Progress inDrosophila genome manipulation

The introduction of cloned and manipulated genetic material into the germline of an experimental organism is one of the most powerful tools of modern biology. In the case of the fruit fly,Drosophila melanogaster, there is also an unparalleled range of sophisticated genetic tools to facilitate subsequent analysis. In consequence,Drosophila remains a most favourable model organism for the dissection of gene structure and functionin vivo. In this review we look at some of the achievements to date inDrosophila genome manipulation, and at what may be possible in the near future.     

Shifting the paradigm in Evolve and Resequence studies: From analysis of single nucleotide polymorphisms to selected haplotype blocks

For almost a decade the combination of whole genome sequencing with experimental evolution (Evolve and Resequence, E&R; Turner, Stewart, Fields, Rice, & Tarone, 2011) has been used to study adaptation in outcrossing organisms. However, complications caused by inversions and hitchhiking variants have prevented this powerful approach from living up to its potential. In this issue of Molecular Ecology, Michalak, Kang, Schou, Garner, and Loeschke (2018), provide an important step ahead by using a population of Drosophila melanogaster devoid of segregating inversions to identify the genetic basis of resistance to five environmental stressors. They further address the challenge of hitchhiking variants by reconstructing selected haplotype blocks. While it is apparent that the haplotype block reconstruction needs further refinements, their work underpins the potential of E&R studies in Drosophila to address fundamental questions in evolutionary biology.     

Eco-genetics of desiccation resistance in Drosophila

Climate change globally perturbs water circulation thereby influencing ecosystems including cultivated land. Both harmful and beneficial species of insects are likely to be vulnerable to such changes in climate. As small animals with a disadvantageous surface area to body mass ratio, they face a risk of desiccation. A number of behavioural, physiological and genetic strategies are deployed to solve these problems during adaptation in various Drosophila species. Over 100 desiccation-related genes have been identified in laboratory and wild populations of the cosmopolitan fruit fly Drosophila melanogaster and its sister species in large-scale and single-gene approaches. These genes are involved in water sensing and homeostasis, and barrier formation and function via the production and composition of surface lipids and via pigmentation. Interestingly, the genetic strategy implemented in a given population appears to be unpredictable. In part, this may be due to different experimental approaches in different studies. The observed variability may also reflect a rich standing genetic variation in Drosophila allowing a quasi-random choice of response strategies through soft-sweep events, although further studies are needed to unravel any underlying principles. These findings underline that D. melanogaster is a robust species well adapted to resist climate change-related desiccation. The rich data obtained in Drosophila research provide a framework to address and understand desiccation resistance in other insects. Through the application of powerful genetic tools in the model organism D. melanogaster, the functions of desiccation-related genes revealed by correlative studies can be tested and the underlying molecular mechanisms of desiccation tolerance understood. The combination of the wealth of available data and its genetic accessibility makes Drosophila an ideal bioindicator. Accumulation of data on desiccation resistance in Drosophila may allow us to create a world map of genetic evolution in response to climate change in an insect genome. Ultimately these efforts may provide guidelines for dealing with the effects of climate-related perturbations on insect population dynamics in the future.     

Chemically InducedDrosophila melanogaster mutants with Changes in Brain Structure

Neurodegenerative human diseases are caused by nerve cell death and anatomical changes in some brain regions. Molecular genetic studies of Drosophila showed that this organism can serve as a valuable test-system for conserved mechanisms underlying human nervous system disorders. Analysis of brain functions is possible when the mutants with disturbed functions are available. In this study, we have developed a unique collection ofDrosophila melanogaster mutants with morphological and neurodegenerative changes in brain structure, which were induced by chemical mutagens.     

Dopamine Dynamics and Signaling in Drosophila: An Overview of Genes,Drugs and Behavioral Paradigms

Changes in dopamine (DA) signaling have been implicated in a number of human neurologic and psychiatric disorders. Similarly, defects in DA signaling in the fruit fly, Drosophila melanogaster, have also been associated with several behavioral defects. As most genes involved in DA synthesis, transport, secretion, and signaling are conserved between species, Drosophila is a powerful genetic model organism to study the regulation of DA signaling in vivo. In this review, we will provide an overview of the genes and drugs that regulate DA biology in Drosophila. Furthermore, we will discuss the behavioral paradigms that are regulated by DA signaling in flies. By analyzing the genes and neuronal circuits that govern such behaviors using sophisticated genetic, pharmacologic, electrophysiologic, and imaging approaches in Drosophila, we will likely gain a better understanding about how this neuromodulator regulates motor tasks and cognition in humans.     

