Death related ced-3/Nedd2-like protein

REGULATION

Transcriptional Regulation

Steroid hormones coordinate multiple cellular changes, yet the mechanisms by which these systemic signals are refined into stage- and tissue-specific responses remain poorly understood. The Drosophila gene Eip93F, more familiarly termed E93 determines the nature of a steroid-induced biological response. E93 mutants possess larval salivary glands that fail to undergo steroid-triggered programmed cell death, and E93 is expressed in cells immediately before the onset of death. E93 protein is bound to the sites of steroid-regulated and cell death genes on polytene chromosomes, and the expression of these genes is defective in E93 mutants. Furthermore, expression of E93 is sufficient to induce programmed cell death. It is proposed that the steroid induction of E93 determines a programmed cell death response during development (Lee, 2000).

The nuclear localization of E93 in larval salivary glands provided an opportunity to determine if E93 binds to the salivary gland polytene chromosomes and, if so, to identify the sites bound by the protein. Salivary glands were dissected 12-14 hr after puparium formation, fixed, squashed, and photographed to acquire accurate cytology of the banding and puffing patterns for mapping. The chromosomes were then stained with affinity-purified E93 antibodies, and these patterns were compared with the original set of photographs to allow accurate mapping of the bound sites. E93 clearly binds to the polytene chromosomes in a reproducible and site-specific manner and is consistently detected at 65 chromosome sites, many of which contain ecdysone-regulated genes or programmed cell death genes. Among these sites are the 74EF and 75B early puffs, which contain the E74 and E75 ecdysone-inducible genes, as well as the 93F puff, which contains E93. In addition, 1B, 21C, 59F, and 99B are bound by E93 and contain the programmed cell death genes dredd, crq, dcp-1, and drICE, respectively. The 2B5 early puff, containing the BR-C ecdysone-inducible gene, and 75CD, containing βFTZ-F1 and the programmed cell death genes rpr, hid, and grim, were not bound by E93. These data indicate that E93 may directly regulate the genes in bound chromosome loci and may either encode a site-specific DNA binding protein or a chromatin-associated protein that functions as a transcriptional regulator (Lee, 2000).

Protein Interactions

Drosophila MyD88 is an adapter in the Toll signaling pathway that associates with both the Toll receptor and the downstream kinase Pelle. Expression of MyD88 in S2 cells strongly induces activity of a Drosomycin reporter gene, whereas a dominant-negative version of MyD88 potently inhibits Toll-mediated signaling. MyD88 associates with the death domain-containing adapter Drosophila Fas-associated death domain-containing protein (FADD), which in turn interacts with the apical caspase Dredd. This pathway links a cell surface receptor to an apical caspase in invertebrate cells and therefore suggests that the Toll-mediated pathway of caspase activation may be the evolutionary ancestor of the death receptor-mediated pathway for apoptosis induction in mammals (Horng, 2002).

A BLAST search of the Drosophila genome identified the sequence encoding MyD88, a Drosophila homolog of human MyD88. Similar to its human homolog, Drosophila MyD88 contains an N-terminal death domain, an intermediate domain, and a TIR domain. However, unlike human MyD88, Drosophila MyD88 contains an additional 81 amino acids preceding the death domain and a 162-aa-long C-terminal region following the TIR domain (Horng, 2002).

Transfection of MyD88 into Drosophila S2 cells potently induces a Drosomycin reporter gene but not an Attacin reporter gene. This preferential ability to induce an antifungal gene is similar to that of Toll 10b, a constitutively active form of Toll, and suggests that MyD88 may be a component of the Toll-Tube-Pelle-Cactus-Dif signaling pathway. Previous studies have demonstrated that Toll-mediated Drosomycin induction requires the nuclear translocation of Dif. Dif is normally retained in the cytoplasm by the IkappaB inhibitor Cactus and is released only in response to signal-dependent degradation of Cactus. To test whether MyD88-mediated Drosomycin induction also depends on Cactus degradation, a Cactus mutant was constructed that contains mutations of the conserved serine residues that, in mammalian IkappaB, are the targets of signal-dependent phosphorylation. A Cactus mutant inhibits Drosomycin induction by MyD88 and, as expected, by Toll. This result indicates that, similar to Toll, MyD88 regulates Drosomycin induction through the Cactus-dependent pathway (Horng, 2002).

