Direct selection of mutants of Drosophila resistant to juvenile hormone analogues

A direct screening technique was devised in which applications of a juvenile hormone analogue (JHA) (RO-20-3600; ENT No. 70357; 6,7-Epoxy-3-methyl-7-ethyl-1-(3,4-[methyl-enedioxy]-(phenoxy)-2-cis/trans-octene), Hoffman-LaRoche) to the food resulted in the death of all the F-1 descendants of mutagenized Drosophila melanogaster males with the exception of those animals which possessed sexlinked mutations affecting the metabolism of JHA. These exceptions would comprise (a), those mutant animals which possess any resistance to the lethal effects of the JHA (JH^R mutants) and/or (b), those mutant animals which have a defective JH metabolism and would normally die but are rescued by the exogenous application of JHA (JH^? mutants). Approximately 95, 850 mutagenized chromosomes were tested in this screen, 99,6% of which were killed by the JHA. Of the surviving 181 fertile males, 6 were found to be JH^R mutants. The statistically significant resistance factors ranged from 1.4X to 21.5X. The analysis of the most resistant mutant strain indicates that it may possibly be a behavioral mutant. The implications of such JH^R mutants are discussed in terms of ‘third generation pesticides’.     

Steroid signaling promotes stem cell maintenance in the Drosophila testis

Stem cell regulation by local signals is intensely studied, but less is known about the effects of hormonal signals on stem cells. In Drosophila, the primary steroid twenty-hydroxyecdysone (20E) regulates ovarian germline stem cells (GSCs) but was considered dispensable for testis GSC maintenance. Male GSCs reside in a microenvironment (niche) generated by somatic hub cells and adjacent cyst stem cells (CySCs). Here, we show that depletion of 20E from adult males by overexpressing a dominant negative form of the Ecdysone receptor (EcR) or its heterodimeric partner ultraspiracle (usp) causes GSC and CySC loss that is rescued by 20E feeding, uncovering a requirement for 20E in stem cell maintenance. EcR and USP are expressed, activated and autonomously required in the CySC lineage to promote CySC maintenance, as are downstream genes ftz-f1 and E75. In contrast, GSCs non-autonomously require ecdysone signaling. Global inactivation of EcR increases cell death in the testis that is rescued by expression of EcR-B2 in the CySC lineage, indicating that ecdysone signaling supports stem cell viability primarily through a specific receptor isoform. Finally, EcR genetically interacts with the NURF chromatin-remodeling complex, which we previously showed maintains CySCs. Thus, although 20E levels are lower in males than females, ecdysone signaling acts through distinct cell types and effectors to ensure both ovarian and testis stem cell maintenance.     

Zinc accumulation in heterozygous mutants of fumble, the pantothenate kinase homologue of Drosophila

Coenzyme A (CoA) functions in the intracellular trafficking of acetyl groups. In humans, mutations in the pantothenate kinase-2 gene, which encodes a key enzyme in CoA biosynthesis, are associated with neurodegeneration and premature death. Diagnosis is based on iron accumulation in the globus pallidus observed by magnetic resonance imaging. We investigated the elemental composition of the fumble mutant, a model of the human disease. Surprisingly, flies carrying a fumble loss-of-function allele had a three-fold increase in total zinc levels per dry weight when compared to control strains, but no change in total iron, copper or manganese levels. Accordingly, zinc supplementation had an adverse impact on the development of fumble mutant larvae, but zinc chelation failed to protect.     

Sensitivity and adaptation in the Drosophila phototransduction and photoreceptor degeneration mutants trp and rdgB

We studied rdgB, a retinal degeneration mutant, and trp, a phototransduction mutant, separately and in combination in Drosophila. First we showed that trp did not block degeneration in white-eyed rdgB mutants. Thus, rdgB was useful in determining the defects which trp caused in the compound eye receptors R7 and R8; this is because rdgB selectively eliminates R1-6 photoreceptors which would, if present, dominate the compound eye responses. R7 and R8 both express the trptransient receptor potential phenotype in trp mutants. The trp mutation does not change receptor spectral sensitivities, nor does it alter the dark stability of R1-6's and R7's metarhodopsins as judged by dark adaptation studies. The dark adaptation is not significantly affected by trp. However, trp slows the dark adaptation of R8 considerably and seems to make the blue-induced inactivation of R1-6 less stable.     