Microarray methods in Drosophila neurobiology

In the relatively short period since their development, DNA microarrays have been used increasingly in the study of genetic and cellular processes, thereby offering a genome-wide approach to gene expression studies. With the advent of genome sequencing programs for organisms from yeast to man, the number of organisms which now have ready-made commercial arrays continues to increase. Here, the principle of DNA microarrays is introduced, with particular attention being given to the role of this technology in studies of the nervous system of the fruitfly Drosophila melanogaster. The importance of experimental design and sample preparation, in line with minimum information about microarray experiment (MIAME) compliance, is emphasised. The technical platforms available to the Drosophila neurobiologist have been illustrated and a brief number of data analysis tools that are readily available reviewed.     

Casein kinase 2, circadian clocks, and the flight from mutagenic light

Circadian clocks play a fundamental role in biology and disease. Much has been learned about the molecular underpinnings of these biological clocks from genetic studies in model organisms, such as the fruit fly, Drosophila melanogaster. Here we review the literature from our lab and others that establish a role for the protein kinase CK2 in Drosophila clock timing. Among the clock genes described thus far, CK2 is unique in its involvement in plant, fungal, as well as animal circadian clocks. We propose that this reflects an ancient, conserved function for CK2 in circadian clocks. CK2 and other clock genes have been implicated in cellular responses to DNA damage, particularly those induced by ultraviolet (UV) light. The finding of a dual function of CK2 in clocks and in UV responses supports the notion that clocks evolved to assist organisms in avoiding the mutagenic effects of daily sunlight.     

DNA organization and polymorphism of a wild-type Drosophila telomere region

Telomeres at the ends of linear chromosomes of eukaryotes protect the chromosome termini from degradation and fusion. While telomeric replication/elongation mechanisms have been studied extensively, the functions of subterminal sequences are less well understood. In general, subterminal regions can be quite polymorphic, varying in size from organism to organism, and differing among chromosomes within an organism. The subterminal regions of Drosophila melanogaster are not well characterized today, and it is not known which and how many different components they contain. Here we present the molecular characterization of DNA components and their organization in the subterminal region of the left arm of chromosome 2 of the Oregon RC wildtype strain of D. melanogaster, including a minisatellite with a 457 bp repeat length. Two distinct polymorphic arrangements at 2L were found and analyzed, supporting the Drosophila telomere elongation model by retrotransposition. The high incidence of terminal chromosome deficiencies occurring in natural Drosophila populations is discussed in view of the telomere structure at 2L.     

Cholera toxin and pertussis toxin substrates and endogenous ADP-ribosyltransferase activity in Drosophila melanogaster

Cholera toxin- and pertussis toxin-catalyzed ADP-ribosylation were used to identify and localize G protein substrates in Drosophila melanogaster and in Manduca sexta. Cholera toxin catalyzes ADP-ribosylation of 37 kDa and 50 kDa polypeptides, but these polypeptides are also substrates for an ADP-ribosyltransferase (EC 2.4.2.30) activity endogenous to the Drosophila extracts. Pertussis toxin modifies 37 kDa and 39 kDa polypeptides in Drosophila homogenates. The pattern of proteolysis of the 39 kDa pertussis toxin substrate is similar to that of mammalian G_o and is influenced by guanyl nucleotide binding. The 39 kDa G_o-like Drosophila and Manduca pertussis toxin substrates are found primarily in neural tissues. These studies provide further evidence that G proteins are present in Drosophila and that this organism can therefore be used to investigate the physiological roles of these enzymes using advanced genetic manipulations.     

Host Species and Environmental Effects on Bacterial Communities Associated with Drosophila in the Laboratory and in the Natural Environment

The fruit fly Drosophila is a classic model organism to study adaptation as well as the relationship between genetic variation and phenotypes. Although associated bacterial communities might be important for many aspects of Drosophila biology, knowledge about their diversity, composition, and factors shaping them is limited. We used 454-based sequencing of a variable region of the bacterial 16S ribosomal RNA gene to characterize the bacterial communities associated with wild and laboratory Drosophila isolates. In order to specifically investigate effects of food source and host species on bacterial communities, we analyzed samples from wild Drosophila melanogaster and D. simulans collected from a variety of natural substrates, as well as from adults and larvae of nine laboratory-reared Drosophila species. We find no evidence for host species effects in lab-reared flies; instead, lab of origin and stochastic effects, which could influence studies of Drosophila phenotypes, are pronounced. In contrast, the natural Drosophila–associated microbiota appears to be predominantly shaped by food substrate with an additional but smaller effect of host species identity. We identify a core member of this natural microbiota that belongs to the genus Gluconobacter and is common to all wild-caught flies in this study, but absent from the laboratory. This makes it a strong candidate for being part of what could be a natural D. melanogaster and D. simulans core microbiome. Furthermore, we were able to identify candidate pathogens in natural fly isolates.     

