Regulation of Hemocytes in Drosophila Requires dappled Cytochrome b5

A major category of mutant hematopoietic phenotypes in Drosophila is melanotic tumors or nodules, which consist of abnormal and overproliferated blood cells, similar to granulomas. Our analyses of the melanotic mutant dappled have revealed a novel type of gene involved in blood cell regulation. The dappled gene is an essential gene that encodes cytochrome b5, a conserved hemoprotein that participates in electron transfer in multiple biochemical reactions and pathways. Viable mutations of dappled cause melanotic nodules and hemocyte misregulation during both hematopoietic waves of development. The sexes are similarly affected, but hemocyte number is different in females and males of both mutants and wild type. Additionally, initial tests show that curcumin enhances the dappled melanotic phenotype and establish screening of endogenous and xenobiotic compounds as a route for analysis of cytochrome b5 function. Overall, dappled provides a tractable genetic model for cytochrome b5, which has been difficult to study in higher organisms.     

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.     

A misexpression screen to identify regulators of Drosophila larval hemocyte development

In Drosophila, defense against foreign pathogens is mediated by an effective innate immune system, the cellular arm of which is composed of circulating hemocytes that engulf bacteria and encapsulate larger foreign particles. Three hemocyte types occur: plasmatocytes, crystal cells, and lamellocytes. The most abundant larval hemocyte type is the plasmatocyte, which is responsible for phagocytosis and is present either in circulation or in adherent sessile domains under the larval cuticle. The mechanisms controlling differentiation of plasmatocytes and their migration toward these sessile compartments are unclear. To address these questions we have conducted a misexpression screen using the plasmatocyte-expressed GAL4 driver Peroxidasin-GAL4 (Pxn-GAL4) and existing enhancer-promoter (EP) and EP yellow (EY) transposon libraries to systematically misexpress approximately 20% of Drosophila genes in larval hemocytes. The Pxn-GAL4 strain also contains a UAS-GFP reporter enabling hemocyte phenotypes to be visualized in the semitransparent larvae. Among 3412 insertions screened we uncovered 101 candidate hemocyte regulators. Some of these are known to control hemocyte development, but the majority either have no characterized function or are proteins of known function not previously implicated in hemocyte development. We have further analyzed three candidate genes for changes in hemocyte morphology, cell-cell adhesion properties, phagocytosis activity, and melanotic tumor formation.     

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing

Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.     

SID-1 is a dsRNA-selective dsRNA-gated channel

Systemic RNAi in Caenorhabditis elegans requires the widely conserved transmembrane protein SID-1 to transport RNAi silencing signals between cells. When expressed in Drosophila S2 cells, C. elegans SID-1 enables passive dsRNA uptake from the culture medium, suggesting that SID-1 functions as a channel for the transport of double-stranded RNA (dsRNA). Here we show that nucleic acid transport by SID-1 is specific for dsRNA and that addition of dsRNA to SID-1 expressing cells results in changes in membrane conductance, which indicate that SID-1 is a dsRNA gated channel protein. Consistent with passive bidirectional transport, we find that the RNA induced silencing complex (RISC) is required to prevent the export of imported dsRNA and that retention of dsRNA by RISC does not seem to involve processing of retained dsRNA into siRNAs. Finally, we show that mimics of natural molecules that contain both single- and double-stranded dsRNA, such as hairpin RNA and pre-microRNA, can be transported by SID-1. These findings provide insight into the nature of potential endogenous RNA signaling molecules in animals.     