For further analyses, various deletion mutants of MyD88 were generated. Two of the deletion mutants, one containing the TIR domain and the C-terminal domain (amino acids 237-537) and another containing the intermediate, TIR, and C-terminal domains (amino acids 176-537), activate the Drosomycin reporter weakly (10-fold) in comparison to full length MyD88, indicating that the intact protein is required for optimal activity. However, the fact that these truncation mutants can still induce signaling is surprising, since they lack the death domain that mediates interactions with downstream signaling components. Moreover, similar analyses of human MyD88 have shown that a combination of the death domain and the intermediate domain is sufficient to induce signaling activity comparable to that of the wild-type protein. An equivalent truncation of dMyD88 (amino acids 1-237) retains no residual activity despite being well expressed, suggesting that there are some differences in domain function between human and Drosophila MyD88 proteins (Horng, 2002).

To determine whether MyD88 is a component of the Toll signaling pathway, attempts were made to identify a deletion mutant that would have dominant-negative activity. Therefore, three MyD88 deletion mutants that do not activate the Drosomycin reporter were tested for their ability to inhibit Toll-mediated Drosomycin induction. The strongest inhibitor was the death domain- and middle domain-containing construct (amino acids 1-237), which at low concentrations potently inhibits Toll-mediated Drosomycin induction in a dose-dependent manner (Horng, 2002).

To order MyD88 in the pathway with respect to Pelle, MyD88 was tested for its ability to be inhibited by PelleN, a dominant-negative form of Pelle that consists of the N-terminal death domain-containing region of Pelle. MyD88, like Toll, is strongly inhibited by PelleN. MyD88, however, does not inhibit Pelle, demonstrating that, similar to the mammalian pathway, MyD88 functions upstream of Pelle (Horng, 2002).

To further establish MyD88 as a component of the Toll pathway, whether MyD88 interacts with Toll was tested by coimmunoprecipitation assays. The TIR domain-containing MyD88 construct is detected in anti-Toll immunoprecipitates. Interestingly, when cotransfected with Toll 10b, MyD88 reproducibly appears as two distinct bands -- a slower migrating upper band that may correspond to phosphorylated MyD88 construct and a faster migrating lower band. The predominant form of MyD88 detected in immunoprecipitates is the faster migrating species. MyD88 therefore associates with Toll, presumably through TIR domains, and is a component of the active receptor complex (Horng, 2002).

Because human MyD88 associates with IRAK through death domains, a likely immediate downstream target of MyD88 is the IRAK homolog Pelle. Interaction between the death domain-containing dMyD88 construct (amino acids 1-237) and Pelle was examined. MyD88 is detected in Pelle immunoprecipitates, indicating that MyD88 interacts with Pelle, presumably through their death domains (Horng, 2002).

These results therefore demonstrate that MyD88 is an adaptor in the Toll signaling pathway downstream of the receptor and upstream of Pelle. From genetic analyses, the adaptor protein Tube has also been implicated to be downstream of Toll and upstream of Pelle in the Toll signaling pathway. The death domain of Tube also interacts with Pelle. Because Tube and MyD88 also contain death domains that could potentially mediate their interaction, tests were performed for association between these two proteins in immunoprecipitation assays; Tube and MyD88 do indeed interact. Therefore, MyD88 and Tube both function as adaptors downstream of Toll, exist in the same active complex along with Pelle, and are probably both involved in the recruitment and/or activation of Pelle. Understanding functional differences between these two adapters will require further analysis (Horng, 2002).

To identify other potential downstream targets of MyD88, a search of the Drosophila genome was performed for other sequences that encode death domain-containing proteins that may interact with MyD88. One such sequence encodes a protein with a death domain as well as a death effector domain and appears to be a homolog of mammalian FADD. This cDNA has been identified and named FADD (Hu, 2000). Whether FADD can interact with MyD88 was tested. Lysates from S2 cells transfected with MyD88 were incubated with anti-Flag beads to immunoprecipitate FADD, and immunoprecipitates were blotted with anti-V5 antibody to look for associated MyD88. A strong band corresponding to MyD88 was observed, indicating that MyD88 can interact with FADD through death domains. Overexpression of FADD in S2 cells, however, does not lead to activation of either the Drosomycin or Attacin reporters (Horng, 2002).

