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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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Genetic perturbations of autophagy and the Tor pathway affect developmental atrophy of persistent muscles. Short hairpin RNAs (UAS-shRNA) and the nuclear UAS-histone-mKO (red) reporter were co-expressed in muscles using the Mef2-Gal4 driver. Another fluorescent reporter MHC-tau-GFP (green) was used to label muscle cell bodies. (a) In a control pupa, persistent muscles in the 3rd abdominal segments undergo atrophy upon head eversion which is defined as time point zero hours. (b) Silencing of Atg9 by RNAi inhibits atrophy, resulting in enlarged muscle fibers compared to control. (c) Silencing of Tor enhances atrophy, leading to thinner fibers. (d) The effects of gene perturbations on muscle fiber diameter can be compared in time-series plots.
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Figure 5: Genetic perturbations of autophagy and the Tor pathway affect developmental atrophy of persistent muscles. Short hairpin RNAs (UAS-shRNA) and the nuclear UAS-histone-mKO (red) reporter were co-expressed in muscles using the Mef2-Gal4 driver. Another fluorescent reporter MHC-tau-GFP (green) was used to label muscle cell bodies. (a) In a control pupa, persistent muscles in the 3rd abdominal segments undergo atrophy upon head eversion which is defined as time point zero hours. (b) Silencing of Atg9 by RNAi inhibits atrophy, resulting in enlarged muscle fibers compared to control. (c) Silencing of Tor enhances atrophy, leading to thinner fibers. (d) The effects of gene perturbations on muscle fiber diameter can be compared in time-series plots.

Mentions: Autophagy, which is negatively regulated by Tor signalling, sequesters cytoplasmic proteins and organelles for lysosomal degradation [31]. In Drosophila metamorphosis, autophagy is believed to control the cell death of larval tissues like salivary gland, midgut and fat bodies [32]. In order to test FMAj in quantifying developmental changes in muscle morphology and explore the genetic control of muscle remodelling, we selected three UAS-shRNA (small hairpin RNA) RNA interference constructs crossed to reporter genes for in vivo time-lapse microscopy and image analysis. The constructs corresponding to Tor, Atg9 and Chromator (Chro) were chosen based on 3 different phenotypes observed in late pupae by stereomicroscopy. Tor RNAi produced smaller, while Atg9 resulted in enlarged muscles (Figure 5). Chro served as control since the muscles were indistinguishable from unperturbed muscles. We acquired 10 time-lapse datasets over 5 days per genotype, each of which contained 240 time points recorded at 30 minute intervals from the prepupal until the pharate adult stage. To monitor developmental changes of DIOMs, we segmented muscle fibers at 5 hours intervals (Figure 5a-c) and determined their areas (not shown) and average diameters (Figure 5d). In mammalian models, cross sectional area or diameter is the main feature to quantify muscle atrophy. In the control animal (Figure 5a), diameter decreased approximately 3-fold from 90 to 30 µm in the first 50 hours AHE. Consistent with its role in promoting growth, Tor silencing (Figure 5c) resulted in smaller muscles throughout metamorphosis, suggesting enhanced atrophy. In contrast, inhibition of the autophagy factor Atg9 did not lead to altered diameter compared to control until 30 hours AHE. An enlargement of the muscle became only apparent in the later stages. Although removal of many larval tissues is believed to be mediated by autophagic cell death, the loss of Atg9 did not cause delay of DEOM histolysis, as was observed in the case of EAST overexpression [9]. The different temporal profiles in muscle remodelling were also seen when comparing populations of muscles from 10 animals per genotype (Figure 6). For each time point, we determined the median diameter of 10-20 muscles. To visualize the range of features, FMAj can display the 25% and 75% percentiles around the median value. Statistical differences between control and the two knockdowns were calculated using the Mann-Whitney U test and plotted beneath the line charts showing muscle diameters. In the case of Atg9 RNAi (Figure 6a) versus control, we observed two phases of remodelling. In the early phase up to 30 hours AHE, when muscle diameters appear similar, the p-values remained above a threshold of 0.01. At 30 hours AHE and later the median diameters diverged, with Atg9 silencing resulting in a suppression of atrophy compared to controls. This divergence was reflected by lower p-values. The time-series plot comparing population medians of controls with Tor RNAi (Figure 6b) revealed a prolonged atrophy phase from 0 to 45 hours AHE in Tor mutants compared to 0-30 hours AHE in controls. As the diameters were discernibly different throughout metamorphosis the p-values remained below the 0.01 threshold. Besides an increase in cell size, Atg9 RNAi also caused a change in shape of muscle cells. From the mid-pupa stage onwards (30 h AHE), we observed that the muscle cells were thicker in central than terminal regions, (Figure 5b) indicating that autophagy is not only attenuated but also unevenly distributed along the longitudinal axis of fibers. In comparison, control muscles were thinner with even diameter (Figure 5a; 70 and 90 hours AHE). To quantify the attenuation of muscle thinning, we calculated elongation, which is defined as the ratio between the difference in length of major and minor axis and length of major axis. In early pupa (0-25 hours AHE) of control and Atg9 RNAi genotypes, elongation showed a steady increase as muscles underwent thinning along their longitudinal axis (Figure 7, top panel). From midpupal stage onwards, the curves of median elongation values continued increasing in controls, while decreasing in Atg9, indicating a suppression of atrophy. This divergence was reflected by a decrease in significance values (Figure 7, bottom panel) determined by the Mann-Whitney U test.


