Knock-in mutagenesis in Drosophila Rdl underscores the critical role of the conserved M3 glycine in mediating the actions of broflanilide and isocycloseram on GABA receptors.

γ-Aminobutyric acid receptors (GABARs) are crucial targets for pest control chemicals, including meta-diamide and isoxazoline insecticides, which act as negative allosteric modulators of insect GABARs. Previous cell-based assays have indicated that amino acid residues in the transmembrane cavity between adjacent subunits of Drosophila RDL GABAR (i.e., Ile276, Leu280, and Gly335) are involved in mediating the action of meta-diamides. In this study, to confirm this result at the organismal level, we employed CRISPR/Cas9-mediated genome editing, generated six transgenic Drosophila strains carrying substitutions in these amino acid residues, and investigated their sensitivity to broflanilide and isocycloseram. Flies homozygous for the I276F mutation did not exhibit any change in sensitivity to the tested insecticides compared to the control flies. Conversely, I276C homozygosity was lethal, and heterozygous flies exhibited ∼2-fold lower sensitivity to broflanilide than the control flies. Flies homozygous for the L280C mutation survived into adulthood but exhibited infertility. Both heterozygous and homozygous L280C flies exhibited ∼3- and âˆ¼20-fold lower sensitivities to broflanilide and isocycloseram, respectively, than the control flies. The reduction in sensitivity to isocycloseram in L280C flies diminished to ∼3-fold when treated with piperonyl butoxide. Flies homozygous for the G335A mutation reached the adult stage. However, they were sterile, had small bodies, and exhibited reduced locomotion, indicating the critical role of Gly335 in RDL function. These flies exhibited markedly increased tolerance to topically applied broflanilide and isocycloseram, demonstrating that the conserved Gly335 is the target of the insecticidal actions of broflanilide and isocycloseram. Considering the significant fitness costs, the Gly335 mutation may not pose a serious risk for the development of resistance in field populations of insect pests. However, more careful studies using insect pests are needed to investigate whether our perspective applies to resistance development under field conditions.

Controlled expression of nicotinic acetylcholine receptor-encoding genes in insects uncovers distinct mechanisms of action of the neonicotinoid insecticide dinotefuran.

Dinotefuran, a neonicotinoid, is a unique insecticide owing to its structure and action. We took two approaches that employed insects with controlled expression of nicotinic acetylcholine receptor (nAChR)-encoding genes to gain insight into the uniqueness of dinotefuran. First, we examined the insecticidal activity of dinotefuran and imidacloprid against brown planthoppers (Nilaparvata lugens), in which the expression of eight (of 13) individual subunit-encoding genes was specifically reduced using RNA interference. Knockdown of the tested gene, except one, resulted in a decrease in sensitivity to imidacloprid, whereas the sensitivity of N. lugens to dinotefuran decreased only when two of the eight genes were knocked down. These findings imply that a major dinotefuran-targeted nAChR subtype may contain specific subunits although imidacloprid acts on a broad range of receptor subtypes. Next, we examined the effects of knockout of Drosophila α1 subunit-encoding gene (Dα1) on the insecticidal effects of dinotefuran and imidacloprid. Dα1-deficient flies (Dα1KO) demonstrated the same sensitivity to dinotefuran as control flies, but a decreased sensitivity to imidacloprid. This difference was attributed to a reduction in imidacloprid-binding sites in Dα1KO flies, whereas the binding of dinotefuran remained unchanged. RNA sequencing analysis indicated that Dα2 expression was specifically enhanced in Dα1KO flies. These findings suggest that changes in Dα1 and Dα2 expression contribute to the differences in the insecticidal activity of dinotefuran and imidacloprid in Dα1KO flies. Overall, our findings suggest that dinotefuran acts on distinct nAChR subtypes.

Drosophila transcription factor NF-Y suppresses transcription of the lipase 4 gene, a key gene for lipid storage.

