such as the embryonic midline glia, and cells with transient functions, such as larval abdominal ganglion neuroblasts (Kimura and Truman, 1990; Truman et al., 1993; Sonnenfeld and Jacobs, 1995; Zhou et al, 1995). As in mammalian development, the extent of cell death in the embryonic Drosophila nervous system is profound; approximately two-thirds of all cells born die before larval hatching (Abrams et al., 1993; White et al., 1994).

The extent of cell death in Drosophila embryos suggests that this process is critical for development of the larval nervous system. However, homozygous Df(3L)H99 mutant embryos that lack cell death exhibit relatively normal organization of the ventral nerve cord despite having a large excess of surviving cells (White et al., 1994; Zhou et al., 1995). Nonetheless, mutations in several genes that are important for PCD result in hypertrophy of the embryonic, larval, or adult CNS, and these mutants often exhibit stage-specific lethality or sterility (Grether et al., 1995; Song et al., 1997; Peterson et al., 2002; Rogulja-Ortmann et al., 2007; Kumar et al., 2009). Removal of dead cells from the CNS also appears to be important for nervous system development, as mutants lacking functional mac-rophages exhibit disruptions in the normal architecture of the embryonic axon scaffold (Sears et al., 2003).

The proportion of neurons that die during the post-embryonic period is lower than during embryogenesis, and the widespread degeneration of larval tissues during metamorphosis in flies does not extend to the CNS. As in Manduca, most neurons in the larva persist rather than die (Truman, 1990). After pupariation, however, many dying neurons can be observed in the ventral CNS in both thoracic and abdominal neuromeres (Truman et al., 1993). The identity of most of these neurons has not been determined, with the exception of several motoneurons identified by retrograde fills of the T2 mesothoracic nerve (Consoulas et al., 2002). In addition, PCD of neuroblasts after pupariation marks the termination of post-embryonic neurogenesis in the ventral nervous system (Truman and Bate, 1988; Truman et al., 1993; Kumar et al., 2009). As in Manduca, identified peptidergic neurons provide a population that can be tracked across metamorphic transitions. Corazonin is an 11-amino acid peptide expressed by 8 pairs of neurons in the ventral nerve cord of larval fruit flies. Detailed studies of the time-course of corazonin disappearance revealed that these neurons undergo PCD 2-6 hours after puparium formation. Because their fate can be readily tracked by anti-corazonin immunolabeling, they were chosen to explore the ecdysteroid-dependent and cell-specific mechanisms of PCD (Choi et al., 2006). These studies revealed that the pulses of ecdysteroids that drive the onset of metamorphosis trigger the death of the corazonin neurons via activation of the EcR nuclear receptor. The EcR gene of Drosophila encodes three ecdysone receptor subunits: EcR-A, EcR-B1, and Ecr-B2 (Talbot et al., 1993). These receptor subunits share common DNA and ligand-binding domains, but have different N-terminal regions. Analysis of loss-of-function mutants for specific EcR isoforms revealed that either EcR-B1 or EcR-B2 can activate the cascade of transcriptional events that results in the PCD of these neurons (Choi et al., 2006).

The use of EcR isoform-specific antibodies also revealed that a high level of expression of EcR-A is correlated with post-eclosion PCD (Robinow et al., 1993). Prior to eclosion, nearly 300 neurons in the ventral ganglia displayed high levels of EcR-A immunoreactivity. During the first 24 hours after adult eclosion, essentially the EcR-A-immunopositive neurons in the ventral ganglia displayed characteristic features of PCD and were basically all absent by the end of this first day. In contrast to the death of the corazonin neurons at the end of larval life described above (Choi et al., 2006), it appears that it is EcR-A expression during the period of ecdysteroid decline at the end of metamorphosis which is critical to the decision of these neurons to commit suicide. A subsequent analysis of EcR-A mutants supported the view that the distinct EcR-isoforms have specific functions during development, and linked EcR-A receptors to PCD in an additional tissue, the larval salivary glands (Davis et al., 2005). Analysis of isoform-specific activities of EcR is an active area of research with a current focus on the regulatory role of heterodimerization partners (Braun et al., 2009).