Dissecting RNA silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level

Short interfering RNA (siRNA)-mediated RNA silencing plays an important role in cellular defence against viral infection and abnormal gene expression in multiple organisms. Many viruses have evolved silencing suppressors for counter-defence. We have developed an RNA silencing system in the protoplasts of Nicotiana benthamiana to investigate the functions of viral suppressors at the cellular level. We showed that RNA silencing against a green fluorescent protein (GFP) reporter gene in the protoplasts could be induced rapidly and specifically by co-transfection with the reporter gene and various silencing inducers [i.e. siRNA, double-stranded RNA (dsRNA) or plasmid encoding dsRNA]. Using this system, we uncovered novel roles of some viral suppressors. Notably, the Cucumber mosaic virus 2b protein, shown previously to function predominantly by preventing the long-distance transmission of systemic silencing signals, was a very strong silencing suppressor in the protoplasts. Some suppressors thought to interfere with upstream steps of siRNA production appeared to also act downstream. Therefore, a viral suppressor can affect multiple steps of the RNA silencing pathway. Our analyses suggest that protoplast-based transient RNA silencing is a useful experimental system to investigate the functions of viral suppressors and further dissect the mechanistic details of the RNA silencing pathway in single cells.     

Cell interactions in the differentiation of a melanotic tumor in Drosophila.

The cellular events in the formation of melanotic tumors in the tu-W mutant larva of Drosophila melanogaster are described. The first step is the differentiation of spherical hemocytes to flattened cells, the lamellocyte variants. Subsequently, the surface of the caudal fat body undergoes changes to which the hemocytes respond by forming cellular capsules. The hemocytes utilize two mechanisms in this process: (1) phagocytosis of small particulate materials escaping from the adipose cells, (2) adhesion to form a multilayered wall of lamellocytes. Differentiating hemocytes in the vicinity of the tumor-forming site extrude membrane-bound vesicles that tend to adhere to the hemocyte surfaces. These vesicles are trapped between the lamellocytes as they pile in layers to form the capsule wall. It is suggested that the vesicles play a role in lamellocyte-to-lamellocyte adhesion during the initial stages of hemocyte aggregation at the tumor-forming site.     

Cell Interactions in the Differentiation of a Melanotic Tumor in Drosophila

The cellular events in the formation of melanotic tumors in the tu-W mutant larva of Drosophila melanogaster are described. The first step is the differentiation of spherical hemocytes to flattened cells, the lamellocyte variants. Subsequently, the surface of the caudal fat body undergoes changes to which the hemocytes respond by forming cellular capsules. The hemocytes utilize two mechanisms in this process: (1) phagocytosis of small participate materials escaping from the adipose cells, (2) adhesion to form a multilayered wall of lamellocytes.
Differentiating hemocytes in the vicinity of the tumor-forming site extrude membrane-bound vesicles that tend to adhere to the hemocyte surfaces. These vesicles are trapped between the lamellocytes as they pile in layers to form the capsule wall. It is suggested that the vesicles play a role in lamellocyte-to-lamellocyte adhesion during the initial stages of hemocyte aggregation at the tumorforming site.     

Developmental analysis of a temperature-sensitive melanotic tumor mutant inDrosophila melanogaster

Summary A sex-linked, temperature-sensitive melanotic tumor mutation inDrosophila melanogaster, tu (1) Sz ^ts, was mapped at 34.3±and localized to bands 10A10-11 of the polytene chromosomes. At 26°Ctu-Sz ^ts larvae develop melanotic tumors whereas 18°C is non-permissive for tumor formation. Tumorigenesis at 26°C involves the encapsulation of abnormal caudal fat body regions by precociously differentiated hemocytes. Low temperature blocks the development of the abnormal adipose cells and the overlying aberrant tissue surfaces but does not inhibit precocious differentiation of the hemocytes to the lamellocytic form. This phenotypic difference at the two temperatures indicates that lamellocyte encapsulation to form melanotic tumors is directed against abnormal tissue surfaces. On the basis of these observations and an earlier study (Rizki and Rizki 1979) we propose that hereditary melanotic tumors inD. melanogaster are a calss of autoimmune disorders in which affected tissue surfaces arouse the body's cellmediated defense response.     

Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells

Double-stranded RNA (dsRNA) fragments are readily internalized and processed by Drosophila S2 cells, making these cells a widely used tool for the analysis of gene function by gene silencing through RNA interference (RNAi). The underlying mechanisms are insufficiently understood. To identify components of the RNAi pathway in S2 cells, we developed a screen based on rescue from RNAi-induced lethality. We identified Argonaute 2, a core component of the RNAi machinery, and three gene products previously unknown to be involved in RNAi in Drosophila: DEAD-box RNA helicase Belle, 26 S proteasome regulatory subunit 8 (Pros45), and clathrin heavy chain, a component of the endocytic machinery. Blocking endocytosis in S2 cells impaired RNAi, suggesting that dsRNA fragments are internalized by receptor-mediated endocytosis. Indeed, using a candidate gene approach, we identified two Drosophila scavenger receptors, SR-CI and Eater, which together accounted for more than 90% of the dsRNA uptake into S2 cells. When expressed in mammalian cells, SR-CI was sufficient to mediate internalization of dsRNA fragments. Our data provide insight into the mechanism of dsRNA internalization by Drosophila cells. These results have implications for dsRNA delivery into mammalian cells.     

Novel DNA binding proteins highly specific to UV-damaged DNA sequences from embryos of Drosophila melanogaster.

Three new proteins which selectively bind to UV-damaged DNA were identified and purified to near homogeneity from UV-irradiated Drosophila melanogaster embryos through several column chromatographies. These proteins, tentatively designated as D-DDB P1, P2 and P3, can be identified as different complex bands in a gel shift assay by using UV-irradiated TC-31 probe DNA. Analysis of the purified D-DDB P1 fraction by native or SDS-polyacrylamide gel electrophoresis and FPLC-Superose 6 gel filtration demonstrated that it is a monomer protein which is a 30 kDa polypeptide. The D-DDB P2 protein is a monopolypeptide with a molecular mass of 14 kDa. Both D-DDB P1 and P2 highly prefer binding to UV-irradiated DNA, and have almost no affinity for non-irradiated DNA. Gel shift assays with either UV-irradiated DNA probes demonstrated that D-DDB P1 may show a preference for binding to (6-4) photoproducts, while D-DDB P2 may prefer binding to pyrimidine dimers. Both these proteins require magnesium ions for binding. D-DDB P1 is an ATP-preferent protein. These findings are discussed in relation to two recently described [Todo and Ryo (1991) Mutat. Res., 273, 85-93; Todo et al. (1993) Nature, 361, 371-374] DNA-binding factors from Drosophila cell extracts. A possible role for these DNA-binding proteins in lesion recognition and DNA-binding proteins in lesion recognition and DNA repair of UV-induced photo-products is discussed.     

利用RNAi技术研究果蝇心脏发育基因的功能

RNAi是近两年发展起来的一种阻抑基因表达的新方法。它通过导入一段与内源基因同源的双链RNA序列（dsRNA）,使内源mRNA降解,从而达到阻抑基因表达的目的。目前已在线虫、果蝇、臭虫、真菌及植物等生物中建立RNAi技术,用于研究某些特定基因或已知基因在特定发育时期的功能。对于难于获得突变体的基因或生物体,RNAi技术尤其有效。虽然果蝇心脏发育基因wingless和tinman在果蝇心脏发育的早期功能已经清楚,它们都与果蝇心脏前体细胞的形成有关,但它们在果蝇心脏发育的后期功能仍有待进一步研究。实验运用RNAi技术,分别将tinman和wingless的dsRNA注入果蝇的早期胚胎,得到了这两个基因的dsRNA干扰表型,与两个基因的突变体表型非常相似,都表现为果蝇心脏前体细胞不能形成或心脏管缺失。尤其是tinman基因的dsRNA,还引起了肠中胚胎层缺失和体壁肌肉组织的紊乱,而wingless基因的dsRNA却只影响心脏的形成,而不影响肠中胚层,说明dsRNA干扰具有非常强的特异性,因而不失为研究果蝇心脏发育基因功能的有效方法。     

Regulator gene of phenyl oxidase activity in Drosophila melanogaster

A recessive lethal mutation l(2)hemo causing the occurrence of melanotic tumors in homozygous Drosophila larvae was found. The study of phenoloxidase (PO) activity revealed that the number of hemocytes with PO activity in homozygous larvae was significantly reduced (0.4 +/- 0.24%), compared to wild-type larvae (6.3 +/- 0.5%). On injury followed by injection with bacterial cells, the formation of melanotic thrombus did not occurred and hemocytes with PO activity were not recorded in homozygotes of line P103. Suppression of the activity of PO isozymes A1 and A3 was detected by means of electrophoretic analysis of homozygotes. According to gene mapping data, the localization of this mutation did not match any structural gene for known PO forms and is therefore related to a regulatory gene controlling the activity of the immune system of Drosophila.     