Genetics of host–parasite interactions: towards a comprehensive dissection of Drosophila resistance to viral infection

One of the major challenges in evolutionary biology is to unravel the genetic basis of adaptation. This issue has been gaining momentum in recent years with the accelerated development of novel genetic and genomic techniques and resources. In this issue of Molecular Ecology, Cogni et al. (2016) address the genetic basis of resistance to two viruses in Drosophila melanogaster using a panel of recombinant inbred lines with unprecedented resolution allowing detection of rare alleles and/or alleles of small effect. The study confirms the role of previously identified genes of major effect and adds novel regions with minor effect to the genetic basis of Drosophila resistance to the Drosophila C virus or the sigma virus. Additional analyses reveal the absence of cross‐resistance and of epistasis between the various genomic regions. This detailed information on the genetic architecture of host resistance constitutes an important step towards the understanding of both the physiology of antiviral immunity and the evolution of host–parasite interactions.     

Pneumocystis carinii: Genetic Diversity and Cell Biology

As an important opportunistic pulmonary pathogen, Pneumocystis carinii has been the focus of extensive research over the decades. The use of laboratory animal models has permitted a detailed understanding of the host–parasite interaction but an understanding of the basic biology of P. carinii has lagged due in large part to the inability of the organism to grow well in culture and to the lack of a tractable genetic system. Molecular techniques have demonstrated extensive heterogeneity among P. carinii organisms isolated from different host species. Characterization of the genes and genomes of the Pneumocystis family has supported the notion that the family comprises different species rather than strains within the genus Pneumocystis and contributed to the understanding of the pathophysiology of infection. Many of the technical obstacles in the study of the organisms have been overcome in the past decade and the pace of research into the basic biology of the organism has accelerated. Biochemical pathways have been inferred from the presence of key enzyme activities or gene sequences, and attempts to dissect cellular pathways have been initiated. The Pneumocystis genome project promises to be a rich source of information with regard to the functional activity of the organism and the presence of specific biochemical pathways. These advances in our understanding of the biology of this organism should provide for future studies leading to the control of this opportunistic pathogen.     

Glycomic studies of Drosophila melanogaster embryos

With the complete genome sequence of Drosophila melanogaster defined a systematic approach towards understanding the function of glycosylation has become possible. Structural assignment of the entire Drosophila glycome during specific developmental stages could provide information that would shed further light on the specific roles of different glycans during development and pinpoint the activity of certain glycosyltransferases and other glycan biosynthetic genes that otherwise might be missed through genetic analyses. In this paper the major glycoprotein N- and O-glycans of Drosophila embryos are described as part of our initial undertaking to characterize the glycome of Drosophila melanogaster. The N-glycans are dominated by high mannose and paucimannose structures. Minor amounts of mono-, bi- and tri-antennary complex glycans were observed with GlcNAc and Galβ1–4GlcNAc non-reducing end termini. O-glycans were restricted to the mucin-type core 1 Galβ1-3GalNAc sequence.     

Genetics on the Fly: A Primer on the Drosophila Model System

Fruit flies of the genus Drosophila have been an attractive and effective genetic model organism since Thomas Hunt Morgan and colleagues made seminal discoveries with them a century ago. Work with Drosophila has enabled dramatic advances in cell and developmental biology, neurobiology and behavior, molecular biology, evolutionary and population genetics, and other fields. With more tissue types and observable behaviors than in other short-generation model organisms, and with vast genome data available for many species within the genus, the fly’s tractable complexity will continue to enable exciting opportunities to explore mechanisms of complex developmental programs, behaviors, and broader evolutionary questions. This primer describes the organism’s natural history, the features of sequenced genomes within the genus, the wide range of available genetic tools and online resources, the types of biological questions Drosophila can help address, and historical milestones.     

Probing the enigma: unraveling glial cell biology in invertebrates

Despite their predominance in the nervous system, the precise ways in which glial cells develop and contribute to overall neural function remain poorly defined in any organism. Investigations in simple model organisms have identified remarkable morphological, molecular, and functional similarities between invertebrate and vertebrate glial subtypes. Invertebrates like Drosophila and Caenorhabditis elegans offer an abundance of tools for in vivo genetic manipulation of single cells or whole populations of glia, ease of access to neural tissues throughout development, and the opportunity for forward genetic analysis of fundamental aspects of glial cell biology. These features suggest that invertebrate model systems have high potential for vastly improving the understanding of glial biology. This review highlights recent work in Drosophila and other invertebrates that reveal new insights into basic mechanisms involved in glial development.     