Diversification of muscle types: Recent insights from Drosophila

Myogenesis is a highly conserved process ending up by the formation of contracting muscles. In Drosophila embryos, myogenesis gives rise to a segmentally repeated array of thirty distinct fibres, each of which represents an individual muscle. Since Drosophila offers a large range of genetic tools for easily testing gene functions, it has become one of the most studied and consequently best-described model organisms for muscle development. Over the last two decades, the Drosophila model system has enabled major advances in our understanding of how the initially equivalent mesodermal cells become competent for entering myogenic differentiation and how each distinct type of muscle is specified. Here we present an overview of Drosophila muscle development with a special focus on the diversification of muscle types and the genes that control acquisition of distinct muscle properties.     

DrosophilaCG10527 mutants are resistant to juvenile hormone and its analog methoprene

Juvenile hormone (JH) is critical for development, metamorphosis, and reproduction in insects. While the physiological importance of JH has been appreciated for decades, its biosynthetic pathway and molecular action remain poorly understood. DrosophilaCG10527 encodes a protein with high homology to crustacean farnesoic acid methyltransferase (FAMeT) that converts farnesoic acid to methyl farnesoate (MF), a precursor of JH, but its in vivo functions remain unclear. Here we report that CG10527 is expressed widely in secondary cells in the male accessory glands, in ovarian follicle cells, and in glial cells in the nervous system. Furthermore, CG10527 is expressed abundantly in the corpora allata where JH is synthesized. To understand the physiological functions of CG10527, we generated specific CG10527 deletions. Phenotypic analysis showed that CG10527 null mutants are fully viable and fertile in both sexes, indicating that CG10527 is not essential for survival and fertility. Surprisingly, CG10527 mutants showed no defects in the biosynthesis of MF and JH. However, CG10527 mutants were 3-5 times more resistant than wild-type flies to topically applied MF and JH as well as the JH analog methoprene at both sub-lethal and lethal doses. Taken together, our data indicate that DrosophilaCG10527 plays little, if any, role in JH biosynthesis but may participate in the JH signaling pathway.     

New yeast/E. coli/Drosophila triple shuttle vectors for efficient generation of Drosophila P element transformation constructs

We have generated a set of novel triple shuttle vectors that facilitate the construction of Drosophila-P-element transformations vectors. These YED-vectors allow the insertion of any kind of sequence at any chosen position due to the presence of a yeast casette which ensures replication and allows for homologous recombination in Saccharomyces cerevisiae. As a proof of principle we generated several reporter constructs and tested them in transgenic flies for expression and correct subcellular localization. YED-vectors can be used for many purposes including promoter analysis or the expression of tagged or truncated proteins. Thus, time-consuming conventional restriction site based multi-step cloning procedures can be circumvented by using the new YED-vectors. The new set of triple shuttle vectors will be highly beneficial for the rapid construction of complex Drosophila transformation plasmids.     

Role of svp in Drosophila Pericardial Cell Growth

The Drosophila dorsal vessel is a segmentally repeated linear organ, in which seven-up (svp) is expressed in two pairs of cardioblasts and two pairs of pericardial cells in each segment. Under the control of hedgehog (hh) signaling from the dorsal ectoderm, svp participates in diversifying cardioblast identities within each segment. In this experiment, the homozygous embryos of svp mutants exhibited an increase in cell size of Eve positive pericardial cells (EPCs) and a disarranged expression pattern, while the cardioblasts pattern of svp-lacZ expression was normal. In the meantime, the DA1 muscle founders were absent in some segments in svp mutant embryos, and the dorsal somatic muscle patterning was also severely damaged in the late stage mutant embryos, suggesting that svp is required for the differentiation of Eve-positive pericardial cells and DA1 muscle founders and may have a role in EPC cell growth.     

Stepwise polarisation of the Drosophila follicular epithelium

The function of epithelial tissues is dependent on their polarised architecture, and loss of cell polarity is a hallmark of various diseases. Here we analyse cell polarisation in the follicular epithelium of Drosophila, an epithelium that arises by a mesenchymal-epithelial transition. Although many epithelia are formed by mesenchymal precursors, it is unclear how they polarise. Here we show how lateral, apical, and adherens junction proteins act stepwise to establish polarity in the follicular epithelium. Polarisation starts with the formation of adherens junctions, whose positioning is controlled by combined activities of Par-3, β-catenin, and Discs large. Subsequently, Par-6 and aPKC localise to the apical membrane in a Par-3-dependent manner. Apical membrane specification continues by the accumulation of the Crumbs complex, which is controlled by Par-3, Par-6, and aPKC. Thus, our data elucidate the genetic mechanisms leading to the stepwise polarisation of an epithelium with a mesenchymal origin.     

