InteractiveFly: GeneBrief

eiger: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - eiger

Synonyms -

Cytological map position - 56F1

Function - ligand

Keywords - apoptosis, JNK pathway

Symbol - eiger

FlyBase ID: FBgn0033483

Genetic map position -

Classification - TNF superfamily; type II membrane protein

Cellular location - surface

NCBI links: Precomputed BLAST | Entrez Gene

Recent literature
Andersen, D. S., et al. (2015). The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth. Nature 522(7557):482-6. PubMed ID: 25874673
Disruption of epithelial polarity is a key event in the acquisition of neoplastic growth. JNK signalling is known to play an important part in driving the malignant progression of many epithelial tumours, although the link between loss of polarity and JNK signalling remains elusive. In a Drosophila genome-wide genetic screen designed to identify molecules implicated in neoplastic growth, this study identified grindelwald (grnd; CG10176), a gene encoding a transmembrane protein with homology to members of the tumour necrosis factor receptor (TNFR) superfamily. This study shows that Grnd mediates the pro-apoptotic functions of Eiger (Egr), the unique Drosophila TNF, and that overexpression of an active form of Grnd lacking the extracellular domain is sufficient to activate JNK signalling in vivo. Grnd also promotes the invasiveness of RasV12/scrib-/- tumours through Egr-dependent Matrix metalloprotease-1 (Mmp1) expression. Grnd localizes to the subapical membrane domain with the cell polarity determinant Crumbs (Crb) and couples Crb-induced loss of polarity with JNK activation and neoplastic growth through physical interaction with Veli (also known as Lin-7). Therefore, Grnd represents the first example of a TNFR that integrates signals from both Egr and apical polarity determinants to induce JNK-dependent cell death or tumour growth.

O'Keefe, L. V., Lee, C. S., Choo, A. and Richards, R. I. (2015). Tumor Suppressor WWOX contributes to the elimination of tumorigenic cells in Drosophila melanogaster. PLoS One 10: e0136356. PubMed ID: 26302329
WWOX is a >1Mb gene spanning FRA16D Common Chromosomal Fragile Site, a region of DNA instability in cancer. Consequently, altered WWOX levels have been observed in a wide variety of cancers. In vitro studies have identified a large number and variety of potential roles for WWOX. Although its normal role in vivo and functional contribution to cancer have not been fully defined, WWOX does have an integral role in metabolism and can suppress tumor growth. Using Drosophila melanogaster as an in vivo model system, this study found that WWOX is a modulator of TNFalpha/Egr-mediated cell death. Altered levels of WWOX can modify phenotypes generated by low level ectopic expression of TNFalpha/Egr and this corresponds to altered levels of Caspase 3 activity. These results demonstrate an in vivo role for WWOX in promoting cell death. This form of cell death is accompanied by an increase in levels of reactive oxygen species, the regulation of can also be modified by altered WWOX activity. It is now hypothesized that, through regulation of reactive oxygen species, WWOX constitutes a link between alterations in cellular metabolism observed in cancer cells and their ability to evade normal cell death pathways. WWOX activity is required for the efficient removal of tumorigenic cells from a developing epithelial tissue. Together these results provide a molecular basis for the tumor suppressor functions of WWOX and the better prognosis observed in cancer patients with higher levels of WWOX activity. Understanding the conserved cellular pathways to which WWOX contributes provides novel possibilities for the development of therapeutic approaches to restore WWOX function in cancer.

Ruan, W., Srinivasan, A., Lin, S., Kara, K. I. and Barker, P. A. (2016). Eiger-induced cell death relies on Rac1-dependent endocytosis. Cell Death Dis 7: e2181. PubMed ID: 27054336
Signaling via tumor necrosis factor receptor (TNFR) superfamily members regulates cellular life and death decisions. A subset of mammalian TNFR proteins, most notably the p75 neurotrophin receptor (p75NTR), induces cell death through a pathway that requires activation of c-Jun N-terminal kinases (JNKs). However the receptor-proximal signaling events that mediate this remain unclear. Drosophila express a single tumor necrosis factor (TNF) ligand termed Eiger (Egr) that activates JNK-dependent cell death. This model was exploited to identify phylogenetically conserved signaling events that allow Egr to induce JNK activation and cell death in vivo. This study reports that Rac1, a small GTPase, is specifically required in Egr-mediated cell death. rac1 loss of function blocks Egr-induced cell death, whereas Rac1 overexpression enhances Egr-induced killing. Vav was identified as a GEF for Rac1 in this pathway, and dLRRK functions were identified as a negative regulator of Rac1 that normally acts to constrain Egr-induced death. Thus dLRRK loss of function increases Egr-induced cell death in the fly. Rac1-dependent entry of Egr into early endosomes was shown to be a crucial prerequisite for JNK activation and for cell death and show that this entry requires the activity of Rab21 and Rab7. These findings reveal novel regulatory mechanisms that allow Rac1 to contribute to Egr-induced JNK activation and cell death.

Agrawal, N., Delanoue, R., Mauri, A., Basco, D., Pasco, M., Thorens, B. and Leopold, P. (2016). The Drosophila TNF Eiger is an adipokine that acts on insulin-producing cells to mediate nutrient response. Cell Metab 23: 675-684. PubMed ID: 27076079
Adaptation of organisms to ever-changing nutritional environments relies on sensor tissues and systemic signals. Identification of these signals would help understand the physiological crosstalk between organs contributing to growth and metabolic homeostasis. This study shows that Eiger, the Drosophila TNF-alpha, is a metabolic hormone that mediates nutrient response by remotely acting on insulin-producing cells (IPCs). In the condition of nutrient shortage, a metalloprotease of the TNF-alpha converting enzyme (TACE) family is active in fat body (adipose-like) cells, allowing the cleavage and release of adipose Eiger in the hemolymph. In the brain IPCs, Eiger activates its receptor Grindelwald, leading to JNK-dependent inhibition of insulin production. Therefore, this study has identified a humoral connexion between the fat body and the brain insulin-producing cells relying on TNF-alpha that mediates adaptive response to nutrient deprivation.

Eiger, the first invertebrate tumor necrosis factor (TNF) superfamily ligand that can induce cell death, was identified in a large-scale gain-of-function screen. Eiger is a type II transmembrane protein with a C-terminal TNF homology domain. It is predominantly expressed in the nervous system. Genetic evidence shows that Eiger induces cell death by activating the Drosophila JNK pathway. Although this cell death process is blocked by Drosophila inhibitor-of-apoptosis protein 1 (DIAP1, Thread), it does not require caspase activity. Genetically, Eiger has been shown to be a physiological ligand for the Drosophila JNK pathway. These findings demonstrate that Eiger can initiate cell death through an IAP-sensitive cell death pathway via JNK signaling (Igaki, 2002).

To discover the cell death triggers encoded in the Drosophila genome, a misexpression screen was conducted using the GAL4/UAS system. The GS vector is a P element-based gene search vector with UAS enhancers. A collection of 5000 lines harboring the GS vector (GS lines) was crossed with an eye-specific GAL4 (GMR-GAL4) strain to screen for genes that generate the reduced-eye phenotype. Six GS lines resulted in greatly reduced eyes in a GAL4-dependent manner; these are called Regg strains (Regg1-6, for reduced-eye generator with a GMR-GAL4 driver) (Igaki, 2002).

The F1 progeny generated by mating the GMR-GAL4 strain and Regg1 (GS9830) strain (GMR-GAL4 driven regg1GS9830) display a strong reduced-eye phenotype when compared with the wild-type eye. To assess whether the small eye phenotype of GMR-GAL4 driven regg1GS9830 flies are generated by the acceleration of cell death, eye discs were stained with acridine orange to detect dying cells. Acridine orange is commonly used as a marker for apoptotic cell death in Drosophila. In GMR-GAL4 driven regg1GS9830 discs, numerous acridine orange-staining cells were observed, indicating that regg1GS9830 induces massive apoptotic cell death, leading to the reduced-eye phenotype. Using the inverse PCR method, the insertion site of the P element in the Regg1GS9830 strain was determined; a predicted gene, CG12919, was discovered adjacent to the insertion site. The GS9830 strain was generated by inserting the GS6 vector, which has a green fluorescent protein (GFP) trailer (UAS-GFP-SV40 terminator) near the 3'P end and a UAS enhancer near the 5'P end, so that a misexpression of vector-flanking sequence occurs at the 5'P side only. As expected, CG12919 was the only gene with an elevated expression level in a GAL4-dependent manner. A Drosophila EST clone, LP03784, which included the nucleotide sequence of CG12919 was sequenced, and the open reading frame (ORF) of a novel gene that has been named eiger (EDA-like cell death trigger) was identified (Igaki, 2002).

To examine whether Eiger is indeed a membrane protein, S2 cells were transfected with expression vectors for an N-terminal hemagglutinin (HA)-tagged Eiger and GFP. In permeabilized cells, cell surface staining was observed in GFP-positive cells by anti-HA immunostaining. When immunostained prior to fixation and permeabilization, however, the staining was not detected, consistent with Eiger being a type II transmembrane protein with an extracellular C-terminus and a cytoplasmic N-terminus (Igaki, 2002).

Ectopic expression of Eiger in the eye results in a reduced-eye phenotype similar to that of Reaper, a Drosophila 'intrinsic' cell death trigger. The effects of Eiger overexpression in other tissues using different GAL4 drivers was further analyzed. When overexpressed in the dorsoventral compartments of the wing discs by a vg-GAL4 driver, Eiger entirely blocks wing formation, whereas Reaper induces only a regional defect. Ectopic expression of Eiger in precursor cells for the external sensory organs by a sca-GAL4 driver results in disorganized macrochaetae in the notum and scutellum, whereas Reaper induces a complete loss of bristles in these regions. In the abdomen, in contrast, sca-GAL4 driven regg1GS9830 results in a severe developmental defect, whereas reaper causes a loss of bristles in the tergum. Thus, Eiger has the potential to induce developmental defects distinct from the defects caused by the cell-autonomous killer protein Reaper (Igaki, 2002).

In mammals, the binding of death ligands such as TNF-alpha and FasL to their receptors triggers the activation of caspase-8, leading to the subsequent caspase-dependent cell death cascade (Ashkenazi, 1998). To assess whether Eiger stimulates similar death signaling in Drosophila, Eiger and caspase-inhibitory proteins such as baculovirus P35, Drosophila inhibitor-of-apoptosis protein 1 (DIAP1) or a dominant-negative form of DRONC (DRONC-DN), a P35-resistant Drosophila apical caspase, were co-expressed. P35 and DRONC-DN exhibit only slight suppressive effects on the Eiger-induced eye phenotype, although they strongly suppressed the Reaper-induced eye ablation. These results indicate that Eiger-induced cell death does not require caspase activity differing from the mammalian 'extrinsic' cell death system. In contrast, the co-expression of DIAP1 strongly suppresses the Eiger-induced eye phenotype, suggesting that DIAP1 can block Eiger-activated death signaling through a mechanism that is independent of caspase inhibition. Whether endogenous DIAP1 negatively regulates the Eiger-induced phenotype was assessed genetically. Whereas heterozygous diap1 mutant flies exhibit a normal eye, GMR-GAL4 driven regg1GS9830 flies with a half dosage of the diap1 gene display a completely ablated eye phenotype compared with the reduced-eye phenotype of GMR-GAL4 driven regg1GS9830 flies, suggesting that DIAP1 is an endogenous inhibitor of Eiger-induced cell death signaling (Igaki, 2002).

To assess whether Eiger does cause caspase activation, the eye disc was stained with (DMe)2R, a caspase substrate that contains only an aspartate residue linked to rhodamine-110. The eye disc from GMR-GAL4 driven reaper flies shows a strong rhodamine-110 fluorescence at the region posterior to the morphogenetic furrow, compared with the eye disc from GMR-GAL4 flies. In the GMR-GAL4 driven regg1GS9830 eye disc, the fluorescence is detected at lower, but still significant, levels in many cells posterior to the furrow. These data suggest that although caspase activation is not essential for cell death execution, Eiger activates both caspase-dependent and -independent signaling pathways (Igaki, 2002).

Thus, Eiger has been identified as a novel cell death trigger molecule in Drosophila. The structure and function of Eiger suggest that the extrinsic cell death-inducing mechanism might be evolutionarily conserved in Drosophila. Genetic evidence reveals that caspase activation is not essential to execute Eiger-induced cell death. The Drosophila extrinsic cell death system might predominantly utilize the caspase-independent pathway, in contrast to the intrinsic cell death system, which is regulated by Reaper, Hid and Grim, and depends completely on caspase activation. Although caspases do take part in the apoptotic effects of most of the mammalian TNF ligand/receptor superfamily members studied so far, there is accumulating evidence that they can also kill the cells in the absence of caspases (Igaki, 2002 and references therein).

The genetic data clearly show that the Eiger-induced small eye phenotype depends strongly on the JNK signaling pathway. In mammals, it has been demonstrated that the JNK pathway is essential for the execution of stress-induced cell death. JNK3, a JNK isoform that is selectively expressed in the nervous system, is required for neuronal cell death caused by excitotoxic stress. The results suggest the possibility that Eiger-induced cell death signaling may be independent of downstream jun expression, similar to the observation that the effect of UV to cause cell death does not require new gene expression. The JNK signaling also mediates heat shock-induced cell death, the execution of which is caspase independent. Furthermore, overexpression of the EDA receptor or TAJ/TROY, a member of the TNF receptor superfamily that exhibits extensive homology to the EDA receptor, results in the activation of the JNK pathway and caspase-independent cell death (Eby, 2000; Kumar, 2001). In some cases, JNK-induced cell death is mediated by the release of mitochondrial apoptogenic factors. Recently, it has been shown that cancer cell death induced by TRAIL, a mammalian TNF superfamily ligand, requires mitochondrial release of Smac. One possible mechanism of Eiger-induced cell death may be JNK-mediated release of mitochondrial caspase-independent cell death factors. In fact, the Drosophila genome also encodes homologs of such molecules: AIF, endo G and HtrA2 (Igaki, 2002 and references therein).

One important feature of Eiger-stimulating cell death signaling is that it can be blocked by DIAP1. It is well understood that IAP family proteins suppress cell death through direct inhibition of caspases. The observations in this study suggest a potential mechanism of IAP that can inhibit caspase-independent cell death. It has been reported that Xenopus cell death induced by TAK1 (see Drosophila TGF-ß activated kinase 1) and TAB1, an activator for TAK1, is blocked by X-chromosome-linked IAP (XIAP). More recently, it has been shown that XIAP attenuates TNF-alpha-mediated JNK activation in HeLa cells and RelA-/- fibroblasts. These findings and the data presented in this study lead to a model in Drosophila in which DIAP1 regulates caspase-dependent and -independent cell death pathways by blocking both the caspases and the JNK signaling (Igaki, 2002).

Loss-of-function study demonstrates that Eiger is a physiological trigger for the JNK pathway in the eye disc. Genetic interaction assays show that Eiger-stimulating cell death signaling is mediated by Msn, dTAK1, Hep and Bsk. Although dominant-negative dTAK1 completely suppresses the Eiger-induced phenotype, it is also possible that many components of MAP kinase pathways expressed as 'dominant negatives' can have a gain-of-function inhibitory activity. In fact, the immune response phenotype of dTAK1 mutants seems to be inconsistent with the idea that dTAK1 participates in the Eiger pathway. Another possible JNKKK family member to mediate Eiger signaling is Slipper. Previous genetic studies in Drosophila have revealed that the JNK signaling pathway regulates epithelial morphogenesis during the process of embryonic dorsal closure, and that it also participates in the control of planar polarity in several tissues. It has also been reported that the JNK signaling regulates cell death to maintain normal morphogenesis of the wing. Eiger might function as a JNK-dependent cell death regulator to facilitate normal morphogenesis of the eye. Further analysis of eiger mutant flies would dissect the physiological role of Eiger in neural development (Igaki, 2002 and references therein).

In mammals, members of the TNF superfamily play crucial roles in the regulation of infections, inflammation, autoimmune diseases and tissue homeostasis. The TNF superfamily ligands bind to their respective receptors leading to the activation of diverse signaling pathways, including the caspase cascade, NF-kappaB, or MAPKs such as JNK or ERK. Thus, TNF-related ligands can trigger either the extrinsic cell death execution, differentiation or proliferation. Although overexpression of Eiger can strongly induce cell death in the Drosophila compound eye, the possibility that Eiger-stimulated signaling may contribute to cellular events other than cell death execution cannot be excluded. In fact, the amino acid sequence of Eiger showed the highest homology (19%) with EDA, a human TNF superfamily ligand, the mutation of which causes impaired ectodermal development. eiger is predominantly expressed in the nervous system, whereas most mammalian TNF/TNF receptor superfamily proteins are expressed in the immune system, raising the possibility that Eiger might regulate proliferation of neural progenitor cells as does TNF-alpha (Arnett, 2001) to maintain normal development of the nervous system. The Drosophila genome has a gene encoding a candidate Eiger receptor with a TNF receptor homology domain and a transmembrane domain (see wengen). In addition, the Drosophila genome also encodes genes for mediating factors such as TNF-receptor-associated factors (TRAFs: see Traf1 and Traf2), FADD and RIP (IMD), all of which may play a role in Eiger/Eiger receptor signaling. For information about Traf1 see Misshappen Protein Interactions section. For information about Traf2 see Pelle Protein Interactions section. Further genetic study of Eiger and its receptor should help elucidate the universal role of TNF/TNF receptor superfamily proteins in normal development, as well as in some pathophysiological conditions (Igaki, 2002).


Acting downstream of Eiger and Wengen, tumor suppressor CYLD regulates JNK-induced cell death in Drosophila

CYLD encodes a tumor suppressor that is mutated in familial cylindromatosis. Despite biochemical and cell culture studies, the physiological functions of CYLD in animal development and tumorigenesis remain poorly understood. To address these questions, Drosophila CYLD (dCYLD) mutant and transgenic flies were generated expressing wild-type and mutant dCYLD proteins. dCYLD is essential for JNK-dependent oxidative stress resistance and normal lifespan. Furthermore, dCYLD regulates TNF-induced JNK activation and cell death through dTRAF2, which acts downstream of the TNF receptor Wengen and upstream of the JNKK kinase dTAK1. dCYLD encodes a deubiquitinating enzyme that deubiquitinates dTRAF2 and prevents dTRAF2 from ubiquitin-mediated proteolytic degradation. These data provide a molecular mechanism for the tumor suppressor function of this evolutionary conserved molecule by indicating that dCYLD plays a critical role in modulating TNF-JNK-mediated cell death (Xue, 2007).

Shortened animal lifespan may result from compromised oxidative stress tolerance. To examine the oxidative stress resistance, 3-day-old flies were challenged with paraquat for a prolonged period of time and their survival rates were measured. It was found that dCYLD mutants exhibited a significant reduction in survival rate as compared with wild-type or heterozygous dCYLD flies after 24 hr or 36 hr of exposure to paraquat, suggesting dCYLD plays a pivotal role in regulating oxidative stress resistance (Xue, 2007).

JNK signaling has been reported to play an important role in regulating oxidative stress resistance and lifespan in Drosophila (Wang, 2003). Ubiquitous expression of Bsk, the Drosophila JNK ortholog, under the control of tubulin promoter, rescues both lifespan and oxidative stress resistance defects in dCYLD mutants, suggesting that dCYLD regulates these two physiological effects through the JNK signaling pathway (Xue, 2007).

This study was extended to other stress conditions and it was found that dCYLD mutants are less resistant to dry starvation (no food and water), a phenotype that has been associated with reduced JNK activity (Wang, 2003). In contrast, dCYLD mutants do not affect animal survival at high and low temperature conditions (Xue, 2007).

