Assessment of Chemical Toxicity in Adult Drosophila Melanogaster.

Human industries generate hundreds of thousands of chemicals, many of which have not been adequately studied for environmental safety or effects on human health. This deficit of chemical safety information is exacerbated by current testing methods in mammals that are expensive, labor-intensive, and time-consuming. Recently, scientists and regulators have been working to develop new approach methodologies (NAMs) for chemical safety testing that are cheaper, more rapid, and reduce animal suffering. One of the key NAMs to emerge is the use of invertebrate organisms as replacements for mammalian models to elucidate conserved chemical modes of action across distantly related species, including humans. To advance these efforts, here, we describe a method that uses the fruit fly, Drosophila melanogaster, to assess chemical safety. The protocol describes a simple, rapid, and inexpensive procedure to measure the viability and feeding behavior of exposed adult flies. In addition, the protocol can be easily adapted to generate samples for genomic and metabolomic approaches. Overall, the protocol represents an important step forward in establishing Drosophila as a standard model for use in precision toxicology.

FlyBase 2.0: the next generation.

FlyBase (flybase.org) is a knowledge base that supports the community of researchers that use the fruit fly, Drosophila melanogaster, as a model organism. The FlyBase team curates and organizes a diverse array of genetic, molecular, genomic, and developmental information about Drosophila. At the beginning of 2018, 'FlyBase 2.0' was released with a significantly improved user interface and new tools. Among these important changes are a new organization of search results into interactive lists or tables (hitlists), enhanced reference lists, and new protein domain graphics. An important new data class called 'experimental tools' consolidates information on useful fly strains and other resources related to a specific gene, which significantly enhances the ability of the Drosophila researcher to design and carry out experiments. With the release of FlyBase 2.0, there has also been a restructuring of backend architecture and a continued development of application programming interfaces (APIs) for programmatic access to FlyBase data. In this review, we describe these major new features and functionalities of the FlyBase 2.0 site and how they support the use of Drosophila as a model organism for biological discovery and translational research.

Model organism data evolving in support of translational medicine.

Model organism databases (MODs) have been collecting and integrating biomedical research data for 30 years and were designed to meet specific needs of each model organism research community. The contributions of model organism research to understanding biological systems would be hard to overstate. Modern molecular biology methods and cost reductions in nucleotide sequencing have opened avenues for direct application of model organism research to elucidating mechanisms of human diseases. Thus, the mandate for model organism research and databases has now grown to include facilitating use of these data in translational applications. Challenges in meeting this opportunity include the distribution of research data across many databases and websites, a lack of data format standards for some data types, and sustainability of scale and cost for genomic database resources like MODs. The issues of widely distributed data and application of data standards are some of the challenges addressed by FAIR (Findable, Accessible, Interoperable, and Re-usable) data principles. The Alliance of Genome Resources is now moving to address these challenges by bringing together expertly curated research data from fly, mouse, rat, worm, yeast, zebrafish, and the Gene Ontology consortium. Centralized multi-species data access, integration, and format standardization will lower the data utilization barrier in comparative genomics and translational applications and will provide a framework in which sustainable scale and cost can be addressed. This article presents a brief historical perspective on how the Alliance model organisms are complementary and how they have already contributed to understanding the etiology of human diseases. In addition, we discuss four challenges for using data from MODs in translational applications and how the Alliance is working to address them, in part by applying FAIR data principles. Ultimately, combined data from these animal models are more powerful than the sum of the parts.

A Short History and Description of Drosophila melanogaster Classical Genetics: Chromosome Aberrations, Forward Genetic Screens, and the Nature of Mutations.

The purpose of this chapter in FlyBook is to acquaint the reader with the Drosophila genome and the ways in which it can be altered by mutation. Much of what follows will be familiar to the experienced Fly Pusher but hopefully will be useful to those just entering the field and are thus unfamiliar with the genome, the history of how it has been and can be altered, and the consequences of those alterations. I will begin with the structure, content, and organization of the genome, followed by the kinds of structural alterations (karyotypic aberrations), how they affect the behavior of chromosomes in meiotic cell division, and how that behavior can be used. Finally, screens for mutations as they have been performed will be discussed. There are several excellent sources of detailed information on Drosophila husbandry and screening that are recommended for those interested in further expanding their familiarity with Drosophila as a research tool and model organism. These are a book by Ralph Greenspan and a review article by John Roote and Andreas Prokop, which should be required reading for any new student entering a fly lab for the first time.

The end of a monolith: Deconstructing the Cnn-Polo interaction.