Use of encapsulated live microalgae to investigate pre-ingestive selection in the oyster Crassostrea gigas

The involvement of algal chemical cues in the pre-ingestive selection of food particles in Crassostrea gigas was studied using a new approach. Live cells of two microalgal species, Nitzschia closterium and Tetraselmis suesica, were separately entrapped in small alginate microcapsules using an emulsification/internal gelation method. Microcapsule size was adjusted to be within the range of particles ingested by oysters. Using this technique, about 80% of microcapsules had a diameter ranging from 21 to 100 μm. The monitoring of entrapped algae showed that phytoplankton cells remained alive and maintained an active growth for at least 24 days. In particle selection bioassays, adult C. gigas were fed a mixture of microcapsules containing the above algae species as well as control empty alginate microcapsules. The comparison of the proportions of each microcapsule type in the diet and in pseudofeces revealed that those containing T. suesica were significantly ingested while those containing N. closterium were preferentially rejected. Since microcapsule material (alginate matrix) prevented physical contacts between algae cells and oyster feeding organs, this study clearly demonstrate that extracellular metabolites produced by microalgae play a crucial role in the pre-ingestive selection of particles in suspension-feeding bivalves.     

A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities

Ubiquilin (UBQLN) plays a crucial role in cellular proteostasis through its involvement in the ubiquitin proteasome system and autophagy. Mutations in the UBQLN2 gene have been implicated in amyotrophic lateral sclerosis (ALS) and ALS with frontotemporal lobar dementia (ALS/FTLD). Previous studies reported a key role for UBQLN in Alzheimer's disease (AD); however, the mechanistic involvement of UBQLN in other neurodegenerative diseases remains unclear. The genome of Drosophila contains a single UBQLN homolog (dUbqn) that shows high similarity to UBQLN1 and UBQLN2; therefore, the fly is a useful model for characterizing the role of UBQLN in vivo in neurological disorders affecting locomotion and learning abilities. We herein performed a phenotypic and molecular characterization of diverse dUbqn RNAi lines. We found that the depletion of dUbqn induced the accumulation of polyubiquitinated proteins and caused morphological defects in various tissues. Our results showed that structural defects in larval neuromuscular junctions, abdominal neuromeres, and mushroom bodies correlated with limited abilities in locomotion, learning, and memory. These results contribute to our understanding of the impact of impaired proteostasis in neurodegenerative diseases and provide a useful Drosophila model for the development of promising therapies for ALS and FTLD.     

Epigenetic regulation of oogenesis and germ stem cell maintenance by the Drosophila histone methyltransferase Eggless/dSetDB1

The Drosophila melanogaster histone lysine methyltransferase (HKMT) Eggless (Egg/dSETDB1) catalyzes methylation of Histone H3 lysine 9 (H3K9), a signature of repressive heterochromatin. Our previous studies showed that H3K9 methylation by Egg is required for oogenesis. Here we analyze a set of EMS-induced mutations in the egg gene, identify the molecular lesions of these mutations, and compare the effects on oogenesis of both strong loss-of-function and weak hypomorphic alleles. These studies show that H3K9 methylation by Egg is required for multiple stages of oogenesis. Mosaic expression experiments show that the egg gene is not required intrinsically in the germ cells for their early differentiation, but is required in the germ cells for their survival past stage 5 of oogenesis. egg is also required in germ stem cells for their maintenance, since egg⁻ germ stem cells initially survive but are not maintained as females age. Mosaic analysis also reveals that the early egg chamber budding defects in egg⁻ ovaries are due to an intrinsic requirement for egg in follicle stem cells and their descendents, and that egg plays a non-autonomous role in somatic cells in the germarium to influence the differentiation of early germ cells.     

