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

Kuleesha Y, Puah WC, Lin F, Wasser M - BMC Bioinformatics (2014)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

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.

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Screenshot of the FMAj user interface. (a) In the left panel, the user can browse through the time-lapse images. Various annotations can be projected onto the images, including anterior-to-posterior and left-to-right body axes, scale bar, time point and contours of segmented ROIs. (b) The top right panel shows image metadata and contains controls for manual annotations. (c) The bottom right panel contains the control elements for manual and semi-automatic segmentation. Annotations like muscle type can be added to selected ROIs. (d) The bottom panel controls view options like color channel display.
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Figure 2: Screenshot of the FMAj user interface. (a) In the left panel, the user can browse through the time-lapse images. Various annotations can be projected onto the images, including anterior-to-posterior and left-to-right body axes, scale bar, time point and contours of segmented ROIs. (b) The top right panel shows image metadata and contains controls for manual annotations. (c) The bottom right panel contains the control elements for manual and semi-automatic segmentation. Annotations like muscle type can be added to selected ROIs. (d) The bottom panel controls view options like color channel display.

Mentions: Figure 1 illustrates the image processing pipeline of FMAj. After starting the application, the user establishes a database connection and selects the root folder of the microscopic images. Once the database is online, the tool automatically downloads the experimental information, such as gene names, stock ids, muscle type, developmental stages and the imaging protocol from the MySQL database. Initially, metadata about image acquisition are extracted from raw image files, while biological details are entered into the database by an expert user. Input images of FMAj are time-series MIPs in RGB format containing two colour channels; with green representing the cytoplasm and red the nuclei of muscle fibers (Figure 2). The tool provides options to view both channels together or separately. The FMAj tool consists of three modules which perform three sequential tasks. The first module captures experimental metadata which are either derived from the images or via manual annotation by the user. An important user input is the definition of the onset of head eversion, which serves as the main reference point (time point = 0 hours) for comparing datasets. The second module performs segmentation of muscle cells and nuclei in a semi-automated fashion. Features of detected ROIs are either calculated (e.g. shape) or annotated by the expert user (e.g. cell nomenclature). The third module performs comparative phenotypic analysis, such as comparing the cell morphology between control and genetically perturbed cells.


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

Kuleesha Y, Puah WC, Lin F, Wasser M - BMC Bioinformatics (2014)

Screenshot of the FMAj user interface. (a) In the left panel, the user can browse through the time-lapse images. Various annotations can be projected onto the images, including anterior-to-posterior and left-to-right body axes, scale bar, time point and contours of segmented ROIs. (b) The top right panel shows image metadata and contains controls for manual annotations. (c) The bottom right panel contains the control elements for manual and semi-automatic segmentation. Annotations like muscle type can be added to selected ROIs. (d) The bottom panel controls view options like color channel display.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4290658&req=5

Figure 2: Screenshot of the FMAj user interface. (a) In the left panel, the user can browse through the time-lapse images. Various annotations can be projected onto the images, including anterior-to-posterior and left-to-right body axes, scale bar, time point and contours of segmented ROIs. (b) The top right panel shows image metadata and contains controls for manual annotations. (c) The bottom right panel contains the control elements for manual and semi-automatic segmentation. Annotations like muscle type can be added to selected ROIs. (d) The bottom panel controls view options like color channel display.
Mentions: Figure 1 illustrates the image processing pipeline of FMAj. After starting the application, the user establishes a database connection and selects the root folder of the microscopic images. Once the database is online, the tool automatically downloads the experimental information, such as gene names, stock ids, muscle type, developmental stages and the imaging protocol from the MySQL database. Initially, metadata about image acquisition are extracted from raw image files, while biological details are entered into the database by an expert user. Input images of FMAj are time-series MIPs in RGB format containing two colour channels; with green representing the cytoplasm and red the nuclei of muscle fibers (Figure 2). The tool provides options to view both channels together or separately. The FMAj tool consists of three modules which perform three sequential tasks. The first module captures experimental metadata which are either derived from the images or via manual annotation by the user. An important user input is the definition of the onset of head eversion, which serves as the main reference point (time point = 0 hours) for comparing datasets. The second module performs segmentation of muscle cells and nuclei in a semi-automated fashion. Features of detected ROIs are either calculated (e.g. shape) or annotated by the expert user (e.g. cell nomenclature). The third module performs comparative phenotypic analysis, such as comparing the cell morphology between control and genetically perturbed cells.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus