The Toll immune-regulated Drosophila protein Fondue is involved in hemolymph clotting and puparium formation

Clotting is critical in limiting hemolymph loss and initiating wound healing in insects as in vertebrates. It is also an important immune defense, quickly forming a secondary barrier to infection, immobilizing bacteria and thereby promoting their killing. However, hemolymph clotting is one of the least understood immune responses in insects. Here, we characterize fondue (fon; CG15825), an immune-responsive gene of Drosophila melanogaster that encodes an abundant hemolymph protein containing multiple repeat blocks. After knockdown of fon by RNAi, bead aggregation activity of larval hemolymph is strongly reduced, and wound closure is affected. fon is thus the second Drosophila gene after hemolectin (hml), for which a knockdown causes a clotting phenotype. In contrast to hml-RNAi larvae, clot fibers are still observed in samples from fon-RNAi larvae. However, clot fibers from fon-RNAi larvae are more ductile and longer than in wt hemolymph samples, indicating that Fondue might be involved in cross-linking of fiber proteins. In addition, fon-RNAi larvae exhibit melanotic tumors and constitutive expression of the antifungal peptide gene Drosomycin (Drs), while fon-RNAi pupae display an aberrant pupal phenotype. Altogether, our studies indicate that Fondue is a major hemolymph protein required for efficient clotting in Drosophila.     

结合GAL4/UAS系统与CyO-GFP平衡子获取表达RNA干扰序列果蝇幼虫并有效干扰Tis11表达

TIS11是转录后调控因子TTP在果蝇中的同源物,在果蝇幼虫免疫、发育和代谢等多种生理过程中都发挥重要作用.为研究TIS11的功能,需要利用GAL4/UAS系统获得整体高干扰效率的Tis11 RNAi果蝇幼虫．为平衡GAL4转基因果蝇高活性启动子的致死效应,需要使用带有成蝇卷翅标记CyO的第二染色体的平衡子,但CyO标记在幼虫中无可见表型,因此无法区分杂交幼虫的基因型．为解决这一问题,引入了带有CyO-GFP标记的平衡子．携带CyO-GFP平衡子的G-Actin果蝇与携带Tis11 RNAi序列的101765果蝇杂交,杂交幼虫可以通过GFP标记进行区分,剔除带有CyO-GFP平衡子的幼虫,从而挑选出表达Tis11 RNAi序列的幼虫,最后经real-time PCR检测所得幼虫具有整体高干扰效率．     

Larval RNAi in Drosophila?

RNA interference (RNAi) has become a common method of gene knockdown in many model systems. To trigger an RNAi response, double-stranded RNA (dsRNA) must enter the cell. In some organisms such as Caenorhabditis elegans, cells can take up dsRNA from the extracellular environment via a cellular uptake mechanism termed systemic RNAi. However, in the fruit fly Drosophila melanogaster, it is widely believed that cells are unable to take up dsRNA, although there is little published data to support this claim. In this study, we set out to determine whether this perception has a factual basis. We took advantage of traditional Gal4/upstream activation sequence (UAS) transgenic flies as well as the mosaic analysis with a repressible cell marker (MARCM) system to show that extracellular injection of dsRNA into Drosophila larvae cannot trigger RNAi in most Drosophila tissues (with the exception of hemocytes). Our results show that this is not due to a lack of RNAi machinery in these tissues as overexpression of dsRNA inside the cells using hairpin RNAs efficiently induces an RNAi response in the same tissues. These results suggest that, while most Drosophila tissues indeed lack the ability to uptake dsRNA from the surrounding environment, hemocytes can initiate RNAi in response to extracellular dsRNA. We also examined another insect, the red flour beetle Tribolium castaneum, which has been shown to exhibit a robust systemic RNAi response. We show that virtually all Tribolium tissues can respond to extracellular dsRNA, which is strikingly different from the situation in Drosophila. Our data provide specific information about the tissues amenable to RNAi in two different insects, which may help us understand the molecular basis of systemic RNAi.     

Fondue and transglutaminase in the Drosophila larval clot

Hemolymph coagulation is vital for larval hemostasis and important in immunity, yet the molecular basis of coagulation is not well understood in insects. Of the larval clotting factors identified in Drosophila, Fondue is not conserved in other insects, but is notable for its effects on the clot's physical properties, a possible function in the cuticle, and for being a substrate of transglutaminase. Transglutaminase is the only mammalian clotting factor found in Drosophila, and as it acts in coagulation in other invertebrates, it is also likely to be important in clotting in Drosophila. Here we describe a Fondue-GFP fusion construct that labels the cuticle and clot, and show that chemical inhibition and RNAi knockdown of the Drosophila transglutaminase gene affect clot properties and composition in ways similar to knockdown of the fon gene. Thus, Fondue appears to be incorporated into the cuticle and is a key transglutaminase substrate in the clot. This is also the first direct functional confirmation that transglutaminase acts in coagulation in Drosophila.     

Influence of RNAi knockdown for E-complex genes on the silkworm proleg development

Larvae of many holometabolous insects possess abdominal appendages called prolegs. Lepidoptera larvae have prolegs in the segments A3-A6. Functions of Lepidoptera hox genes on these abdominal appendages development is still a controversial issue. In this article, we report the use of double strand RNA (dsRNA)-mediated interference (RNAi) to dissect the function of some hox genes, specifically E-complex genes Ubx, abd-A, and Abd-B, in the ventral appendage development of the Lepidoptera silkworm, Bombyx mori. We found that Ubx RNAi caused leg identity in A1 segment, abd-A RNAi caused severe defect of abdominal prolegs and Abd-B RNAi allowed proleg identity in more posterior abdominal segments. These results confirm that Lepidoptera hox genes Ubx and Abd-B have evolved the repressing function to ventral appendage development, which is similar to those of Drosophila. However, Lepidoptera abd-A might have been modified distinctively during evolution, and has important roles in directing the development of prolegs.     

Yantar, a conserved arginine-rich protein is involved in Drosophila hemocyte development

To identify novel factors involved in Drosophila hematopoiesis, we screened a collection of lethal recessive mutations that also affected normal hemocyte composition in larvae. We present the characterization of the gene yantar (ytr) for which we isolated null and hypomorphic mutations that were associated with severe defects in hemocyte differentiation and proliferation; ytr is predominantly expressed in the hematopoietic tissue during larval development and encodes an evolutionary conserved protein which is predominantly localized in the nucleus. The hematopoietic phenotype in ytr mutants is consistent with a defect or block in differentiation of precursor hemocytes: mutant larvae have enlarged lymph glands (LGs) and have an excess of circulating hemocytes. In addition, many cells exhibit both lamellocyte and crystal cell markers. Ytr function has been preserved in evolution as hematopoietic specific expression of the Drosophila or mouse Ytr proteins rescue the differentiation defects in mutant hemocytes.     

Coagulation and survival in Drosophila melanogaster fondue mutants

Drosophila larval coagulation factors have been identified in vitro. Better understanding of insect hemolymph coagulation calls for experiments in vivo. We have characterized a fondue (fon) mutation and null alleles isolated by imprecise excision of a transposable element. Loss of fon was pupal lethal, but adults could be recovered by expressing the UAS::fonGFP construct of Lindgren et al. (2008). Despite their lethality, fon mutations did not affect larval survival after wounding either when tested alone or in combination with a mutation in the hemolectin clotting factor gene. This reinforces the idea of redundant hemostatic mechanisms in Drosophila larvae, and independent pleiotropic functions of the fondue protein in coagulation and a vital process in metamorphosis.     