Vertebrate Homologue of Drosophila GAGA Factor

Polycomb group (PcG) and trithorax group (trxG) proteins are chromatin-mediated regulators of a number of developmentally important genes including the homeotic genes. In Drosophila melanogaster, one of the trxG members, Trithorax like (Trl), encodes the essential multifunctional DNA binding protein called GAGA factor (GAF). While most of the PcG and trxG genes are conserved from flies to humans, a Trl-GAF homologue has been conspicuously missing in vertebrates. Here, we report the first identification of c-Krox/Th-POK as the vertebrate homologue of GAF on the basis of sequence similarity and comparative structural analysis. The in silico structural analysis of the zinc finger region showed preferential interaction of vertebrate GAF with GAGA sites similar to that of fly GAF. We also show by cross-immunoreactivity studies that both fly and vertebrate GAFs are highly conserved and share a high degree of structural similarity. Electrophoretic mobility shift assays show that vertebrate GAF binds to GAGA sites in vitro. Finally, in vivo studies by chromatin immunoprecipitation confirmed that vertebrate GAF binds to GAGA-rich DNA sequences present in hox clusters. Identification of vertebrate GAF and the presence of its target sites at various developmentally regulated loci, including hox complexes, highlight the evolutionarily conserved components involved in developmental mechanisms across the evolutionary lineage and answer a long-standing question of the presence of vertebrate GAF.     

Behavioral dissection of Drosophila larval phototaxis

A behavior generally comprises multiple processes. Analyzing these processes helps to reveal more characteristics of the behavior. In this report, light/dark choice-based Drosophila larval phototaxis is analyzed with a simplistic mathematical model to reveal a fast phase and a slow phase response that are involved. Larvae of the strain w¹¹¹⁸, which is photophobic in phototaxis tests, prefer darkness to light in an immediate light/dark boundary passing test and demonstrate a significant reduction in motility in the dark condition during phototaxis tests. For tim⁰¹ larvae, which show neutral performance in phototaxis tests, larvae unexpectedly prefer light to darkness in the immediate light/dark boundary passing test and demonstrate no significant motility alteration in the dark condition. It is proposed that Drosophila larval phototaxis is determined by a fast phase immediate light/dark choice and an independent slow phase light/dark-induced motility alteration that follows.     

Effect of myonuclear number and mitochondrial fusion on Drosophila indirect flight muscle organization and size

Mechanisms involved in establishing the organization and numbers of fibres in a muscle are not completely understood. During Drosophila indirect flight muscle (IFM) formation, muscle growth is achieved by both incorporating hundreds of nuclei, and hypertrophy. As a result, IFMs provide a good model with which to understand the mechanisms that govern overall muscle organization and growth. We present a detailed analysis of the organization of dorsal longitudinal muscles (DLMs), a subset of the IFMs. We show that each DLM is similar to a vertebrate fascicle and consists of multiple muscle fibres. However, increased fascicle size does not necessarily change the number of constituent fibres, but does increase the number of myofibrils packed within the fibres. We also find that altering the number of myoblasts available for fusion changes DLM fascicle size and fibres are loosely packed with myofibrils. Additionally, we show that knock down of genes required for mitochondrial fusion causes a severe reduction in the size of DLM fascicles and fibres. Our results establish the organization levels of DLMs and highlight the importance of the appropriate number of nuclei and mitochondrial fusion in determining the overall organization, growth and size of DLMs.     

Kinetics and models of the Drosophila heat-shock system

The puffing of Drosophila heat-shock genes after (1) a step-wise temperature increase (“heat-shock”); (2) recovery from anaerobiosis; and (3) incubation with uncoupling reagents was expressed as percent of the maximal size and normalized to the time scale. Data were taken from the literature and new measurements. In addition, puffing was measured after a 30-min temperature pulse and after two 30-min pulses. The latter experiment revealed a second, smaller increase in puff-size. Data on RNA and protein synthesis in Drosophila cells were collected from the literature and also normalized. From the available data, a feed-back control system is derived that consists of a controlled variable x, possibly a metabolic function of the mitochondria, interacting with an activator molecule which exists in an active (A⁺) and an inactive (A^?) configuration. A⁺ activates the heat-shock genes which in turn produce their mRNA (y) and proteins (z) which then change the controlled variable x into a new steady state. A modified version of this model assumes a feed-back control of the heat-shock proteins on the activator molecule. A mathematical model of this system (Goodwin, 1965) was simulated by computer and compared with the experimental results.