Gene silencing of the tick protective antigens, Bm86, Bm91 and subolesin, in the one-host tick Boophilus microplus by RNA interference

The use of RNA interference (RNAi) to assess gene function has been demonstrated in several three-host tick species but adaptation of RNAi to the one-host tick, Boophilus microplus, has not been reported. We evaluated the application of RNAi in B. microplus and the effect of gene silencing on three tick-protective antigens: Bm86, Bm91 and subolesin. Gene-specific double-stranded (dsRNA) was injected into two tick stages, freshly molted unfed and engorged females, and specific gene silencing was confirmed by real time PCR. Gene silencing occurred in injected unfed females after they were allowed to feed. Injection of dsRNA into engorged females caused gene silencing in the subsequently oviposited eggs and larvae that hatched from these eggs, but not in adults that developed from these larvae. dsRNA injected into engorged females could be detected by quantitative real-time RT-PCR in eggs 14 days from the beginning of oviposition, demonstrating that unprocessed dsRNA was incorporated in the eggs. Eggs produced by engorged females injected with subolesin dsRNA were abnormal, suggesting that subolesin may play a role in embryonic development. The injection of dsRNA into engorged females to obtain gene-specific silencing in eggs and larvae is a novel method which can be used to study gene function in tick embryogenesis.     

The Drosophila lymph gland as a developmental model of hematopoiesis

Drosophila hematopoiesis occurs in a specialized organ called the lymph gland. In this systematic analysis of lymph gland structure and gene expression, we define the developmental steps in the maturation of blood cells (hemocytes) from their precursors. In particular, distinct zones of hemocyte maturation, signaling and proliferation in the lymph gland during hematopoietic progression are described. Different stages of hemocyte development have been classified according to marker expression and placed within developmental niches: a medullary zone for quiescent prohemocytes, a cortical zone for maturing hemocytes and a zone called the posterior signaling center for specialized signaling hemocytes. This establishes a framework for the identification of Drosophila blood cells, at various stages of maturation, and provides a genetic basis for spatial and temporal events that govern hemocyte development. The cellular events identified in this analysis further establish Drosophila as a model system for hematopoiesis.     

The lethal prune/Killer-of-prune interaction of Drosophila causes a syndrome resembling human neurofibromatosis (NF1).

The eye color mutant prune (pn) of Drosophila melanogaster shows a lethal interaction with the Killer-of-prune (K-pn) allele of the abnormal wing disc (awd) locus. The awd gene is the Drosophila homologue of the mammalian tumor metastasis gene nm23, and it has been postulated that pn encodes a protein with similarity to a GAP, a GTPase-activating protein. Such GAPs potentially control Ras-like proteins, which are important molecular switches. However, there is only a low sequence homology with the genes for human GAP and neurofibromatosis (NF1), and with yeast IRA1 and IRA2, and there is no evidence for the functional significance of this homologization. I now show that pn mutations lower the concentrations of larval pteridines, and that this phenomenon is enhanced by two orders of magnitude by the lethal interaction between pn and awdK-pn. These gradual effects on the pteridin concentrations indicate a corresponding drop of the pools of free GTP, and favor the involvement of GTP-binding proteins. In addition, cytology reveals a considerable hypertrophy of the neuroglia and the perineurium of the larval brain. Furthermore, the lymph glands of the larvae are highly abnormal and form melanotic (pseudo)tumors upon ageing of the larvae. These pseudotumors consist predominantly of lamellocytes which are part of the cellular defence system of Drosophila. These observations most likely indicate hyperactivity of a Ras-like protein which becomes manifest in cell types equivalent to the cell types affected by human neurofibromatosis (NF1). Thus, it is very suggestive to regard the synthetic lethal system prune/Killer-of-prune as the Drosophila model for human neurofibromatosis.