Molecular Mechanisms of Neuronal Death

PCD research in Drosophila is a large and active field that can no longer be adequately summarized in a single topic. We focus here on selected topics related to the death of neurons and glial cells, covering both apoptosis and autophagy. Accounts of the control of neuron number by neuroblast apoptosis in the ventral nerve cord and the brain can be found in Peterson et al. (2002), Bello et al. (2003, 2007), Kumar et al. (2009), and Siegrist et al. (2010). Descriptions of the regulation of cell survival by cell intrinsic mechanisms in sensory neuron lineages can be found in Spana and Doe (1996) and Orgogozo et al. (2002). These studies have provided insights into the regulation of apoptosis and other cell fates by asymmetric cell division (Hatzold and Conradt, 2008; Zhong, 2008).

The Drosophila cell death "machinery" for apoptosis The basic molecular machinery of apoptosis includes both initiator and executioner caspases, a Ced (cell death abnormal)-4/Apaf (apoptosome associated factor)-1 ortholog, and Ced-9/Bcl-2 protein family members (Tittel and Steller, 2000; Vernooy et al, 2000) (Table 2). These proteins all have critical functions in apoptosis that were initially defined by studies in the nematode Caenorhabditis elegans (Horvitz et al., 1994; Liu and Hengartner, 1999). Caspases are considered the cellular executioners because they activate proteolytic enzymes that digest cellular structural elements; Apaf-1 factors form complexes called apoptosomes that regulate caspase activity, and Bcl-2 family proteins regulate the integrity of mitochondrial membranes.

Table 2 Drosophila Cell Death Regulators

Family

Proteins

Caspases

Dronc

Dcp-1

Drice

Dredd

Strica/Dream

Decay

Damm

Caspase inhibitor

DIAP1/Thread

DIAP2

Bcl-2 family

Drob-1/Debcl/Dborg-1/Dbok

Buffy/Dborg-2

IAP inhibitors

Reaper

Hid

Grim

Sickle

Jafrac2

Apaf-1/Ded 4 family

Dark/HAC-1/Dapaf-1

An important difference between apoptosis in C. elegans, Drosophila, and mammals is the role of mitochondria (and, hence, an important role for Bcl-2 family members). In mammals, mitochondria undergo membrane permeability changes in dying cells and serve as critical sources of several pro-apoptotic factors, including cytochrome c and Smac/ Diablo. However, while mitochondria have clearly been implicated in apoptotic pathways in flies and worms, the importance and extent of their contribution is less certain (see, for example, Colin et al., 2009; Krieser and White, 2009). In flies, the major arbiter and point of regulation for cell survival decisions appears to be DIAP1 (Hay, 2000; Vaux and Silke, 2005; Orme and Meier, 2009).

In flies, worms, and mammals, caspases serve as the effectors of PCD. These specialized cysteine proteases cleave at aspartate or glutamate residues within enzymatic or structural substrate proteins to promote the dismantling of a cell (Cooper et al., 2009; Feinstein-Rotkopf and Arama, 2009). Caspases are initially synthesized as inactive zymogens that contain either a long or a short prodo-main, and a large and small subunit. Proteolytic cleavage of caspases in dying cells results in formation of an active heterotetramer comprised of two large and two small sub-nits. Heterotetramers derived from long prodomain zyo-mogens correspond to initiator caspases, and these cleave short prodomain zymogens to generate active effector caspases that ultimately dismantle diverse cellular substrates. Thus, a cascade of caspase activities defines apop-totic cell deaths. The Drosophila genome encodes seven caspases (Table 2), including three long prodomain initiator caspases such as Dronc (Drosophila nedd2-like caspase, similar to caspase 9), and four short prodomain effector caspases such as Drice (similar to caspase-3) and DCP-1 (death caspase 1; similar to caspase-7). Dronc and Drice are now recognized as key elements of the core of the Drosophila caspase-dependent cell death machinery (Hay and Guo, 2006). Mutant Drosophila embryos lacking zygotic dronc gene product display significantly reduced levels of apoptosis in many cell populations, including the developing ventral nerve cord (Chew et al., 2004). During metamorphosis, targeted expression of the baculovirus pan-caspase inhibitor p35 blocks the death of the larval corazonin neurons of the ventral nerve cord (Choi et al., 2006). Interestingly, a recent report links the regulation of Dronc activity (via phosphorylation at S130) to cellular metabolism by demonstration that increases in NADPH inhibit this caspase while inhibition of NADPH production triggers apoptosis (Yang et al., 2010); this relationship between NADPH and Dronc activity was first demonstrated in Drosophila S2 cells, but was also found in neurons in the developing CNS. Focal activation of caspases plays a role in neurite pruning in Drosophila, which provides a surprising and elegant tool for sculpting the nervous system and refining synaptic connections (Williams et al., 2006).