The CCAAT motif-binding factor NF-Y consists of three different subunits, NF-YA, NF-YB, and NF-YC. Although it is suggested that NF-Y activity is essential for normal tissue homeostasis, survival, and metabolic function, its precise role in lipid metabolism is not clarified yet. In Drosophila, eye disc specific knockdown of Drosophila NF-YA (dNF-YA) induced aberrant morphology of the compound eye, the rough eye phenotype in adults and mutation of the lipase 4 (lip4) gene suppressed the rough eye phenotype. RNA-seq analyses with dNF-YA knockdown third instar larvae identified the lip4 gene as one of the genes that are up-regulated by the dNF-YA knockdown. We identified three dNF-Y-binding consensuses in the 5'flanking region of the lip4 gene, and a chromatin immunoprecipitation assay with the specific anti-dNF-YA IgG demonstrated dNF-Y binding to this genomic region. The luciferase transient expression assay with cultured Drosophila S2 cells and the lip4 promoter-luciferase fusion genes with and without mutations in the dNF-Y-binding consensuses showed that each of the three dNF-Y consensus sequences negatively regulated lip4 gene promoter activity. Consistent with these results, qRT-PCR analysis with the dNF-YA knockdown third instar larvae revealed that endogenous lip4 mRNA levels were increased by the knockdown of dNF-YA in vivo. The specific knockdown of dNF-YA in the fat body with the collagen-GAL4 driver resulted in smaller oil droplets in the fat body cells. Collectively, these results suggest that dNF-Y is involved in lipid storage through its negative regulation of lip4 gene transcription.

DREF plays multiple roles during Drosophila development.

DREF was originally identified as a transcription factor that coordinately regulates the expression of DNA replication- and proliferation-related genes in Drosophila. Subsequent studies demonstrated that DREF is involved in tumor suppressor pathways including p53 and Hippo signaling. DREF also regulates the expression of genes encoding components of the JNK and EGFR pathways during Drosophila development. DREF itself is under the control of the TOR pathway during cell and tissue growth responding to nutrition. Recent studies revealed that DREF plays a role in chromatin organization including insulator function, chromatin remodeling, and telomere maintenance. DREF is also involved in the regulation of genes related to mitochondrial biogenesis, linking it to cellular proliferation. Thus, DREF is now emerging as not only a transcription factor, but also a multi-functional protein. In this review, we summarize current advances in studies on the novel functions of Drosophila DREF.

NF-Y in invertebrates.

Both Drosophila melanogaster and Caenorhabditis elegans (C. elegans) are useful model organisms to study in vivo roles of NF-Y during development. Drosophila NF-Y (dNF-Y) consists of three subunits dNF-YA, dNF-YB and dNF-YC. In some tissues, dNF-YC-related protein Mes4 may replace dNF-YC in dNF-Y complex. Studies with eye imaginal disc-specific dNF-Y-knockdown flies revealed that dNF-Y positively regulates the sevenless gene encoding a receptor tyrosine kinase, a component of the ERK pathway and negatively regulates the Sensless gene encoding a transcription factor to ensure proper development of R7 photoreceptor cells together with proper R7 axon targeting. dNF-Y also controls the Drosophila Bcl-2 (debcl) to regulate apoptosis. In thorax development, dNF-Y is necessary for both proper Drosophila JNK (basket) expression and JNK signaling activity that is responsible for thorax development. Drosophila p53 gene was also identified as one of the dNF-Y target genes in this system. C. elegans contains two forms of NF-YA subunit, CeNF-YA1 and CeNF-YA2. C. elegans NF-Y (CeNF-Y) therefore consists of CeNF-YB, CeNF-YC and either CeNF-YA1 or CeNF-YA2. CeNF-Y negatively regulates expression of the Hox gene egl-5 (ortholog of Drosophila Abdominal-B) that is involved in tail patterning. CeNF-Y also negatively regulates expression of the tbx-2 gene that is essential for development of the pharyngeal muscles, specification of neural cell fate and adaptation in olfactory neurons. Negative regulation of the expression of egl-5 and tbx-2 by CeNF-Y provides new insight into the physiological meaning of negative regulation of gene expression by NF-Y during development. In addition, studies on NF-Y in platyhelminths are also summarized. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.

Drosophila DOCK family protein sponge regulates the JNK pathway during thorax development.

The dedicator of cytokinesis (DOCK) family proteins that are conserved in a wide variety of species are known as DOCK1-DOCK11 in mammals. The Sponge (Spg) is a Drosophila counterpart to the mammalian DOCK3. Specific knockdown of spg by pannir-GAL4 or apterous-GAL4 driver in wing discs induced split thorax phenotype in adults. Reduction of the Drosophila c-Jun N-terminal kinase (JNK), basket (bsk) gene dose enhanced the spg knockdown-induced phenotype. Conversely, overexpression of bsk suppressed the split thorax phenotype. Monitoring JNK activity in the wing imaginal discs by immunostaining with anti-phosphorylated JNK (anti-pJNK) antibody together with examination of lacZ expression in a puckered-lacZ enhancer trap line revealed the strong reduction of the JNK activity in the spg knockdown clones. This was further confirmed by Western immunoblot analysis of extracts from wing discs of spg knockdown fly with anti-pJNK antibody. Furthermore, the Duolink in situ Proximity Ligation Assay method detected interaction signals between Spg and Rac1 in the wing discs. Taken together, these results indicate Spg positively regulates JNK pathway that is required for thorax development and the regulation is mediated by interaction with Rac1.

Drosophila RecQ5 is involved in proper progression of early spermatogenesis.

RecQ5, a member of the conserved RecQ DNA helicase family, is required for the maintenance of genome stability. The human RECQL5 gene is expressed ubiquitously in almost all tissues, with strong expression in the testes (Shimamoto et al., 2000). However, it remains to be elucidated in which cells RecQ5 is expressed and how RecQ5 functions in the testes. In this present study we analyzed the expression of RecQ5 in Drosophila testes. The RecQ5 protein was specifically expressed in germline cells in larval, pupal, and adult testes. Drosophila RecQ5 was localized in nuclei of male germline stem cells, spermatogoniablasts, spermatogonia, and early spermatocytes. As growth of the early spermatocyte proceeded, the amount of RecQ5 increased in the nuclei. However, before maturation of the spermatocyte, the level of RecQ5 declined. Thus, RecQ5 expression was regulated. Furthermore, we compared recq5 mutant testes with the wild-type ones. The most conspicuous alterations were swelling of the apical region of and an increase in the number of spermatocytes in the recq5 testis, suggesting a relative accumulation of spermatocytes in the recq5 mutant testes. Therefore, Drosophila RecQ5 may contribute to the proper progression from germline stem cells to spermatocytes for maintenance of genome stability.

The Drosophila DOCK family protein sponge is involved in differentiation of R7 photoreceptor cells.

The Drosophila sponge (spg)/CG31048 gene belongs to the dedicator of cytokinesis (DOCK) family genes that are conserved in a wide variety of species. DOCK family members are known as DOCK1-DOCK11 in mammals. Although DOCK1 and DOCK2 involve neurite elongation and immunocyte differentiation, respectively, the functions of other DOCK family members are not fully understood. Spg is a Drosophila homolog of mammalian DOCK3 and DOCK4. Specific knockdown of spg by the GMR-GAL4 driver in eye imaginal discs induced abnormal eye morphology in adults. To mark the photoreceptor cells in eye imaginal discs, we used a set of enhancer trap strains that express lacZ in various sets of photoreceptor cells. Immunostaining with anti-Spg antibodies and anti-lacZ antibodies revealed that Spg is localized mainly in R7 photoreceptor cells. Knockdown of spg by the GMR-GAL4 driver reduced signals of R7 photoreceptor cells, suggesting involvement of Spg in R7 cell differentiation. Furthermore, immunostaining with anti-dpERK antibodies showed the level of activated ERK signal was reduced extensively by knockdown of spg in eye discs, and both the defects in eye morphology and dpERK signals were rescued by over-expression of the Drosophila raf gene, a component of the ERK signaling pathway. Furthermore, the Duolink in situ Proximity Ligation Assay method detected interaction signals between Spg and Rap1 in and around the plasma membrane of the eye disc cells. Together, these results indicate Spg positively regulates the ERK pathway that is required for R7 photoreceptor cell differentiation and the regulation is mediated by interaction with Rap1 during development of the compound eye.

dNF-YB plays dual roles in cell death and cell differentiation during Drosophila eye development.

Nuclear transcription factor Y (NF-Y) is well characterized in eukaryotes. It consists of three different subunits, NF-YA, NF-YB and NF-YC, all of which are required for formation of the NF-Y complex and DNA-binding. There is a high homology in NF-YB among Drosophila species with 75% identity and 95% similarity overall, especially in the histone-fold motif (HFM) (95% identity and 100% similarity). In the present study, specific knockdown of Drosophila NF-YB (dNF-YB) in eye imaginal discs induced a rough eye phenotype in adults and this phenotype was the result of induction of caspase-dependent apoptosis followed by apoptosis-induced proliferation. Furthermore, knockdown specifically inhibited R7 photoreceptor cell differentiation, independent of the apoptotic function. dNF-YB and dNF-YA indeed form complexes in vivo where they impair R7 photoreceptor cell differentiation by down regulating the mitogen-activated protein kinase (MAPK) pathway. Expression of the sev gene, or the D-raf gene, a downstream component of the MAPK cascade, could rescue the rough eye phenotype and the loss of R7 signals in dNF-YB knockdown flies. The death executioner Bcl-2 (debcl) is the homolog of Bcl-2 in Drosophila melanogaster and its promoter contains four dNF-Y-binding consensus sequences which play positive roles in promoter activity. In chromatin immunoprecipitation assays with anti-dNF-YB antibody and S2 cells, the debcl gene promoter region containing the NF-Y consensus was effectively amplified in immunoprecipitates by polymerase chain reaction. Taken together, these results indicate that dNF-Y regulates debcl gene expression.

Transcription factor NF-Y is involved in differentiation of R7 photoreceptor cell in Drosophila.

The CCAAT motif-binding factor NF-Y consists of three different subunits, NF-YA, NF-YB and NF-YC. Knockdown of Drosophila NF-YA (dNF-YA) in eye discs with GMR-GAL4 and UAS-dNF-YAIR resulted in a rough eye phenotype and monitoring of differentiation of photoreceptor cells by LacZ expression in seven up-LacZ and deadpan-lacZ enhancer trap lines revealed associated loss of R7 photoreceptor signals. In line with differentiation of R7 being regulated by the sevenless (sev) gene and the MAPK cascade, the rough eye phenotype and loss of R7 signals in dNF-YA-knockdown flies were rescued by expression of the sev gene, or the D-raf gene, a downstream component of the MAPK cascade. The sev gene promoter contains two dNF-Y-binding consensus sequences which play positive roles in promoter activity. In chromatin immunoprecipitation assays with anti-dNF-YA antibody and S2 cells, the sev gene promoter region containing the NF-Y consensus was effectively amplified in immunoprecipitates from transgenic flies by polymerase chain reaction, indicating that dNF-Y is necessary for appropriate sev expression and involved in R7 photoreceptor cell development.

Drosophila DREF acting via the JNK pathway is required for thorax development.

The Drosophila Jun N-terminal kinase (JNK) gene basket (bsk) promoter contains a DNA replication-related element (DRE)-like sequence, raising the possibility of regulation by the DNA replication-related element-binding factor (DREF). Chromatin immunoprecipitation assays with anti-DREF IgG showed the bsk gene promoter region to be effectively amplified. Luciferase transient expression assays revealed the DRE-like sequence to be important for bsk gene promoter activity, and knockdown of DREF decreased the bsk mRNA level and the bsk gene promoter activity. Furthermore, knockdown of DREF in the notum compartment of wing discs by pannier-GAL4 and UAS-DREFIR resulted in a split thorax phenotype. Monitoring of JNK activity in the wing disc by LacZ expression in a puckered (puc)-LacZ enhancer trap line revealed the reduction in DREF knockdown clones. These findings indicate that DREF is involved in regulation of Drosophila thorax development via actions on the JNK pathway.

XNP/dATRX interacts with DREF in the chromatin to regulate gene expression.

The ATRX gene encodes a chromatin remodeling protein that has two important domains, a helicase/ATPase domain and a domain composed of two zinc fingers called the ADD domain. The ADD domain binds to histone tails and has been proposed to mediate their binding to chromatin. The putative ATRX homolog in Drosophila (XNP/dATRX) has a conserved helicase/ATPase domain but lacks the ADD domain. In this study, we propose that XNP/dATRX interacts with other proteins with chromatin-binding domains to recognize specific regions of chromatin to regulate gene expression. We report a novel functional interaction between XNP/dATRX and the cell proliferation factor DREF in the expression of pannier (pnr). DREF binds to DNA-replication elements (DRE) at the pnr promoter to modulate pnr expression. XNP/dATRX interacts with DREF, and the contact between the two factors occurs at the DRE sites, resulting in transcriptional repression of pnr. The occupancy of XNP/dATRX at the DRE, depends on DNA binding of DREF at this site. Interestingly, XNP/dATRX regulates some, but not all of the genes modulated by DREF, suggesting a promoter-specific role of XNP/dATRX in gene regulation. This work establishes that XNP/dATRX directly contacts the transcriptional activator DREF in the chromatin to regulate gene expression.

The warts gene as a novel target of the Drosophila DRE/DREF transcription pathway.

