Transgenerational inheritance of wing development defects in Drosophila melanogaster induced by cadmium.

The transgenerational inheritance of phenotype induced by environmental factors is a new focus in epigenetic research. In this study, Drosophila melanogaster (F0) was cultured in the medium containing cadmium (Cd, 4.5 mg/kg) from eggs to adults, and offspring (F1-F4) were continuously kept in standard medium (without cadmium). The phenotype analysis showed that cadmium induced developmental defects on wings and apoptosis in the wing disc cells of Drosophila (F0). The wing defects were transmitted for at least four generations even without Cd afterwards. And the effect on the mRNA expression of wing development related genes (shg, omb, F-actin, Mekk1) can be maintained for at least two or three generations. More importantly, under cadmium stress, the post-translational modification (PTM) on the histones H3K4me3 in the third instar larvae and ovaries or testes of adult flies increased significantly, while the levels of H3K9me3 and H3K27me3 decreased significantly. The expression of histone methylation related genes (dSet-1, ash1, Lsd1) increased significantly and these changes can be transmitted to offspring from one or two generations in ovaries or testes. These results suggest that the phenotypic defects of wings caused by cadmium can be inherited to the offspring, and this transgenerational inheritance effect may be related to the epigenetic regulation of histone methylation. Therefore, the adaptability of offspring should be considered when evaluating the toxicity and environmental risk of cadmium.

Silence of yki by miR-7 regulates the Hippo pathway.

The Hippo signaling pathway governs organ size via coordinating cell proliferation and apoptosis, and its dysregulation causes congenital diseases and cancers. The homeostasis of Hippo pathway is achieved through multiple post translational modifications. Through Drosophila genetic screening, we found that miRNAs were also involved in Hippo pathway regulation. Here, we showed that overexpression of miR-7 resulted in small wings, which were neutralized by miR-7-sponge (miR-7-sp) co-expression. Mechanistically, miR-7 inhibited the expression of Hippo pathway target genes. Epistatic analyses revealed that miR-7 modulated Hippo pathway through the transcriptional cofactor Yorkie (Yki). Consistently, overexpression of miR-7 decreased Yki protein. We further found a seed sequence of miR-7 in the yki 3'-UTR region. In addition, we discovered that miR-7 was a transcriptional target of Yki. Thus, a negative feedback loop existed for fine tuning Hippo pathway activity. Taken together, our findings uncovered a novel mechanism by which Yki was silenced by miR-7 for Hippo pathway regulation.

Role of Chitin Deacetylase 1 in the Molting and Metamorphosis of the Cigarette Beetle Lasioderma serricorne.

Chitin deacetylases (CDAs) are chitin-modifying enzymes known to play vital roles in insect metamorphosis and development. In this study, we identified and characterized a chitin deacetylase 1 gene (LsCDA1) from the cigarette beetle Lasioderma serricorne. LsCDA1 contains a 1614 bp open reading frame encoding a protein of 537 amino acids that includes domain structures typical of CDAs. LsCDA1 was mainly expressed in the late larval and late pupal stages. In larval tissues, the highest level of LsCDA1 was detected in the integument. The expression of LsCDA1 was induced by 20-hydroxyecdysone (20E) in vivo, and it was significantly suppressed by knocking down the expression of ecdysteroidogenesis genes and 20E signaling genes. RNA interference (RNAi)-aided silencing of LsCDA1 in fifth-instar larvae prevented the larval-pupal molt and caused 75% larval mortality. In the late pupal stage, depletion of LsCDA1 resulted in the inhibition of pupal growth and wing abnormalities, and the expression levels of four wing development-related genes (LsDY, LsWG, LsVG, and LsAP) were dramatically decreased. Meanwhile, the chitin contents of LsCDA1 RNAi beetles were significantly reduced, and expressions of three chitin synthesis pathway genes (LsTRE1, LsUAP1, and LsCHS1) were greatly decreased. The results suggest that LsCDA1 is indispensable for larval-pupal and pupal-adult molts, and that it is a potential target for the RNAi-based control of L. serricorne.