The fly genome encodes two Bcl-2 family members, Drob-1/Debcl/dBorg-1/dBok and Buffy/dBorg-2, that possess the Bcl-2 homology (BH) domains BH1, BH2, and BH3 (Brachmann et al., 2000; Colussi et al., 2000; Zhang et al., 2000; Quinn et al., 2003). Both of these proteins resemble the pro-apoptotic mammalian Bok protein, and act to promote apoptosis (Igaki and Miura, 2004). This difference supports the view that flies are less dependent upon mitochondrial disruption for the activation of caspases than are mammals (Wang and Youle, 2009). This observation provokes speculation concerning the evolution of cell death mechanisms, and raises the question: what factors inhibit death in insect cells?

Role of IAP and RHG family proteins in apoptosis The major anti-apoptotic proteins in Drosophila are the IAPs (inhibitors of apoptosis proteins), which function by binding to and inactivating caspases (Hay, 2000; Vaux and Silke, 2005; Steller, 2008; Orme and Meier, 2009). IAPs were first identified from the Cydia pomonella granulosis virus, and found to inhibit apoptosis in infected host cells (Crook et al., 1993). Subsequently, a large number of viral and cellular IAPs have been identified in divergent species that function as potent caspase repressors and inhibitors of PCD. IAPs all contain one or more copies of a 70-aa baculovirus IAP repeat (BIR) domain, typically located in the central or N-terminal region of the protein. In addition, they contain a 50-aa RING (Really Interesting New Gene) domain, generally situated towards the C-terminal region of the protein. The sequences of the BIR and RING domains both resemble zinc fingers, and serve as critical protein-protein interaction domains. The BIR domains can directly associate with procaspases and caspases, and inhibit their activation or activities (Vaux and Silke, 2005; Steller, 2008; Orme and Meier, 2009). The RING domain possesses ubiquitin E3 ligase activity that can target bound caspases for polyubiquitination and degradation via the 26S proteasome (reviewed by Bergmann, 2010). In addition, IAPs bound to caspases are themselves targeted to proteasome-dependent proteolysis via the N-end rule pathway of protein turnover (Ditzel et al., 2003). The IAPs therefore can efficiently reduce cellular caspase levels, and the interplay between caspases and IAPs is a key determinant of cell survival. The levels of IAPS themselves are also under rigid regulation. In particular, the RHG proteins bind not only to the BIR domains of IAPs and displace bound caspases, but also promote IAP auto-ubiquitination (Yang et al., 2000; Yoo et al., 2002). The release and de-repression of bound caspases as well as increased turnover of IAPs act together to strongly promote PCD.

The dependence on IAP proteins for cellular survival provides a key regulatory point for the regulation of apop-tosis. The BIR domains of DIAP1 bind both caspases and the Grim-Reaper proteins. In particular, the BIR2 domain of DIAP1 associates both with Dronc and Reaper, Hid, Grim, and Sickle. The BIR1 domain binds to Drice and DCP-1 and also to Reaper and Grim, and, to some extent, Hid. Thus, the two BIR domains exhibit distinct abilities to bind to both specific caspases and RHG proteins (see, for example, Zachariou et al., 2003). In surviving cells, DIAP1 binds and inhibits caspases, thereby repressing cell death (Figure 5). In contrast, in cells that receive signals to die, Grim-Reaper proteins are expressed and compete with caspases for DIAP1 binding. The displacement of caspases from DIAP1 results in increased levels of proteolytically active enzymes that promote cell death. Cell survival decisions, therefore, are determined by the interactions among the pro-apoptotic RHG proteins, the anti-apoptotic IAPs, and caspases.