To further examine the role of dCYLD in regulating JNK signaling in animal development, the genetic interactions between dCYLD and Eiger (Egr), the Drosophila ortholog of TNF that triggers the JNK pathway, was tested. Ectopic expression of Egr, under the control of the GMR promoter (GMR > Egr) and using the Gal4/UAS binary system, induces JNK activation and cell death in the developing eye that results in vastly reduced adult eye size. The Egr-induced JNK activation and small-eye phenotype was suppressed modestly by deleting one copy of dCYLD and suppressed soundly by removing both copies. The strong suppression of the Egr eye phenotype in homozygous dCYLD mutants was partially reverted by adding one copy of dCYLDRes. These results indicate that dCYLD is required for Egr-triggered JNK activation and cell death (Xue, 2007).

dCYLD encodes a protein of 640 amino acids, containing in its N terminal portion a cytoskeleton-associated protein (CAP) domain that is present in proteins associated with microtubules and the cytoskeletal network, two ubiquitin carboxyl-terminal hydrolases (UCH) domains that are commonly associated with deubiquitinating enzyme activity, and three CXXC zinc-finger (ZF) motifs with potential protein-protein interaction ability. To functionally characterize these motifs, UAS transgenes were generated expressing the wild-type or three mutant versions of dCYLD that delete the CAP domain, the two UCH domains, or the three ZF motifs. When expressed under the control of the GMR promoter, neither the full-length nor the dCYLD mutants displayed any detectable phenotype. When introduced into the GMR > Egr; dCYLD−/− background, wild-type dCYLD released the suppression of the Egr eye phenotype, confirming that the suppressive effect was due to the loss of dCYLD functions. In contrast, dCYLDΔUCH had no effect on the suppression of the Egr eye phenotype, and dCYLDΔCAP could only partially relieve this suppression, implying that the UCH domains are necessary for dCYLD functions and that the CAP domain is essential for dCYLD to execute its full activity in vivo. Interestingly, expression of dCYLDΔZF completely abolished the suppression effect, suggesting that the ZF motifs are dispensable in dCYLD regulation of Egr-induced cell death (Xue, 2007).

Ubiquitous expression of the full-length dCYLD, but not dCYLDΔUCH, rescues both shortened lifespan and hypersensitivity to paraquat in dCYLD mutants, suggesting that the deubiquitinating activity is indispensable for dCYLD to regulate JNK-dependent oxidative stress resistance and lifespan (Xue, 2007).

TNF receptor-associated factors (TRAFs) are important adaptor proteins that bind to TNF receptors and relay TNF signals to the JNK and NF-κB pathways in mammals. In Drosophila, Egr signal is mediated exclusively by the JNK pathway. However, the role of Drosophila TRAF proteins in Egr-JNK signaling remains unclear. The Drosophila genome encodes two TRAFs: dTRAF1, the TRAF2 ortholog; and dTRAF2, the TRAF6 ortholog. To determine the role of dTRAF1 and dTRAF2 in Egr-JNK signaling, the effects were examined of loss-of-function mutations and RNAi-mediated downregulation of dTRAF1 or dTRAF2 on the Egr eye phenotype. The Egr-induced small-eye phenotype was not suppressed by either deletion of one copy of the dTRAF1 gene or coexpression of a dTRAF1 RNAi. In contrast, the Egr eye phenotype was suppressed strongly by removing half of the dosage of dTRAF2 and suppressed completely by deleting the dTRAF2 gene. Consistently, coexpression of a dTRAF2 RNAi significantly suppressed the Egr eye phenotype. In agreement with genetic data, dTRAF2 exhibited a much stronger physical interaction with Wgn and dTAK1 than did dTRAF1 . Together, the above results point to dTRAF2, but not dTRAF1, as the adaptor protein that mediates Egr signaling in Drosophila (Xue, 2007).

To investigate the physiological functions of dCYLD and dTRAF2 in JNK activation, the expression pattern of puckered (puc) was checked in dCYLD or dTRAF2 mutants. puc encodes a JNK phosphatase whose expression is positively regulated by the JNK pathway, and thus, the puc-LacZ expression of the pucE69 enhancer-trap allele can be used as a readout of JNK activity in vivo. puc is weakly expressed in wild-type third-instar eye discs, and can be detected by prolonged staining. It has been previously shown that puc expression posterior to the morphogenetic furrow (MF) depends on endogenous Egr signaling. This study found that such expression patterns are reduced dramatically in dCYLD mutants and dTRAF2 RNAi animals. In contrast, puc expression in the disc margin, which is independent of Egr signaling, was not affected. GMR > Egr strongly activated puc transcription posterior to the MF. This ectopic Egr-induced puc expression was largely blocked by loss of dCYLD or by expression of dTRAF2 RNAi. Taken together, these observations indicate that both dCYLD and dTRAF2 are physiologically required by the endogenous JNK pathway (Xue, 2007).

The role of CYLD in modulating JNK signaling in mammalian cells has remained controversial. Consistent with the current observation, it was reported that JNK activity diminished in Cyld−/− thymocytes, which implies that CYLD is physiologically required for JNK activation. However, CYLD was also reported to negatively regulate JNK signaling in culture cells and macrophages. Thus, CYLD could positively or negatively regulate JNK signaling in a cell-type-specific manner (Xue, 2007).

To genetically map the epistasis of dCYLD and dTRAF2 in the Egr-JNK pathway, the genetic interaction between dTAK1 (JNKKK) and dCYLD or dTRAF2 was examined in the developing eyes. Expression of dTAK1 under the control of the GMR promoter resulted in pupa lethality, while dTAK1 expression under the control of the sevenless (sev) promoter (sev > dTAK1) induced extensive cell death in larval eye discs and gave rise to rough eyes with a reduced size. Loss of dCYLD or dTRAF2, or coexpression of a dTRAF2 RNAi, had no effect on the sev > dTAK1 phenotype, while removal of one copy of hep (JNKK) or bsk (JNK) partially suppressed the sev > dTAK1 phenotype, suggesting that dCYLD and dTRAF2 operate upstream of dTAK1 in the Egr-JNK pathway (Xue, 2007).

Ectopic Egr expression in the dorsal thorax driven by the potent pannier-GAL4 driver resulted in pupa lethality. However, when reared at 18°C, these animals survived to adulthood, presumably due to lessened Egr expression caused by reduced Gal4 activity, and produced a small-scutellum phenotype. This phenotype could be suppressed by RNAi inactivation of JNK signaling components, e.g., wgn, dTRAF2, dTAK1, hep, or bsk, suggesting that the phenotype is caused by activation of JNK signaling. Ectopic expression of dCYLD driven by pannier-GAL4 produced a similar but weaker phenotype, which could be fully suppressed by the coexpression of an RNAi of dTRAF2 and dTAK1, but not that of wgn. These results indicate that dCYLD functions downstream of Wgn, but upstream of dTRAF2 and dTAK1, in modulating JNK signaling (Xue, 2007).

RNAi-mediated downregulation of dTRAF2, but not dTRAF1, resulted in compromised oxidative stress resistance and shortened lifespan, suggesting that the role of dTRAF2 in dCYLD-JNK signaling has been conserved in different physiological contexts (Xue, 2007).

Previous studies have reported that the Shark tyrosine kinase and Src42A regulate JNK signaling in epidermal closure during embryogenesis and metamorphosis. However, null mutants for egr and dCYLD are fully viable and do not display the epidermal closure defect, implying that Shark and Src42A act in parallel to Egr and dCYLD in modulating JNK signaling. Consistent with this interpretation, loss of shark or src42A failed to suppress the GMR > Egr or pnr > dCYLD phenotype. In addition, it was found that loss of the transcription factor dFOXO could suppress both GMR > Egr and pnr > dCYLD phenotypes, suggesting that dFOXO acts downstream of dCYLD in JNK signaling. Consistent with this observation, dFOXO is required downstream of JNK in modulating cell death, oxidative stress resistance, and lifespan (Xue, 2007).

Overexpression of CYLD in mammalian tissue culture cells negatively regulates NF-κB signaling by deubiquitinating TRAF2/6. dCYLD, like its mammalian counterpart, contains two UCH deubiquitinating domains. Indeed, genetic analysis revealed that the UCH deubiquitinating domains are crucial for the in vivo functions of dCYLD. Furthermore, genetic epistasis data show that dCYLD acts upstream of dTRAF2 in the JNK pathway. Thus, it was hypothesized that dCYLD might act in the JNK pathway to deubiquitinate and subsequently stabilize dTRAF2 by preventing its ubiquitination-mediated proteolytic degradation. To examine this hypothesis in vivo, a FLAG-tagged dTRAF2 transgene (GMR > FLAG-dTRAF2) was introduced into dCYLD mutants and transgenic flies. Proteins were extracted from the heads of these flies for biochemical analyses. It was found that loss of dCYLD resulted in a significant reduction in dTRAF2 protein level, while the ubiquitination of dTRAF2 was markedly enhanced. Both changes were suppressed by dCYLDRes. Consistently, overexpression of dCYLD, but not dCYLDΔUCH, increased dTRAF2 protein level and decreased its ubiquitination. These results show that dCYLD functions as a deubiquitinating enzyme that deubiquitinates dTRAF2 and promotes dTRAF2 accumulation in vivo (Xue, 2007).

Polyubiquitination chains are usually formed on two lysine residues, K48 and K63. It is generally believed that the K48-linked polyubiquitination mediates proteasome-dependent protein degradation, while the K63-linked polyubiquitination mediates endocytosis and signal transduction. Previous work in mammalian culture cells has implicated that CYLD encodes a deubiquitinating enzyme that preferentially cleaves K63-linked polyubiquitin chain from its target proteins for NF-κB signaling. However, a recent in vivo study in Cyld−/− mice has reported that CYLD could regulate the stability of its target protein by effectively removing K48-linked polyubiquitin chain in thymocytes. Interestingly, JNK activity also diminished in Cyld−/− thymocytes. Thus, the role of CYLD in regulating protein stability and positively modulating JNK signaling could be conserved in mammals (Xue, 2007).

CYLD mutations in human patients cause dramatic skin tumors. However, the physiological function of CYLD and the mechanism underlying CYLD deficiency-induced tumorigenesis remain largely unknown. By generating the null dCYLD mutation and dCYLD transgenic animals and performing genetic analysis, this study has shown that dCYLD is a critical regulatory component of the JNK signaling pathway. Genetic epistasis and biochemical analysis further reveal that dCYLD modulates JNK signaling by deubiquitinating dTRAF2 and thus preventing dTRAF2 from ubiquitination-mediated proteolytic degradation. Loss of dCYLD results in augmented ubiquitination and degradation of dTRAF2, which renders cells resistant to apoptosis triggered by JNK signaling. Deregulation of apoptosis has been implicated as a major cause of tumorigenesis. Consistently, mice deficient for both jnk1 and jnk2 were resistant to apoptosis induced by UV irradiation, anisomycin, and MMS, and jnk1−/− mice exhibited enhanced skin tumor development, a phenotype that is pathogenically similar to cylindromatosis in CYLD human patients. Together these data argue that modulation of JNK signaling could be a conserved mechanism underlying familial cylindromatosis in CYLD patients (Xue, 2007).

Notch and Mef2 synergize to promote proliferation and metastasis through JNK signal activation in Drosophila

Genetic analyses in Drosophila revealed a synergy between Notch and the pleiotropic transcription factor Mef2 (myocyte enhancer factor 2), which profoundly influences proliferation and metastasis. This study shows that these hyperproliferative and invasive Drosophila phenotypes are attributed to upregulation of eiger, a member of the tumour necrosis factor superfamily of ligands, and the consequent activation of Jun N-terminal kinase signalling, which in turn triggers the expression of the invasive marker MMP1. Expression studies in human breast tumour samples demonstrate correlation between Notch and Mef2 paralogues and support the notion that Notch-MEF2 synergy may be significant for modulating human mammary oncogenesis (Pallavi, 2012).

A genetic modifier screen was undertaken in Drosophila and a number of genetic modifiers of Notch signals were identified that affect proliferation. Further examination of one of these modifiers, Mef2, established that its synergy with Notch signals directly triggers expression of the Drosophila JNK pathway ligand eiger, consequently activating JNK signalling that profoundly influences proliferation and metastatic behaviour. It might perhaps be worth noting that metastatic behaviour in Drosophila may not be completely equivalent to mammalian metastasis, notwithstanding the fact that they share molecular signatures, for example, MMP activation (Pallavi, 2012).

Cancer is characterized by the deregulation of the balance between differentiation, proliferation and apoptosis; thus, it is not surprising that the Notch signalling pathway, which plays a central role in all these developmental events, is increasingly implicated in oncogenic events. The rationale of this study is based on the fact that synergy between Notch and other genes is key in understanding how Notch signals contribute to oncogenesis. It remains a remarkable fact that while activating mutations in the Notch receptor have been associated with >50% of T-cell lymphoblastic leukemias (T-ALLs), a search for mutations in other cancers, despite a few suggestive reports, remains essentially unfruitful. Yet, correlative studies have linked Notch activity with a broad spectrum of human cancers and work in mice suggests that while Notch activation promotes proliferation, it is the synergy between Notch and other factors that eventually leads to cancer. Similar synergies have been identified before but the extraordinary complexity of the gene circuitry that modulates the Notch pathway suggests that more such relationships will be uncovered as exemplified by the discovery of Mef2 as a Notch synergistic partner affecting proliferation (Pallavi, 2012).

The transcription factor Mef2 plays an essential role in myogenic differentiation, but several studies have also shown a broad pleiotropic role of Mef2. Mef2 can integrate signals from several signalling cascades through chromatin remodelling factors and other transcriptional regulators to control differentiation events. This study extends the functionality of Mef2 by uncovering the profound effect it can have on proliferation and metastatic cell migration in synergy with Notch signals (Pallavi, 2012).

This is not the first study to link Mef2 with Notch. They have been linked before in the context of myogenesis both in Drosophila and in vertebrates. A ChIP-on-chip analysis of Mef2 target regions identified several Notch pathway components as potential Mef2 targets during Drosophila myogenesis. In human myoblasts, Mef2C was suggested to bind directly to the intracellular domain of Notch via the ankyrin repeat region, suppressing Mef2C-induced myogenic differentiation. Mef2 has also been reported to interact with the Notch coactivator MAML1 and suppress differentiation (Pallavi, 2012).

While upregulation of Mef2 alone does not show overt proliferation effects, these analyses demonstrate that in vivo it can activate MMP1. Even though Mef2 was ectopically expressed in the whole wing pouch, MMP1 expression was confined around the D/V boundary, where endogenous Notch signals are active. This effect of Mef2 overexpression depends on Notch signals, a notion corroborated by the fact that inhibiting Notch activity by RNAi reverses the effects of Mef2 on MMP1 (Pallavi, 2012).

The polarity gene scribble cooperates with Ras signalling to upregulate the JNK pathway, promoting invasiveness and hyperplasticity. However, the synergy seen in this study appears to be scribble independent. The fact that both the scribbled/Ras and the Notch/Mef2 metastatic pathways converge at the level of JNK signal activation suggests that JNK is a crucial regulator of oncogenic behaviour, which is controlled by inputs from multiple signals. Even though there is little evidence that twist activates JNK signalling, it is a crucial regulator of epithelial-to-mesenchymal transition and metastasis and has also been independently linked to both Mef2 and Notch in myogenesis. However, it is noted that the Notch-Mef2 synergy seems to be independent of twist, as Twist cannot replace Mef2 in the synergistic relationship (Pallavi, 2012).

Numerous reports link JNK signalling to normal developmental events requiring cell movement and to metastatic phenomena both in Drosophila and in vertebrates. JNK signals seem to be crucial for controlling gene activities involved in epithelial integrity and the observations from Drosophila suggest that the Nact and Mef2 synergy may be important in JNK-linked carcinogenesis. A role for Notch in controlling JNK signals has been reported previously. While studies carried out using breast cancer samples can only be correlative now, the observations suggest that metastatic breast tumours harbour higher levels of Notch and Mef2 paralogue pairs, consistent with observations in Drosophila (Pallavi, 2012).

Although the majority of studies on Mef2 are focused on muscle development/differentiation, some intriguing links between Mef2C and leukaemias are noteworthy. MEF2C and Sox4 synergize to cause myeloid leukaemia in mice. Analysis of T-ALL patient samples revealed increased levels of Mef2C; however, Mef2C alone could not cause cellular transformation of NIH3T3 cells, but it could do so in the presence of RAS or myc. Given the role of Notch in T-ALL it will be important to examine how activated Notch mutations, often the causative oncogenic mutation, correlate with Mef2 family members. The functional differences between the different Mef2 homologues in humans are not well understood and the specific role each may play in the Notch synergy remains to be elucidated (Pallavi, 2012).

This analysis clearly indicates that, in Drosophila, the underlying molecular mechanism of the Notch/Mef2 synergy relies on the direct upregulation of expression of the prototypical TNF ligand egr through the binding of Mef2 and Su(H), the effector of Notch signals, to regulatory sequences on the egr promoter. In Drosophila, egr is the only JNK ligand while in humans, the superfamily is large and includes the cytokines TNFα (TNF), TRAIL and RANKL which have been associated with tumour progression in numerous human cancers including breast. RANKL plays a key role in bone metastasis of breast cancer, and is the target of a therapeutically effective monoclonal antibody. In breast cancer cells, TNFα, which can signal through several pathways, including JNK and NF-κB, affects proliferation and promotes invasion and metastasis (Pallavi, 2012).

In human breast cancer, clinical relapse after initial treatment is almost always accompanied by metastatic spread and it is almost invariably lethal. ER- tumours tend to respond well to first-line chemotherapy, but a significant subset of these tumours recur. Recurrent ER- tumours are typically resistant to chemotherapy and radiation, and are highly lethal. The current data suggest that ER- tumours that recur but not ER- tumours that do not recur show significant positive correlation between NOTCH1 and all four MEF2 paralogues. Further, the data show that even within the recurrent subset, NOTCH1 expression predicts poor survival but MEF2 expression does not. While these observations do not establish causality, they are consistent with the hypothesis that NOTCH1/MEF2 coexpression identifies a set of breast cancers that are more likely to relapse, and that MEF2 genes act as NOTCH cofactors rather than independently of NOTCH (Pallavi, 2012).

In conclusion, this study in Drosophila uncovers a new functional role for Mef2, which in synergy with Notch affects proliferation and metastasis. Mechanistically, this synergy relies on the direct upregulation of the JNK pathway ligand eiger. The correlation analysis and tumour staining of human cancer samples suggests that the observations in Drosophila may well be valid in humans, defining Notch-Mef2 synergy as a critical oncogenic parameter, one that may be associated with metastatic behaviour, emphasizing the value of model systems in gaining insight into human pathobiology (Pallavi, 2012).

Oncogenic Ras stimulates Eiger/TNF exocytosis to promote growth

Oncogenic mutations in Ras deregulate cell death and proliferation to cause cancer in a significant number of patients. Although normal Ras signaling during development has been well elucidated in multiple organisms, it is less clear how oncogenic Ras exerts its effects. Furthermore, cancers with oncogenic Ras mutations are aggressive and generally resistant to targeted therapies or chemotherapy. This study identified the exocytosis component Sec15 as a synthetic suppressor of oncogenic Ras in an in vivo Drosophila mosaic screen. Oncogenic Ras elevates exocytosis and promotes the export of the pro-apoptotic ligand Eiger (Drosophila TNF). This blocks tumor cell death and stimulates overgrowth by activating the JNK-JAK-STAT non-autonomous proliferation signal from the neighboring wild-type cells. Inhibition of Eiger/TNF exocytosis or interfering with the JNK-JAK-STAT non-autonomous proliferation signaling at various steps suppresses oncogenic Ras-mediated overgrowth. These findings highlight important cell-intrinsic and cell-extrinsic roles of exocytosis during oncogenic growth and provide a new class of synthetic suppressors for targeted therapy approaches (Chabu, 2014).