The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells

Drosophila melanogaster is a widely used model organism for the molecular dissection of mitosis in animals. However, despite the popularity of this system, no studies have been published on the ultrastructure of Drosophila kinetochores and kinetochore fibers (K-fibers) in somatic cells. To amend this situation, we used correlative light (LM) and electron microscopy (EM) to study kinetochores in cultured Drosophila S2 cells during metaphase, and after colchicine treatment to depolymerize all microtubules (MTs). We find that the structure of attached kinetochores in S2 cells is indistinct, consisting of an amorphous inner zone associated with a more electron-dense peripheral surface layer that is approximately 40–50 nm thick. On average, each S2 kinetochore binds 11±2 MTs, in contrast to the 4–6 MTs per kinetochore reported for Drosophila spermatocytes. Importantly, nearly all of the kinetochore MT plus ends terminate in the peripheral surface layer, which we argue is analogous to the outer plate in vertebrate kinetochores. Our structural observations provide important data for assessing the results of RNAi studies of mitosis, as well as for the development of mathematical modelling and computer simulation studies in Drosophila and related organisms.Electronic supplementary material Supplementary material is available for this article at and is accessible to authorized users.     

Rab11 regulates JNK and Raf/MAPK‐ERK signalling pathways during Drosophila wing development

Developmental signalling pathways are regulated by intracellular vesicle trafficking in multicellular organisms. In our earlier communication, we have shown that mutation in Rab11 (a subfamily of the Ypt/Rab gene family) results in the activation of JNK signalling pathways in Drosophila eye. Here, we report that Rab11 regulates JNK and Raf/MAPK‐ERK signalling pathways during Drosophila wing development. Using immunofluorescence and immunohistochemical analyses, we show that overexpression of Rab11 in mutant wing imaginal disc cells triggers the induction of apoptosis and activation of JNK and ERK. Further, using a genetic approach it has been shown that Rab11 interacts with the components of these pathways during Drosophila wing development. In addition to this, in Rab11 mutant wing imaginal discs JNK activity was monitored using puc^E69, a P‐lacZ enhancer‐trap line inserted in puckered (puc). A strong induction of puc in Rab11 mutant wing imaginal disc cells provided a strong support that Rab11 regulates the JNK signalling pathway during Drosophila wing development.     

BMP signaling dynamics in the follicle cells of multiple Drosophila species

The dorsal anterior region of the follicle cells (FCs) in the developing Drosophila egg gives rise to the respiratory eggshell appendages. These tubular structures display a wide range of qualitative and quantitative variations across Drosophila species, providing a remarkable example of a rapidly evolving morphology. In D. melanogaster, the bone morphogenetic protein (BMP) signaling pathway is an important regulator of FCs patterning and dorsal appendages morphology. To explore the mechanisms underlying the diversification of eggshell patterning, we analyzed BMP signaling in the FCs of 16 Drosophila species that span 45 million years of evolution. We found that the spatial patterns of BMP signaling in the FCs are dynamic and exhibit a range of interspecies' variations. In most of the species examined, the dynamics of BMP signaling correlate with the expression of the type I BMP receptor thickveins (tkv). This correlation suggests that interspecies' variations of tkv expression are responsible for the diversification of BMP signaling during oogenesis. This model was supported by genetic manipulations of tkv expression in the FCs of D. melanogaster that successfully recapitulated the signaling diversities found in the other species. Our results suggest that regulation of receptor expression mediates spatial diversification of BMP signaling in Drosophila oogenesis, and they provide insight into a mechanism underlying the evolution of eggshell patterning.     

In situ mechanical analysis of myofibrillar perturbation and aging on soft, bilayered Drosophila myocardium

Drosophila melanogaster is a genetically malleable organism with a short life span, making it a tractable system in which to study mechanical effects of genetic perturbation and aging on tissues, e.g., impaired heart function. However, Drosophila heart-tube studies can be hampered by its bilayered structure: a ventral muscle layer covers the contractile cardiomyocytes. Here we propose an atomic force microscopy-based analysis that uses a linearized-Hertz method to measure individual mechanical components of soft composite materials. The technique was verified using bilayered polydimethylsiloxane. We further demonstrated its biological utility via its ability to resolve stiffness changes due to RNA interference to reduce myofibrillar content or due to aging in Drosophila myocardial layers. This protocol provides a platform to assess the mechanics of soft biological composite systems and, to our knowledge, for the first time, permits direct measurement of how genetic perturbations, aging, and disease can impact cardiac function in situ.