IAP regulation is a conserved mechanism for controlling cell survival (Figure 5). In mammals, Smac/Diablo and Omi/Htr2A are mitochondrial proteins released with cytochrome c in dying cells (Du et al., 2000; Suzuki, 2001; Verhagen and Vaux, 2002). Cytoplasmic Smac/Diablo and Omi/Htr2A associates with X-linked inhibitor of apoptosis (XIAP) to prevent it from binding and thereby inhibiting caspase-9. Strikingly, the binding of Smac/ Diablo and Omi/Htr2A to IAPs is mediated through an N-terminal tetrapeptide sequence that is conserved in the Grim-Reaper RHG motif, implying conserved modes of action for the fly and mammalian proteins (Chai et al., 2000; Srinivasula et al., 2000, 2002). As expression of IAPs is upregulated in many types of tumors, interest is intense regarding the possibility that either these endogenous inhibitors or related synthetic compounds can be exploited to develop new therapies for cancer and other diseases (Fulda, 2007; Gyrd-Hansen and Meier, 2010).

Regulation of embryonic glial cell survival in Drosophila One important mechanism for regulating the survival of neurons and glia within the vertebrate nervous system involves the actions of trophic factors (Raff et al., 1993). These pro-survival molecules are synthesized in restricted amounts by target tissues, and permit winnowing of innervating cells to ensure matching of interacting cell populations. Such molecules appear to be involved in regulating glial cell survival in Drosophila embryos.

IAP inhibition is a conserved mechanism of regulation of apoptosis. In Drosophila, expression of the grim-reaper, jafrac2, or dOmi/Hrt2A genes is activated in doomed cells, and the corresponding proteins bind to DIAP1 and block its ability to inhibit caspases. This results in active caspases that degrade cellular proteins to mediate apoptosis. In mammals, pro-apoptotic stimuli induce the release of Smac/Diabl, Omi/Hrt2A, as well as other pro-apoptotic factors, including cytochrome c, from the mitochondria. Cytoplasmic Smac/Diablo or Omi/Hrt2A binds XIAP and represses its caspase-inhibitory actions, thereby promoting apoptosis. In both flies and mammals, the orthologous Dark and Apaf-1 proteins as well as pro-apoptotic members of the Ced-9/Bcl-2 family promote activation of inhibitor caspases.

Figure 5 IAP inhibition is a conserved mechanism of regulation of apoptosis. In Drosophila, expression of the grim-reaper, jafrac2, or dOmi/Hrt2A genes is activated in doomed cells, and the corresponding proteins bind to DIAP1 and block its ability to inhibit caspases. This results in active caspases that degrade cellular proteins to mediate apoptosis. In mammals, pro-apoptotic stimuli induce the release of Smac/Diabl, Omi/Hrt2A, as well as other pro-apoptotic factors, including cytochrome c, from the mitochondria. Cytoplasmic Smac/Diablo or Omi/Hrt2A binds XIAP and represses its caspase-inhibitory actions, thereby promoting apoptosis. In both flies and mammals, the orthologous Dark and Apaf-1 proteins as well as pro-apoptotic members of the Ced-9/Bcl-2 family promote activation of inhibitor caspases.

Approximately 10% of all cells within the Drosophila nervous system are glial cells. Neurons and glia display complex signaling relationships during development. For example, the PCD of neurons de-represses division of glial cells, leading to glial cell proliferation (Kato et al., 2009). Among the best-characterized glial cells are the embryonic longitudinal and midline glia of the CNS. In both these lineages, glial cells undergo extensive apoptosis during embryogenesis (Sonnenfeld and Jacobs, 1995; Zhou et al., 1995; Kinrade et al., 2001). The survival of subsets of these cell populations is governed by trophic actions of EGF-related ligands and activation of the Ras/MAP kinase pathway via the EGF receptor homolog (EGFR).

During embryogenesis, a single, laterally positioned glioblast precursor gives rise to approximately 10 longitudinal glia in each hemisegment of the ventral nerve cord. These glial cells migrate medially, contact pioneer longitudinal axons, and ultimately ensheathe the longitudinal nerve bundles (Hidalgo and Booth, 2000). Coincident with the onset of axon/glial contact, many of these longitudinal glia undergo apoptosis, suggesting that as the glia contact the axons, they become dependent upon them for survival (Kinrade et al., 2001). Consistent with this notion, ablation of pioneer and other neurons results in a decrease in longitudinal glia (Hidalgo et al., 2001). Thus, apoptosis determines the final numbers of longitudinal glia during embryogenesis, and axon-derived factors are required for longitudinal glial cell survival. One of these factors is Vein (Vn), a Drosophila neuregulin homolog that contains both an IgG domain and an EGF domain (Schnepp et al., 1996). The vein gene is expressed in a subset of neurons within the embryonic CNS including the midline precursor 2 (MP2) pioneer neurons and the Ventral Unpaired Median (VUM) neurons of the CNS midline (Hidalgo et al., 2001). Vein mutants exhibit ectopic apoptosis of longitudinal glial cells, which is also observed for RNAi-mediated knockdown of vein gene product in either all neurons or the MP2 neurons. Vein is a secreted ligand for Drosophila EGFR, and EGFR-mediated activation of the Ras/MAP kinase pathway is essential for longitudinal glial cell survival. EGFR is transiently expressed in a subset of these glia, suggesting that Vein is secreted from axons to promote survival of these glial cells.