Role of the ABC transporter Mdr49 in Hedgehog signaling and germ cell migration.

Coalescence of the embryonic gonad in Drosophila melanogaster requires directed migration of primordial germ cells (PGCs) towards somatic gonadal precursor cells (SGPs). It was recently proposed that the ATP-binding cassette (ABC) transporter Mdr49 functions in the embryonic mesoderm to facilitate the transmission of the PGC attractant from the SGPs; however, the precise molecular identity of the Mdr49-dependent guidance signal remained elusive. Employing the loss- and gain-of-function strategies, we show that Mdr49 is a component of the Hedgehog (hh) pathway and it potentiates the signaling activity. This function is direct because in Mdr49 mutant embryos the Hh ligand is inappropriately sequestered in the hh-expressing cells. Our data also suggest that the role of Mdr49 is to provide cholesterol for the correct processing of the Hh precursor protein. Supporting this conclusion, PGC migration defects in Mdr49 embryos are substantially ameliorated by a cholesterol-rich diet.

Decanalization of wing development accompanied the evolution of large wings in high-altitude Drosophila.

In higher organisms, the phenotypic impacts of potentially harmful or beneficial mutations are often modulated by complex developmental networks. Stabilizing selection may favor the evolution of developmental canalization--that is, robustness despite perturbation--to insulate development against environmental and genetic variability. In contrast, directional selection acts to alter the developmental process, possibly undermining the molecular mechanisms that buffer a trait's development, but this scenario has not been shown in nature. Here, we examined the developmental consequences of size increase in highland Ethiopian Drosophila melanogaster. Ethiopian inbred strains exhibited much higher frequencies of wing abnormalities than lowland populations, consistent with an elevated susceptibility to the genetic perturbation of inbreeding. We then used mutagenesis to test whether Ethiopian wing development is, indeed, decanalized. Ethiopian strains were far more susceptible to this genetic disruption of development, yielding 26 times more novel wing abnormalities than lowland strains in F2 males. Wing size and developmental perturbability cosegregated in the offspring of between-population crosses, suggesting that genes conferring size differences had undermined developmental buffering mechanisms. Our findings represent the first observation, to our knowledge, of morphological evolution associated with decanalization in the same tissue, underscoring the sensitivity of development to adaptive change.

The RNA binding KH domain of Spoonbill depletes pathogenic non-coding spinocerebellar ataxia 8 transcripts and suppresses neurodegeneration in Drosophila.

Spinocerebellar ataxia 8 (SCA8) pathogenesis is a resultant of gain-of-function machinery that primarily results at the RNA level. It has been reported that expanded non-coding CTG trinucleotide repeat in the ATXN8OS transcripts leads to SCA8 coupled neurodegeneration. Targeted depletion of pathogenic SCA8 transcripts is a viable therapeutic approach. In this report we have focused on the suppression of toxic RNA gain-of-function associated with SCA8. We report suppression of SCA8 associated neurodegeneration by KH RNA binding domain of Spoonbill. KH domain suppresses pathogenic SCA8 associated phenotype in adult flies. Ectopic expression of KH domain leads to massive reduction in the number and size of SCA8 RNA foci. We show that Spoonbill interacts with toxic SCA8 transcripts via its KH domain and promotes its depletion. Till date, no attempts have been made for therapeutic intervention of SCA8 pathogenesis. Further characterization of Spoonbill KH domain may aid us in designing peptide based therapeutics for SCA8 associated neurodegeneration.

Reduced glutathione biosynthesis in Drosophila melanogaster causes neuronal defects linked to copper deficiency.