A complex containing the CCR4 and CAF1 proteins is involved in mRNA deadenylation in Drosophila

The CCR4-NOT complex is the major enzyme catalyzing mRNA deadenylation in Saccharomyces cerevisiae. We have identified homologs for almost all subunits of this complex in the Drosophila genome. Biochemical fractionation showed that the two likely catalytic subunits, CCR4 and CAF1, were associated with each other and with a poly(A)-specific 3' exonuclease activity. In Drosophila, the CCR4 and CAF1 proteins were ubiquitously expressed and present in cytoplasmic foci. Individual knock-down of several potential subunits of the Drosophila CCR4-NOT complex by RNAi in tissue culture cells led to a lengthening of bulk mRNA poly(A) tails. Knock-down of two individual subunits also interfered with the rapid deadenylation of Hsp70 mRNA during recovery from heat shock. Similarly, ccr4 mutant flies had elongated bulk poly(A) and a defect in Hsp70 mRNA deadenylation. A minor increase in bulk poly(A) tail length was also observed in Rga mutant flies, which are affected in the NOT2 subunit. The data show that the CCR4-NOT complex is conserved in Drosophila melanogaster and plays a role in general and regulated mRNA deadenylation.     

Ancestral functions of Delta/Notch signaling in the formation of body and leg segments in the cricket Gryllus bimaculatus

Delta/Notch signaling controls a wide spectrum of developmental processes, including body and leg segmentation in arthropods. The various functions of Delta/Notch signaling vary among species. For instance, in Cupiennius spiders, Delta/Notch signaling is essential for body and leg segmentation, whereas in Drosophila fruit flies it is involved in leg segmentation but not body segmentation. Therefore, to gain further insight into the functional evolution of Delta/Notch signaling in arthropod body and leg segmentation, we analyzed the function of the Delta (Gb'Delta) and Notch (Gb'Notch) genes in the hemimetabolous, intermediate-germ cricket Gryllus bimaculatus. We found that Gb'Delta and Gb'Notch were expressed in developing legs, and that RNAi silencing of Gb'Notch resulted in a marked reduction in leg length with a loss of joints. Our results suggest that the role of Notch signaling in leg segmentation is conserved in hemimetabolous insects. Furthermore, we found that Gb'Delta was expressed transiently in the posterior growth zone of the germband and in segmental stripes earlier than the appearance of wingless segmental stripes, whereas Gb'Notch was uniformly expressed in early germbands. RNAi knockdown of Gb'Delta or Gb'Notch expression resulted in malformation in body segments and a loss of posterior segments, the latter probably due to a defect in posterior growth. Therefore, in the cricket, Delta/Notch signaling might be required for proper morphogenesis of body segments and posterior elongation, but not for specification of segment boundaries.     

Neurexin-1 is required for synapse formation and larvae associative learning in Drosophila

Neurexins are highly polymorphic cell-surface adhesive molecules in neurons. In cultured mammalian cell system, they were found to be involved in synaptogenesis. Here, we report for the first time that Drosophila neurexin is required for synapse formation and associative learning in larvae. Drosophila genome encodes a single functional neurexin (CG7050; Neurexin-1 or Nrx-1), which is a homolog of vertebrate alpha-neurexin. Neurexin-1 is expressed in central nervous system and highly enriched in synaptic regions of the ventral ganglion and brain. Neurexin-1 null mutants are viable and fertile, but have shortened lifespan. The synapse number is decreased in central nervous system in Neurexin-1 null mutants. In addition, Neurexin-1 null mutants exhibit associative learning defect in larvae.     

Drosophila Amphiphysin is a Post-Synaptic Protein Required for Normal Locomotion but Not Endocytosis

Clathrin-mediated endocytosis is required to recycle synaptic vesicles for fast and efficient neurotransmission. Amphiphysins are thought to be multiprotein adaptors that may contribute to this process by bringing together many of the proteins required for endocytosis. Their in vivo function, however, has yet to be determined. Here, we show that the Drosophila genome encodes a single amphiphysin gene that is broadly expressed during development. We also show that, unlike its vertebrate counterparts, Drosophila Amphiphysin is enriched postsynaptically at the larval neuromuscular junction. To determine the role of Drosophila Amphiphysin, we also generated null mutants which are viable but give rise to larvae and adults with pronounced locomotory defects. Surprisingly, the locomotory defects cannot be accounted for by alterations in the morphology or physiology of the neuromuscular junction. Moreover, using stimulus protocols designed to test endocytosis under moderate and extreme vesicle cycling, we could not detect any defect in the neuromuscular junction of the amphiphysin mutant. Taken together, our findings suggest that Amphiphysin is not required for viability, nor is it absolutely required for clathrin-mediated endocytosis. However, Drosophila Amphiphysin function is required in both larvae and adults for normal locomotion.     