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

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

Genetic perturbations of autophagy and the Tor pathway affect developmental atrophy of persistent muscles. Short hairpin RNAs (UAS-shRNA) and the nuclear UAS-histone-mKO (red) reporter were co-expressed in muscles using the Mef2-Gal4 driver. Another fluorescent reporter MHC-tau-GFP (green) was used to label muscle cell bodies. (a) In a control pupa, persistent muscles in the 3rd abdominal segments undergo atrophy upon head eversion which is defined as time point zero hours. (b) Silencing of Atg9 by RNAi inhibits atrophy, resulting in enlarged muscle fibers compared to control. (c) Silencing of Tor enhances atrophy, leading to thinner fibers. (d) The effects of gene perturbations on muscle fiber diameter can be compared in time-series plots.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Genetic perturbations of autophagy and the Tor pathway affect developmental atrophy of persistent muscles. Short hairpin RNAs (UAS-shRNA) and the nuclear UAS-histone-mKO (red) reporter were co-expressed in muscles using the Mef2-Gal4 driver. Another fluorescent reporter MHC-tau-GFP (green) was used to label muscle cell bodies. (a) In a control pupa, persistent muscles in the 3rd abdominal segments undergo atrophy upon head eversion which is defined as time point zero hours. (b) Silencing of Atg9 by RNAi inhibits atrophy, resulting in enlarged muscle fibers compared to control. (c) Silencing of Tor enhances atrophy, leading to thinner fibers. (d) The effects of gene perturbations on muscle fiber diameter can be compared in time-series plots.
Mentions: Autophagy, which is negatively regulated by Tor signalling, sequesters cytoplasmic proteins and organelles for lysosomal degradation [31]. In Drosophila metamorphosis, autophagy is believed to control the cell death of larval tissues like salivary gland, midgut and fat bodies [32]. In order to test FMAj in quantifying developmental changes in muscle morphology and explore the genetic control of muscle remodelling, we selected three UAS-shRNA (small hairpin RNA) RNA interference constructs crossed to reporter genes for in vivo time-lapse microscopy and image analysis. The constructs corresponding to Tor, Atg9 and Chromator (Chro) were chosen based on 3 different phenotypes observed in late pupae by stereomicroscopy. Tor RNAi produced smaller, while Atg9 resulted in enlarged muscles (Figure 5). Chro served as control since the muscles were indistinguishable from unperturbed muscles. We acquired 10 time-lapse datasets over 5 days per genotype, each of which contained 240 time points recorded at 30 minute intervals from the prepupal until the pharate adult stage. To monitor developmental changes of DIOMs, we segmented muscle fibers at 5 hours intervals (Figure 5a-c) and determined their areas (not shown) and average diameters (Figure 5d). In mammalian models, cross sectional area or diameter is the main feature to quantify muscle atrophy. In the control animal (Figure 5a), diameter decreased approximately 3-fold from 90 to 30 µm in the first 50 hours AHE. Consistent with its role in promoting growth, Tor silencing (Figure 5c) resulted in smaller muscles throughout metamorphosis, suggesting enhanced atrophy. In contrast, inhibition of the autophagy factor Atg9 did not lead to altered diameter compared to control until 30 hours AHE. An enlargement of the muscle became only apparent in the later stages. Although removal of many larval tissues is believed to be mediated by autophagic cell death, the loss of Atg9 did not cause delay of DEOM histolysis, as was observed in the case of EAST overexpression [9]. The different temporal profiles in muscle remodelling were also seen when comparing populations of muscles from 10 animals per genotype (Figure 6). For each time point, we determined the median diameter of 10-20 muscles. To visualize the range of features, FMAj can display the 25% and 75% percentiles around the median value. Statistical differences between control and the two knockdowns were calculated using the Mann-Whitney U test and plotted beneath the line charts showing muscle diameters. In the case of Atg9 RNAi (Figure 6a) versus control, we observed two phases of remodelling. In the early phase up to 30 hours AHE, when muscle diameters appear similar, the p-values remained above a threshold of 0.01. At 30 hours AHE and later the median diameters diverged, with Atg9 silencing resulting in a suppression of atrophy compared to controls. This divergence was reflected by lower p-values. The time-series plot comparing population medians of controls with Tor RNAi (Figure 6b) revealed a prolonged atrophy phase from 0 to 45 hours AHE in Tor mutants compared to 0-30 hours AHE in controls. As the diameters were discernibly different throughout metamorphosis the p-values remained below the 0.01 threshold. Besides an increase in cell size, Atg9 RNAi also caused a change in shape of muscle cells. From the mid-pupa stage onwards (30 h AHE), we observed that the muscle cells were thicker in central than terminal regions, (Figure 5b) indicating that autophagy is not only attenuated but also unevenly distributed along the longitudinal axis of fibers. In comparison, control muscles were thinner with even diameter (Figure 5a; 70 and 90 hours AHE). To quantify the attenuation of muscle thinning, we calculated elongation, which is defined as the ratio between the difference in length of major and minor axis and length of major axis. In early pupa (0-25 hours AHE) of control and Atg9 RNAi genotypes, elongation showed a steady increase as muscles underwent thinning along their longitudinal axis (Figure 7, top panel). From midpupal stage onwards, the curves of median elongation values continued increasing in controls, while decreasing in Atg9, indicating a suppression of atrophy. This divergence was reflected by a decrease in significance values (Figure 7, bottom panel) determined by the Mann-Whitney U test.

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