The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth

Disruption of epithelial polarity is a key event in the acquisition of neoplastic growth. JNK signalling is known to play an important part in driving the malignant progression of many epithelial tumours, although the link between loss of polarity and JNK signalling remains elusive. In a Drosophila genome-wide genetic screen designed to identify molecules implicated in neoplastic growth, this study identified grindelwald (grnd; CG10176), a gene encoding a transmembrane protein with homology to members of the tumour necrosis factor receptor (TNFR) superfamily. This study shows that Grnd mediates the pro-apoptotic functions of Eiger (Egr), the unique Drosophila TNF, and that overexpression of an active form of Grnd lacking the extracellular domain is sufficient to activate JNK signalling in vivo. Grnd also promotes the invasiveness of RasV12/scrib-/- tumours through Egr-dependent Matrix metalloprotease-1 (Mmp1) expression. Grnd localizes to the subapical membrane domain with the cell polarity determinant Crumbs (Crb) and couples Crb-induced loss of polarity with JNK activation and neoplastic growth through physical interaction with Veli (also known as Lin-7). Therefore, Grnd represents the first example of a TNFR that integrates signals from both Egr and apical polarity determinants to induce JNK-dependent cell death or tumour growth (Andersen, 2015).

A genome-wide screen was carried to identify molecules that are required for neoplastic growth. The condition used for this screen was the disc-specific knockdown of avalanche, also known as syntaxin 7), a gene encoding a syntaxin that functions in the early step of endocytosis2. avl-RNAi results in ectopic Wingless (Wg) expression, neoplastic disc overgrowth, and a 2-day delay in larva-to-pupa transition. A collection of 10,100 transgenic RNA interference (RNAi) lines were screened for their ability to rescue the pupariation delay, and 121 candidate genes were identified. Interestingly, only eight candidate genes also rescued ectopic Wg expression and neoplastic overgrowth. These included five lines targeting core components of the JNK pathway (Bendless, Tab2, Tak1, Hemipterous and Basket. Using a puckered enhancer trap (puc-lacZ) as a readout for JNK activity, it was confirmed that JNK signalling is highly upregulated in avl-RNAi discs. One of the remaining lines targets CG10176, a gene encoding a transmembrane protein. Reducing expression of CG10176 by using two different RNAi lines was as efficient as tak1 silencing to restore normal Wg pattern and suppresses JNK signalling and neoplastic growth in the avl-RNAi background. Sequence analysis of GC10176 identified a cysteine-rich domain (CRD) in the extracellular part with homology to vertebrate TNFRs harbouring a glycosphingolipid-binding motif (GBM) characteristic of many TNFRs including Fas. CG10176 was named grindelwald (grnd) , after a village at the foot of Eiger, a Swiss mountain that lent its name to the unique Drosophila TNF, Egr. Immunostaining and subcellular fractionation of disc extracts confirmed that Grnd localizes to the membrane. Moreover, co-immunoprecipitation experiments showed that both Grnd full-length and Grnd-intra, a form lacking its extracellular domain, directly associate with Traf2, the most upstream component of the JNK pathway. This interaction is disrupted by a single amino acid substitution within a conserved Traf6-binding motif (human TRAF6 is the closest homologue to Traf2. Overexpression of Grnd-intra, but not full-length Grnd, is sufficient to induce JNK signalling, ectopic Wg expression and apoptosis, and Grnd-intra-induced apoptosis is efficiently suppressed in a hep (JNKK) mutant background, confirming that Grnd acts upstream of the JNK signalling cascade (Andersen, 2015).

The Drosophila TNF Egr activates JNK signalling and triggers cell death or proliferation, depending on the cellular context. Therefore tests were performed to see whether Grnd is required for the small-eye phenotype generated by Egr-induced apoptosis in the retinal epithelium (via Egr overexpression). Inhibition of JNK signalling by reducing tak1 or traf2 expression, or by overexpressing puckered, blocks Egr-induced apoptosis and rescues the small-eye phenotype. In contrast to a previous report, RNAi silencing of wengen (wgn) , a gene encoding a presumptive receptor for Egr, does not rescue the small-eye phenotype. Furthermore, the small-eye phenotype is not modified in a wgn-null mutant background, confirming that Wgn is not required for Egr-induced apoptosis in the eye. By contrast, reducing grnd levels partially rescues the Egr-induced small-eye phenotype, producing a 'hanging-eye' phenotype that is not further rescued in a wgn-knockout mutant background. A similar phenotype was previously reported as a result of non-autonomous cell death induced by a diffusible form of Egr. This suggests that Grnd prevents Egr from diffusing outside of its expression domain. Co-immunoprecipitation experiments show that both full-length Grnd and Grnd-extra, a truncated form of Grnd lacking the cytoplasmic domain, associate with Egr through its TNF-homology domain. Although Grnd-extra can bind Egr, it cannot activate JNK signalling. Therefore, it was reasoned that Grnd-extra expression might prevent both cell-autonomous and non-autonomous apoptosis by trapping Egr and preventing its diffusion and binding to endogenous Grnd. Indeed, GMR-Gal4-mediated expression of grnd-extra fully rescues the Egr small-eye phenotype. To confirm that the removal of Grnd induces Egr-mediated non-autonomous cell death, wing disc clones were generated expressing egr alone, egr + tak1 RNAi, or egr + grnd RNAi. As expected, reducing tak1 levels in egr-expressing clones prevents their elimination by apoptosis. Similarly, reducing grnd levels prevents autonomous cell death, but also induces non-autonomous apoptosis. This suggests that Egr, like its mammalian counterpart TNF-α, can be processed into a diffusible form in vivo whose interaction with Grnd limits the potential to act at a distance. Flies carrying homozygous (grndMinos/Minos) or transheterozygous (grndMinos/Df) combinations of a transposon inserted in the grnd locus express no detectable levels of Grnd protein and are equally resistant to Egr-induced cell death. In addition, grndMinos/Minos mutant flies are viable and display no obvious phenotype, suggesting that Grnd, like Egr, participates in a stress response to limit organismal damage. Collectively, these data demonstrate that Grnd is a new Drosophila TNF receptor that mediates most, if not all, Egr-induced apoptosis (Andersen, 2015).

TNFs probably represent a danger signal produced in response to tissue damage to rid the organism of premalignant tissue or to facilitate wound healing. Disc clones mutant for the polarity gene scribbled (scrib) induce an Egr-dependent response resulting in the elimination of scrib mutant cells by JNK-mediated apoptosis. To test the requirement for Grnd in this process, scrib-RNAi and scrib-RNAi + grnd-RNAi clones obtained 72 h after heat shock induction were compared. As expected, scrib-RNAi cells undergo apoptosis and detach from the epithelium. By contrast, scrib-RNAi clones with reduced grnd expression survive, indicating that Grnd is required for Egr-dependent elimination of scrib-RNAi cells. Similar results were obtained by generating scrib mutant clones in the eye disc (Andersen, 2015).

In both mammals and flies, TNFs are double-edged swords that also have the capacity to promote tumorigenesis in specific cellular contexts. Indeed, scrib minus eye disc cells expressing an activated form of Ras (RasV12) exhibit a dramatic tumour-like overgrowth and metastatic behaviour, a process that critically relies on Egr. RasV12/scrib-/- metastatic cells show a strong accumulation of Grnd and Mmp1, and invade the ventral nerve cord. Primary tumour cells reach peripheral tissues such as the fat body and the gut, where they form micro-metastases expressing high levels of Grnd. Reducing grnd levels in RasV12/scrib-/- clones is sufficient to restore normal levels of Mmp1 and abolish invasiveness in a way similar to that observed in an egr mutant background. Therefore, Grnd is required for the Egr-induced metastatic behaviour of RasV12/scrib-/- tumorous cells. Similarly, reducing grnd, but not wgn levels, strongly suppresses Mmp1 expression in RasV12/dlg-RNAi cells and limits tumour invasion, indicating that Wgn does not have a major role in the progression of these tumours (Andersen, 2015).

Perturbation of cell polarity is an early hallmark of tumour progression in epithelial cells. In contrast to small patches of polarity-deficient cells, for example, scrib mutant clones, organ compartments or animals fully composed of polarity-deficient cells become refractory to Egr-induced cell death and develop epithelial tumours. The formation of these tumours requires JNK/MAPK signalling, but not Egr, suggesting Egr-independent coupling between loss of polarity and JNK/MAPK-dependent tumour growth. In line with these observations, it was noticed that, in contrast to Grnd, Egr is not required to drive neoplastic growth in avl-RNAi conditions. This suggests that, in addition to its role in promoting Egr-dependent functions, Grnd couples loss of polarity with JNK-dependent growth independently of Egr. Disc immunostainings revealed that Grnd co-localizes with the apical determinant Crb in the marginal zone, apical to the adherens junction protein E-cadherin (E-cad) and the atypical protein kinase C (aPKC). In avl-RNAi discs, Grnd and Crb accumulate in a wider apical domain. Apical accumulation of Crb is proposed to be partly responsible for the neoplastic growth induced by avl knockdown, since overexpression of Crb or a membrane-bound cytoplasmic tail of Crb (Crb-intra) mimics the avl-RNAi phenotype. Therefore whether Grnd might couple the activity of the Crb complex with JNK-mediated neoplastic growth was examined. Indeed, reducing grnd levels, but not wgn, in ectopic crb-intra discs suppresses neoplastic growth as efficiently as inhibiting the activity of the JNK pathway. Notably, Yki activation is not rescued in these conditions, illustrating the ability of Crb-intra to promote growth independently of Grnd by inhibiting Hippo signalling through its FERM-binding motif (FBM). Indeed, neoplastic growth and polarity defects induced by a form of Crb-intra lacking its FBM (CrbΔFBM-intra) are both rescued by Grnd silencing. As expected, the size of ectopic crbΔFBM-intra;grnd-RNAi discs is reduced compared to the size of ectopic crb-intra; grnd-RNAi discs (Andersen, 2015).

Crb, Stardust (Sdt; PALS1 in humans), and Pals1-associated tight junction protein (Patj) make up the core Crb complex, which recruits the adaptor protein Veli (MALS1-3 in humans). In agreement with previous yeast two-hybrid data, this study found that Grnd binds directly and specifically to the PDZ domain of Veli through a membrane-proximal stretch of 28 amino acids in its intracellular domain. Grnd localization is unaffected in crb and veli RNAi mutant clones. However, reducing veli expression rescues the patterning defects and disc morphology of ectopic crb-intra mutant cells, suggesting that Grnd couples Crb activity with JNK signalling through its interaction with Veli. Interestingly, aPKC-dependent activation of JNK signalling also depends on Grnd. aPKC is capable of directly binding and phosphorylating Crb, which is important for Crb function. This suggests that aPKC, either directly or through Crb phosphorylation, activates Grnd-dependent JNK signalling in response to perturbation of apico-basal polarity (Andersen, 2015).

These data are consistent with a model whereby Grnd integrates signals from Egr, the unique fly TNF, and apical polarity determinants to induce JNK-dependent neoplastic growth or apoptosis in a context-dependent manner. Recent work reveals a correlation between mammalian Crb3 expression and tumorigenic potential in mouse kidney epithelial cells. The conserved nature of the Grnd receptor suggests that specific TNFRs might carry out similar functions in vertebrates, in which the link between apical cell polarity and tumour progression remains elusive (Andersen, 2015).

Extracellular reactive oxygen species drive apoptosis-induced proliferation via Drosophila macrophages

Apoptosis-induced proliferation (AiP) is a compensatory mechanism to maintain tissue size and morphology following unexpected cell loss during normal development, and may also be a contributing factor to cancer and drug resistance. In apoptotic cells, caspase-initiated signaling cascades lead to the downstream production of mitogenic factors and the proliferation of neighboring surviving cells. In epithelial cells of Drosophila imaginal discs, the Caspase-9 ortholog Dronc drives AiP via activation of Jun N-terminal kinase (JNK); however, the specific mechanisms of JNK activation remain unknown. This study shows that caspase-induced activation of JNK during AiP depends on an inflammatory response. This is mediated by extracellular reactive oxygen species (ROSs) generated by the NADPH oxidase Duox in epithelial disc cells. Extracellular ROSs activate Drosophila macrophages (hemocytes), which in turn trigger JNK activity in epithelial cells by signaling through the tumor necrosis factor (TNF) ortholog Eiger. It is proposed that in an immortalized ('undead') model of AiP, in which the activity of the effector caspases is blocked, signaling back and forth between epithelial disc cells and hemocytes by extracellular ROSs and TNF/Eiger drives overgrowth of the disc epithelium. These data illustrate a bidirectional cell-cell communication pathway with implication for tissue repair, regeneration, and cancer (Fogarty, 2016).

The role of ROSs as a regulated form of redox signaling in damage detection and damage response is becoming increasingly clear. This study has shown that in Drosophila, extracellular ROSs generated by the NADPH oxidase Duox drive compensatory proliferation and overgrowth following hid-induced activation of the initiator caspase Dronc in developing epithelial tissues. At least one consequence of ROS production is the activation of hemocytes at undead epithelial disc tissue. Furthermore, the work implies that extracellular ROS and hemocytes are part of the feedback amplification loop between Hid, Dronc, and JNK that occurs during stress-induced apoptosis. Finally, hemocytes release the TNF ligand Eiger, which promotes JNK activation in epithelial disc cells (Fogarty, 2016).

This work helps to understand why JNK activation occurs mostly in apoptotic/undead cells but occasionally also in neighboring surviving cells. Because the data indicate that hemocytes trigger JNK activation in epithelial cells, the location of hemocytes on the imaginal discs determines which epithelial cells receive the signal for JNK activation. Nevertheless, the possibility is not excluded that there is also an autonomous manner of Dronc-induced JNK activation in undead/apoptotic cells (Fogarty, 2016).

In the context of apoptosis, hemocytes engulf and degrade dying cells. However, there is no evidence that hemocytes have this role in the undead AiP model. No Caspase-3 (CC3) material is observed in hemocytes attached to undead tissue. Therefore, the role of hemocytes in driving proliferation is less clear and likely context dependent. In Drosophila embryos, hemocytes are required for epidermal wound healing, but this is a nonproliferative process. With respect to tumor models in Drosophila, much of the research to date has focused on the tumor-suppressing role of hemocytes and the innate immune response. However, a few reports have implicated hemocytes as tumor promoters in a neoplastic tumor model. Consistently, in the undead model of AiP, this study found that hemocytes have an overgrowth- and tumor-promoting role. Therefore, the state of the damaged tissue and the signals produced by the epithelium may have differential effects on hemocyte response (Fogarty, 2016).

In a recent study, ROSs were found to be required for tissue repair of wing imaginal discs in a regenerative (p35-independent) model of AiP, consistent with the current work. Although a role of hemocytes was not investigated in this study, it should be noted that p35-independent AiP models do not cause overgrowth, whereas undead ones such as the ey>hid-p35 AiP model do. It is therefore possible that ROSs in p35-independent AiP models are necessary for tissue repair independent of hemocytes, whereas ROSs in conjunction with ROS-activated hemocytes in undead models mediate the overgrowth of the affected tissue. Future work will clarify the overgrowth-promoting function of hemocytes. These considerations are reminiscent of mammalian systems, where many solid tumors are known to host alternatively activated (M2) tumor-associated macrophages, which promote tumor growth and are associated with a poor prognosis (Fogarty, 2016).

Because tumors are considered 'wounds that do not heal', the undead model of AiP is seen as a tool to probe the dynamic interactions and intercellular signaling events that occur in the chronic wound microenvironment. Future studies will investigate the specific mechanisms of hemocyte-induced growth and the tumor-promoting role of inflammation in Drosophila as well as roles of additional tissue types, such as the fat body, on modulating tumorous growth (Fogarty, 2016).

Protein Interactions

Wengen has been identified as the first member of the Drosophila tumor necrosis factor receptor (TNFR) superfamily. Wengen is a type III membrane protein with conserved cysteine-rich residues (TNFR homology domain) in the extracellular domain, a hallmark of the TNFR superfamily. Wengen mRNA is expressed at all stages of Drosophila development. The small-eye phenotype caused by an eye-specific overexpression of a Drosophila TNF superfamily ligand, Eiger, is dramatically suppressed by downregulation of Wengen using RNA interference. In addition, Wengen and Eiger physically interact with each other through their TNFR homology domain and TNF homology domain, respectively. These results suggest that Wengen can act as a component of a functional receptor for Eiger. This identification of Wengen and further genetic analysis should provide increased understanding of the evolutionarily conserved roles of TNF/TNFR superfamily proteins in normal development, as well as in some pathophysiological conditions (Kanda, 2002).

In a Drosophila dominant-modifier screen using the chromosomal deficiency lines that covered more than 70% of the genome, several lines were obtained that suppress the small-eye phenotype caused by Eiger overexpression (GMR>eigerregg1). Through the analysis of these deficiency lines, a line, Df(1)E128/FM7c, was identified in which the deficiency spans the coding region of a predicted gene, CG6531. CG6531 encodes a protein with a cysteine-rich domain (TNFR homology domain), the hallmark of the TNFR superfamily. This gene was named wengen for a village at the foot of Switzerland's Mt. Eiger. Analysis of the wengen nucleotide sequence revealed an open reading frame of 343 amino acids with a predicted relative molecular mass of 40 kDa. Alignment analysis revealed that Wengen harbors a TNFR homology domain in the extracellular region and a membrane-spanning region without signal sequence. This is a characteristic of the type III membrane protein of TNFR superfamily (extracellular N terminus, intracellular C terminus, lacking a signal peptide). The TNFR homology domain of Wengen has significant structural and amino acid homology with the TNFR domains of human EDAR (hXEDAR) and human TNFR1 (hTNFR1). The TNFR homology domains of Wengen, hXEDAR, and hTNFR1 share the topologically distinctive modules (termed A1 and B2 module, except for the TNFR homology domain of hTNFR1 [amino acids 168-195], which is composed of the A1 and C2 modules). To investigate whether Wengen is indeed a type III membrane protein like the other TNFR superfamily members, its subcellular localization was examined. S2 cells were transiently transfected with expression vectors for C-terminally HA-tagged Wengen (Wengen-HA) and GFP. In GFP-positive cells Wengen-HA is detected on the surface membrane with anti-HA antibody when the S2 cells were permeabilized. These data suggest that the Wengen C-terminal domain is indeed cytoplasmic. These results suggest that Wengen is a member of the type III TNFR superfamily. The expression of wengen was examined in flies. RT-PCR analysis revealed that wengen mRNA is expressed at all stages of development. Its putative ligand, Eiger, is also expressed at all stages of Drosophila development (Kanda, 2002).

To assess whether Wengen is required for Eiger to induce the small-eye phenotype, RNA interference (RNAi) was used to down-regulate the endogenous expression of Wengen. A head-to-head inverted repeat construct for wengen, pUAS-wengen-IR, was generated, and its ability to knock down the wengen expression was examined. Co-transfection of pUAS-HA-wengen together with pUAS-wengen-IR into S2 cells dramatically reduces Wengen expression but has no effect on the expression of HA-CARD, suggesting that wengen-IR works as a specific inhibitor of Wengen expression. To assess the biological functions of Wengen in Drosophila, transgenic flies were generated that misexpress wengen-IR in the developing retina. The small-eye phenotype induced by the eye-specific ectopic expression of Eiger (GMR>eigerregg1) was suppressed by the coexpression of wengen-IR. These results strongly suggest that Wengen is required as a functional transducer of Eiger signaling (Kanda, 2002).

The physical interactions between Wengen and Eiger were assessed using various deletion mutants. Immunoprecipitation assays revealed that full-length Wengen and Eiger physically interact with each other. Eiger interacts with WengenDeltacyt (lacking the cytoplasmic domain) but not with WengenDeltaTNFR (lacking the homology TNFR domain). In addition, full-length Wengen could not interact with Eiger DeltaTNF. These results suggest that Wengen can interact with Eiger, and this interaction is mediated through the TNFR homology domain of Wengen and the TNF homology domain of Eiger (Kanda, 2002).

