The authors would like to thank members of the Translational Genomics Group and Kathryn J Hanson for the feed-back and the improvements on this protocol. This project was carried out with research grant SAF2015-64500-R, which includes European Regional Development Funds, awarded to R.A by the Ministerio de Economia y Competitividad.

1. Fixation and resin embedding
<ol>
	<li>Anesthetize the flies with carbon dioxide (CO2) or via hypothermia using an ice block. Use a dissecting microscope (with low magnification to see the whole fly) and scissors to remove the legs, wings, head and the terminal part of abdomen to facilitate the penetration of the fixative. Careful handling of the carcasses is required at this step to avoid the deformation of the thorax. 
	NOTE: Start with at least 12 flies per genotype to ensure a sufficient number of properly processed individuals at the end of the procedure.</li>
	<li>Transfer the carcasses to a 1.5 mL tube on ice (placing a maximum of 6 individuals per tube) containing 200 &#181;L of solution 1. 
	NOTE: The carcasses cannot remain in the solution 1 for more than 20 min.
	<ol>
		<li>For solution 1, mix components in these volume proportions: &#188; 4% paraformaldehyde, &#188; 8% glutaraldehyde, &#188; 0.2 M Na2HPO4 and &#188; 0.2 M NaH2PO4.</li>
	</ol>
	</li>
	<li>Add 200 &#181;L of solution 2 and incubate on ice for 30 min.
	<ol>
		<li>Solution 2: Mix solution 1 and OsO4 in a 1:1 (v/v) proportion. 
		CAUTION: OsO4 is extremely toxic. Manipulate it in a fume hood with appropriate safety and disposal measures.</li>
	</ol>
	</li>
	<li>Remove all the liquid. Add 200 &#181;L of solution 2 and incubate on ice for 1-2 h.</li>
	<li>Remove the solution and dehydrate the samples through a graded ethanol series as follows: once in 30 %, 50%, 70%, 90%, and twice in absolute ethanol; 5 min each. Add 1 mL of ethanol to cover the samples. 
	NOTE: Once the tissue is placed in 90% ethanol, the subsequent steps can be performed at room temperature.</li>
	<li>Incubate the samples twice in propylene oxide for 10 min each incubation. 
	CAUTION: Propylene oxide is toxic, use it in a fume hood.</li>
	<li>Replace the propylene oxide by a 1:1 (v/v) mixture of epoxy resin and propylene oxide and incubate at room temperature overnight. 
	CAUTION: The resin is extremely toxic in a liquid state.The resin is available as a set of four components (A, B, C and D).
	<ol>
		<li>For epoxy resin, add in a disposable beaker 54 g of resin (A), 44.5 g of hardener (B), 2.5 g of accelerator (C) and 10 g of plasticizer (D). Combine all the resin components in a fume hood. Store the resin in aliquots at -20 &#176;C (for up to 6 months) or at -80 &#176;C (for several years).</li>
		<li>To discard the resin, bake it at 70 &#176;C for 24 h because solid resin is not toxic. Discard all the previous solutions in the Heavy Metals Group according to appropriate international procedures, because solutions can contain traces of OsO4.</li>
	</ol>
	</li>
	<li>Replace the previous mixture with 100% resin, and incubate the samples for 4 h.</li>
	<li>Transfer the carcasses to plastic molds containing new resin (one carcass in each well of the mold). Use a sharpened wooden stick or needle to transfer and orient the carcasses in the wells and leave the resin to polymerize overnight in a Pasteur oven at 70 &#176;C. 
	NOTE: To ensure appropriate orientation of the carcasses for posterior trimming and sectioning, the carcasses should be lying on their long side, with the anterior part of the carcass very near to the edge of the well.</li>
</ol>
2. Trimming and Sectioning of Blocks
<ol>
	<li>Remove the polymerized resin blocks from the molds. Use razor blades to trim away the resin, form a trapezoidal shape surrounding the carcass and give appropriate orientation in the microtome. Start sectioning all the carcasses after the transverse suture to obtain comparable results. 
	NOTE: The section must be perpendicular to the anteroposterior axis of the DLMs to trim the sample correctly ( Figure 3).</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56179/56179fig3.jpg" /> 