Glutathione (GSH) is a tripeptide often considered to be the master antioxidant in cells. GSH plays an integral role in cellular redox regulation and is also known to have a role in mammalian copper homeostasis. In vitro evidence suggests that GSH is involved in copper uptake, sequestration and efflux. This study was undertaken to further investigate the roles that GSH plays in neuronal copper homeostasis in vivo, using the model organism Drosophila melanogaster. RNA interference-mediated knockdown of the Glutamate-cysteine ligase catalytic subunit gene (Gclc) that encodes the rate-limiting enzyme in GSH biosynthesis was utilised to genetically deplete GSH levels. When Gclc was knocked down in all neurons, this caused lethality, which was partially rescued by copper supplementation and was exacerbated by additional knockdown of the copper uptake transporter Ctr1A, or over-expression of the copper efflux transporter ATP7. Furthermore, when Gclc was knocked down in a subset of neuropeptide-producing cells, this resulted in adult progeny with unexpanded wings, a phenotype previously associated with copper dyshomeostasis. In these cells, Gclc suppression caused a decrease in axon branching, a phenotype further enhanced by ATP7 over-expression. Therefore, we conclude that GSH may play an important role in regulating neuronal copper levels and that reduction in GSH may lead to functional copper deficiency in neurons in vivo. We provide genetic evidence that glutathione (GSH) levels influence Cu content or distribution in vivo, in Drosophila neurons. GSH could be required for binding Cu imported by Ctr1A and distributing it to chaperones, such as Mtn, CCS and Atox1. Alternatively, GSH could modify the copper-binding and transport activities of Atox1 and the ATP7 efflux protein via glutathionylation of copper-binding cysteines.

An inwardly rectifying K+ channel is required for patterning.

Mutations that disrupt function of the human inwardly rectifying potassium channel KIR2.1 are associated with the craniofacial and digital defects of Andersen-Tawil Syndrome, but the contribution of Kir channels to development is undefined. Deletion of mouse Kir2.1 also causes cleft palate and digital defects. These defects are strikingly similar to phenotypes that result from disrupted TGFß/BMP signaling. We use Drosophila melanogaster to show that a Kir2.1 homolog, Irk2, affects development by disrupting BMP signaling. Phenotypes of irk2 deficient lines, a mutant irk2 allele, irk2 siRNA and expression of a dominant-negative Irk2 subunit (Irk2DN) all demonstrate that Irk2 function is necessary for development of the adult wing. Compromised Irk2 function causes wing-patterning defects similar to those found when signaling through a Drosophila BMP homolog, Decapentaplegic (Dpp), is disrupted. To determine whether Irk2 plays a role in the Dpp pathway, we generated flies in which both Irk2 and Dpp functions are reduced. Irk2DN phenotypes are enhanced by decreased Dpp signaling. In wild-type flies, Dpp signaling can be detected in stripes along the anterior/posterior boundary of the larval imaginal wing disc. Reducing function of Irk2 with siRNA, an irk2 deletion, or expression of Irk2DN reduces the Dpp signal in the wing disc. As Irk channels contribute to Dpp signaling in flies, a similar role for Kir2.1 in BMP signaling may explain the morphological defects of Andersen-Tawil Syndrome and the Kir2.1 knockout mouse.

BCAS2 is essential for Drosophila viability and functions in pre-mRNA splicing.

Here, we show that dBCAS2 (CG4980, human Breast Carcinoma Amplified Sequence 2 ortholog) is essential for the viability of Drosophila melanogaster. We find that ubiquitous or tissue-specific depletion of dBCAS2 leads to larval lethality, wing deformities, impaired splicing, and apoptosis. More importantly, overexpression of hBCAS2 rescues these defects. Furthermore, the C-terminal coiled-coil domain of hBCAS2 binds directly to CDC5L and recruits hPrp19/PLRG1 to form a core complex for splicing in mammalian cells and can partially restore wing damage induced by knocking down dBCAS2 in flies. In summary, Drosophila and human BCAS2 share a similar function in RNA splicing, which affects cell viability.

Wingless gene cloning and its role in manipulating the wing dimorphism in the white-backed planthopper, Sogatella furcifera.