Mammalian FADD is recruited to the tumor necrosis factor receptor complex through homophilic death domain interactions with the adapter TNFR-associated death domain-containing protein (TRADD). In turn, FADD recruits procaspase-8 through homophilic death effector domain associations. It is speculated that Drosophila FADD may likewise recruit a Drosophila caspase to the Toll receptor complex. A potential candidate caspase is Dredd, an apical caspase with a long prodomain shown to be essential for induction of antibacterial genes. Indeed, analysis of immunoprecipitated lysates from cells cotransfected with Drosophila FADD, and either full length Dredd or the death effector domain of Dredd showed strong association of Dredd with FADD. A second study (Hu, 2000) has also shown interaction of dFADD with Dredd (Horng, 2002).

Thus Drosophila MyD88 is an adapter in the Toll signaling pathway. MyD88 associates with both Toll and Pelle and functions upstream of Pelle. Tube is known from genetic studies to be an adapter in the Toll pathway that functions upstream of Pelle. Why Toll should signal through MyD88 and Tube, two receptor-proximal adapters with seemingly similar functions, is not yet clear. MyD88 associates with the receptor Toll as well as the downstream adapter FADD, which in turn interacts with the apical caspase Dredd. Because caspases are essential executioners of the apoptotic machinery in organisms from nematodes to mammals, and because Dredd has been shown to be involved in apoptosis during Drosophila development, it is possible that Toll-1 or some of the other eight Tolls that exist in Drosophila may induce apoptosis (or another Dredd-dependent pathway) through the MyD88/dFADD/Dredd pathway in a cell-type specific and/or developmental stage-specific manner. The pathway comprised of Toll, MyD88, dFADD, and Dredd would be the first description of a pathway in invertebrates that links a cell surface receptor to an apical caspase. Such a pathway, if it exists, would enable extracellular stimuli, perhaps ligands secreted by other cells during development or pathogen-derived products during infection, to instruct invertebrate cells to undergo cell death. In addition, the Toll/MyD88/dFADD/Dredd pathway is remarkably similar to that activated by the receptors of the tumor necrosis factor receptor (TNFR) superfamily in mammals, in which FADD-mediated recruitment of caspase-8 leads to induction of apoptosis. Since the Drosophila genome does not encode any cell surface receptors homologous to TNFRs, it appears that the Toll/MyD88/dFADD/Dredd pathway is the evolutionary ancestor of the mammalian death receptor pathways. This possibility is further supported by the recent finding that human TLR2 can induce apoptosis through the MyD88/FADD/Caspase-8 pathway (Horng, 2002).

DEVELOPMENTAL BIOLOGY

Embryonic

The earliest expression of DREDD mRNA in young embryos is detected prior to the onset of zygotic transcription, indicating that DREDD mRNA is maternally derived. Low ubiquitous levels of DREDD mRNA expression are also observed in the middle stages of embryogenesis (through stage 10/11). However, during stage 11 and beyond, a distinct accumulation of DREDD mRNA is observed in spatial and temporal patterns that are strikingly coincident with well-documented patterns of programmed cell death (PCD) in the embryo. Conspicuous expression of DREDD mRNA coincides with the initial appearance of dying cells (stage 11) in the subepidermis of the gnathal segments, the clypeolabrum, and the caudal tip of the retracting germ band. During stage 13, punctate DREDD accumulation occurs in dying cells distributed around the supraesophageal ganglia and in the dorsal ridge. During stage 16, when most if not all PCD is confined to the central nervous system, DREDD mRNA expression also occurs throughout the brain and ventral cord in patterns coincident with PCD in this tissue. Accumulation of DREDD mRNA in the ventral cord at this time occurs in cells that are positioned at the midline and examples of asymmetric staining are also evident. While mRNAs occurring in the ventral cord are not macrophage associated (circulating hemocytes do not enter the ventral nerve cord), hybridization signals in other tissue are clearly associated with phagocytic macrophages (Chen, 1998b).

Multiple apoptotic caspase cascades are required in nonapoptotic roles for Drosophila spermatid individualization

Spermatozoa are generated and mature within a germline syncytium. Differentiation of haploid syncytial spermatids into single motile sperm requires the encapsulation of each spermatid by an independent plasma membrane and the elimination of most sperm cytoplasm, a process known as individualization. Apoptosis is mediated by caspase family proteases. Many apoptotic cell deaths in Drosophila utilize the REAPER/HID/GRIM family proapoptotic proteins. These proteins promote cell death, at least in part, by disrupting interactions between the caspase inhibitor DIAP1 and the apical caspase DRONC, which is continually activated in many viable cells through interactions with ARK, the Drosophila homolog of the mammalian death-activating adaptor APAF-1. This leads to unrestrained activity of DRONC and other DIAP1-inhibitable caspases activated by DRONC. This study demonstrates that ARK- and HID-dependent activation of DRONC occurs at sites of spermatid individualization and that all three proteins are required for this process. dFADD, the Drosophila homolog of mammalian FADD, an adaptor that mediates recruitment of apical caspases to ligand-bound death receptors, and its target caspase DREDD are also required. A third apoptotic caspase, DRICE, is activated throughout the length of individualizing spermatids in a process that requires the product of the driceless locus, which also participates in individualization. These results demonstrate that multiple caspases and caspase regulators, likely acting at distinct points in time and space, are required for spermatid individualization, a nonapoptotic process (Huh, 2004; full text of article).