Figure 3. Dorsoventral sections of resin-embedded adult thoraces of the Myotonic Dystrophy model flies. In (A), the trimming of the sample was not correct as the section in the dorsal-right part of the image was not completely transversal to the muscles. As a consequence, the three muscle bundles on the upper right side seem bigger than the ones of the left side. This mistake would result in an overestimation of the muscle area. (B)&#160;Section from a correctly trimmed resin block. Scale bar: 200 &#181;m.
<ol>
	<li>Cut 1.5-&#181;m thick sections with an ultramicrotome and transfer the sections into water drops over gelatinized slides.
	<ol>
		<li>Obtain the sections from the mesothorax segment for all the samples to obtain comparable results.</li>
	</ol>
	</li>
	<li>Place the slides containing the sections on a heating block at 70-80 &#176;C until the H2O evaporates. At this point, the samples can be observed with the OsO4 contrast (Figure 4A).</li>
</ol>
3. Staining IFM sections
<ol>
	<li>Cover the sections with enough toluidine blue for approximately 30 s on the heating block.
	<ol>
		<li>For toluidine blue solution, dissolve 1% of toluidine blue and 1% of borax in H2O.</li>
	</ol>
	</li>
	<li>Rinse with H2O and leave the slides on a warm plate to evaporate the water. Repeat the staining with toluidine blue if the contrast needs to be enhanced (Figure 4B).</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56179/56179fig4.jpg" /> 
Figure 4. Dorsoventral sections of resin-embedded adult thoraces with different contrast stainings. Both panels show images of DM1 model flies fed with Abp1 as in Figure 2D. (A)&#160;Image of a section with the OsO4 contrast. (B)&#160;Section stained with toluidine blue allows better visualization of the muscle bundles. Scale bar: 200 &#181;m.
4. Mounting IFM sections
<ol>
	<li>Dry the slides with the heating block until the H2O evaporates.</li>
	<li>Put 1 or 2 drops of mounting medium on the sections and put on a coverslip. 
	Caution: Carry out this step in a fume hood because the mounting medium is toxic.</li>
</ol>
5. Acquisition of images and quantification of muscle area
<ol>
	<li>Take the images at low magnification so that the whole set of DLM muscles is in focus. We normally use 10X.
	<ol>
		<li>For statistical analysis take at least 5 serial images per fly and analyze at least 5 flies per experimental group.</li>
	</ol>
	</li>
	<li>Select the image containing the whole muscles (Figure 5A). Check the axis or orientation of the section and discard the samples with inappropriate orientation, which would distort the results (compare Figures 3A/ 3B to distinguish between badly and well-oriented sections, respectively).</li>
	<li>Use ImageJ software to select an area (in pixels) containing only the DLMs (Figure 5A/5B). For comparison between different experimental groups, the reference area to be used would always be the fly with the biggest muscles. In all the images of the different genotypes to compare, select an area containing DLMs of equal dimensions to the reference area (Figure 5B).</li>
	<li>Binarize the images with the Image-J command: Process/Binary/Make binary and delete the areas or pixels that do not correspond to the muscles of interest (Figure 5C).
	<ol>
		<li>In the case that artefactual black spots appear in areas other than muscle, delete them with the command Drawing tools and select the eraser symbol.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56179/56179fig5.jpg" /> 
Figure 5. Image analysis procedure for muscle area determination. (A)&#160;Transversal section of Myotonic Dystrophy type 1 model fly thorax with a rectangle containing the DLM. (B) Selected area with only the indirect flight muscles and the intestine (*). (C)&#160;Binarized image. The black areas correspond to the muscles of interest. The intestine has been erased for an accurate quantification of muscle area. Scale bar: 200 &#181;m.
<ol start="5">
	<li>Quantify percentage of pixels corresponding to muscle tissue (black pixels) out of total with the command: Analyze/Measurement and select the option area fraction. This percentage of area is an estimation of the DLM muscular mass of each fly.</li>
</ol>