This study has identified the first Drosophila member of the TNFR superfamily, Wengen. Most of the genes for the TNFR superfamily encode type I or III membrane proteins with one or more extracellular ligand-binding domains and a cytoplasmic region that activates cell functions. In general, the extracellular domain of this family of proteins shows a relatively low level of sequence conservation, despite sharing a common fundamental structure. The cytoplasmic regions of the receptors show considerably more diversity in sequence and size than the extracellular regions. There are no common intracellular motifs found in all members of the TNFR superfamily except for some domains such as the TRAF2-binding domain [(P/S/A/T)X(Q/E)E or PXQXXD], which is required for both NF-kappaB activation and JNK activation, or a domain of ~80 amino acids called the 'death domain', for caspase activation. However, the amino acid sequence of Wengen reveals that it has neither a TRAF2-binding domain nor a death domain in the cytoplasmic region, suggesting that there should be another mechanism to transduce signals (Kanda, 2002).

Whereas Eiger can stimulate the JNK pathway, the stimulation of the JNK pathway in response to the overexpression of Wengen in S2 cells or the Drosophila compound eye could not be detected. It is possible that because the amount of ligand is limited, overexpression of Wengen is not sufficient to activate the downstream signals. It is also possible that intracellular adapter proteins, which are required for transducing signals, are not expressed or limited in Wengen expressing cells. Otherwise, Wengen may require one or more co-receptors that transduce signals to the cytoplasm. For instance, heteromeric receptor complex is used to transduce Hedgehog signaling. Hedgehog binds to its receptor Patched, and then the inhibitory function of Patched against its binding partner, Smoothened, is cancelled. In this way, Hedgehog signaling is transduced into the cells. Because the heteromeric complex of receptors has never been reported to transduce TNF family signaling, it is possible that Eiger/Wengen may use the novel type of TNF signaling mechanisms. In any case, further genetic and biochemical studies of Eiger/Wengen should help to elucidate the unique signaling mechanisms that include the caspase-independent pathway triggered by Eiger (Kanda, 2002).

Eiger triggers apoptosis by acting through the JNK pathway

Much of what is known about apoptosis in human cells stems from pioneering genetic studies in the nematode C. elegans. However, one important way in which the regulation of mammalian cell death appears to differ from that of its nematode counterpart is in the employment of TNF and TNF receptor superfamilies. No members of these families are present in C. elegans, yet TNF factors play prominent roles in mammalian development and disease. The cloning and characterization of Eiger, a unique TNF homolog in Drosophila, is described. Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution (Moreno, 2002).

Analysis of the Drosophila genome sequence reveals a single predicted transcript that encodes a type II membrane protein with structural similarities to members of the TNF superfamily. This protein is referred to as Eiger, in memory of the numerous mountaineers that have been killed by the Eiger Nordwand, the 'wall of death.' The Eiger protein contains a cytoplasmic domain, a transmembrane region between amino acid residues 36 and 62, and an extracellular domain of 353 amino acids. The C-terminal TNF homology domain (THD) of Eiger shows comparable homology to several human TNF family members (20%–25% identity). In situ hybridization has revealed a weak expression in imaginal discs with a pronounced pattern in the eye (Moreno, 2002).

Like a subset of human TNF ligands, Eiger can induce caspase-dependent apoptosis. Targeted expression of Eiger in the eyes and wings of Drosophila causes a severe ablation of these organs, and Eiger-expressing cell clones are rapidly eliminated. Both of these effects can be suppressed by coexpression of the pan-caspase inhibitor p35 (Moreno, 2002).

Caspase-8 is the key initiator caspase of death ligand-induced apoptosis in mammals. Upon stimulation by TNF, the adaptor protein FADD recruits and aggregates several molecules of procaspase-8 that mutually cleave and activate each other. Due to the involvement of an extracellular ligand, this pathway has been referred to as the 'extrinsic death pathway'. DREDD is the Drosophila caspase most similar to caspase-8 and has been shown to physically interact with Drosophila FADD. Surprisingly, complete removal of DREDD function fails to block Eiger-induced apoptosis, indicating that Eiger triggers cell death by a DREDD/caspase-8-independent pathway (Moreno, 2002).

Another pathway activated by mammalian TNF death factors is the JNK pathway, although its role in inducing apoptosis upon TNF signaling is less well defined. JNK signaling in Drosophila is reflected by the expression levels of puckered (puc), a gene encoding a dual-specificity phosphatase that forms a negative feedback loop by downregulating the activity of JNK. High levels of puc expression are induced by Eiger. Due to its function as a negative regulator of JNK, Puc can also be used as a powerful means to repress JNK activity if overexpressed by a constitutive promoter that is no longer dependent on this activity. Coexpression of Eiger and Puc completely blocks Eiger activity, strikingly reverting the eye and wing phenotypes to wild-type and blocking Eiger-induced elimination of cell clones. Forced expression of Puc does not prevent all forms of cell death. For example, when tested in the situation of polyglutamine repeat-induced neurodegeneration, which is also caused by apoptosis, coexpression of puc has no discernible protective effect. Taken together, these results are interpreted as firm evidence that Eiger induces the JNK signaling pathway and that Eiger-induced apoptosis is critically dependent on JNK activity (Moreno, 2002).

Consistent with this interpretation, several components of the Drosophila JNK pathway are rate limiting in mediating or preventing Eiger-induced apoptosis. The removal of one wild-type copy of either DTRAF1 (encoding the homolog of human TRAF2), misshapen (encoding a Ste20 kinase that binds to DTRAF1), or basket (encodes Drosophila JNK) suppresses Eiger-induced apoptosis. Conversely, animals heterozygous for a mutation in puc display an enhanced phenotype (Moreno, 2002).

The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).

Unlike the situation in mammals, Drosophila TNF appears to activate a linear pathway to induce apoptosis, involving JNK and the apoptosome as landmark components. Thus, in Drosophila, the signaling module formed by FADD and DREDD appears to be dedicated to signaling by the Toll/IMD system, whereas, in mammals, this 'extrinsic' apoptotic pathway can be activated both by some members of the TNF superfamily and by ligands of Toll-like receptors. It is proposed that, at some point during vertebrate evolution, the Toll signaling modules, comprising at least the FADD/caspase-8 branch, but presumably also the NF-kappaB pathway, were coopted by some members of the TNF/TNFR superfamilies, expanding their ancestral function of JNK-mediated induction of apoptosis. Other members of the mammalian TNF/TNFR superfamilies may have retained the primordial Eiger mode of signaling with a linear 'JNK/apoptosome-only' output (Moreno, 2002).

Eiger and its receptor, Wengen, comprise a TNF-like system in Drosophila

In mammals, members of the tumor necrosis factor (TNF) family play an important role in the regulation of cellular proliferation, differentiation and programmed cell death. This study describes isolation and characterization of an orthologous ligand/receptor axis in Drosophila. The ligand, designated Eiger, is a type II membrane glycosylated protein, which can be cleaved at residue 145 and released from the cell surface as a soluble factor, thereby representing the first potential cytokine to be described in Drosophila. Eiger exists in two alternatively spliced isoforms, Eiger long (Eiger-L) and Eiger short (Eiger-s), both of which are expressed throughout development and in the adult. A novel Drosophila member of the TNF receptor family, designated Wengen, is a type I membrane protein that can physically interact with the recently described TRAF2 homolog dTRAF2. Both Eiger and Wengen are expressed in distinctive patterns during embryogenesis and Eiger is responsive to genotoxic stress. Forced expression of Eiger-L, Eiger-s or Wengen, caused apoptotic cell death which could be rescued by caspase inhibitors or the JNK phosphatase Puckered. In addition, Eiger-induced cell killing is attenuated by RNAi-mediated suppression of Wengen. These results illustrate that Eiger and Wengen represent proximal components of an evolutionarily conserved TNF-like signaling pathway in Drosophila (Kauppila, 2003).

The Drosophila genomic database was searched for sequences with homology to the extracellular domain of human TNFR1, and a candidate sequence was identified. This sequence encoded a cDNA of 993 nt and was identical to the sequence of a recently isolated Drosophila receptor, designated Wengen (Kanda, 2002). Sequence analysis revealed that the extracellular domain of Wengen contained a single cysteine-rich pseudorepeat with significant homology to other members of the TNFR family. However, Wengen possessed a unique cytoplasmic domain with no sequence homology to any TNFR family member (Kauppila, 2003).

It was reported that Wengen is a type III membrane protein with a single hydrophobic transmembrane domain (Kanda, 2002). However, sequence analysis revealed that, in addition to the transmembrane domain between residues 202 and 222, Wengen cDNA also encoded for a hydrophobic stretch of amino acids between residues 54 and 59, which could potentially represent a signal peptide. Such a topology is characteristic of type I membrane proteins, including most mammalian receptors of the TNFR family. In order to determine whether Wengen is a type I or type III membrane protein, several constructs were generated with a Flag epitope tag placed at different locations in the deduced open reading frame. Construct Wengen-A positions the Flag epitope tag at the N-terminus of the full-length Wengen protein-coding region. The construct designated Wengen-B encoded a murine Igkappa chain signal peptide followed by the Flag epitope tag and amino acids 14-343 of Wengen. Construct C resembled construct B in topology except that it included the Wengen protein-coding region downstream of the hydrophobic stretch of amino acids representing the putative signal peptide (i.e. amino-acid residues 60-343). A construct was generated encoding amino acids 1-318 of human XEDAR fused to the murine Igkappa chain signal peptide and the Flag epitope. XEDAR is a recently isolated receptor of the TNFR family and a well-characterized type III membrane protein. The above constructs were transfected into 293 T cells along with a GFP reporter construct, and the expression of the Flag epitope tag was analysed on the surface of GFP-expressing cells using FACS analysis. If Wengen corresponds to a type I membrane protein, the epitope tag will be absent from the mature proteins predicted from constructs A and B since it is positioned upstream of the signal peptide cleavage site in these plasmids and, as such, the Flag tag would not be detected on the surface of the cells expressing these constructs (Wengen-A and Wengen-B). In contrast, the Flag epitope tag will be expected to be expressed on the surface of cells expressing the construct C since, in this construct, the Flag epitope is located downstream of the heterologous signal peptide. The cell surface expression of the Flag epitope was not detected in the case of cells transfected with constructs A and B, but readily detected Flag-positive cells in the construct C transfectants. Hence, the epitope was found on the surface of cells only if it was positioned on the C-terminal side of the predicted Wengen signal peptide. In parallel studies with a known type 1 receptor, cell surface expression of the Flag tag was also detected in the case of cells transfected with the comparable Flag-XEDAR construct. Collectively, the above results lend strong support for the hypothesis that Wengen is a type I membrane protein (Kauppila, 2003).

To begin a functional characterization of Eiger and Wengen, the embryonic expression patterns of these genes were examined by in situ hybridization. Eiger mRNA was detected in early embryonic stages in a pattern that was highly localized to the dorsal surface of pregastrulating embryos. Localization of mRNAs to the dorsal side probably reflects maternally derived transcripts that become positioned during oogenesis and could reflect an important role in early pattern formation. By contrast, expression of Wengen appears to be very low or undetectable in pregastrulating embryos. During gastrulation, Wengen-positive cells are detected in the inner layer of embryonic tissue corresponding to the presumptive mesoderm while, at the same stage, Eiger expression occurs in the epidermal layer of the embryo and is most prominent at the surface of dorsal folds that will later form the amnioserosa. At germ band extended stages, Wengen transcripts continue to accumulate in the mesodermal segments of the embryo while its ligand, Eiger, is prominent in the adjacent neurogenic region. In later-staged embryos (stages 15/16) both Eiger and Wengen are detected in subsets of cells within the condensing nerve cord (Kauppila, 2003).

Since TNF and its receptors are implicated in some vertebrate models of damage-induced cell death, tests were performed to see whether Eiger or Wengen might be responsive during genotoxic stress. Eiger RNAs were consistently induced by gamma radiation between two- and fourfold while, in these same samples, levels of Wengen transcripts were unchanged. These findings were confirmed using RT-PCR methods and also it was also determined that both Eiger-s and Eiger-L isoforms are radiation responsive (Kauppila, 2003).

Using transient transfection assays, Wengen and Eiger were tested for apoptosis induction in Drosophila S2 cells. Both the long and short isoforms of Eiger ligand are equally effective at promoting cell death (~40% survival). Similar levels of cell death are provoked by the receptor, Wengen. No overt synergistic effects were detected in cotransfections of Eiger together with Wengen, but cell death induced by both isoforms of the ligand -- as well as the receptor -- were partially reversed by caspase inhibitor peptides, zDEVD and zVAD (Kauppila, 2003).

To test the hypothesis that Wengen is a receptor for Eiger, the effects of dsRNA-mediated silencing of Wengen upon Eiger-induced cell killing were tested. Silencing of Wengen effectively attenuates cell killing triggered by either of the Eiger isoforms. Moreover, these effects are clearly target specific since dsRNA-mediated silencing of either hid or rpr had no influence upon Eiger-induced cell killing. As expected, in converse experiments, it was found that silencing of Eiger had no influence upon Wengen-induced cell killing. Together, these results support a ligand/receptor relationship for Eiger and Wengen (Kauppila, 2003).

Since the JNK phosophatase Puckered is an effective suppressor of Eiger-dependent phenotypes in the animal (Igaki, 2002), the effects of forced puckered expression were examined in cultured cells. This phosphatase clearly protected against cell killing via the Eiger/Wengen axis. In these assays, Puckered had pronounced suppressive effects upon killing by Wengen and the short isoform of Eiger and significant -- but less potent effects -- against the long Eiger isoform. These effects were specific for Eiger/Wengen signaling since, in parallel assays, it was found that puckered had no effect upon levels of cell killing elicited by grim or rpr (Kauppila, 2003).

Each of the two recently published studies on Eiger found only a single isoform, each of which differ by five amino-acid residues. This report has clarified that both isoforms are authentic and expressed at the mRNA level. However, so far no differential expression of these two isoforms during embryogenesis or following genotoxic stress has been discovered. In addition, using the assays undertaken, no overt difference was found in killing activity conferred by the two isoforms. However, the above results are not surprising since the difference between the two isoforms of Eiger is located outside the TNF homology domain, which is usually the main determinant of receptor binding (Kauppila, 2003).

Unlike mammalian TNF family receptors, only a single cysteine-rich domain is present in Wengen. It is conceivable that multiple copies of cysteine-rich domains contribute to increase in affinity and/or specificity of receptor-ligand interaction. Consistent with the above hypothesis, no significant physical interactions were found between soluble Eiger and Wengen (Kauppila, 2003).

The in vivo roles of Eiger and its receptor during development await further characterization but the expression patterns, particularly for the ligand Eiger, raise intriguing possibilities. Eiger is highly localized to a narrow stripe along the dorsal surface of pregastrulating embryos at a time coincident with specification of ventral cell fates via the Toll/Dorsal pathway. Few precedents exist for this unusual transcript distribution. One such precedent, zen, is localized to the dorsal surface as a consequence of repression by dorsal proteins and, in a recent genome-wide analysis, the Eiger locus was also identified as dorsal target. Together with the fact that Eiger can be cleaved to form a soluble ligand, these observations raise the possibility that Eiger may function during early embryonic patterning along the dorsal ventral axis and/or in the specification of dorsal structures such as the amnioserosa. In subsequent embryonic stages, Eiger and Wengen are expressed in nonoverlapping but neighboring tissues. For example, in germ-band-extended embryos, Wengen is expressed in mesodermal tissues, while Eiger is expressed in the adjacent neurogenic ectoderm. Hence, it is possible that TNF-like signaling occurs among these tissues as they develop in the embryo (Kauppila, 2003).

Both Eiger and Wengen triggered cell death which could be blocked by Puckered, an inhibitor of the Drosophila JNK pathway. These results confirm earlier reports from Igaki (2002) and Moreno (2002) who also demonstrated a requirement for JNK signaling. Interestingly, a mammalian TNFR family member has been described that also lacks a death domain, TAJ, that similarly activates the JNK pathway and promotes cell death (Eby, 2000). Thus, it seems plausible that Wengen and TAJ could share evolutionarily conserved mechanisms to trigger cell death. However, unlike TAJ, Eiger- and Wengen-induced cell death was partially blocked by caspase inhibitors. Further characterization of Eiger/Wengen-induced cell death may shed light on this novel evolutionary conserved pathway of apoptosis and open up new therapeutic opportunities for the treatment of cancer (Kauppila, 2003).

Secreted bacterial effectors and host-produced Eiger/TNF drive death in a Salmonella-infected fruit fly: Mutations in eiger delay the lethality of Salmonella infection

Death by infection is often as much due to the host's reaction as it is to the direct result of microbial action. This study identifies genes in both the host and microbe that are involved in the pathogenesis of infection and disease in Drosophila challenged with Salmonella enterica serovartyphimurium. Wild-type Salmonella causes a lethal systemic infection when injected into the hemocoel of Drosophila. Deletion of the gene encoding the secreted bacterial effector Salmonella leucine-rich (PslrP) changes an acute and lethal infection to one that is persistent and less deadly. A model is proposed in which Salmonella secreted effectors stimulate the fly and thus cause an immune response that is damaging both to the bacteria and, subsequently, to the host. In support of this model, mutations in the fly gene eiger, a TNF homolog, are shown to delay the lethality of Salmonella infection. These results suggest that Salmonella-infected flies die from a condition that resembles TNF-induced metabolic collapse in vertebrates. This idea provides a new model to study shock-like biology in a genetically manipulable host. In addition, it allows the study of difference in pathways followed by a microbe when producing an acute or persistent infection (Brandt, 2005).

Attempts were made to infect Drosophila with Salmonella by feeding bacteria to flies and by injecting bacteria into the hemocoel. Flies were resistant to feeding, but direct injection of approximately 10,000 bacterial cells resulted in an infection that caused death in 7-9 d, while injection of sterile medium led to a mean time of death of approximately 20 d (Brandt, 2005).

The most thoroughly characterized immune response in the fly is the humoral immune response, which involves the secretion of antimicrobial peptides into the hemocoel by an organ called the fat body. Two highly conserved signaling systems, the Toll and IMD pathways, have been shown to control the transcription of the antimicrobial peptide genes. The IMD pathway is implicated in raising a response to gram-negative bacteria such as Salmonella. Flies homozygous for an imd mutation succumbed to Salmonella infections rapidly, dying within 12 h of infection. Bacterial numbers exceeded 100,000,000 per fly as the flies died, and Salmonella in dying flies were found circulating in the hemolymph. Circulating bacteria were not seen in wild-type flies. In contrast to imd homozygotes, flies homozygous for a mutation in Dif, a transcription factor in the Toll pathway, did not die rapidly. This experiment shows that mutations in the IMD but not the Toll pathway sensitize the fly to Salmonella. Further, these results suggest that the fly's humoral immune response limits the growth of free Salmonella in the hemocoel (Brandt, 2005).

In mammals and birds, Salmonella is largely an intracellular pathogen. To determine whether Salmonella was indeed growing in a protected intracellular niche in the fly, the in vivo sensitivity of Salmonella to gentamicin, an antibiotic that does not cross host-cell plasma membranes was measured. This experiment showed that bacteria that had been in the fly for 7 d were protected from the antibiotic and suggests that the bacteria were located in an intracellular location (Brandt, 2005).

In other bacterial infection models in the fly, death of the host occurs only when bacterial numbers increase beyond 106 bacteria per fly. This is the case for infections both of wild-type flies with pathogens such as Pseudomonas aeruginosa and L. monocytogenes and of mutant flies with nonpathogenic bacteria such as Escherichia coli. This is not the case for Salmonella in a wild-type fly. To determine the growth characteristics of Salmonella in Drosophila, bacteria were injected over a 1,000-fold dilution range; 7 d after infection, flies were homogenized and plated. At this time, bacteria were found to cover only a 5-fold range. Bacteria injected at low densities grew to levels between 10,000 and 100,000 bacteria per fly, but those injected at higher levels did not pass this threshold. This final number is at least 1,000 fold lower than the number of bacteria found during fatal L. monocytogenes and E. coli infections in the fly. Salmonella thus reaches a population ceiling in flies, and the flies die containing relatively small numbers of bacteria (Brandt, 2005).