Effects of Mutation or Deletion

Ectopic death of retinal cells results from ectopic expression of rpr and grim in eye discs. Reduction of the level of Dredd in Drosophila eyes reduces the level of ectopic cell death. Heterozygosity at the Dredd locus suppresses apoptosis in transgenic models of reaper- and grim-induced cell killing, demonstrating that levels of Dredd product can modulate signaling triggered by these death activators (Chen, 1998b).

The Drosophila innate immune system discriminates between pathogens and responds by inducing the expression of specific antimicrobial peptide-encoding genes through distinct signaling cascades. Fungal infection activates NF-kappaB-like transcription factors via the Toll pathway, which also regulates innate immune responses in mammals. The pathways that mediate antibacterial defenses, however, are less defined. Loss-of-function mutations are reported in the caspase encoding gene dredd, which block the expression of all genes that code for peptides with antibacterial activity. These mutations also render flies highly susceptible to infection by Gram-negative bacteria. These results demonstrate that Dredd regulates antibacterial peptide gene expression, and it is proposed that Dredd, Immune Deficiency and the P105-like rel protein Relish define a pathway that is required to resist Gram-negative bacterial infections (Leulier, 2000).

To identify genes that control Drosophila antibacterial immune responses, a screen was carried out for mutations on the X chromosome that affect the expression of the antibacterial peptide gene diptericin after bacterial infection. Among 2500 EMS mutagenized lines, five viable, recessive mutations (named B118, F64, L23, D55, D44) were isolated of a gene that is required for the expression of a diptericin-GFP reporter gene in larvae after bacterial infection. In addition, Northern blot analysis shows that adults homozygous for each of the five alleles do not express the diptericin gene after bacterial injection. The B118 allele was mapped to cytological region 1B9-1B13 on the proximal tip of the X chromosome and a small deficiency, Df(1)R194, was identified that does not complement B118. Deficiency Df(1)R194 spans four previously identified genes: rpL36, l(1)1Bi, dredd and su(s). Several results demonstrate that B118 is a mutation in dredd: (1) B118 is allelic to a viable P element insertion (EP-1412) inserted 50 bp upstream of dredd coding sequences; (2) the two genes flanking dredd, su(s) and l(1)1Bi complement B118; (3) a small deficiency, Df(1)dredd^D3, which was generated by imprecise P element excision, and which removes dredd and affects the 5' upstream sequences of su(s), blocks diptericin expression after bacterial infection, and (4) a P element insertion, P[dredd⁺], containing 7.6 kb of genomic DNA, including dredd but not su(s) and l(1)1Bi , fully restores diptericin expression in B118 flies. All five dredd EMS mutations block diptericin expression after infection to the same degree as Df(1)dredd^D3, indicating that they are probably null alleles. The P element insertion in line EP-1412 generates a strong hypomorphic dredd mutation since a small amount of diptericin expression is detectable after infection (Leulier, 2000).

dredd encodes an apical caspase and is an effector of the apoptosis activators reaper, grim and hid. One or more dredd transcripts are specifically enriched in cells programmed to die and dredd overexpression induces apoptosis in SL2 cells. In mammals, the closest dredd homologs are caspases 8 and 10, which mediate apoptosis induced by members of the tumor necrosis factor receptor family. Caspases are produced as inactive zymogens termed pro-caspases; when activated, mature caspases catalyze the proteolytic cleavage of death substrates that are associated with apoptosis. The isolation of mutations in dredd that block diptericin expression after infection demonstrate that Dredd also regulates immune responses. In addition, a dredd-lacZ reporter gene is constitutively expressed in all adult and larval tissues including the fat body, the major immuno-responsive tissue in insects. Infection does not, however, appear to increase dredd expression levels (Leulier, 2000).

