Cell cycle phase classification in 3D in vivo microscopy of Drosophila embryogenesis

10th International Conference on Bioinformatics - 1st ISCB Asia Joint Conference 2011, InCoB 2011/ISCB-Asia 2011: Bioinformatics - Proceedings from Asia Pacific Bioinformatics Network (APBioNet)12SUPPL. 13

Live imaging of muscle histolysis in Drosophila metamorphosis

Background: The contribution of programmed cell death (PCD) to muscle wasting disorders remains a matter of debate. Drosophila melanogaster metamorphosis offers the opportunity to study muscle cell death in the context of development. Using live cell imaging of the abdomen, two groups of larval muscles can be observed, doomed muscles that undergo histolysis and persistent muscles that are remodelled and survive into adulthood. Method: To identify and characterize genes that control the decision between survival and cell death of muscles, we developed a method comprising in vivo imaging, targeted gene perturbation and time-lapse image analysis. Our approach enabled us to study the cytological and temporal aspects of abnormal cell death phenotypes. Results: In a previous genetic screen for genes controlling muscle size and cell death in metamorphosis, we identified gene perturbations that induced cell death of persistent or inhibit histolysis of doomed larval muscles. RNA interference (RNAi) of the genes encoding the helicase Rm62 and the lysosomal Cathepsin-L homolog Cysteine proteinase 1 (Cp1) caused premature cell death of persistent muscle in early and mid-pupation, respectively. Silencing of the transcriptional co-repressor Atrophin inhibited histolysis of doomed muscles. Overexpression of dominant-negative Target of Rapamycin (TOR) delayed the histolysis of a subset of doomed and induced ablation of all persistent muscles. RNAi of AMPKα, which encodes a subunit of the AMPK protein complex that senses AMP and promotes ATP formation, led to loss of attachment and a spherical morphology. None of the perturbations affected the survival of newly formed adult muscles, suggesting that the method is useful to find genes that are crucial for the survival of metabolically challenged muscles, like those undergoing atrophy. The ablation of persistent muscles did not affect eclosion of adult flies. Conclusions: Live imaging is a versatile approach to uncover gene functions that are required for the survival of muscle undergoing remodelling, yet are dispensable for other adult muscles. Our approach promises to identify molecular mechanisms that can explain the resilience of muscles to PCD.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

FMAj: a tool for high content analysis of muscle dynamics in Drosophila metamorphosis

Background: 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. 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. However, time-lapse microscopy generates sizeable image data that require new tools for high throughput image analysis. Results: We performed targeted gene perturbation in muscles and acquired 3D time-series images of muscles in metamorphosis using laser scanning confocal microscopy. 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. The image analysis pipeline of FMAj contains three modules. 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 second module performs segmentation and feature extraction of muscle cells and nuclei. Users can provide annotations to the detected objects, such as muscle identities and anatomical information. The third module performs comparative quantitative analysis of muscle phenotypes. We applied our tool to the phenotypic characterization of two atrophy related genes that were silenced by RNA interference. 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. FMAj enabled us to monitor the progression of atrophic and hypertrophic phenotypes of individual muscles throughout metamorphosis. Conclusions: We designed a new tool to visualize and quantify morphological changes of muscles in time-lapse images of Drosophila metamorphosis. 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. Extending our approach to a genome-wide scale has the potential to identify new genes involved in muscle size regulation.ASTAR (Agency for Sci., Tech. and Research, S’pore)Published versio

Quantitative microscopy uncovers ploidy changes during mitosis in live Drosophila embryos and their effect on nuclear size

<div>Supplementary Data for manuscript</div><div>"Quantitative microscopy uncovers ploidy changes during mitosis in live Drosophila embryos and their effect on nuclear size".</div><div>- 4 Movies</div><div>- Custom image analysis software: einSTA, DyVis3D</div><div>- manuals and sample data sets for image analysis software </div><p><b></b></p

Oral Administration and Selective Uptake of Polymeric Nanoparticles in <i>Drosophila</i> Larvae as an <i>in Vivo</i> Model

In this article, <i>Drosophila</i> larvae are applied as an <i>in vivo</i> model to investigate the transport and uptake of polymeric nanoparticles in the larval digestive tract after oral administration. After feeding the larvae with food containing bare and chitosan-coated Poly­(d,l-lactic-<i>co</i>-glycolic acid) (PLGA) nanoparticles encapsulated with BODIPY, time-lapse imaging of live larvae is used to monitor the movement of fluorescent nanoparticles in the anterior, middle, and posterior midgut of the digestive tract. Also, the dissection of the digestive tract enables the analysis of cellular uptake in the midgut. Bare PLGA nanoparticles travel through the whole midgut smoothly while chitosan-coated PLGA nanoparticles have a long retention time in the posterior midgut. We identify that this retention occurs in the posterior segment of the posterior midgut, and it is termed as the retention segment. During transport in the midgut, chitosan-coated nanoparticles pass through the near-neutral anterior midgut and become highly positively charged when entering into the highly acidic middle midgut. After traveling through the near-neutral anterior segment of the posterior midgut, chitosan-coated nanoparticles have a long retention time of ∼10 h in the retention segment, indicating that the chitosan coating greatly enhances mucoadhesive ability and promotes cellular uptake in this part of the midgut. The dynamic behavior of orally administered nanoparticles in <i>Drosophila</i> larvae is in agreement with studies in other animal models. A <i>Drosophila</i> larva has the potential to evolve into a low-cost drug screening model through real time imaging, which will accelerate the development of improved nanoparticle formulations for oral drug delivery