To determine the location of bacteria within the fly, flies infected with Salmonella expressing green fluorescent protein (GFP) were examined. Bacteria carrying the plasmid containing the macrophage-inducible gene (pmig-1), which induces GFP expression upon entry into the phagosome, or the strain smo22, which constitutively expresses GFP, were injected into larvae and flies. GFP-expressing Salmonella could be seen within hemocytes bled from infected larvae. Hemocytes in adults are mostly sessile and cannot be easily removed from the fly. However, these cells can be observed through the cuticle, and clusters of these cells are found on the dorsal surface of the abdomen, along the dorsal vessel. Salmonella were found associated with these cells. The distribution of bacteria seen using the two GFP constructs was similar. This experiment suggests that, similar to the situation in mice, Salmonella are found within phagocytes. Furthermore, the Salmonella in fly hemocytes induced the expression of GFP from a Salmonella promoter normally induced upon phagocytosis by a mouse macrophage. This demonstrates that a Salmonella infection-related gene is induced even when the bacteria are infecting an animal at 29°C instead of 37°C (Brandt, 2005).

Salmonella is naturally infectious to mice through an oral route and causes a systemic disease resembling human typhoid fever. Salmonella crosses the gut epithelium by entering and then killing M cells of the Peyer's patch. Once across the epithelial barrier, Salmonella infect macrophages and spread to the mesenteric lymph nodes and subsequently to other organs. S. typhimurium has two type III secretory apparatuses (TTSAs) that translocate effector proteins across both bacterial and host membranes into the host cell cytoplasm. One TTSA encoded by Salmonella pathogenicity island 1 (SPI1) plays an important role in cell entry, whereas a second TTSA, SPI2, alters the intracellular environment of the host cell to permit Salmonella growth. Salmonella likely does not find itself in the hemolymph of Drosophila in nature, but by placing it there some questions can be answered that are difficult to approach using other techniques (Brandt, 2005).

Salmonella carrying mutations in a gene encoding a regulator of virulence, phosphatase P (phoP), or blocking the function of either SPI1 (orgA::Tn10) or SPI2 (ssrA::miniTn5), were injected into flies. All of these mutants killed flies more slowly than wild-type bacteria. However, analysis of bacterial growth within infected flies revealed a more complicated story. PhoP mutants produced infections that were less lethal than wild-type Salmonella, but the infecting bacteria grew to the same levels seen in wild-type infections. In contrast, a SPI1 or SPI2 mutant caused little death, and the bacteria grew to an average of 149,000 cfu/fly (SPI2 mutants) instead of the average of 40,000 cfu per fly found in wild-type infections. Thus, the level of bacterial growth of this mutant is almost 4-fold higher than for wild-type Salmonella. This suggests that if Salmonella cannot manipulate Drosophila cells by secreting effectors, the survival of both pathogen and host is dramatically improved (Brandt, 2005).

To determine which of the known TTSA effector proteins contributed to this phenotype, flies were injected with Salmonella carrying in-frame deletions of these genes as well as several others suspected to be involved in virulence in the fly. Deletions of spiC, Salmonella secreted effector A (sseA), sseB, sseC, and sseD all resembled a SPI2 knockout as expected because the proteins encoded by these genes are implicated in building the SPI2 translocation machinery. Deletion of the gene encoding the Salmonella leucine-rich repeat-containing protein (PSlrP) produced a phenotype similar to that of the knockout of the entire SPI2 TTSA in terms of fly death. Wild-type virulence could be restored to the slrP mutant by expressing slrP, controlled by its native promoter, in trans on a single-copy plasmid, p.slrP. Because SPI1 and SPI2 bacteria accumulate to higher levels than wild-type strains, in the fruit fly, growth was measured of slrP-knockout bacteria. Mutation of slrP did not significantly alter the growth of Salmonella. These experiments suggest that slrP is a determinant of Salmonella virulence in the fruit fly, but that mutation of slrP alone is not sufficient to alter Salmonella growth. The protein encoded by slrP is suspected to be a specific effector translocated directly into the host cytoplasm. Its function there remains unknown (Brandt, 2005).

The results obtained during fly infection differ from what is found in mammalian and avian hosts. SPI1 is required by Salmonella for breaching the gut barrier but is dispensable if the bacteria are introduced systemically by intraperitoneal or intravenous injection. Both phoP- and SPI2-mutant Salmonella are highly attenuated even when injected into mammalian hosts; host survival increases, and the mutant bacteria do not readily replicate and are cleared during infections. In the fly, not only does host survival increase during infection by SPI1 or SPI2 mutants but, paradoxically, the bacteria are not cleared and replicate better than their wild-type parents. This is an interesting difference, because it separates mere bacterial presence from pathogenesis and disease. It remains to be determined why the fly does not clear the mutant bacteria in the manner that is seen in the mammalian host (Brandt, 2005).

The analysis of adult hemocytes in the fly is difficult, because good molecular markers for them are lacking, and the cells are sessile and rare enough to make them difficult to find regularly in tissue sections. Therefore a functional assay was used to monitor the behavior of these infected cells by determining their ability to carry out one of their definitive behaviors, phagocytosis. Flies were infected with wild-type and mutant Salmonella and then assayed for the phagocytic capacity of the hemocytes on their dorsal surface. Fluorescein isothiocyanate (FITC)-labeled dead E. coli were injected into flies and incubated for 60 min to allow time for phagocytosis to occur. Trypan blue was then injected into the flies to quench the fluorescence of extracellular bacteria. Uptake of the E. coli was then monitored by observing the flies under FITC illumination with a dissecting microscope. At day 1 post-Salmonella infection, hemocytes infected with wild-type, SPI1-mutant, SPI2-mutant, and SlrP bacteria showed similar phagocytic activities. Following 7 d of infection, flies infected with wild-type or slrP-mutant bacteria had greatly reduced numbers of phagocytic cells. In contrast, phagocytes remained active during the course of infection by SPI1 or SPI2 mutants. This experiment is interpreted to show that the wild-type Salmonella infection results in death of phagocytes or a reduced capacity for phagocytosis. This reduction is not dependent on SlrP function (Brandt, 2005).

In mammals, cytokines such as TNF and interferon relay information about infection through the body. Cytokine expression, in particular TNF expression, is responsible directly and indirectly for a significant degree of the pathology observed during microbial infection. Flies have one known TNF-like protein, encoded by the gene eiger. Overexpression of eiger can induce cell death, but no phenotype had been identified for loss-of-function mutants thus far. Wild-type and eiger- flies were infected with Salmonella to determine whether this fly TNF homolog plays a role in the pathogenesis of Salmonella infections in Drosophila. The mean time to death was lengthened by 3 d in the two eiger mutants tested. This experiment suggests that eiger/TNF signaling during a Salmonella infection contributes to the rate of death of the fly. Accumulation of Salmonella does not differ between eiger mutants and the background fly strain. This suggests that eiger mutants do not directly affect Salmonella growth, but do markedly affect host survival. RNA transcripts of eiger were not significantly altered during Salmonella infection. This suggests that if eiger is regulated during Salmonella infection, the transcriptional changes are too small to be seen relative to expression in whole flies or the changes are posttranscriptional (Brandt, 2005).

In human disease, morbidity and mortality are often the result of immune responses to invading pathogens rather than to direct action of the pathogens themselves. Fever, inflammation, and shock are examples of such processes. In plants, a model is emerging in which host cells monitor important cell functions and respond to their perturbations by pathogens. It is suggested that infection caused by Salmonella in the fly has attributes of both models. Salmonella growth appears to be restricted to hemocytes by action of the humoral immune response. Perhaps the manipulation of these hemocytes by secreted effectors induces additional immune responses that limit bacterial growth. This resembles what is seen in a resistant plant infected by a bacterium expressing the appropriate avirulence protein. Such proteins are secreted into the plant cell's cytoplasm via a TTSA, and they alter the host cell's physiology, presumably to make growth conditions for the bacteria more favorable. Resistant plants can sense this physiological change and raise a type of immune reaction called a hypersensitive response. In the case of Drosophila, it is suggested that the fly's immune response not only limits the growth of the pathogen but also damages the fly and ultimately leads to its death. When Salmonella is prevented from using its secreted effectors, the bacteria survive in the phagocyte and grow to higher numbers, possibly because the bacteria are not being attacked as intensely by the host. The result is that there are more bacteria in the fly and host death is greatly delayed. These two properties appear to be separable. The mutations in eiger suggest that there are signaling events that increase the death rate in the fly but do not alter the numbers of Salmonella. These experiments provide a new genetic model to explore microbial choice of pathogenic lifestyles in addition to a potential new genetic model for the study of TNF-induced metabolic collapse (Brandt, 2005).


The spatial patterns of eiger expression during embryogenesis and in larval tissues were analyzed by in situ hybridization. Weak signals were detected in pre-blastoderm embryos (stage 1-3), indicating a low level of maternal contribution. After stage 10, eiger transcripts are predominantly detected in the nervous system of the embryos. In the third-instar larva, eiger is strongly expressed in the brain hemispheres and at the morphogenetic furrow in the eye disc; it is also expressed at significant levels in many cells posterior to the furrow. Double staining of the eye disc with the eiger RNA probe and an anti-ELAV antibody, which recognizes terminally differentiated neuronal nuclei, has revealed that eiger is strongly expressed in the proliferating cells at the furrow. RT-PCR analysis demonstrated that eiger mRNA is expressed at all stages of Drosophila development (Igaki, 2002).

Conserved metabolic energy production pathways govern Eiger/TNF-induced nonapoptotic cell death

Caspase-independent cell death is known to be important in physiological and pathological conditions, but its molecular regulation is not well-understood. Eiger is the sole fly ortholog of TNF. The ectopic expression of Eiger in the developing eye primordium caused JNK-dependent but caspase-independent cell death. To understand the molecular basis of this Eiger-induced nonapoptotic cell death, a large-scale genetic screen was performed in Drosophila for suppressors of the Eiger-induced cell death phenotype. It was found that molecules that regulate metabolic energy production are central to this form of cell death: it was dramatically suppressed by decreased levels of molecules that regulate cytosolic glycolysis, mitochondrial β-oxidation of fatty acids, the tricarboxylic acid cycle, and the electron transport chain. Importantly, reducing the expression of energy production-related genes did not affect the cell death triggered by proapoptotic genes, such as reaper, hid, or debcl, indicating that the energy production-related genes have a specific role in Eiger-induced nonapoptotic cell death. It was also found that energy production-related genes regulate the Eiger-induced cell death downstream of JNK. In addition, Eiger induced the production of reactive oxygen species in a manner dependent on energy production-related genes. Furthermore, this cell death machinery was shown to be involved in Eiger's physiological function, because decreasing the energy production-related genes suppressed Eiger-dependent tumor suppression, an intrinsic mechanism for removing tumorigenic mutant clones from epithelia by inducing cell death. This result suggests a link between sensitivity to cell death and metabolic activity in cancer (Kanda, 2011).

At least some nonapoptotic cell deaths can be categorized as necroptosis, in which cells undergo nonapoptotic cell death under apoptosis-deficient conditions when treated with agonistic ligands of death receptors, such as TNFα, FasL, or TRAIL (Christofferson, 2010; see The signaling complexes induced by TNFα to mediate NF-kappaB activation, apoptosis and necroptosis.). The Eiger-induced cell death shares features with necroptosis in that it is triggered by TNF family proteins, produces ROS, and is caspase-independent. Furthermore, the Drosophila homolog of a tumor suppressor protein, Cylindromatosis, one of the essential regulators of necroptosis (Wang, 2008; Hitomi, 2008), has been shown to regulate JNK activation in Eiger-induced cell death signaling (Xue, 2007). However, despite the high conservation of most of the apoptotic machinery, blast search analysis has not identified Drosophila homologs of receptor-interacting protein (RIP) 1 or RIP3, the essential kinases for inducing necroptosis (Kanda, 2011 and references therein).

Oxidative stress could be induced downstream of the activated JNK pathway. However, it was also found that the knockdown of energy production-related genes such as cyt.c-d or GAPDH2 did not suppress the dTAK1- or HepCA-induced cell death phenotype. This finding could be caused by the overexpression of dTAK1- or HepCA-induced additional pathways, because dTAK1 or HepCA overexpression were found to induced JNK activation much more strongly than Eiger overexpression. Because dTAK1 also functions downstream of Imd in the innate immune response, Imd-related signaling was another possible mechanism for mediating Eiger signaling. Therefore, the genetic interaction between Eiger-induced cell death and Imd or Imd-related genes was examined. However, no significant interactions between them were found. Therefore, it would be interesting to examine if other proteins can substitute for the functions of RIP1 or RIP3 in Eiger signaling or if fly TNF signaling uses other mechanisms to induce nonapoptotic cell death (Kanda, 2011).

It has been reported that TNF-induced nonapoptotic cell death leads to the RIP3-dependent activation of glycogen phosphorylase, which is the rate-limiting enzyme in the degradation of glycogen and therefore, the key molecule for regulating energy production (Zhang, 2009). In this context, ROS can be generated by the production of excess energy. This finding could explain why the down-regulation of energy production-related genes suppressed Eiger-induced cell death. However, it was also observed that the amount of ATP in the eye antenna imaginal disc decreased when Eiger was overexpressed, and this decrease in ATP was cancelled by the knockdown of bsk, CPTI, pgk, or cyt.c-d. This finding suggests that the activation of energy production by Eiger signaling could also trigger another mechanism that decreases the tissue ATP level. It is possible that the tissue loses ATP simply because of the massive cell death caused by Eiger expression. Alternatively, the work by Temkin (2006) has shown that treatment with TNFα and the caspase inhibitor zVAD not only induces nonapoptotic cell death with ROS production but also decreases ATP because of the inhibition of adenine nucleotide translocase (ANT) by RIP1. A similar ANT-dependent inhibition mechanism could be involved in Eiger-induced cell death. Because neither RIP1 nor RIP3 has yet been identified in Drosophila, the mechanism by which the total ATP is regulated in Eiger-induced cell death remains to be elucidated (Kanda, 2011).

When scrib or dlg mutant cells are induced as clones in otherwise WT eye imaginal discs, most of these mutant cells are eliminated by Eiger–JNK-dependent cell death during development (Cordero, 2010; Igaki, 2009). This cell death could involve a caspase-independent mechanism, because the elimination of mutant cells is not fully suppressed by the overexpression of p35 compared with the blockage of JNK signaling. Thus, the mode of cell death triggered in scrib mutant clones is analogous to the mode of cell death triggered by the overexpression of Eiger in imaginal discs. These observations suggest that the regulation of energy production could be a crucial determinant of the susceptibility of tumor cells to cytotoxic stimuli (Kanda, 2011).

Interestingly, tumor cells frequently produce ATP by glycolysis in the cytosol rather than in the mitochondria, which is known as the Warburg effect. Because mitochondrial energy production generates cytotoxic ROS, cancer cells might increase their resistance to cytotoxic stimuli by reducing mitochondrial energy production. In this sense, mitochondrial energy production could act as a tumor suppressor. In fact, subunits of a TCA cycle enzyme, Succinate dehydrogenase (Sdh; SdhB, SdhC, and SdhD), are reported to be classical tumor suppressors in pheochromocytoma or paraganglioma. Furthermore, a specific isoform of pyruvate kinase, which is involved in glycolysis, is necessary for cellular metabolism to shift to aerobic glycolysis and the promotion of tumorigenesis. Similarly, the activity of pyruvate dehydrogenase (PDH), which links the glycolytic pathway to the TCA cycle by transforming pyruvate to acetyl-CoA, is suppressed in cancer cells, whereas the reactivation of PDH induces cell death in a solid tumor cell line and xenografts. Interestingly, this study found that the knockdown of Drosophila PDH (which is encoded by CG7010) strongly suppressed Eiger-induced cell death. Furthermore, the down-regulation of genes involved in glycolysis and the β-oxidation of fatty acids significantly suppressed the elimination of scrib cells from imaginal epithelia. These observations suggest that the regulation of cellular energy production or even the source of energy could be critical for controlling the susceptibility of cancer cells to cytotoxic stimuli such as TNFα (Kanda, 2011).

Apoptotic cells can induce non-autonomous apoptosis through the TNF pathway

Apoptotic cells can produce signals to instruct cells in their local environment, including ones that stimulate engulfment and proliferation. This study identified a novel mode of communication by which apoptotic cells induce additional apoptosis in the same tissue. Strong induction of apoptosis in one compartment of the Drosophila wing disc causes apoptosis of cells in the other compartment, indicating that dying cells can release long-range death factors. Eiger, the Drosophila tumor necrosis factor (TNF) homolog, was identified as the signal responsible for apoptosis-induced apoptosis (AiA). Eiger is produced in apoptotic cells and, through activation of the c-Jun N-terminal kinase (JNK) pathway, is able to propagate the initial apoptotic stimulus. During coordinated cell death of hair follicle cells in mice, TNF-alpha is expressed in apoptotic cells and is required for normal cell death. AiA provides a mechanism to explain cohort behavior of dying cells that is seen both in normal development and under pathological conditions (Perez-Garijo, 2013)

It is becoming clear that apoptosis is not a passive phenomenon where dying cells merely die and are silently removed from the tissues. Instead, apoptotic cells have the capacity to produce proliferative signals, such as Wg and Dpp, thus serving as a crucial driving force in wound healing, regeneration and tumor formation in a variety of different organisms. This study demonstrates that signaling by apoptotic cells is not limited to the production of proliferative signals: dying cells can generate pro-apoptotic signals that induce apoptosis in neighboring cells. The induction of this apoptosis is triggered by Eiger, the ortholog of TNF in Drosophila, which in turn activates the JNK pathway and leads to cell death in a non-autonomous manner. Furthermore, evidence is provided for the existence of AiA under physiological conditions in mice. During catagen, the regressive phase of the hair cycle, apoptotic cells in the lower and transient portion of the HF also express TNF-α. Significantly, TNF-α is required for coordinated cell death in the HF. Taken together, these results suggest that AiA plays an important physiological role for the coordination of cohort cell death (Perez-Garijo, 2013)

These experiments demonstrate that induction of apoptosis in one compartment results in induction of non-autonomous apoptosis in the neighboring compartment. This is true under many different conditions: both when undead cells (expressing rpr/hid and baculovirus caspase inhibitor p35) were generated or upon induction of genuine apoptosis (expressing rpr/hid alone); once there is ectopic expression of mitogens that leads to excessive proliferation and growth or while blocking mitogenic production or growth of the compartment (Perez-Garijo, 2013)

One intriguing observation is that this non-autonomous cell death usually displays a pattern consisting of two groups of cells in the wing pouch. One possible explanation for this is that the affected cells are the most susceptible to the death signal. In fact, the regions of the wing pouch where the non-autonomous cell death is observed are also more prone to cell death as a response to different apoptotic stimuli, such as irradiation or hid over-expression (Perez-Garijo, 2013)

Another possibility to explain why cell death is observed at a distance would be that dying cells are producing other signals that inhibit apoptosis. This protective signal would diffuse only short range, and in this way the distance of cells to the border would determine the ratio between the pro-apoptotic and the protective signal, tipping the balance in favor of death or survival. In fact, it has been shown that cells neighboring apoptotic cells downregulate Hippo pathway and consequently activate Diap1. Another good candidate for such an anti-apoptotic signal would be Wg, as it is expressed in an opposite pattern from the non-autonomous apoptosis and is also diffusing from apoptotic cells in the posterior compartment. However, this study attempted to modify Wg levels in different ways and no changes were observed in the apoptosis pattern (Perez-Garijo, 2013)