The five dredd alleles all contain point mutations that affect different regions of the dredd protein. Alleles B118, D55 and F64 generate either premature stop codons or frameshift changes in the Dredd prodomain. D44 has a missense mutation in sequences encoding the first death effector domain (DED), a region thought to mediate protein-protein interactions. In the protein encoded by allele L23, a tryptophan (W) in the caspase domain is replaced by an arginine (R) residue. The strong phenotype of alleles D44 and L23 indicates that Dredd domains affected in these alleles are essential for Dredd function in immunity (Leulier, 2000).

The isolation of dredd mutations that block diptericin expression enabled the characterization of dredd's role in mediating Drosophila antimicrobial host defense as well as dredd's relationship to other genes that function in this response. Pricking adult flies with a mixture of Gram-positive and Gram-negative bacteria activates the expression of all the genes that encode antimicrobial peptides in Drosophila. In the dredd^B118 mutant, however, mixed Gram-positive/Gram-negative infections only induce the expression of the antifungal gene drosomycin and the gene coding for Metchnikowin, which has both antifungal and antibacterial activity; diptericin, cecropin A, attacin A and defensin are expressed at <5% of wild-type levels and metchnikowin is expressed at 50% of the wild-type level. Antimicrobial gene expression is similarly affected in flies homozygous for rel^E20, a strong or null mutant allele of relish and imd, although most of the antibacterial genes are expressed at slightly higher levels in imd flies. By contrast, a mutation in the spz gene, which blocks Toll activation, reduces drosomycin induction by mixed Gram-negative/Gram-positive bacterial infection and reduces the induction of some of the antibacterial genes (defensin, attacin, cecropin A). These data demonstrate that mutations in dredd are phenotypically similar to mutations in imd and relish, and that these three genes regulate all Drosophila antibacterial peptide gene expression (Leulier, 2000).

To define further the roles of imd, dredd and relish in activating metchnikowin and drosomycin after different types of bacterial infection, metchnikowin and drosomycin expression were quantified in different mutant backgrounds 6 h after infection with either Gram-negative Escherichia coli or Gram-positive Micrococcus luteus bacteria. The dredd^B118 and rel^E20 mutations strongly reduce metchnikowin and drosomycin induction by Gram-negative bacterial infections, while the imd mutation has a weak effect; by contrast, metchnikowin and drosomycin are expressed at close to wild-type levels in the imd, dredd^B118 and rel^E20 mutants after Gram-positive bacterial infection. It is concluded, therefore, that dredd and relish play a greater role in inducing metchnikowin and drosomycin after Gram-negative bacterial infection than after Gram-positive bacterial infection (Leulier, 2000).

The observation that drosomycin and metchnikowin expression is almost completely abolished in imd;Toll double mutants suggests that Gram-positive bacterial infection triggers the expression of metchnikowin and drosomycin via the Toll pathway. In agreement, this analysis shows that mutations in spz affect drosomycin gene expression more strongly after Gram-positive than after Gram-negative bacterial infection, and that the constitutive activation of the Toll pathway in the Tl^10b mutant leads to drosomycin expression in the absence of dredd activity. metchnikowin, however, is still expressed to a high level in spz mutants after Gram-positive bacterial infection, indicating that metchnikowin induction by Gram-positive bacterial infection may also be mediated in part by the Imd pathway (Leulier, 2000).

The susceptibility to microbial infection observed in dredd, imd, relish, spz and imd;spz mutants is correlated with the expression pattern of antimicrobial genes in these mutants. dredd^B118, rel^E20 and imd;spz^rm7 adults are highly susceptible to bacterial infection by Gram-negative bacteria, and imd adults are slightly less susceptible. These survival results confirm that the activation of defense responses to Gram-negative bacterial infection require imd, dredd and relish. Only the imd;spz^rm7 double mutants, however, are highly susceptible to bacterial infection by Gram-positive bacteria, indicating that resistance to Gram-positive bacteria is regulated by both the Toll and Imd pathways. Finally, only spz^rm7 and imd;spz^rm7 mutants are highly sensitive to natural infection by the entomopathogenic fungus Beauveria bassiana or injection of Aspergillus fumigatus spores, confirming that responses to fungi are largely activated by the Toll pathway (Leulier, 2000).