In Drosophila melanogaster a functional pericentriolar matrix (PCM) at mitotic centrosomes requires Centrosomin-Long Form (Cnn-LF) proteins. Moreover, tissue culture cells have shown that the centrosomal localization of both Cnn-LF and Polo kinase are co-dependent, suggesting a direct interaction. Our recent study found Cnn potentially binds to and is phosphorylated by Polo kinase at 2 residues encoded by Exon1A, the initiating exon of a subset of Cnn isoforms. These interactions are required for the centrosomal localization of Cnn-LF in syncytial embryos and a mutation of either phosphorylation site is sufficient to block localization of both mutant and wild-type Cnn when they are co-expressed. Immunoprecipitation experiments show that Cnn-LF interacts directly with mitotically activated Polo kinase and requires the 2 phosphorylation sites in Exon1A. These IP experiments also show that Cnn-LF proteins form multimers. Depending on the stoichiometry between functional and mutant peptides, heteromultimers exhibit dominant negative or positive trans-complementation (rescue) effects on mitosis. Additionally, following the completion of meiosis, Cnn-Short Form (Cnn-SF) proteins are required for polar body formation in embryos, a process previously shown to require Polo kinase. These findings, when combined with previous work, clearly demonstrate the complexity of cnn and show that a view of cnn as encoding a single peptide is too simplistic.

Comparative analysis of the transcriptome across distant species.

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters.

Diversity of miRNAs, siRNAs, and piRNAs across 25 Drosophila cell lines.

We expanded the knowledge base for Drosophila cell line transcriptomes by deeply sequencing their small RNAs. In total, we analyzed more than 1 billion raw reads from 53 libraries across 25 cell lines. We verify reproducibility of biological replicate data sets, determine common and distinct aspects of miRNA expression across cell lines, and infer the global impact of miRNAs on cell line transcriptomes. We next characterize their commonalities and differences in endo-siRNA populations. Interestingly, most cell lines exhibit enhanced TE-siRNA production relative to tissues, suggesting this as a common aspect of cell immortalization. We also broadly extend annotations of cis-NAT-siRNA loci, identifying ones with common expression across diverse cells and tissues, as well as cell-restricted loci. Finally, we characterize small RNAs in a set of ovary-derived cell lines, including somatic cells (OSS and OSC) and a mixed germline/somatic cell population (fGS/OSS) that exhibits ping-pong piRNA signatures. Collectively, the ovary data reveal new genic piRNA loci, including unusual configurations of piRNA-generating regions. Together with the companion analysis of mRNAs described in a previous study, these small RNA data provide comprehensive information on the transcriptional landscape of diverse Drosophila cell lines. These data should encourage broader usage of fly cell lines, beyond the few that are presently in common usage.

Diversity and dynamics of the Drosophila transcriptome.

Animal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites.

Probing the boundaries of orthology: the unanticipated rapid evolution of Drosophila centrosomin.

The rapid evolution of essential developmental genes and their protein products is both intriguing and problematic. The rapid evolution of gene products with simple protein folds and a lack of well-characterized functional domains typically result in a low discovery rate of orthologous genes. Additionally, in the absence of orthologs it is difficult to study the processes and mechanisms underlying rapid evolution. In this study, we have investigated the rapid evolution of centrosomin (cnn), an essential gene encoding centrosomal protein isoforms required during syncytial development in Drosophila melanogaster. Until recently the rapid divergence of cnn made identification of orthologs difficult and questionable because Cnn violates many of the assumptions underlying models for protein evolution. To overcome these limitations, we have identified a group of insect orthologs and present conserved features likely to be required for the functions attributed to cnn in D. melanogaster. We also show that the rapid divergence of Cnn isoforms is apparently due to frequent coding sequence indels and an accelerated rate of intronic additions and eliminations. These changes appear to be buffered by multi-exon and multi-reading frame maximum potential ORFs, simple protein folds, and the splicing machinery. These buffering features also occur in other genes in Drosophila and may help prevent potentially deleterious mutations due to indels in genes with large coding exons and exon-dense regions separated by small introns. This work promises to be useful for future investigations of cnn and potentially other rapidly evolving genes and proteins.

The generation of chromosomal deletions to provide extensive coverage and subdivision of the Drosophila melanogaster genome.