The Hippo tumor suppressor pathway in Drosophila represses expression of DIAP1 and Cyclin E via inactivation of the transcription co-activator Yorkie, resulting in cell cycle arrest and induction of apoptosis. The warts (wts) gene is well known as a core kinase in this pathway, but its transcriptional regulation has yet to be clarified. In Drosophila, DREF binds to a target sequence named DRE (5'-TATCGATA) and regulates transcription of cell proliferation-related genes containing the DRE sequence in their promoter regions. Here we found half reduction of the wts gene dose to enhance the DREF-induced rough eye phenotype, suggesting a DREF genetic interaction with the Hippo pathway in vivo. Three DREs indentified in the wts gene promoter region exhibited strong promoter activity with a luciferase transient expression assay in Drosophila S2 cells, this decreasing under DREF-RNAi conditions. In addition, knockdown of DREF in S2 cells reduced the level of endogenous wts mRNA. Chromatin immunoprecipitation assays with anti-DREF antibody revealed that DREF binds specifically to the wts gene promoter region containing DREs in vivo. These results indicate that the DRE/DREF pathway is required for transcriptional regulation of the wts gene, indicating a novel link between the DRE/DREF and the Hippo pathways.

Targeted overexpression of Drosophila transglutaminase-B induced characteristic phenotypes in a manner similar to transglutaminase-a.

The Drosophila transglutaminase gene (CG7356) encodes two transglutaminases, dTG-A and dTG-B. To understand the roles of dTG-B during the development of the fly, we examined phenotypes induced through ectopic expression of dTG-B. Overexpression of dTG-B induced rough eye and extra wing crossvein phenotypes. These phenotypes were similar to those observed in the case of targeted overexpression of dTG-A.

NF-Y transcriptionally regulates the Drosophila p53 gene.

The p53 protein is important in multicellular organisms, where it regulates the cell cycle and thus functions as a tumor suppressor that contributes to preventing cancer. However, molecular regulation of p53 gene expression is not fully understood. NF-YA is a subunit of the NF-Y trimeric complex, a transcription factor that binds to CCAAT motifs in the promoter regions of a variety of genes playing key roles in cell cycle regulation. We have identified four potential Drosophila NF-Y (dNF-Y)-binding sites located in the 5'-flanking region of the Drosophila p53 (dmp53) gene. Chromatin immunoprecipitation analyses using anti-dNF-YA antibodies confirmed that dNF-YA binds specifically to the genomic region containing CCAAT boxes in the dmp53 gene promoter in vivo. Furthermore, the thorax disclosed phenotype of dNF-YA knockdown flies can be enhanced by dmp53 mutation. In addition, the level of dmp53 mRNA was found to be decreased in the dNF-YA knockdown cells and transient expression of the luciferase gene revealed that wild-type dmp53 gene promoter activity is much stronger than mutated promoter activity in S2 cells. The requirement of CCAAT boxes for dmp53 promoter activity was further confirmed by expression of EGFP in various tissues from transgenic flies carrying wild-type and CCAAT box-mutated versions of dmp53 promoter-GFP fusion genes. These results taken together indicate that dNF-Y is necessary for dmp53 gene promoter activity.

Overexpression of transglutaminase in the Drosophila wing imaginal disc induced an extra wing crossvein phenotype.

To determine the roles of Drosophila transglutaminase-A (dTG-A), we examined a phenotype induced through ectopic expression of dTG-A. Overexpression of dTG-A in the wing imaginal disc induced an extra wing crossvein phenotype. This phenotype was suppressed by crossing with epidermal growth factor receptor (Egfr) signaling pathway mutant flies. These results indicate that this phenotype, induced by dTG-A, is related to enhancement of the Egfr signaling pathway.

Over-expression of transglutaminase in the Drosophila eye imaginal disc induces a rough eye phenotype.

Transglutaminases (TGs) catalyze the cross-linking of proteins and are involved in various biological processes in mammals. In invertebrates, except for the involvement in the hemolymph clotting, the functions of TG have not been revealed. Drosophila has a single TG gene (CG7356), from which two kinds of mRNAs (dTG-RA and dTG-RB) are formed. RT-PCR analyses indicated that both dTGs-RA and -RB are synthesized in all the developmental stages tested. To reveal the roles of dTG during the development, we examined a phenotype induced through the ectopic expression of dTG by using a GAL4-UAS targeted expression system. Over-expression of dTG-A in the eye imaginal disc of larva induced a rough eye phenotype in adult compound eyes. Co-expression of P35, an inhibitor of apoptosis, suppressed the rough eye phenotype, suggesting that the rough eye phenotype induced by the over-expression of dTG-A in the eye imaginal disc is due to the occurrence of apoptosis. The rough eye phenotype induced by the over-expression of dTG-A was suppressed by the crossing with mutant fly lines lacking Drosophila JNK gene basket (bsk) or Drosophila JNKK gene hemipterous. FLP-out experiments using an enhancer trap line showed that the over-expression of dTG-A in the eye imaginal disc increased the puckered enhancer activity, a reporter of Bsk activity. These results suggested that the rough eye phenotype induced by the over-expression of dTG-A is related to an enhancement of JNK signaling pathway.