In contrast, in physiological conditions such as the coordinated cell death of hair follicle (HF) cells observed in mice, it would be expected that signaling between apoptotic cells would occur at a much shorter range, probably affecting the immediate neighbors. In any case, the observation that TNF-α is exclusively detected in apoptotic cells and the fact that its inhibition leads to desynchronization of the HF cycle strongly suggests that AiA can be a mechanism to coordinate cell death within a tissue (Perez-Garijo, 2013)

In these experimental systems, AiA requires both the TNF and JNK signaling pathways. Eiger is produced by apoptotic cells in the posterior compartment of the wing disc and it activates JNK in cells of the neighboring compartment, inducing them to die. Downregulation of Eiger in the posterior compartment or JNK in the anterior compartment is able to suppress AiA. However, it remains to be elucidated whether Eiger directly diffuses to the cells in the anterior compartment, or if some other mechanism is responsible for the activation of JNK in dying cells in the anterior compartment. Recently, it was shown that, upon wounding, JNK activity can be propagated at a distance through a feed-forward loop (Wu, 2010). Significantly, AiA is not restricted to the Drosophila wing disc. Evidence was obtained for a role of TNF-α-mediated AiA during the destruction of the hair follicle (HF) in catagen, the regressive phase of the hair cycle. TNF-α plays a known role to promote cell death and has been previously implicated in HF progression, wound healing and regeneration. However, the cellular source of TNF-α remained unknown and it was previously not appreciated that apoptotic cells can be the source of these signals. These results suggest that AiA and at least some of the underlying mechanism have been conserved in evolution to promote coordinated cell death (Perez-Garijo, 2013)

The observation that apoptotic cells can signal to other cells in their environment and instruct them to die has potentially many important implications. On the one hand, there are situations where propagation of an apoptotic stimulus may be a useful mechanism to achieve the rapid and coordinated death of large cell populations. The experiments in mice show that this can be the case during the catagen phase of the HF cycle. There are many other examples of cell death being used during development to sculpt tissues and organs, including the removal of structures during metamorphosis (tadpole tail, larval organs in insects, elimination of inappropriate sex organs in mammals, deletion of the amnioserosa during insect embryogenesis) and the separation of digits through apoptosis of the interdigital webbing in many vertebrates. In all these cases, AiA may facilitate cohort behavior and contribute to the rapid and complete elimination of large fields of cells (Perez-Garijo, 2013)

Propagation of cell death may also be an efficient way to prevent infection. It is known that cells respond to viral infection by entering apoptosis and in this way impede the replication of the virus. The process of AiA would extend apoptosis to the neighboring cells, preventing also their infection and thus avoiding the spread of the virus (Perez-Garijo, 2013)

However, propagation of apoptosis may be detrimental in pathological conditions where excessive cell death underlies the etiology of the disease. This may be the case for neurodegenerative disorders, hepatic diseases, cardiac infarction, etc. In all these cases it remains to be studied whether extensive amounts of apoptosis that are observed in the affected tissues are a direct consequence of cell damage in an autonomous manner or if part of the cell loss could be attributed to a process of propagation through AiA (Perez-Garijo, 2013)

Finally, AiA may play a role in cancer. It is known that radiotherapy in humans can induce biological effects in non-irradiated cells at a considerable distance, a phenomenon called radiation-induced bystander effect. The current findings provide a possible explanation for some of these effects. Therefore, large-scale induction of apoptosis by AiA may contribute to successful cancer therapy. TNF family proteins are being used as models for drug development aimed to treat cancer). Furthermore, Eiger, the only TNF member in Drosophila, has a known role in the elimination of pre-tumoral scrib- clones. In addition, cell competition induces cell death even in aggressive scrib-RasV12 tumors, raising the possibility that AiA is induced during tumor initiation, which may affect the tumor microenvironment and ultimately tumor growth. It is well known that TNF can play both tumor-promoting and tumor-suppressing roles, but AiA has not been investigated in this context. Future studies will shed new light on the relevance of signaling by apoptotic cells and the implications of this signaling mechanism in different scenarios (Perez-Garijo, 2013)

A genetic network conferring canalization to a bistable patterning system in Drosophila

To achieve the 'constancy of the wild-type,' the developing organism must be buffered against stochastic fluctuations and environmental perturbations. This phenotypic buffering has been theorized to arise from a variety of genetic mechanisms and is widely thought to be adaptive and essential for viability. In the Drosophila blastoderm embryo, staining with antibodies against the active, phosphorylated form of the bone morphogenetic protein (BMP) signal transducer Mad, pMad, or visualization of the spatial pattern of BMP-receptor interactions reveals a spatially bistable pattern of BMP signaling centered on the dorsal midline. This signaling event is essential for the specification of dorsal cell fates, including the extraembryonic amnioserosa. BMP signaling is initiated by facilitated extracellular diffusion that localizes BMP ligands dorsally. BMP signaling then activates an intracellular positive feedback circuit that promotes future BMP-receptor interactions. This study identified a genetic network comprising three genes that canalizes this BMP signaling event. The BMP target eiger (egr) acts in the positive feedback circuit to promote signaling, while the BMP binding protein encoded by crossveinless-2 (cv-2) antagonizes signaling. Expression of both genes requires the early activity of the homeobox gene zerknullt (zen). Two Drosophila species lacking early zen expression have high variability in BMP signaling. These data both detail a new mechanism that generates developmental canalization and identify an example of a species with noncanalized axial patterning (Gavin-Smith, 2013).

This study has identified a genetic network that acts as a phenotypic stabilizer of a spatially bistable patterning process. The minimal bistable systems allowed by theory require a nonlinear activation rate and a linear degradation rate. It is believed that the identified network defined in this study represents the minimal genetic components required for bistability of BMP signaling in D. melanogaster. In turn, bistability canalizes dorsal patterning. During amnioserosa specification, egr provides positive feedback, conferring nonlinearity, while cv-2> acts as a linear negative regulator of the signaling pathway. The loss of both components reveals the inherent noise of facilitated extracellular diffusion of BMP ligands, as without egr and cv-2, embryos manifest a huge range of signaling domain breadth and intensity. The data also reveal that amnioserosa specification in D. melanogaster is robust on multiple levels, with different mechanisms ensuring robustness in various Drosophila species (Gavin-Smith, 2013).

First, egr or bsk RNAi embryos have normal amounts of amnioserosa and minimal embryonic lethality despite the 2-fold reduction in signaling intensity. This demonstrates that amnioserosa specification is robust to decreases of BMP signaling and the wild-type level of BMP signaling in D. melanogaster is much higher than necessary. Second, the D. melanogaster embryo can tolerate at least a 250% increase or a 20% decrease in amnioserosa cell number without compromising viability. Lastly, the variability in amnioserosa cell number in D. yakuba embryos is equivalent to that in D. melanogaster embryos, indicating that amnioserosa specification in D. yakuba is robust against variable BMP signaling intensity. Therefore, in D. yakuba embryos, either less BMP signaling is required to direct amnioserosa specification or a second mechanism downstream of BMP signaling intensity maintains robust amnioserosa specification (Gavin-Smith, 2013).

Finally, as a counterpoint to the predicted ubiquity and selective maintenance of developmental canalization, D. santomea has been identified as a noncanalized wildtype species. D. santomea both has highly variable cell fate specification and is not robust to genetic variants found in its wild population. The identification of this noncanalized species may permit further investigation of the evolutionary factors allowing for this diversity in developmental trajectories (Gavin-Smith, 2013).

Drosophila homologs of mammalian TNF/TNFR-related molecules regulate segregation of Miranda/Prospero in neuroblasts

During neuroblast (NB) divisions, cell fate determinants Prospero (Pros) and Numb, together with their adaptor proteins Miranda (Mira) and Partner of Numb, localize to the basal cell cortex at metaphase and segregate exclusively to the future ganglion mother cells (GMCs) at telophase. In inscuteable mutant NBs, these basal proteins are mislocalized during metaphase. However, during anaphase/telophase, these mutant NBs can partially correct these earlier localization defects and redistribute cell fate determinants as crescents to the region where the future GMC 'buds' off. This compensatory mechanism has been referred to as 'telophase rescue'. The Drosophila homolog of the mammalian tumor-necrosis factor (TNF) receptor-associated factor (TRAF1) and Eiger (Egr), the homolog of the mammalian TNF, are required for telophase rescue of Mira/Pros. TRAF1 localizes as an apical crescent in metaphase NBs and this apical localization requires Bazooka (Baz) and Egr. The Mira/Pros telophase rescue seen in inscuteable mutant NBs requires TRAF1. These data suggest that TRAF1 binds to Baz and acts downstream of Egr in the Mira/Pros telophase rescue pathway (Wang, 2006).

In telophase NBs, segregation of cell fate determinants, such as Pros, into future GMCs, is critical for their proper development. Telophase rescue appears to be one of the safeguard mechanisms that acts to ensure that GMCs inherit the cell fate determinants and adopt the correct cell identity when the mechanisms, which normally operate during NB divisions, fail (e.g., in insc mutant). Telophase rescue is a phenomenon for which the underlying mechanism involved remains largely unknown. The current data demonstrate that TRAF1 and Egr are two members of the Insc-independent telophase rescue pathway specific for Mira/Pros (Wang, 2006).

Although it is apically enriched in mitotic NBs and can directly interact with Baz in vitro, TRAF1 does not seem to be involved with the functions normally associated with the apical complex proteins. One distinct feature of TRAF1 differs from the other known apical proteins is its localization pattern; it is cytoplasmic in interphase and the apical crescent is prominent only at metaphase. In contrast, proteins of the apical complex are largely undetectable during interphase and form distinct apical crescents, starting from late interphase or early prophase. The protein localization difference between TRAF1 and other apical proteins suggests that TRAF1 and apical proteins are not always colocalized during mitosis. If TRAF1 is a bona fide member of the apical complex, the localization defects of other apical proteins are expected to be observed in TRAF1 mutant, as well as mislocalization of basal proteins, which was not detect. In addition, no spindle orientation or geometry defects were observed in the absence of TRAF1. Based on these observations, it is concluded that TRAF1 is not involved with the functions normally associated with the apical complex proteins (Wang, 2006).

The in vitro GST fusion protein pull-down assay suggests that TRAF1 may physically bind to Baz. This result is consistent with genetic data, indicating that TRAF1 acts downstream of baz and that its apical localization requires baz. These observations are consistent with the view that TRAF1 is recruited to the apical cortex by apical Baz in mitotic NBs. Baz, even at very low levels, can recruit TRAF1 to the apical cortex of the mitotic NBs. For example, in insc mutant NBs, TRAF1 remains apical probably owing to the low levels of Baz that remain localized to the apical cortex. This speculation is supported by Mira/Pros telophase rescue data, which clearly demonstrate that the telophase rescue seen in insc mutant NBs is severely damaged in baz mutant, suggesting that the Baz function required for Mira/Pros (and Pon/Numb) telophase rescue is intact in insc mutant NBs (Wang, 2006).

It has been shown that Pins/Gαi asymmetric cortical localization can be induced at metaphase by the combination of astral microtubules, kinesin Khc-73 and Dlg in the absence of Insc; this coincides with the observation that TRAF1 also forms tight crescent only at metaphase in both WT and insc mutant NBs. Does TRAF1 apical crescent formation also require the functions of astral microtubules, kinesin Khc-73 and Dlg? The data do not favor this hypothesis based on the following observations. (1) In TE35BC-3, a small deficiency uncovering sna family genes insc is not expressed but Pins and Gαi are asymmetrically localized, indicating that the astral microtubules, kinesin Khc-73 and Dlg pathway remain functional. TRAF1 is delocalized and is uniformly cortical in this deficiency line. (2) Similarly, in egr insc NBs, TRAF1 is cytoplasmic whereas the functions of astral microtubules, kinesin Khc-73 and Dlg are intact. (3) In egr NBs TRAF1 is cytoplasmic, whereas the apical complex is normal and astral microtubules, kinesin Khc-73 and Dlg are present. (4) TRAF1 apical localization remains unchanged in dlg mutant NBs. Based on these observations, it is concluded that TRAF1 apical localization is unlikely to share similar mechanism with Pins and Gαi and is likely to be independent of astral microtubules, kinesin Khc-73 and Dlg. TRAF1 apical localization appears to specifically require Egr and Baz (Wang, 2006).

In TRAF1 insc double-mutant embryos, the complete segregation of Mir/Pros into future GMCs occurs only in about 12% of the total population, and in the remaining NBs, only a fraction of Mira/Pros segregate into future GMCs as indicated by the Mira 'tail' extending into the future NBs at telophase. As it is difficult to address the global effect of this partial segregation of Mira/Pros on GMC specification in TRAF1 insc double mutant, focus was place on a well-defined GMC, GMC4-2a in NB4-2 lineage, to evaluate this issue. It is assumed that as long as the RP2 neuron (progeny of GMC4-2a, Even-skipped (Eve)-positive) was identified in a particular hemisegment, the GMC cell fate of GMC4-2a in that hemisegment should have been correctly specified. In insc mutants, almost all hemisegments contain RP2s, indicating that GMC4-2a has adopted the correct GMC cell fate in 99% of the total hemisegments. When TRAF1 insc double-mutant embryos were stained with anti-Eve, it was found the frequency of loss of Eve-positive RP2 neuron increased (to 8%) in late embryos, suggesting that about 8% of the GMCs in TRAF1 insc double mutant did not inherit sufficient Pros to specify the GMC fate in these embryos. The relatively low frequency (8%) of mis-specification of GMCs suggests that the threshold amount of Pros protein needed is sufficiently low such that just a partial inheritance of Pros, even when telophase rescue is compromised, is sufficient for most GMCs to be correctly specified (Wang, 2006).

Although Mira/Pros and Pon/Numb share similar basal localization patterns in insc NBs, further removal of either TRAF1 or Egr compromised telophase rescue only for Mira/Pros, but not for Pon/Numb. This difference between Mira/Pros and Pon/Numb indicates that the detailed mechanisms of basal localization and segregation of Mira/Pros differ from those of Pon/Numb, which is consistent with the observations that the dynamics of Mira/Pros and Pon/Numb localization early in mitosis are different and the basal localization for Mira/Pros and Pon/Numb requires different regions of the Insc coding sequence (Wang, 2006).

Dlg/Lgl/Scrib are required for correct basal localization of Mira/Pros and Pon/Numb in mitotic NBs. Dlg has been shown to be involved in the Mira telophase rescue. In dlg insc double-mutant NBs, not only was spindle geometry symmetric but Mira telophase rescue was also affected. It would be interesting to know if Dlg belongs to the same pathway as TRAF1 and Egr and if Dlg is also involved in Pon/Numb telophase rescue (Wang, 2006).

Two other members of the TRAF family have also been identified in Drosophila: DTRAF2 (DTRAF6) and DTRAF3. In contrast to the specific and strong expression of TRAF1 in the embryonic NBs, only low levels of ubiquitous signals similar to the control background were seen in the NBs with DTRAF2 and DTRAF3 probes. It is likely that DTRAF2 and DTRAF3 are not expressed in NBs and do not play an important role in Mira/Pros telophase rescue pathway as the Mira/Pros telophase rescue is dramatically compromised in TRAF1 insc and egr insc NBs (Wang, 2006).

In mammals, the TNF pathway works as a typical receptor-mediated signal transduction pathway. TNFR is a key player in transducing external signal to the cytoplasm. In the Drosophila compound eyes, ectopic Egr, Wgn and TRAF1 seem to work in a similar receptor-mediated signal pathway to induce apoptosis through the activation of the JNK pathway. Does the same Egr, Wgn and TRAF1 receptor-mediated signal pathway play a role in Mira/Pros telophase rescue? If it does, the coexpression of Egr, Wgn and TRAF1 might be expected to be seen in dividing NBs, along with the potential interaction between TRAF1 and the cytoplasmic domain of Wgn. Three observations argue against this hypothesis: (1) wgn is not expressed in embryonic NBs but in the mesoderm. (2) The domain analysis suggests that the Drosophila Wgn cytoplasmic domain is unique with no sequence homology to any mammalian TNFR family members and has neither a TRAF-binding domain nor a death domain, which is required for the interaction between TNFR and TRAF in mammals. (3) More informatively, Wgn knockdown by a UAS head-to-head inverted repeat construct of wgn (UAS-wgn-IR) driven by a strong maternal driver, mata-gal4 V32A, in WT embryos did not affect TRAF1 apical localization. These observations are consistent with the view that the receptor Wgn may not be involved in Mira/Pros telophase rescue or is redundant in this pathway. If this is the case, then how do TRAF1 and Egr function in Mira/Pros telophase rescue? It has been reported that TRAFs associate with numerous receptors other than the TNFR superfamily in mammals. It is speculated that Egr and TRAF1 may adopt an alternative receptor in NBs for Mira/Pros telophase rescue. However, until an anti-Wgn antibody and wgn mutant alleles are available, the possibility that Wgn is involved in Mira/Pros telophase rescue cannot be ruled out (Wang, 2006).

Neuronal programmed cell death induces glial cell division in the adult Drosophila brain

Although mechanisms that lead to programmed cell death (PCD) in neurons have been analysed extensively, little is known about how surrounding cells coordinate with it. This study shows that neuronal PCD in the Drosophila brain induces glial cell division. PCD in neurons and cell division in glia were identified as occurring in a consistent spatiotemporal manner in adult flies shortly after eclosion. Glial division was suppressed when neuronal PCD was inhibited by ectopic expression of the caspase inhibitor gene p35, indicating their causal relationship. Glia also responded to neural injury in a similar manner: both stab injury and degeneration of sensory axons in the brain caused by antennal ablation induced glial division. Eiger, a tumour necrosis factor superfamily ligand, appears to be a link between developmental PCD/neural injury and glial division, as glial division is attenuated in eiger mutant flies. Whereas PCD soon after eclosion occurred in eiger mutants as in the wild type, excess neuronal PCD was observed 2 days later, suggesting a protective function for Eiger or the resulting glial division against the endogenous PCD. In older flies, between 6 and 50 days after adult eclosion, glial division was scarcely observed in the intact brain. Moreover, 8 days after adult eclosion, glial cells no longer responded to brain injury. These results suggest that the life of an adult fly can be divided into two phases: the first week, as a critical period for neuronal cell death-associated glial division, and the remainder (Kato, 2009).

This study found that neurons in specific areas of the Drosophila brain undergo PCD over several days after adult eclosion (AAE). Similar to the PCD in the ventral nerve cord, dying neurons strongly express EcRA and also express the pro-apoptotic genes rpr and grim, but not hid. These findings suggest that the same ecdysone-mediated developmental mechanism is utilised for eliminating unnecessary neurons in the brain and the ventral nerve cord (Kato, 2009).

Neuronal PCD induces glial cell division. Most of the cells that incorporated BrdU were glia. A very small number of BrdU-positive cells that were both REPO-negative and ELAV-negative. A conceivable candidate is neural stem cells (neuroblasts). However, this is unlikely because BrdU-positive neurons have never been observed. The identity of this novel cell type remains to be investigated (Kato, 2009).

That neuronal PCD occurs in essentially all individuals was indicated by the fact that strongly EcRA-positive cells were observed in most flies at 6 hours AAE. In spite of this, not all the antennal nerves were found associated with BrdU-positive cells. This discrepancy might be due to the technical difficulty of labelling cells with BrdU for long periods: some of the BrdU-incorporating cells might have died as BrdU is potentially toxic (Kato, 2009).

In the imaginal wing disc, preventing cell death itself with p35, but leaving the caspase signalling pathway intact, increases proliferation. This was not the case for glial division in the brain because glial cells did not divide when cell death was suppressed by p35. Apparently, a different molecular mechanism triggers the glial response. Eiger, a TNF superfamily ligand, was found to be involved in this process. Glial division in both intact and injured brains was attenuated in eiger mutants, and ectopic expression of eiger in glia rescued this phenotype. The rescue, however, was not complete, and glial expression of eiger alone did not induce ectopic glial division. This might be because (1) spatiotemporal expression of eiger is required in glia, (2) expression of eiger in the neurons might also be important, or (3) factors other than Eiger are also involved in this process (Kato, 2009).