BACKGROUND: Chromosomal deletions are used extensively in Drosophila melanogaster genetics research. Deletion mapping is the primary method used for fine-scale gene localization. Effective and efficient deletion mapping requires both extensive genomic coverage and a high density of molecularly defined breakpoints across the genome. RESULTS: A large-scale resource development project at the Bloomington Drosophila Stock Center has improved the choice of deletions beyond that provided by previous projects. FLP-mediated recombination between FRT-bearing transposon insertions was used to generate deletions, because it is efficient and provides single-nucleotide resolution in planning deletion screens. The 793 deletions generated pushed coverage of the euchromatic genome to 98.4%. Gaps in coverage contain haplolethal and haplosterile genes, but the sizes of these gaps were minimized by flanking these genes as closely as possible with deletions. In improving coverage, a complete inventory of haplolethal and haplosterile genes was generated and extensive information on other haploinsufficient genes was compiled. To aid mapping experiments, a subset of deletions was organized into a Deficiency Kit to provide maximal coverage efficiently. To improve the resolution of deletion mapping, screens were planned to distribute deletion breakpoints evenly across the genome. The median chromosomal interval between breakpoints now contains only nine genes and 377 intervals contain only single genes. CONCLUSIONS: Drosophila melanogaster now has the most extensive genomic deletion coverage and breakpoint subdivision as well as the most comprehensive inventory of haploinsufficient genes of any multicellular organism. The improved selection of chromosomal deletion strains will be useful to nearly all Drosophila researchers.

The developmental transcriptome of Drosophila melanogaster.

Drosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.

Genome-wide analysis of promoter architecture in Drosophila melanogaster.

Core promoters are critical regions for gene regulation in higher eukaryotes. However, the boundaries of promoter regions, the relative rates of initiation at the transcription start sites (TSSs) distributed within them, and the functional significance of promoter architecture remain poorly understood. We produced a high-resolution map of promoters active in the Drosophila melanogaster embryo by integrating data from three independent and complementary methods: 21 million cap analysis of gene expression (CAGE) tags, 1.2 million RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) reads, and 50,000 cap-trapped expressed sequence tags (ESTs). We defined 12,454 promoters of 8037 genes. Our analysis indicates that, due to non-promoter-associated RNA background signal, previous studies have likely overestimated the number of promoter-associated CAGE clusters by fivefold. We show that TSS distributions form a complex continuum of shapes, and that promoters active in the embryo and adult have highly similar shapes in 95% of cases. This suggests that these distributions are generally determined by static elements such as local DNA sequence and are not modulated by dynamic signals such as histone modifications. Transcription factor binding motifs are differentially enriched as a function of promoter shape, and peaked promoter shape is correlated with both temporal and spatial regulation of gene expression. Our results contribute to the emerging view that core promoters are functionally diverse and control patterning of gene expression in Drosophila and mammals.

The transcriptional diversity of 25 Drosophila cell lines.

Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are "off" and survival/growth pathways "on." Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common "cell line" gene expression pattern.

A new resource for characterizing X-linked genes in Drosophila melanogaster: systematic coverage and subdivision of the X chromosome with nested, Y-linked duplications.

Interchromosomal duplications are especially important for the study of X-linked genes. Males inheriting a mutation in a vital X-linked gene cannot survive unless there is a wild-type copy of the gene duplicated elsewhere in the genome. Rescuing the lethality of an X-linked mutation with a duplication allows the mutation to be used experimentally in complementation tests and other genetic crosses and it maps the mutated gene to a defined chromosomal region. Duplications can also be used to screen for dosage-dependent enhancers and suppressors of mutant phenotypes as a way to identify genes involved in the same biological process. We describe an ongoing project in Drosophila melanogaster to generate comprehensive coverage and extensive breakpoint subdivision of the X chromosome with megabase-scale X segments borne on Y chromosomes. The in vivo method involves the creation of X inversions on attached-XY chromosomes by FLP-FRT site-specific recombination technology followed by irradiation to induce large internal X deletions. The resulting chromosomes consist of the X tip, a medial X segment placed near the tip by an inversion, and a full Y. A nested set of medial duplicated segments is derived from each inversion precursor. We have constructed a set of inversions on attached-XY chromosomes that enable us to isolate nested duplicated segments from all X regions. To date, our screens have provided a minimum of 78% X coverage with duplication breakpoints spaced a median of nine genes apart. These duplication chromosomes will be valuable resources for rescuing and mapping X-linked mutations and identifying dosage-dependent modifiers of mutant phenotypes.

A molecularly defined duplication set for the X chromosome of Drosophila melanogaster.