The dredd immune phenotype is similar to the relish and imd phenotypes; it is predicted that the Imd, Dredd and Relish proteins function in a common signaling pathway that regulates antibacterial peptide gene expression. Based on the respective activites of Dredd as a caspase and Relish as a transcriptional transactivator, it is also hypothesized that Dredd functions upstream of Relish in the control of antimicrobial gene expression. This hypothesis is supported by the observation that Dredd is required for Relish activation via endoproteolytic cleavage. It is believed that the weaker effects of the imd mutation on antibacterial gene expression place the imd gene product at an early stage of the antibacterial cascade where multiple responses, some of which bypass imd, trigger the activation of the pathway. Alternatively, the imd mutation may represent a hypomorphic allele (Leulier, 2000).

Caspases were originally identified as effectors of apoptosis, but there is increasing evidence that caspases also function in other physiological processes. Recent studies suggest that the recruitment of the caspase-8 precursor to the TNF-R1 signaling complex either activates NF-kappaB through a Traf2-, RIP-, NIK- and IKK-dependent pathway or, after proteolytic processing of caspase-8, induces apoptosis. The data indicate that Dredd, a close homolog of caspase-8, may also have dual functions in NF-kappaB signaling and apoptosis in Drosophila. Further biochemical analysis is necessary to determine whether Dredd participates directly in Relish activation or functions further upstream (Leulier, 2000).

Deciphering the mechanisms that enable Drosophila to differentiate between pathogens and mount specific immune responses is essential for understanding innate immunity. Recent studies indicate that the Toll pathway is mainly activated in response to fungal and Gram-positive bacterial infection. Several observations suggest that imd, dredd and relish mediate most of the responses to Gram-negative bacterial infection: (1) these genes regulate the antimicrobial peptide genes that are most highly induced by Gram-negative bacterial infection; (2) dredd and relish control the induction of metchnikowin and drosomycin after Gram-negative bacterial infection, and (3) these three genes are required for resistance to Gram-negative bacterial infection. A model is proposed whereby antimicrobial gene expression in Drosophila adults is regulated by a balance of inputs from the Toll pathway and the Imd pathway, which includes Imd, Dredd and Relish, and that these two pathways are differentially activated by different classes of microorganisms. Identifying the receptors that discriminate between invading microbes and stimulate these pathways presents an exciting challenge in the study of innate immunity (Leulier, 2000).

Coexpression of an active-site C408A mutant of the fly apical caspase, Dredd [producing Dredd(C/A)], substantially attenuates cell killing triggered by Apaf-1-related-killer (Ark). In contrast, a comparable C211A mutation in the putative effector caspase drICE [producing drICE(C/A)] did not have similar effects even though it was prominently expressed. Therefore, Ark-mediated cell killing is generally not suppressed by the coexpression of mutant caspases, and the effect of Dredd(C/A) is specific. These data indicated that the Dredd mutant might exert a dominant-negative effect through a physical interaction with Ark. Whether Ark associates with Dredd was tested. A strong interaction between these proteins was detected when using either Ark(1-411) or the full-length protein. Similar tests with a comparable mutant form of drICE showed no evidence for an interaction between this caspase and Ark. These results do not address the question of whether a cofactor is necessary to regulate the Ark-Dredd interaction, since apoptotic SL2 cells may contain other proteins needed for their association. Nevertheless, Ark specifically interacts with the apical caspase Dredd but not with the effector caspase drICE. These data raise parallels to the binding observed between counterparts in the worm (CED-4 and CED-3) and in mammals (Apaf-1 and caspase-9) (Rodriguez, 1999).

The imd gene acts upstream of DmIKKgamma and the Caspase Dredd to activate the expression of antibacterial peptide genes. The dominant effect on antibacterial peptide genes of overexpression of imd driven by the hs-GAL4 driver (even in the absence of heat shock) serves to establish the epistatic relationships of imd with other genes in the pathway. In dredd and kenny mutant flies, expression of all antibacterial peptide genes in response to bacterial challenge, as well as survival to infection, is strongly impaired. The overexpression of imd is unable to confer challenge-independent expression of diptericin in dredd^D55 and kenny¹ mutant backgrounds. Furthermore, it does not restore immune inducibility of the diptericin gene in these mutants. It is noteworthy that drosomycin expression is not affected in these flies. These results demonstrate that imd acts upstream of both dredd and kenny to activate the antibacterial peptide genes during an immune response. In keeping with these results, transcription of the attacin-luciferase reporter gene induced by overexpression of imd in S2 cells is abolished by an ird⁵ (IKKß homolog) dominant-negative expression construct (Georgel, 2001).

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date revised: 25 May 2007

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