What, then, could be the role of glial division upon developmental PCD? In the Drosophila rdgBKS222 mutant, glial cells in the compound eye fill the voids that were formed by axonal degeneration. Similarly, dividing glial cells in the brain might contribute to structural support after neural loss. Another possibility is that glial cells protect neural tissue by removing dead cells and/or by secreting trophic factors. The observation that the lack of Eiger, and thus the lack of glial division, led to the increase in neuronal PCD supports this hypothesis (Kato, 2009).

The Drosophila adult brain shows a similar injury response to that of vertebrates: expression of β amyloid protein precursor-like (APPL) and activation of c-Jun N-terminal kinases (JNKs) are induced. Neurons fail to regenerate in response to injury, and glial cells in the antennal lobe change their morphology upon antennal ablation. Glial division, by contrast, has not been demonstrated in the fly brain. Here, evidence is provided that glia also divide upon injury and that Eiger mediates this process (Kato, 2009).

The glial division observed in the fly brain, however, seems to be much less extensive than that observed in vertebrates. A notable difference is in the variety of the dividing glial cell types. Whereas astrocytes, microglia and oligodendrocyte precursor cells proliferate in vertebrates, only a subset of glial cells around the neuropile is likely to respond in the Drosophila brain. As drastic glial proliferation upon neural injury, which causes a glial scar, is involved in the inhibition of neural recovery in vertebrates, the Drosophila nervous system, with its much restricted level of glial division, should provide an interesting model system for investigating the responses of neurons to injury, including neural recovery (Kato, 2009).

In vertebrates, TNFα is involved in the inflammatory response against neural lesions and plays multiple roles in such as the induction of cell death, cell survival and proliferation through the JNK and NFkappaB pathways. In Drosophila, overexpression of eiger in the imaginal discs appears to cause caspase-dependent cell death through the JNK pathway via Wengen, the sole known TNF receptor. However, Eiger is not required for caspase-dependent cell death caused by ionising radiation of the imaginal discs, even though irradiation induces the expression of eiger. Eiger is known to contribute in vivo to the proper localisation of determinant during the asymmetric division of neuroblasts (Wang, 2006). Wengen, however, does not seem to be involved in this process, suggesting the existence of as yet unknown receptors for Eiger. In this study, RNA interference of wengen did not appear to cause defects in glial division. Further investigation is required to understand the pathways downstream of Eiger in glial cell division, as well as in various other Eiger-mediated phenomena (Kato, 2009).

A surprising finding of this study is that there is a critical period of glial division. Both PCD- and injury-induced glial division occur only during the first 8 days AAE. Glial cells that are distant from the antennal nerve, which do not normally divide in the adult brain, retain the ability to respond to brain stab. This competence is lost as the flies grow older. Interestingly, there is temporal coincidence between the competence of glial division and neural plasticity. The application of certain odorants leads to an increase in the volume of particular glomeruli only during 2-5 days, but not after 8 days, AAE. Considering the possible role of glia in trophic function and structural support, glial division might be actively involved in brain plasticity. The temporal coincidence suggests that the adult stage of Drosophila can be divided into two phases: the first week AAE, as the critical period in which glial division against neural loss and plasticity of the antennal lobe neurons can be observed, and the rest of the adult life, during which these events do not occur (Kato, 2009).

This study has identified the first example in which developmental PCD triggers glial cell division. Important similarities were revealed between the glial response to PCD and to neural injury and between the glial response in insects and vertebrates after injury. The model system introduced in this study serves as a convenient platform for analysing novel types of neuron-glia interaction during recovery of the brain after PCD and injury, as well as how stage-dependent glial competence is controlled (Kato, 2009).

Intrinsic tumor suppression and epithelial maintenance by endocytic activation of Eiger/TNF signaling in Drosophila

Oncogenic alterations in epithelial tissues often trigger apoptosis, suggesting an evolutionary mechanism by which organisms eliminate aberrant cells from epithelia. In Drosophila imaginal epithelia, clones of cells mutant for tumor suppressors, such as scrib or dlg, lose their polarity and are eliminated by cell death. This study shows that Eiger, the Drosophila tumor necrosis factor (TNF), behaves like a tumor suppressor that eliminates oncogenic cells from epithelia through a local endocytic JNK-activation mechanism. In the absence of Eiger, these polarity-deficient clones are no longer eliminated; instead, they grow aggressively into tumors. In scrib clones endocytosis is elevated, which translocates Eiger to endocytic vesicles and leads to activation of apoptotic JNK signaling. Furthermore, blocking endocytosis prevents both JNK activation and cell elimination. These data indicate that TNF signaling and the endocytic machinery could be components of an evolutionarily conserved fail-safe mechanism by which animals protect against neoplastic development (Igaki, 2009).

Clones of cells mutant for Drosophila tumor suppressor genes, such as scrib or dlg, are eliminated from imaginal discs, suggesting an evolutionarily conserved fail-safe mechanism that eliminates oncogenic cells from epithelia. This study reports that this elimination of mutant cells is accomplished by endocytic activation of Eiger/TNF signaling. Eiger is a conserved member of the TNF superfamily in Drosophila, but its physiological function has been elusive. Although ectopic overexpression of Eiger can trigger apoptosis, flies deficient for eiger develop normally and exhibit no morphological or cell death defect. This study shows that Eiger is required for the elimination of oncogenic mutant cells from imaginal epithelia. This not only provides an explanation for previous unexplained observations, but also argues that Eiger is a putative intrinsic tumor suppressor in a fashion similar to mammalian p53 or ATM, which causes no phenotype when mutated, but protects animals as tumor suppressors when their somatic cells are damaged (Igaki, 2009).

The intrinsic tumor suppression found in scrib mutant clones was also observed in dlg mutant clones, suggesting that this is a mechanism triggered by loss of epithelial basolateral determinants. Intriguingly, it was found that mutant clones of salvador, the hippo pathway tumor suppressor, are not susceptible to similar effect of Eiger. These data suggest that the Eiger-JNK pathway behaves as an intrinsic tumor suppressor that eliminates cells with disrupted cell polarity (Igaki, 2009).

It is intriguing that Eiger's tumor suppressor-like function is dependent on endocytosis. The data show that Eiger is translocated to endosomes through endocytosis and activates JNK signaling in these vesicles. Moreover, blocking endocytosis abolishes both JNK activation and Eiger-dependent cell elimination. Endocytic activation of signal transduction has been observed for EGF and β2-adrenergic receptor signaling in mammalian cells. After endocytosis, these ligand/receptor complexes localize to endosomes, where they meet adaptor or scaffold proteins that recruit downstream signaling components. Therefore, the endocytic activation of Eiger/TNF-JNK signaling might also be achieved by the recruitment of its downstream signaling complex to the endosomes, possibly through a scaffold protein that resides in endosomes. Recent studies in Drosophila have shown that components of the endocytic pathway -- vps25, erupted, and avalanche -- function as tumor suppressors (Lu, 2005; Moberg, 2005; Thompson, 2005; Vaccari, 2005). Furthermore, mutations in endocytosis proteins have been reported in human cancers. Thus, deregulation of endocytosis may contribute to tumorigenesis. This study provides new mechanistic insights into the role of endocytosis in tumorigenesis (Igaki, 2009).

Mammalian TNF superfamily consists of at least 19 members. While many have been shown to play important roles in immune responses, hematopoiesis, and morphogenesis, the physiological functions for other members have yet to be determined. Mechanisms that eliminate damaged or oncogenic cells from epithelial tissues are essential for multicellular organisms, especially for long-lived mammals like humans. The tumor suppressor role of Eiger might have evolved for host defense or elimination of dying/damaged cells, such as cancerous cells, very early in animal evolution. Given that components of the Eiger tumor suppressor-like machinery (such as Eiger, endocytic pathway components, and JNK pathway components) are conserved from flies to humans, it is also possible that Eiger and its mammalian counterparts are components of an evolutionarily conserved fail-safe by which animals maintain their epithelial integrity to protect against neoplastic development (Igaki, 2009).

Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae

Heightened nociceptive (pain) sensitivity is an adaptive response to tissue damage and serves to protect the site of injury. Multiple mediators of nociceptive sensitization have been identified in vertebrates, but the complexity of the vertebrate nervous system and tissue-repair responses has hindered identification of the precise roles of these factors. This study established a new model of nociceptive sensitization in Drosophila larvae, in which UV-induced tissue damage alters an aversive withdrawal behavior. UV-treated larvae develop both thermal hyperalgesia, manifested as an exaggerated response to noxious thermal stimuli, and thermal allodynia, a responsiveness to subthreshold thermal stimuli that are not normally perceived as noxious. Allodynia is dependent upon a tumor necrosis factor (TNF) homolog, Eiger, released from apoptotic epidermal cells, and the TNF receptor, Wengen, expressed on nociceptive sensory neurons. These results demonstrate that cytokine-mediated nociceptive sensitization is conserved across animal phyla and set the stage for a sophisticated genetic dissection of the cellular and molecular alterations responsible for development of nociceptive sensitization in sensory neurons (Babcock, 2009).

The cytokine TNF-α and its receptors have been implicated in both cell death and nociceptive sensitization in mammals. Indeed, exposure to UV light causes human keratinocytes to synthesize and release TNF. The Drosophila genome contains one clear homolog of the TNF ligand and TNF receptor, and this study examined whether these genes are responsible for alterations in nociceptive sensitivity. Larvae with null mutations in eiger, the gene encoding the TNF-α homolog, were exposed to UV radiation and, 24 hr later, were stimulated with the highest normally subthreshold temperature, 38°C, and their withdrawal responses were recorded. It was found that larvae transheterozygous for independent eiger null alleles showed a complete absence of thermal allodynia. Larval epidermal-specific expression of a UAS-eigerIR transgene also led to a complete absence of thermal allodynia, suggesting that the epidermis is the source of the TNF that mediates sensitization. Expression of UAS-eigerIR in sensory neurons via the ppk1.9-Gal4 driver did not affect development of allodynia, indicating that Eiger derived from sensory neurons does not contribute to sensitization (Babcock, 2009).

One possibility suggested by the epidermal requirement for Eiger in nociceptive sensitization is that the primary function of Eiger released from UV-treated epidermal cells is to initiate apoptosis, which then leads to production of factors that mediate the development of allodynia. Three lines of evidence argue against this model. (1) Blocking TNF signaling through epidermal expression of an RNAi transgene targeting the Drosophila TNF receptor, Wengen, does not block allodynia. This RNAi transgene has potent on-target effects: it efficiently blocks Eiger-induced cell death in the developing Drosophila eye disc. (2) Larvae transheterozygous for eiger null mutations have a normal epidermal apoptosis response to UV irradiation. (3) To test whether Eiger directly targets nociceptive sensory neurons after UV-induced tissue damage, expression of Wengen was knocked down by using a nociceptive sensory-neuron-specific Gal4 driver (ppk1.9-Gal4). Larvae expressing UAS-wengenIR in the class IV sensory neurons recently shown to be sufficient for nociception displayed normal nociceptive responses but failed to develop thermal allodynia after UV treatment, suggesting that Eiger does not act through the production of signaling intermediates such as prostaglandins or other cytokines. Importantly, neither eiger null mutants nor any combination of Gal4 and UAS transgenes affected developmental progression through the larval stages to pupariation (Babcock, 2009).

Finally, tests were performed to see whether ectopic expression of Eiger is sufficient to induce allodynia even in the absence of UV irradiation. Pan-epidermal overexpression of Eiger is lethal, suggesting either that the ligand is sequestered from its receptor in the untreated epidermis or that it is produced and/or released locally in response to irradiation. It was found, however, that viable larvae could be obtained by ectopically expressing Eiger from nociceptive sensory neurons via ppk1.9-Gal4. In these experiments, only larvae carrying both the Gal4 insert and the Eiger overexpression transgene developed allodynia even in the absence of UV irradiation. Taken together, these results indicate that a conserved cytokine and cytokine receptor module directly mediates a subset of nociceptive sensitization responses in Drosophila larvae (Babcock, 2009).

This study has introduce a new model of nociceptive sensitization in Drosophila larvae. In this model, UV radiation activates the apical apoptotic caspase Dronc and results in the production and/or release of Eiger from epidermal cells. Secreted or released Eiger then binds to its receptor, Wengen, on nociceptive sensory neurons underlying the epidermal sheet and lowers the threshold of the behavioral response. The complete absence of thermal allodynia upon tissue-specific knockdown of epidermal TNF (Eiger) or sensory neuron TNFR (Wengen) presented in this study suggests that TNF is a crucial mediator of this sensitization in Drosophila larvae and that its effects are direct. TNF signaling has been implicated in nociceptive sensitization in vertebrates, and there is evidence for both direct and indirect effects of this cytokine. Some studies suggest that the primary role of TNF is to mediate the production or release of secondary neuromodulatory factors (Interleukin-1β, prostaglandins) that then sensitize nearby nociceptive sensory neurons, whereas other studies have suggested that soluble TNF can directly alter the firing properties of these cells. An important experimental test of the relevance of the proposed model to vertebrate systems would probably involve assessing development of thermal allodynia in mice harboring a skin-specific knockout of TNF-alpha or a sensory-neuron-specific knockout of TNF receptors (Babcock, 2009).

Does the TNF/TNFR signaling module represent an ancestral danger-signaling system? Vertebrate TNFs have been implicated in immune responses to pathogens and toxins and in immune modulation of nervous-system function after tissue insult. TNF-family ligands have been identified in several invertebrates, including Drosophila, molluscs, and the urochordate, Ciona savignyi. In addition to their role in nociceptive sensitization described in this study, other functional data on the role of invertebrate TNFs show that Drosophila Eiger is required for combating extracellular pathogens but can also, as in vertebrates, lead to infection-induced pathologies. Thus, as in vertebrates, invertebrate TNF family ligands have both immunological and neuromodulatory functions, which may be interconnected. It is speculated that the TNF signaling system evolved as a general initiator of cell-type-specific responses to a variety of pathogens and other noxious insults. In vertebrates, TNF can act both directly on sensory neurons and indirectly by stimulating production of other nociceptive mediators, but the current data suggest that in organisms with simpler nervous and immune systems, direct effects might predominate. It will be interesting to test whether damage-induced nociceptive sensitization in urochordates (Ciona) or lower vertebrates employs a direct or indirect mode of TNF signaling and, conversely, whether damage-induced sensitization can occur in even simpler organisms, such as Cnidaria or Ctenophores, that have only a simple neural net but do possess TNF ligand and receptor homologs (Babcock, 2009).

The system established in this study provides a vehicle for the application of a new and complementary set of experimental approaches -- including the powerful toolkit of Drosophila genetics -- to the problem of nociceptive sensitization. Genetic dissection of the sensitization response described in this study should lead to identification of the specific downstream events by which sensory neuron sensitivity is altered after engagement of the TNF receptor. The robustness of the sensitization response and the clear separation of allodynia (Dronc- and TNF-dependent) and hyperalgesia (Dronc-independent) in this system suggest that there may be conserved mechanistic differences mediating the onset of these responses. Given the evolutionary conservation of genes mediating most aspects of neuronal development and function, it is expected that this system will broadly inform the understanding of both the onset and, potentially, the aberrant persistence of nociceptive sensitization in chronic pain syndromes (Babcock, 2009).

Drosophila eiger mutants are sensitive to extracellular pathogens

Eiger, the Drosophila tumor necrosis factor homolog, contributes to the pathology induced by infection with Salmonella typhimurium. It was of interest to discover whether eiger is always detrimental in the context of infection or if it plays a role in fighting some types of microbes. Wild-type and eiger mutant flies were challenged with a collection of facultative intracellular and extracellular pathogens, including a fungus and Gram-positive and Gram-negative bacteria. The response of eiger mutants divided these microbes into two groups: eiger mutants are immunocompromised with respect to extracellular pathogens but show no change or reduced sensitivity to facultative intracellular pathogens. Hence, eiger helps fight infections but also can cause pathology. It is proposed that eiger activates the cellular immune response of the fly to aid clearance of extracellular pathogens. Intracellular pathogens, which can already defeat professional phagocytes, are unaffected by eiger (Schneider, 2007; Full text of article).

Why does eiger affect different microbes in different ways? It is argued that E. coli is removed from flies so rapidly and via so many mechanisms that the effects of eiger cannot be measured easily because of redundancy. In contrast, during an infection with a real pathogen, it may be easier to measure changes in immunity because the fly is fighting hard for survival and its immune mechanisms are not acting in a redundant fashion. Extracellular pathogens are clearly fought by the fly using eiger-dependent mechanisms because the loss of eiger results in a deeply sensitive phenotype. It is intriguing that flies lacking eiger are no worse at fighting intracellular pathogens. This suggests that these intracellular pathogens are normally immune to the effects of wild-type eiger. Intracellular pathogens like M. marinum, L. monocytogenes, and S. typhimurium use different virulence mechanisms for growing inside cells, but the common thread is that they can survive in professional phagocytes. It is suggested that eiger function somehow alters hemocytes to increase their potency against microbes. The reduced ability of eiger mutant hemocytes to phagocytose S. aureus compared to wild-type hemocytes supports this hypothesis. It is predicted that this change in potency is effective against pathogens that grow extracellularly but not against pathogens that have already developed methods of defeating phagocytes (Schneider, 2007).

It is proposed that where eiger signaling does not help fight infection, eiger can cause pathology. This eiger-induced pathology may be linked to the immune function of eiger (for example, the induced immune response may be energetically wasteful or directly toxic). Alternatively, eiger-induced pathology may be separable from the immune function (for example, eiger could cause something like muscle wasting in the fly, as has been suggested for tumor necrosis factor in vertebrates) (Schneider, 2007).

Genetic screens that monitored AMP synthesis have been productive and filled in the Toll and imd pathways but did not reveal eiger signaling. The role eiger plays in innate immunity cannot be measured using a nonpathogenic microbe like E. coli. This study demonstrates that there are important immune mechanisms at work in the fly that are difficult to see using simple endpoints like antimicrobial gene expression; however, studies with microbes that can cause disease in wild-type flies -- real pathogens -- can reveal these physiologies (Schneider, 2007).


Many mammalian TNF superfamily proteins activate both the NF-kappaB and the JNK pathway, and activation of the latter pathway facilitates cell death (Davis, 2000). To examine whether Eiger activates the JNK pathway, the genetic interactions of Eiger with the components of the Drosophila JNK cascade were examined. The reduced-eye phenotype induced by Eiger is strongly suppressed in basket (bsk), a heterozygous mutant of Drosophila JNK. In addition, overexpression of a dominant-negative form of Bsk almost completely suppresses the eye phenotype. Moreover, heterozygosity at the hemipterous (hep) locus, which encodes Drosophila JNKK, suppresses the reduced-eye phenotype much as does bsk, and its hemizygosity (null background) rescues the phenotype almost completely. Furthermore, the co-expression of a dominant-negative form of dTAK1 (TGF-ß activated kinase 1; Drosophila JNKKK) also rescues the Eiger-induced phenotype completely. Misshapen (Msn) is a MAPKKKK that is genetically upstream of the JNK pathway in Drosophila. A half dosage of the msn gene strongly suppresses the Eiger-induced phenotype. Heterozygosity of Drosophila jun, a target of the JNK pathway, did not show any genetic interaction with Eiger, raising the possibility that the Eiger-stimulated death-inducing JNK pathway may not require new transcripts that are controlled by Jun (Igaki, 2002).