BACKGROUND: Wingless gene (Wg) plays a fundamental role in regulating the segment polarity and wing imaginal discs of insects. The rice planthoppers have an obvious wing dimorphism, and the long- and short-winged forms exist normally in natural populations. However, the molecular characteristics and functions of Wg in rice planthoppers are poorly understood, and the relationship between expression level of Wg and wing dimorphism has not been clarified. RESULTS: In this study, wingless gene (Wg) was cloned from three species of rice planthopper, Sogatella furcifera, Laodelphgax striatellus and Nilaparvata lugens, and its characteristics and role in determining the wing dimorphism of S. furcifera were explored. The results showed that only three different amino acid residuals encoded by Wg were found between S. furcifera and L. striatellus, but more than 10 residuals in N. lugens were different with L. striatellus and S. furcifera. The sequences of amino acids encoded by Wg showed a high degree of identity between these three species of rice planthopper that belong to the same family, Delphacidae. The macropterous and brachypterous lineages of S. furcifera were established by selection experiment. The Wg mRNA expression levels in nymphs were significantly higher in the macropterous lineage than in the brachypterous lineage of S. furcifera. In macropterous adults, the Wg was expressed mainly in wings and legs, and less in body segments. Ingestion of 100 ng/µL double-stranded RNA of Wg from second instar nymphs led to a significant decrease of expression level of Wg during nymphal stage and of body weight of subsequent adults. Moreover, RNAi of Wg resulted in significantly shorter and deformative wings, including shrunken and unfolded wings. CONCLUSION: Wg has high degree of identity among three species of rice planthopper. Wg is involved in the development and growth of wings in S. furcifera. Expression level of Wg during the nymphal stage manipulates the size and pattern of wings in S. furcifera.

EGFR signaling attenuates Groucho-dependent repression to antagonize Notch transcriptional output.

Crosstalk between signaling pathways is crucial for the generation of complex and varied transcriptional networks. Antagonism between the EGF-receptor (EGFR) and Notch pathways in particular is well documented, although the underlying mechanism is poorly understood. The global corepressor Groucho (Gro) and its transducin-like Enhancer-of-split (TLE) mammalian homologs mediate repression by a myriad of repressors, including effectors of the Notch, Wnt (Wg) and TGF-beta (Dpp) signaling cascades. Given that there are genetic interactions between gro and components of the EGFR pathway (ref. 9 and P.H. et al., unpublished results), we tested whether Gro is at a crossroad between this and other pathways. Here we show that phosphorylation of Gro in response to MAPK activation weakens its repressor capacity, attenuating Gro-dependent transcriptional silencing by the Enhancer-of-split proteins, effectors of the Notch cascade. Thus, Gro is a new junction between signaling pathways, enabling EGFR signaling to antagonize transcriptional output by Notch and potentially other Gro-dependent pathways.

Wing vein abnormality analysis in honeybee (Apis mellifera L. 1758) populations from Iran.

The insect wing is one of the most important characteristics that allowed insects to occupy most of the habitats on the planet. Honeybee wings has been the subject of studies on the venation abnormalities. A total of 424 honeybees from 14 locations were collected and all four wings were removed and examined for 19 abnormalities on the forewings and 6 abnormalities on the hindwings. In general, supernumerary veins were the most common abnormalities seen and abnormalities no. 23, 2, 6, 1, 5, 21, 10, 13 had the highest and abnormalities no. 11, 17, 18, 19, 20, and 25 had the lowest frequencies. All of the abnormalities had similar frequencies in the right and left wings in the population. In terms of correlation between 25 abnormalities, abnormality pairs AB3-AB13, AB6-AB7, AB7-AB8, AB10-AB12, AB16-AB17 on the forewing and AB2-AB23, AB12-AB20, AB12-AB24, AB13-AB21, AB16-AB25, and AB17-AB25 between the forewing and hindwing show significant positive correlations and abnormality pairs AB4-AB5, AB7-AB15 and AB8-AB9 on the forewing show significant negative correlations with each other. In terms of the differential occurrence of abnormalities , a few locations differed significantly from other locations. This study provides some insights into the nature of these abnormalities on the honeybee wings.

Gene-specific targeting of the histone chaperone asf1 to mediate silencing.