We describe a molecularly defined duplication kit for the X chromosome of Drosophila melanogaster. A set of 408 overlapping P[acman] BAC clones was used to create small duplications (average length 88 kb) covering the 22-Mb sequenced portion of the chromosome. The BAC clones were inserted into an attP docking site on chromosome 3L using ΦC31 integrase, allowing direct comparison of different transgenes. The insertions complement 92% of the essential and viable mutations and deletions tested, demonstrating that almost all Drosophila genes are compact and that the current annotations of the genome are reasonably accurate. Moreover, almost all genes are tolerated at twice the normal dosage. Finally, we more precisely mapped two regions at which duplications cause diplo-lethality in males. This collection comprises the first molecularly defined duplication set to cover a whole chromosome in a multicellular organism. The work presented removes a long-standing barrier to genetic analysis of the Drosophila X chromosome, will greatly facilitate functional assays of X-linked genes in vivo, and provides a model for functional analyses of entire chromosomes in other species.

Insights into aging through measurements of the Drosophila proteome as a function of temperature.

Drosophila melanogaster is used as a model system to investigate protein changes associated with the aging process under conditions that alter organism lifespan. Changes in the proteome are assessed at various ages in populations of Oregon-R adult males that have mean lifetimes of 47 and 111 days at 28 and 18°C, respectively. Peptide hits detected from strong-cation-exchange and reversed-phase liquid chromatography coupled to tandem mass spectrometry analysis are employed to examine patterns in relative protein expression. Thirty-three proteins were identified as having similar patterns of expression at both temperatures investigated when scaling the organism age to lifespan. In addition, the proteins ferritin 2 light chain homologue and larval serum protein 1ß were identified in relatively high abundance and displayed distinctly different patterns of expression between the two temperatures. Overall, the results support the notion that aspects of the aging process may be preprogrammed at the protein level.

New research resources at the Bloomington Drosophila Stock Center.

The Bloomington Drosophila Stock Center (BDSC) is a primary source of Drosophila stocks for researchers all over the world. It houses over 27,000 unique fly lines and distributed over 160,000 samples of these stocks this past year. This report provides a brief overview of significant recent events at the BDSC with a focus on new stock sets acquired in the past year, including stocks for phiC31 transformation, RNAi knockdown of gene expression, and SNP and quantitative trait loci discovery. We also describe additions to sets of insertions and molecularly defined chromosomal deficiencies, the creation of a new Deficiency Kit, and planned additions of X chromosome duplication sets.

Stable isotope labeling and label-free proteomics of Drosophila parkin null mutants.

Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra and formation of intracytoplasmic Lewy bodies (LBs). Loss-of-function mutations in parkin which encodes an E3 ubiquitin protein ligase contribute to a predominant cause of a familial form of PD termed autosomal recessive juvenile Parkinsonism (AR-JP). Drosophila parkin null mutants display muscle degeneration and mitochondrial dysfunction, providing an animal model to study Parkin-associated molecular pathways in PD. To define protein alterations involved in Parkin pathogenesis, we performed quantitative proteomic analyses of Drosophila parkin null mutants and age-matched controls utilizing both global internal standard technology (GIST) and extracted ion chromatogram peak area (XICPA) label-free approaches. A total of 375 proteins were quantified with a minimum of two peptide identifications from the combination of the XICPA and GIST measurements applied to two independent biological replicates. Sixteen proteins exhibited significant alteration. Seven of the dysregulated proteins are involved in energy metabolism, of which six were down-regulated. All five proteins involved in transporter activity exhibited higher levels, of which larval serum protein 1alpha, larval serum protein 1beta, larval serum protein 1gamma, and fat body protein 1 showed >10-fold up-regulation and substantially higher level of fat body protein 1 was confirmed by Western blot analysis. These findings suggest that abnormalities in energy metabolism and protein transporter activity pathways may be associated with the pathogenesis of Parkin-associated AR-JP.

Centrosomin: a complex mix of long and short isoforms is required for centrosome function during early development in Drosophila melanogaster.

Centrosomin (Cnn) is a required core component in mitotic centrosomes during syncytial development and the presence of Cnn at centrosomes has become synonymous with fully functional centrosomes in Drosophila melanogaster. Previous studies of Cnn have attributed this embryonic function to a single isoform or splice variant. In this study, we present new evidence that significantly increases the complexity of cnn. Rather than a single isoform, Cnn function can be attributed to two unique classes of proteins that comprise a total of at least 10 encoded protein isoforms. We present the initial characterization of a new class of Cnn short isoforms required for centrosome function during gametogenesis and embryogenesis. We also introduce new evidence for a complex mix of Cnn isoforms present during early embryogenesis. Finally, we reexamine cnn mutations, in light of the short isoforms, and find previously overlooked differences attributable to allele-specific mutant phenotypes. This study addresses several questions surrounding Cnn function at the centrosome during embryogenesis and shows that cnn function cannot be ascribed to a single protein.