过表达 PINK1改善SCA3/MJD转基因果蝇模型的线粒体功能

脊髓小脑性共济失调3型（SCA3/MJD）,是一种因致病基因MJD1编码区内CAG异常重复扩增所致的常染色体显性遗传迟发性神经退行性疾病. 已知PINK1蛋白可通过抗氧化稳定线粒体,阻止帕金森疾病的发生,但其在SCA3/MJD中的作用尚不清楚. 本文旨在探索过表达PINK1对SCA3/MJD转基因果蝇模型的保护作用.本研究利用Mhc-Gal4启动子表达致病蛋白质片段（MJDtr-Q78）获得SCA3/MJD果蝇模型,分别运用过表达PINK1和RNA干扰PINK1研究其在SCA3/MJD果蝇模型中的功能.结果显示,疾病模型组翅膀异常率增高,线粒体呈过度融合状态,ATP值降低;PINK1 RNA干扰组翅膀异常率明显增高,线粒体呈显著过度融合状态,ATP值明显降低;PINK1过表达组翅膀异常率明显降低,线粒体清晰、完整,ATP值明显升高.本文的结果提示, 过表达PINK1对SCA3/MJD转基因果蝇模型起保护作用,而RNA干扰PINK1表达加重SCA3/MJD转基因果蝇模型病情.PINK1在SCA3/MJD果蝇模型中的功能可能通过改善细胞内线粒体功能实现.     

KNOCKDOWN OF ATPsyn‐b CAUSED LARVAL GROWTH DEFECT AND MALE INFERTILITY IN Drosophila

The ATPsyn‐b encoding for subunit b of ATP synthase in Drosophila melanogaster is proposed to act in ATP synthesis and phagocytosis, and has been identified as one of the sperm proteins in both Drosophila and mammals. At present, its details of functions in animal growth and spermatogenesis have not been reported. In this study, we knocked down ATPsyn‐b using Drosophila lines expressing inducible hairpin RNAi constructs and Gal4 drivers. Ubiquitous knockdown of ATPsyn‐b resulted in growth defects in larval stage as the larvae did not grow bigger than the size of normal second‐instar larvae. Knockdown in testes did not interrupt the developmental excursion to viable adult flies, however, these male adults were sterile. Analyses of testes revealed disrupted nuclear bundles during spermatogenesis and abnormal shaping in spermatid elongation. There were no mature sperm in the seminal vesicle of ATPsyn‐b knockdown male testes. These findings suggest us that ATPsyn‐b acts in growth and male fertility of Drosophila.     

Drosophila damaged DNA binding protein 1 contributes to genome stability in somatic cells

The damaged DNA-binding protein (DDB) complex consists of a heterodimer of p127 (DDB1) and p48 (DDB2) subunits and is believed to have a role in nucleotide excision repair (NER). We used the GAL4-UAS targeted expression system to knock down DDB1 in wing imaginal discs of Drosophila. The knock-down was achieved in transgenic flies using over-expression of inverted repeat RNA of the D-DDB1 gene [UAS-D-DDB1(650)-dsRNA]. As a consequence of RNA interference (RNAi), the fly had a shrunken wing phenotype. The wing spot test showed induced genome instability in transgenic flies with RNAi knock-down of D-DDB1 in wing imaginal discs. When Drosophila larvae with RNAi knock-down of D-DDB1 in wing imaginal discs were treated with the chemical mutagen methyl methanesulfonate (MMS), the frequency of flies with a severely shrunken wing phenotype increased compared to non-treated transgenic flies. These results suggested that DDB1 plays a role in the response to DNA damaged with MMS and in genome stability in Drosophila somatic cells.