The histone chaperone Asf1 assists in chromatin assembly and remodeling during replication, transcription activation, and gene silencing. However, it has been unclear to what extent Asf1 could be targeted to specific loci via interactions with sequence-specific DNA-binding proteins. Here, we show that Asf1 contributes to the repression of Notch target genes, as depletion of Asf1 in cells by RNAi causes derepression of the E(spl) Notch-inducible genes. Conversely, overexpression of Asf1 in vivo results in decreased expression of target genes and produces phenotypes that are strongly modified (enhanced and suppressed) by mutations affecting the Notch pathway, but not by mutations in other signaling pathways. Asf1 can be coprecipitated with the DNA-binding protein Su(H) and the corepressor Hairless and interacts directly with two components of this complex, Hairless and SKIP. Thus, in addition to playing more general roles in chromatin dynamics, Asf1 is directed via interactions with sequence-specific complexes to mediate silencing of specific target genes.

Mercury is a direct and potent γ-secretase inhibitor affecting Notch processing and development in Drosophila.

Prenatal exposure to mercury causes neurodevelopmental disorders and neurological pathologies in infants, such as microcephaly and mental retardation. Despite critical importance, the molecular interactions leading to mercury toxicity are yet to be elucidated. We first used a cell-free assay to investigate mercury effects on purified γ-secretase activity. Next, we treated adult Drosophila melanogaster with mercury and collected control and mercury-treated embryos, which were subjected to mild hypotonic protein extraction, or immunostained to reveal nervous system morphology. Embryos left to develop into adults were examined for wing phenotypes. Relative to control metals, we found that mercury strongly inhibits in vitro γ-secretase processing of both amyloid-ß precursor protein (APP) and Notch. Mercury inhibited APP and Notch cleavage in a dose-dependent manner, with IC(50) values of 50-125 nM, and is therefore comparable in potency to benchmark γ-secretase inhibitors. Immunoblot analysis of embryonic protein extracts showed that mercury inhibits Notch cleavage by γ-secretase in vivo. This is accompanied by severe neurodevelopmental abnormalities in embryos and adult wing-notching phenotypes. Our findings provide first evidence that mercury is a direct and potent γ-secretase inhibitor and suggest that inhibition of γ-secretase and disruption of the Notch developmental pathway potentially contribute to mercury-induced toxicity in the nervous system.

New evidence that deformed wing virus and black queen cell virus are multi-host pathogens.

The host-range breadth of pathogens can have important consequences for pathogens' long term evolution and virulence, and play critical roles in the emergence and spread of the new diseases. Black queen cell virus (BQCV) and Deformed wing virus (DWV) are the two most common and prevalent viruses in European honey bees, Apis mellifera. Here we provide the evidence that BQCV and DWV infect wild species of honey bees, Apis florea and Apis dorsata. Phylogenetic analyses suggest that these viruses might have moved from A. mellifera to wild bee species and that genetic relatedness as well as the geographical proximity of host species likely play an important role in host range of the viruses. The information obtained from this present study can have important implication for understanding the population structure of bee virus as well as host-virus interactions.

[Role of the Drosophila melanogaster hrs gene in wing formation].

The Hrs (hepatocyte growth factor receptor tyrosine kinase substrate) protein is an endosomal protein whose function is to transport receptor tyrosine kinases from early endosomes to lysosomes. Since receptor tyrosine kinases are involved in various signaling pathways, HSR defects lead to various malformations. A study of the role of the hrs gene in wing development in Drosophila confirmed that the gene is involved in the formation of the D/V boundary of the wing imaginal disk and suggested a new role in wing vein refinement for the gene. Structural analysis of the hrs gene transcripts indicated that transcript B is responsible for vein refinement.

Genetic basis of wing morphogenesis in Drosophila: sexual dimorphism and non-allometric effects of shape variation.

