Protein synthesis and cell growth are promoted by a pathway consisting of the insulin-like growth factor IGF1, the kinase Akt and the mammalian target of rapamycin ( mTOR). Muscle mass is regulated by balancing protein synthesis and degradation which can either occur via ubiquitin mediated proteolysis or autophagy. In order to discover pharmacological cures for human muscle wasting disorders like cachexia and sarcopenia the regulation of skeletal muscle mass has been studied extensively. Muscle wasting occurs in ageing, immobility and disease. Extending our approach to a genome-wide scale has the potential to identify new genes involved in muscle size regulation. Our in vivo imaging experiments revealed that evolutionarily conserved genes involved in Tor signalling and autophagy, perform similar functions in regulating muscle mass in mammals and Drosophila. We designed a new tool to visualize and quantify morphological changes of muscles in time-lapse images of Drosophila metamorphosis. FMAj enabled us to monitor the progression of atrophic and hypertrophic phenotypes of individual muscles throughout metamorphosis. Reduction of Drosophila Tor (Target of Rapamycin) expression resulted in enhanced atrophy compared to control, while inhibition of the autophagy factor Atg9 caused suppression of atrophy and enlarged muscle fibers of abnormal morphology. We applied our tool to the phenotypic characterization of two atrophy related genes that were silenced by RNA interference. The third module performs comparative quantitative analysis of muscle phenotypes. Users can provide annotations to the detected objects, such as muscle identities and anatomical information. The second module performs segmentation and feature extraction of muscle cells and nuclei. The first module assists in adding annotations to time-lapse datasets, such as genotypes, experimental parameters and temporal reference points, which are used to compare different datasets. The image analysis pipeline of FMAj contains three modules. To quantify the phenotypic effects of gene perturbations, we designed the Fly Muscle Analysis tool (FMAj) which is based on the ImageJ and MySQL frameworks for image processing and data storage, respectively.
We performed targeted gene perturbation in muscles and acquired 3D time-series images of muscles in metamorphosis using laser scanning confocal microscopy. However, time-lapse microscopy generates sizeable image data that require new tools for high throughput image analysis. Thanks to the ability to perform live imaging of muscle development in transparent pupae and the power of genetics, metamorphosis in Drosophila can be used as a model to study the regulation of skeletal muscle mass. During metamorphosis in Drosophila melanogaster, larval muscles undergo two different developmental fates one population is removed by cell death, while the other persistent subset undergoes morphological remodeling and survives to adulthood.
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