A novel Drosophila Girdin-like protein is involved in Akt pathway control of cell size.

The Akt signaling pathway is well known to regulate cell proliferation and growth. Girdin, a novel substrate of Akt, plays a crucial role in organization of the actin cytoskeleton and cell motility under the control of Akt. We here identified a novel Girdin-like protein in Drosophila (dGirdin), which has two isoforms, dGirdin PA and dGirdin PB. dGirdin shows high homology with human Girdin in the N-terminal and coiled-coil domains, while diverging at the C-terminal domain. On establishment of transgenic fly lines, featuring knockdown or overexpression of dGirdin in vivo, overexpression in the wing disc cells induced ectopic apoptosis, implying a role in directing apoptosis. Knockdown of dGirdin in the Drosophila wing imaginal disc cells resulted in reduction of cell size. Furthermore, this was enhanced by half reduction of the Akt gene dose, suggesting that Akt positively regulates dGirdin. In the wing disc, cells in which dGirdin was knocked down exhibited disruption of actin filaments. From these in vivo analyses, we conclude that dGirdin is required for actin organization and regulation of appropriate cell size under control of the Akt signaling pathway.

Identification of the Drosophila Mes4 gene as a novel target of the transcription factor DREF.

The Mes4 gene has been identified as one of the maternal Dorsal target genes in Drosophila. In the present study, we found a DNA replication-related element (DRE, 5'-TATCGATA) in the Mes4 promoter recognized by the DRE-binding factor (DREF). Luciferase transient expression assays in S2 cells using Mes4 promoter-luciferase fusion plasmids revealed that the DRE sequence is essential for Mes4 promoter activity. Requirement of DRE for Mes4 promoter activity was further confirmed by anti-beta-galactosidase antibody-staining of various tissues from transgenic flies carrying Mes4 promoter-lacZ fusion genes. Furthermore, wild type Mes4 promoter activity was decreased by 40% in DREF-depleted S2 cells. These results indicate that DREF positively regulates Mes4 gene expression. Band mobility shift analyses using Kc cell nuclear extracts further indicated that the DRE sequence in the Mes4 promoter is especially important for binding to DREF. Moreover, specific binding of DREF to the involved genomic region could be demonstrated by chromatin immunoprecipitation assays using anti-DREF antibodies. These results, taken together, indicate that the DRE/DREF system activates transcription of the Mes4 gene. In addition, knockdown of the Mes4 gene in wing imaginal discs using the GAL4-UAS system caused an atrophied wing phenotype, suggesting that Mes4 is required for wing morphogenesis.

Relevant expression of Drosophila heme oxygenase is necessary for the normal development of insect tissues.

Heme oxygenase (HO) is a rate-limiting step of heme degradation, which catalyzes the conversion of heme into biliverdin, iron, and CO. HO has been characterized in micro-organisms, insects, plants, and mammals. The mammalian enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli. The present study reports the use of RNA-interference (RNAi) to suppress HO in the multicellular eukaryote Drosophila. Eye imaginal disc-specific suppression of the Drosophila HO homolog (dHO) conferred serious abnormal eye morphology in adults. Deficiency of the dHO protein resulted in increased levels of iron and heme in larvae. The accumulation of iron was also observed in the compound eyes of dHO-knockdown adult flies. In parallel with the decrease of dHO, the expression of delta-aminolevulinic acid synthase, the first enzyme of the heme-biosynthetic pathway, in larvae was decreased markedly, suggesting that heme biosynthesis was totally suppressed by dHO-deficiency. The activation of caspase-3 occurred in eye imaginal discs of dHO-knockdown flies, indicating the occurrence of apoptosis in the discs. On the other hand, the overexpression of dHO resulted in a weak but significant rough eye phenotype in adults. Taken together, considering that dHO is not a stress-inducible protein, the expression of dHO can be tightly regulated at developmental stages and the relevant expression is necessary for the normal development of tissues in Drosophila.