BACKGROUND: The Drosophila wing represents a particularly appropriate model to investigate the developmental control of phenotypic variation. Previous studies which aimed to identify candidate genes for wing morphology demonstrated that the genetic basis of wing shape variation in D. melanogaster is composed of numerous genetic factors causing small, additive effects. In this study, we analyzed wing shape in males and females from 191 lines of D. melanogaster, homozygous for a single P-element insertion, using geometric morphometrics techniques. The analysis allowed us to identify known and novel candidate genes that may contribute to the expression of wing shape in each sex separately and to compare them to candidate genes affecting wing size which have been identified previously using the same lines. RESULTS: Our results indicate that more than 63% of induced mutations affected wing shape in one or both sexes, although only 33% showed significant differences in both males and females. The joint analysis of wing size and shape revealed that only 19% of the P-element insertions caused coincident effects on both components of wing form in one or both sexes. Further morphometrical analyses revealed that the intersection between veins showed the smallest displacements in the proximal region of the wing. Finally, we observed that mutations causing general deformations were more common than expected in both sexes whereas the opposite occurred with those generating local changes. For most of the 94 candidate genes identified, this seems to be the first record relating them with wing shape variation. CONCLUSIONS: Our results support the idea that the genetic architecture of wing shape is complex with many different genes contributing to the trait in a sexually dimorphic manner. This polygenic basis, which is relatively independent from that of wing size, is composed of genes generally involved in development and/or metabolic functions, especially related to the regulation of different cellular processes such as motility, adhesion, communication and signal transduction. This study suggests that understanding the genetic basis of wing shape requires merging the regulation of vein patterning by signalling pathways with processes that occur during wing development at the cellular level.

In vivo manipulation of heparan sulfate structure and its effect on Drosophila development.

Heparan sulfate proteoglycans (HSPGs) participate in a wide range of biological processes through interactions with a number of ligand proteins. The nature of these interactions largely depends on the heparan sulfate (HS) moiety of HSPGs, which undergoes a series of modifications by various HS-modifying enzymes (HSMEs). Although the effects of alterations in a single HSME on physiological processes have started to be studied, it remains elusive how a combination of these molecules control the structure and function of HS. Here we systematically manipulated the HS structures and analyzed their effect on morphogenesis and signaling, using the genetically tractable model organism, Drosophila. We generated transgenic fly strains overexpressing HSMEs alone or in combination. Unsaturated disaccharide analyses of HS showed that expression of various HSMEs generates distinct HS structures, and the enzymatic activities of HSMEs are influenced by coexpression of other HSMEs. Furthermore, these transgenic HSME animals showed a different extent of lethality, and a subset of HSMEs caused specific morphological defects due to defective activities of Wnt and bone morphogenetic protein signaling. There is no obvious relationship between HS unsaturated disaccharide composition and developmental defects in HSME animals, suggesting that other structural factors, such as domain organization or sulfation sequence, might regulate the function of HS.

Individual behaviour in firebugs (Pyrrhocoris apterus).

The concept of animal personalities has recently become of major interest as researchers began to wonder why animals within a given population show consistent behaviour across situations and contexts, what led to the evolution of such behavioural inflexibility and what mechanisms might underlie the phenomenon. A recent model explains individual differences in a population as the result of trade-off between present and future reproduction. We tested this model on the two wing morphs, i.e. short-winged (brachypterous) and long-winged (macropterous) specimens of the firebug (Pyrrhocoris apterus). Since it has been already demonstrated that the two wing morphs differ in their life-history strategies, this species is an ideal subject to test whether the specimens with different life-history strategies have different personalities as well. The results show that individuals behave consistently over time and across contexts, meaning observed bugs do have personalities. We also have found that in females, the two wing morphs have different personalities supporting the theoretical predictions, i.e. winged ones, which are supposed to have lower future reproductive value, are braver and more exploratory. We found no difference between the morphs in males. Differences in reproductive investment might explain this discrepancy between the sexes.

Negative correlation between Nosema ceranae spore loads and deformed wing virus infection levels in adult honey bee workers.

Interactions between pathogens might contribute to honey bee colony losses. Here we investigated if there is an association between the microsporidian Nosema ceranae and the deformed wing virus (DWV) in different body sections of individual honey bee workers (Apis mellifera ligustica) under exclusion of the vector Varroa destructor. Our data provide correlational evidence for antagonistic interactions between the two pathogens in the midgut of the bees.