Similar to the Vein-dependent longitudinal glia, survival of the midline glia is also promoted by an EGF family member, the TGF-a homolog Spitz (Bergmann et al., 2002). Midline glia are essential for proper formation of the axon scaffold, as migrating midline glia contact, separate, and ultimately ensheathe the anterior and posterior axon commissures (Klambt et al., 1991). The midline glia are normally in close contact with commissural axons, suggesting that glial-axon contact may be important for midline glial cell survival. Observations made in commis-sureless mutants, where the commissures fail to form, are consistent with this notion; in these mutants, the isolated midline glial cells that fail to contact axons undergo apop-tosis (Sonnenfeld and Jacobs, 1995). Ultrastructural and genetic analyses indicate that the midline glia undergo apoptosis, which reduces their number from an initial set of nine cells to three cells in each segment of the mature ventral nerve cord (Sonnenfeld and Jacobs, 1995; Zhou et al., 1995). This apoptosis is dependent upon cas-pases and the actions of multiple Grim-Reaper proteins. Simultaneous loss of reaper, hid, and grim gene expression blocks all midline glial cell death, and results in the survival of the nine midline glia per segment. The loss of hid and grim results in approximately seven to eight mid-line glia per segment, while the loss of hid alone results in six midline glia (Zhou et al., 1997; Bergmann et al., 2002). Thus, hid expression is required for the death of three midline glia, and reaper and grim are together essential for the death of the other three midline glia. Both hid gene transcription and activity of Hid protein in the midline glia are regulated by proteins in the EGF signaling pathway, including Spitz and EGFR (Kurada and White, 1998). The loss of midline glia in spitz mutants is rescued in spitz;hid double mutants, implying that the pro-apototic functions of Hid are normally opposed by the prosurvival functions of Spitz.

The Spitz protein appears to be an axon-derived trophic factor required for midline glial survival (Bergmann et al., 2002), as the loss of midline glia in spitz mutants is rescued by targeted expression of the transmembrane Spitz precursor protein in commissural neurons, and the number of surviving midline glia can be modulated by controlling the levels of Spitz activity. The model that has emerged is that axon-derived Spitz protein signals the midline glia via EGFR, resulting in activation of the Ras/MAP kinase pathway. Hid activity is subsequently downregulated via phosphorylation, which permits midline glial survival. Taken together, the actions of Vein in the longitudinal glia and Spitz in the midline glia indicate that trophic survival mechanisms are utilized to match the sizes of interacting populations of neurons and glial cells in invertebrates as well as vertebrates.

The Drosophila cell death "machinery" for autophagy Studies in Drosophila are now contributing significantly to our understanding of the molecular mechanisms of autophagy (Chang and Neufeld, 2010; Ryoo and Baehrecke, 2010; Figure 6). In animal cells, autophagy is regulated by the class I and class III phosphatidylinositol 3-kinase pathways (PI3K). Metabolic signals such as the binding of insulin to its receptor activate PI3K, which ultimately releases an autophagy inhibitor, TOR (target of rapamycin), from inhibition. The activation of TOR in turn activates Atg-family proteins that regulate the formation of the autophagosome (Ryoo and Baehrecke, 2010).

