Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.
For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae , that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type–related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.
The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts.DOI: http://dx.doi.org/10.7554/eLife.12068.001
Systematic genetic approaches have provided deep insight into the molecular and cellular mechanisms that operate in simple unicellular organisms. For multicellular organisms, however, the pleiotropy of gene function has largely restricted such approaches to the study of early embryogenesis. With the availability of genome-wide transgenic RNA interference (RNAi) libraries in Drosophila, it is now possible to perform a systematic genetic dissection of any cell or tissue type at any stage of the lifespan. Here we apply these methods to define the genetic basis for formation and function of the Drosophila muscle. We identify a role in muscle for 2,785 genes, many of which we assign to specific functions in the organization of muscles, myofibrils or sarcomeres. Many of these genes are phylogenetically conserved, including genes implicated in mammalian sarcomere organization and human muscle diseases.
Localization of bicoid (bcd) messenger RNA to the anterior pole of the Drosophila oocyte requires the exuperantia ( exu), swallow (swa) and staufen (stau) genes. We show here that Swa protein transiently co-localizes with bcd RNA in mid-oogenesis. Swa also localizes to the anterior pole of the oocyte in the absence of bcd RNA. This localization does not require Exu, but depends on intact microtubules. In mutant ovaries with duplicated polarity of microtubules, Swa and bcd RNA are ectopically localized at the posterior pole, as well as being present at the anterior pole. We identify dynein light chain-1 (Ddlc-1), a component of the minus-end-directed microtubule motor cytoplasmic dynein, as a Swa-binding protein. We propose that Swa acts as an adaptor for the dynein complex and thereby enables dynein to transport bcd RNA along microtubules to their minus ends at the anterior pole of the oocyte.
Genetic mutants are invaluable for understanding the development, physiology and behaviour of Drosophila. Modern molecular genetic techniques enable the rapid generation of large numbers of different mutants. To phenotype these mutants sophisticated microscopy techniques are required, ideally allowing the 3D-reconstruction of the anatomy of an adult fly from a single scan. Ultramicroscopy enables up to cm fields of view, whilst providing micron resolution. In this paper, we present ultramicroscopy reconstructions of the flight musculature, the nervous system, and the digestive tract of entire, chemically cleared, drosophila in autofluorescent light. The 3D-reconstructions thus obtained verify that the anatomy of a whole fly, including the filigree spatial organization of the direct flight muscles, can be analysed from a single ultramicroscopy reconstruction. The recording procedure, including 3D-reconstruction using standard software, takes no longer than 30 min. Additionally, image segmentation, which would allow for further quantitative analysis, was performed.
Flying insects oscillate their wings at high frequencies of up to 1,000 Hz and produce large mechanical forces of 80 W per kilogram of muscle. They utilize a pair of perpendicularly oriented indirect flight muscles that contain fibrillar, stretch-activated myofibres. In contrast, all other, more slowly contracting, insect body muscles have a tubular muscle morphology. Here we identify the transcription factor Spalt major (Salm) as a master regulator of fibrillar flight muscle fate in Drosophila. salm is necessary and sufficient to induce fibrillar muscle fate. salm switches the entire transcriptional program from tubular to fibrillar fate by regulating the expression and splicing of key sarcomeric components specific to each muscle type. Spalt function is conserved in insects evolutionarily separated by 280 million years. We propose that Spalt proteins switch myofibres from tubular to fibrillar fate during development, a function potentially conserved in the vertebrate heart--a stretch-activated muscle sharing features with insect flight muscle.
Our results demonstrate that force-resistant attachment enables a stark tension increase in the myotendinous system. Subsequently, this tension increase triggers simultaneous myofibril self-assembly throughout the entire muscle fiber. As myofibril and sarcomeric architecture as well as their molecular components are evolutionarily conserved, we propose a similar tension-based mechanism to regulate myofibrillogenesis in vertebrates.
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