Puckered (Puc) is a dual-specificity phosphatase, the expression of which is induced by the Drosophila JNK pathway to inactivate Bsk, so that puc expression can be used to monitor the extent of activation of the JNK pathway. To confirm that the JNK pathway is actually activated by Eiger, puc expression level was assessed in the eye disc of GMR-GAL4 activated regg1GS9830 flies using a puc-LacZ enhancer-trap allele. The strong induction of puc-LacZ was observed in the region posterior to the morphogenetic furrow of the eye disc compared with the control eye disc. Furthermore, Western blot analysis with an anti-phospho-JNK antibody has revealed that Bsk is phosphorylated by Eiger overexpression. These genetic and biochemical data led to a model in which Eiger activates Msn, thereby triggering the JNK signaling pathway, sequentially stimulating dTAK1, Hep and Bsk. Using RT-PCR analysis, whether Eiger could stimulate the NF-kappaB pathway was tested; however, no obvious upregulation of the antimicrobial peptide genes, the target genes of the Drosophila NF-kappaB pathway, was detected (Igaki, 2002).

To analyze the physiological role of Eiger, a loss-of-function mutant for eiger was generated by imprecise excision of the P element in the Regg1GS9830 strain. Nine mutants were isolated harboring deletions that removed parts of the eiger genomic sequences, but leaving wild-type sequences downstream of the P element intact. egr1 and egr3 mutants were homozygous viable with greatly reduced eiger expressions. In the eye disc of the puc-LacZ enhancer-trap line, endogenous JNK activity could be detected in the region posterior to the morphogenetic furrow by long-time (20 h) X-gal staining. The puc-LacZ expression is dramatically reduced in the eye disc of the eiger mutant. Notably, the JNK activities at the marginal region of the eye disc, where endogenous eiger expression was not detected, were not affected in the eiger mutant. These findings indicate that Eiger functions as a physiological ligand for the Drosophila JNK pathway (Igaki, 2002).

Genetic and microarray analyses have been used to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila melanogaster. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member EIGER. Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. These results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways (Brodsky, 2004).

Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila

Apparent defects in cell polarity are often seen in human cancer. However, the underlying mechanisms of how cell polarity disruption contributes to tumor progression are unknown. Using a Drosophila genetic model for Ras-induced tumor progression, a molecular link has been shown between loss of cell polarity and tumor malignancy. Mutation of different apicobasal polarity genes activates c-Jun N-terminal kinase (JNK) signaling and downregulates the E-cadherin/β-catenin adhesion complex, both of which are necessary and sufficient to cause oncogenic RasV12-induced benign tumors in the developing eye to exhibit metastatic behavior. Furthermore, activated JNK and Ras signaling cooperate in promoting tumor growth cell autonomously, since JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras. The finding that such context-dependent alterations promote both tumor growth and metastatic behavior suggests that metastasis-promoting mutations may be selected for based primarily on their growth-promoting capabilities. Similar oncogenic cooperation mediated through these evolutionarily conserved signaling pathways could contribute to human cancer progression (Igaki, 2006).

Most human cancers originate from epithelial tissues. These epithelial tumors, except for those derived from squamous epithelial cells, normally exhibit pronounced apicobasal polarity. However, these tumors commonly show defects in cell polarity as they progress toward malignancy. Although the integrity of cell polarity is essential for normal development, how cell polarity disruption contributes to the signaling mechanisms essential for tumor progression and metastasis is unknown. To address this, a recently established Drosophila model of Ras-induced tumor progression triggered by loss of cell polarity has been used. This fly tumor model exhibits many aspects of metastatic behaviors observed in human malignant cancers, such as basement membrane degradation, loss of E-cadherin expression, migration, invasion, and metastatic spread to other organ sites (Pagliarini, 2003). In the developing eye tissues of these animals, loss of apicobasal polarity is induced by disruption of evolutionarily conserved cell polarity genes such as scribble (scrib), lethal giant larvae (lgl), or discs large (dlg), three polarity genes that function together in a common genetic pathway, as well as other cell polarity genes such as bazooka, stardust, or cdc42. Oncogenic Ras (RasV12), a common alteration in human cancers, causes noninvasive benign overgrowths in these eye tissues (Pagliarini, 2003). Loss of any one of the cell polarity genes somehow strongly cooperates with the effect of RasV12 to promote excess tumor growth and metastatic behavior. However, on their own, clones of scrib mutant cells are eliminated during development in a JNK-dependent manner; expression of RasV12 in these mutant cells prevents this cell death (Igaki, 2006).

To better quantify the metastatic behavior of tumors in different mutant animals, the analysis focused on invasion of the ventral nerve cord (VNC), a process in which tumor cells leave the eye-antennal discs and optic lobes (the areas where they were born) and migrate to and invade a different organ, the VNC. It was further confirmed that the genotypes associated with the invasion of the VNC in this study also resulted in the presence of secondary tumor foci at distant locations, although the number and size of these foci were highly variable (Pagliarini, 2003; Igaki, 2006).

In analyzing the global expression profiles of noninvasive and invasive tumors induced in Drosophila developing eye discs, it was observed that expression of the JNK phosphatase puckered (puc) was strongly upregulated in the invasive tumors. Upregulation of puc represents activation of the JNK pathway in Drosophila. Therefore an enhancer-trap allele, puc-LacZ, was used to monitor the activation of JNK signaling in invasive tumor cells. Strong ectopic JNK activation was present in invasive tumors, while only a slight expression of puc was seen in restricted regions of RasV12-induced noninvasive overgrowth. Intriguingly, more intense JNK activation was seen in tumor cells located in the marginal region of the eye-antennal disc and tumor cells invading the VNC. Analysis of clones of cells with a cell polarity mutation alone revealed that JNK signaling was activated by mutation of cell polarity genes. Notably, JNK signaling was not activated in a strictly cell-autonomous fashion. JNK activation in these cells was further confirmed by anti-phospho-JNK antibody staining that detects activated JNK (Igaki, 2006).

To examine the contribution of JNK activation to metastatic behavior, the JNK pathway was blocked by overexpressing a dominant-negative form of Drosophila JNK (BskDN). As previously reported (Pagliarini, 2003), clones of cells mutant for scrib, lgl, or dlg do not proliferate as well as wild-type clones, while combination of these mutations with RasV12 expression resulted in massive and metastatic tumors. Strikingly, inhibition of JNK activation by BskDN completely blocked the invasion of the VNC, as well as secondary tumor foci formation. Drosophila has two homologs of TRAF proteins (DTRAF1 and DTRAF2), which mediate signals from cell surface receptors to the JNK kinase cascade in mammalian systems. It was found that RNAi-mediated inactivation of DTRAF2, but not DTRAF1, in the tumors strongly suppressed their metastatic behavior. Inactivation of dTAK1, a Drosophila JNK kinase kinase (JNKKK), or Hep, a JNKK, also suppressed metastatic behavior. Drosophila has two known cell surface receptors that act as triggers for the JNK pathway, Wengen (TNF receptor) and PVR (PDGF/VEGF receptor). Intriguingly, it was found that RNAi-mediated inactivation of Wengen partially suppressed tumor invasion. Inactivation of PVR, in contrast, did not show any suppressive effect on metastatic behavior. It was also found that the metastatic behavior of RasV12-expressing tumors that were also mutated for one of three other cell polarity genes, bazooka, stardust, or cdc42, was also blocked by BskDN. These data indicate that loss of cell polarity contributes to metastatic behavior by activating the evolutionarily conserved JNK pathway (Igaki, 2006).

Next, whether JNK activation is sufficient to trigger metastatic behavior in RasV12-induced benign tumors was examined. Two genetic alterations can be used to activate JNK in Drosophila. First, JNK signaling can be activated by overexpression of Eiger, a Drosophila TNF ligand. While mammalian TNF superfamily proteins activate both the JNK and NFκB pathways, Eiger has been shown to specifically activate the JNK pathway through dTAK1 and Hep. Indeed, the eye phenotype caused by Eiger overexpression could be reversed by blocking JNK through Bsk-IR (Bsk-RNAi). Second, overexpression of a constitutively activated form of Hep (HepCA) can also activate JNK signaling. However, the eye phenotype caused by HepCA overexpression was only slightly suppressed by Bsk-IR, suggesting that HepCA overexpression may have additional effects other than JNK activation. Therefore Eiger overexpression was used to activate JNK in RasV12-induced benign tumors, and it was found that the RasV12+Eiger-expressing tumor cells did not result in the invasion of the VNC. This indicates that loss of cell polarity must induce an additional downstream effect(s) essential for metastatic behavior. A strong candidate for the missing event is downregulation of the E-cadherin/catenin adhesion complex, since this complex is frequently downregulated in malignant human cancer cells and is also downregulated by loss of cell polarity genes in Drosophila invasive tumors (Pagliarini, 2003). In addition, it has been recently reported that higher motility of mammalian scrib knockdown cells can be partially rescued by overexpression of E-cadherin-catenin fusion protein, suggesting a role of E-cadherin in preventing polarity-dependent invasion. Furthermore, overexpression of E-cadherin blocks metastatic behavior of RasV12/scrib−/− tumors (Pagliarini, 2003), indicating that loss of E-cadherin is essential for inducing tumor invasion in this model. It was found that loss of the Drosophila E-cadherin homolog shotgun (shg), combined with the expression of both RasV12 and Eiger, induced the invasion of the VNC. Intriguingly, loss of shg in RasV12-expressing clones also showed a weak invasive phenotype at lower penetrance. In agreement with the essential role of JNK in tumor invasion, clones of shg−/− cells weakly upregulated puc expression. It was further found that JNK activation in dlg−/− clones is not blocked by overexpression of E-cadherin, suggesting that mechanism(s) other than loss of E-cadherin also exist for inducing JNK activation downstream of cell polarity disruption. The metastatic behavior of RasV12+Eiger/shg−/− tumors was completely blocked by coexpression of BskDN, indicating a cell-autonomous requirement of JNK activation for this process. Furthermore, it was found that loss of the β-catenin homolog armadillo also induced metastatic behavior in RasV12-induced benign tumors. In contrast, overexpression of HepCA in RasV12/shg−/− cells resulted in neither enhanced tumor growth nor metastatic behavior. Together, these results suggest that, although the RasV12+Eiger/shg−/− does not completely phenocopy the effect of RasV12/scrib−/−, activation of JNK signaling and inactivation of the E-cadherin/catenin complex are the downstream components of cell polarity disruption that trigger metastatic behavior in RasV12-induced benign tumors (Igaki, 2006).

eiger encodes a protein of 409 amino acids with a C-terminal TNF homology domain and a hydrophobic transmembrane domain, indicating that Eiger is the first Drosophila member of the TNF superfamily. The absence of a signal peptide suggests that Eiger is a type II membrane protein, which is typical of the members of the TNF ligand family. The sequence of the C-terminal TNF domain of Eiger shows highest homology with human EDA-A2 (28% identity), and also shows significant homology with all known TNF superfamily members including RANKL, CD40L, FasL, APRIL, TWEAK, TNF-alpha and TRAIL (Igaki, 2002).

For information on vertebrate TNF and its interaction with the JNK pathway, search PubMed for information on tumor necrosis factor and the JNK pathway

Functional evolution of a morphogenetic gradient

Bone Morphogenetic Proteins (BMPs) pattern the dorsal-ventral axis of bilaterian embryos; however, their roles in the evolution of body plan are largely unknown. This study examined their functional evolution in fly embryos. BMP signaling specifies two extraembryonic tissues, the serosa and amnion, in basal-branching flies such as Megaselia abdita, but only one, the amnioserosa, in Drosophila melanogaster. The BMP signaling dynamics are similar in both species until the beginning of gastrulation, when BMP signaling broadens and intensifies at the edge of the germ rudiment in Megaselia, while remaining static in Drosophila. This study shows that the differences in gradient dynamics and tissue specification result from evolutionary changes in the gene regulatory network that controls the activity of a positive feedback circuit on BMP signaling, involving the tumor necrosis factor alpha homolog eiger. These data illustrate an evolutionary mechanism by which spatiotemporal changes in morphogen gradients can guide tissue complexity (Kwan, 2016).


Search PubMed for articles about Drosophila eiger

Andersen, D. S., Colombani, J., Palmerini, V., Chakrabandhu, K., Boone, E., Rothlisberger, M., Toggweiler, J., Basler, K., Mapelli, M., Hueber, A. O. and Leopold, P. (2015). The Drosophila TNF receptor Grindelwald couples loss of cell polarity and neoplastic growth. Nature. PubMed ID: 25874673

Arnett, H. A., Mason, J., Marino, M., Suzuki, K., Matsushima, G. K. and Ting, J. P. (2001). TNFalpha promotes proliferation of oligodendrocyte progenitors and remyelination. Nature Neurosci. 4: 1116-1122. 11600888

Ashkenazi, A. and Dixit, V. M. (1998). Death receptors: signaling and modulation. Science 281: 1305-1308. 9721089

Babcock, D. T., Landry, C. and Galko, M. J. (2009). Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae. Curr. Biol. 19(10): 799-806. PubMed Citation: 19375319

Brandt, S. M., et al. (2005). Secreted bacterial effectors and host-produced Eiger/TNF drive death in a Salmonella-infected fruit fly. PLoS Biology 2(12): e418. 15562316

Brodsky, M. H., et al. (2004). Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage. Mol. Cell. Biol. 24: 1219-1231. 1472996

Chabu, C. and Xu, T. (2014). Oncogenic Ras stimulates Eiger/TNF exocytosis to promote growth. Development 141(24):4729-39. PubMed ID: 25411211

Christofferson, D. E. and Yuan, J. (2010). Necroptosis as an alternative form of programmed cell death. Curr. Opin. Cell Biol. 22: 263–268. PubMed Citation: 20045303

Cordero, J. B., et al. (2010). Oncogenic Ras diverts a host TNF tumor suppressor activity into tumor promoter. Dev. Cell 18: 999–1011. PubMed Citation: 20627081

Davis, R. J. (2000). Signal transduction by the JNK group of MAP kinases. Cell 103: 239-252. 11057897

Eby, M. T., et al. (2000). TAJ, a novel member of the tumor necrosis factor receptor family, activates the c-Jun N-terminal kinase pathway and mediates caspase-independent cell death. J. Biol. Chem., 275: 15336-15342. 10809768

Fogarty, C. E., Diwanji, N., Lindblad, J. L., Tare, M., Amcheslavsky, A., Makhijani, K., Bruckner, K., Fan, Y. and Bergmann, A. (2016). Extracellular reactive oxygen species drive apoptosis-induced proliferation via Drosophila macrophages. Curr Biol 26(5):575-84. PubMed ID: 26898463

Gavin-Smyth, J., Wang, Y. C., Butler, I. and Ferguson, E. L. (2013). A genetic network conferring canalization to a bistable patterning system in Drosophila. Curr Biol 23: 2296-2302. PubMed ID: 24184102

Hitomi, J., et al. (2008). Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell 135: 1311–1323. PubMed Citation: 19109899

Igaki, T., et al. (2002). Eiger, a TNF superfamily ligand that triggers the Drosophila JNK pathway. EMBO J. 21: 3009-3018. 12065414

Igaki, T., et al. (2006). Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila. Curr. Biol. 16: 1139-1146. 16753569

Igaki, T., et al. (2009). Intrinsic tumor suppression and epithelial maintenance by endocytic activation of Eiger/TNF signaling in Drosophila. Dev. Cell 16: 458-465. PubMed Citation: 19289090

Kanda, H., Igaki, T., Kanuka, H., Yagi, T. and Miura, M. (2002). Wengen, a member of the Drosophila tumor necrosis factor receptor superfamily, is required for Eiger signaling. J. Biol. Chem. 277: 28372-28375. PubMed citation; Online text

Kanda, H., et al. (2011). Conserved metabolic energy production pathways govern Eiger/TNF-induced nonapoptotic cell death. Proc. Natl. Acad. Sci. 108(47):18977-82. PubMed Citation: 22065747

Kato, K., Awasaki, T. and Ito, K. (2009). Neuronal programmed cell death induces glial cell division in the adult Drosophila brain. Development 136: 51-59. PubMed Citation: 19019992

Kauppila, S., et al. (2003). Eiger and its receptor, Wengen, comprise a TNF-like system in Drosophila. Oncogene 22(31): 4860-7. PubMed citation: 12894227

Kumar, A., Eby, M. T., Sinha, S., Jasmin, A. and Chaudhary, P. M. (2001). The ectodermal dysplasia receptor activates the nuclear factor-B, JNK and cell death pathways and binds to ectodysplasin A. J. Biol. Chem. 276: 2668-2677. 11035039

Kwan, C. W., Gavin-Smyth, J., Ferguson, E. L. and Schmidt-Ott, U. (2016). Functional evolution of a morphogenetic gradient. Elife 5 [Epub ahead of print]. PubMed ID: 28005004

Lu H. and Bilder, D. (2005). Endocytic control of epithelial polarity and proliferation in Drosophila. Nat. Cell Biol. 7: 1232-1239. PubMed Citation: 16258546

Moberg, K. H., et al. (2005). Mutations in erupted, the Drosophila ortholog of mammalian tumor susceptibility gene 101, elicit non-cell-autonomous overgrowth. Dev. Cell 9: 699-710. PubMed Citation: 16256744

Moreno, E., Yan, M. and Basler, K. (2002). Evolution of TNF signaling mechanisms: JNK-dependent apoptosis triggered by Eiger, the Drosophila homolog of the TNF superfamily. Curr. Biol. 12: 1263-1268. 12176339

Pagliarini, R. A. and Xu. T. (2003), A genetic screen in Drosophila for metastatic behavior. Science 302: 1227-1231. 14551319

Pallavi, S. K., Ho, D. M., Hicks, C., Miele, L. and Artavanis-Tsakonas, S. (2012). Notch and Mef2 synergize to promote proliferation and metastasis through JNK signal activation in Drosophila. EMBO J. 31(13): 2895-907. PubMed Citation: 22580825

Perez-Garijo, A., Fuchs, Y. and Steller, H. (2013). Apoptotic cells can induce non-autonomous apoptosis through the TNF pathway. Elife 2: e01004. PubMed ID: 24066226

Schneider, D. S., et al. (2007). Drosophila eiger mutants are sensitive to extracellular pathogens. PLoS Pathog. 3(3): e41. PubMed Citation: 17381241

Temkin, V., Huang, Q., Liu, H., Osada, H. and Pope, R. M. (2006). Inhibition of ADP/ATP exchange in receptor-interacting protein-mediated necrosis. Mol. Cell Biol. 26: 2215–2225. PubMed Citation: 16507998

Thompson, B. J. et al. (2005). Tumor suppressor properties of the ESCRT-II complex component Vps25 in Drosophila. Dev. Cell 9: 711-720. PubMed Citation: 16256745

Vaccari, T. and Bilder, D. (2005). The Drosophila tumor suppressor vps25 prevents nonautonomous overproliferation by regulating notch trafficking, Dev. Cell 9: 687-698. PubMed Citation: 16256743

Wang, H., Cai, Y., Chia, W. and Yang, X. (2006). Drosophila homologs of mammalian TNF/TNFR-related molecules regulate segregation of Miranda/Prospero in neuroblasts. EMBO J. 25(24): 5783-93. PubMed citation; Online text

Wang, L., Du, F. and Wang, X. (2008). TNF-alpha induces two distinct caspase-8 activation pathways. Cell 133: 693–703. PubMed Citation: 18485876

Wang, W., Bohmann, D. and Jasper, H. (2003). JNK signaling confers tolerance to oxidative stress and extends lifespan in Drosophila, Dev. Cell 5: 811-816. PubMed citation: 14602080

Wu, M., Pastor-Pareja, J. C. and Xu, T. (2010). Interaction between Ras(V12) and scribbled clones induces tumour growth and invasion. Nature 463: 545-548. PubMed ID: 20072127

Xue, L., et al. (2007). Tumor suppressor CYLD regulates JNK-induced cell death in Drosophila. Dev. Cell 13: 446-454. PubMed citation: 17765686

Zhang, D. W., et al. (2009). RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 325: 332–336. PubMed Citation: 19498109

Biological Overview

date revised: 2 February 2014

Home page: The Interactive Fly © 2017 Thomas Brody, Ph.D.