Simplified schematic overviews of established pathways for induction of autophagy in Drosophila. Other pathways have also been described. (A) Autophagy can be induced by nutrient limitation through inhibition of TOR, which negatively regulates autophagy in well-fed organisms. Rheb and TSC1/2 control the activity of TOR, both acting downstream of PI3K and AKT. (B) Developmental autophagy as described in this topic is triggered by ecdysone signaling that it is time to eliminate tissues or organs. Note that in this scenario activation of EcR by 20E inhibits PI3K, providing an opportunity for cross-talk between these signaling pathways. (For clarity, the steps in the pathway linking P13K with autophagy are omitted in panel (B)). This form of autophagy can involve activation of known cell death genes, including caspases in some cell types. 20E, 20-hydroxyecdysone; AKT,a serine/threonine protein kinase; chico, Drosophila homolog of mammalian IRS1-IRS4, the insulin receptor substrate adaptor proteins; E93, an early ecdysone response gene that encodes a pipsqueak domain transcription factor; EcR, ecdysone receptor (nuclear hormone receptor); InR, Drosophila homolog of the mammalian insulin receptor; PI3K, phosphoinositide 3-kinase; Rheb, "Ras homolog enriched in brain," a GTP-binding protein (GTPase); TOR, "target of rapamycin," a serine/threonine kinase that belongs to the phosphoinositide-3-kinase-related kinase (PIKK) family of checkpoint kinases and is a part of a signaling pathway that regulates growth; TSC1/2, tuberous sclerosis complex proteins that function as GTPase activating proteins against Rheb; USP, Drosophila homolog of RXR that forms a heterodimer with EcR to form the functional insect ecdysteroid receptor.

Figure 6 Simplified schematic overviews of established pathways for induction of autophagy in Drosophila. Other pathways have also been described. (A) Autophagy can be induced by nutrient limitation through inhibition of TOR, which negatively regulates autophagy in well-fed organisms. Rheb and TSC1/2 control the activity of TOR, both acting downstream of PI3K and AKT. (B) Developmental autophagy as described in this topic is triggered by ecdysone signaling that it is time to eliminate tissues or organs. Note that in this scenario activation of EcR by 20E inhibits PI3K, providing an opportunity for cross-talk between these signaling pathways. (For clarity, the steps in the pathway linking P13K with autophagy are omitted in panel (B)). This form of autophagy can involve activation of known cell death genes, including caspases in some cell types. 20E, 20-hydroxyecdysone; AKT,a serine/threonine protein kinase; chico, Drosophila homolog of mammalian IRS1-IRS4, the insulin receptor substrate adaptor proteins; E93, an early ecdysone response gene that encodes a pipsqueak domain transcription factor; EcR, ecdysone receptor (nuclear hormone receptor); InR, Drosophila homolog of the mammalian insulin receptor; PI3K, phosphoinositide 3-kinase; Rheb, "Ras homolog enriched in brain," a GTP-binding protein (GTPase); TOR, "target of rapamycin," a serine/threonine kinase that belongs to the phosphoinositide-3-kinase-related kinase (PIKK) family of checkpoint kinases and is a part of a signaling pathway that regulates growth; TSC1/2, tuberous sclerosis complex proteins that function as GTPase activating proteins against Rheb; USP, Drosophila homolog of RXR that forms a heterodimer with EcR to form the functional insect ecdysteroid receptor.

This same pathway functions in Drosophila, with insulin-like pep-tides and insulin-like receptor corresponding to the mammalian insulin and insulin receptor, respectively. Other autophagy pathways have also been described in Drosophila. The first is a pathway in which autophagy results from an ecdysteroid signal (Figure 6); the second is part of the cellular response to oxidative stress in which Atg-family genes are transcriptionally activated by Jun-N-terminal kinase (JNK) signaling (Chang and Neufeld, 2010).

Autophagy and metamorphosis of neuro-muscular systems A surprising phenotype is associated with mutations in Drosophila Atg-family genes (Shen and Ganetzky, 2010). In Drosophila, the number of boutons associated with each neuromuscular junction can be counted by immunolabeling synapses with markers such as anti-Synaptotagmin or anti-Nervous wreck. When bouton numbers were compared between wild type flies and flies with loss-of-function Atg mutations, the mutants displayed significantly reduced bouton numbers, by up to 50%. Bouton numbers can be increased by pro-autophagic genetic manipulations. The site at which autophagic processes regulate normal development of the neuromuscular junction appears to be the motoneuron, rather than the muscle target (Shen and Ganetzky, 2009). Reduced expression of the Highwire protein appears to be instrumental in the regulation of synaptic development via autophagy. Highwire is an E3 ubiquitin ligase previously shown to reduce the formation of synapses (Wan et al., 2000). The substrate specificity of autophagic pathways in motoneurons is astonishing. Whether this is a singular mechanism for the regulation of synaptic plasticity or the first of many "selective autophagy" targets remains to be discovered.

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