<ol>
	<li>Fernandes, J., Bate, M., Vijayraghavan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+indirect+flight+muscles+of+Drosophila.">Development of the indirect flight muscles of Drosophila.</a> Development. 113, 67-77 (1991).</li><li>Fernandes, J. J., Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+the+dorsal+longitudinal+flight+muscles+(DLM)+of+Drosophila:+insights+from+the+ablation+of+larval+scaffolds.">Patterning the dorsal longitudinal flight muscles (DLM) of Drosophila: insights from the ablation of larval scaffolds.</a> Development. 122, 3755-3763 (1996).</li><li>Hartenstein, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atlas+of+Drosophila+Development.">Atlas of Drosophila Development.</a> Atlas Drosoph. Dev. , 1-57 (1993).</li><li>Tomlinson, A., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fate+in+the+Drosophila+ommatidium.">Cell fate in the Drosophila ommatidium.</a> Dev. Biol. 123, 264-275 (1987).</li><li>Griffith, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmium+Tetroxide+And+Its+Applications.">Osmium Tetroxide And Its Applications.</a> Platin Met Rev. 18, 94-96 (1974).</li><li>Bird, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myotonic+Dystrophy+Type+1.">Myotonic Dystrophy Type 1.</a> GeneReviews. 1, 1-23 (1993).</li><li>Bargiela, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+autophagy+and+apoptosis+contribute+to+muscle+atrophy+in+a+myotonic+dystrophy+type+1+Drosophila+model.">Increased autophagy and apoptosis contribute to muscle atrophy in a myotonic dystrophy type 1 Drosophila model.</a> Dis Model Mech. 8, 679-690 (2015).</li><li>Llamusi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscleblind,+BSF+and+TBPH+are+mislocalized+in+the+muscle+sarcomere+of+a+Drosophila+myotonic+dystrophy+model.">Muscleblind, BSF and TBPH are mislocalized in the muscle sarcomere of a Drosophila myotonic dystrophy model.</a> Dis. Model. Mech. 6, 184-196 (2012).</li><li>Garc&#237;a-L&#243;pez, A., Llamus&#237;, B., Orz&#225;ez, M., P&#233;rez-Pay&#225;, E., Artero, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+discovery+of+a+peptide+that+prevents+CUG-RNA+hairpin+formation+and+reverses+RNA+toxicity+in+myotonic+dystrophy+models.">In vivo discovery of a peptide that prevents CUG-RNA hairpin formation and reverses RNA toxicity in myotonic dystrophy models.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 11866-11871 (2011).</li><li>van der Plas, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Dystrophin+is+required+for+integrity+of+the+musculature.">Drosophila Dystrophin is required for integrity of the musculature.</a> Mech. Dev. 124, 617-630 (2007).</li><li>Lloyd, T. E., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann New York Acad Sci. , 1184 (2010).</li><li>Babcock, D. T., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+method+for+accurate+and+rapid+measurement+of+flight+performance+in+Drosophila.">An improved method for accurate and rapid measurement of flight performance in Drosophila.</a> J. Vis. Exp. , e51223 (2014).</li></ol>

No conflicts of interest declared.

It has been demonstrated that Drosophila melanogaster is a useful model to study human neuromuscular diseases7,10,11, including Myotonic Dystrophies, which are characterized by the appearance of muscular atrophy. The protocol presented here is a useful tool for quantifying the muscle degeneration caused by the onset or progression of a particular disease in a fly model. For example, it is also possible to follow and qua...

The thorax of the fruit fly contains two different classes of flight muscles, which are functionally, physiologically and anatomically distinct. These muscles are: the indirect flight muscles (IFM), which are composed of dorso-longitudinal (DLM) and dorso-ventral (DVM) muscles (Figure 1), and the synchronous flight control muscles1,2. These muscles together generate the elevated mechanical power required for flight. The size, distribution and rostro-caudal disposition of the IFMs allow an easy orientation for transversal sectioning3 (Figure 2A). For this reason, we have selected these muscles to study muscle atrophy in Drosophila melanogaster.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56179/56179fig1.jpg" /> 
Figure 1. Diagram of the thorax of the fruit fly showing the indirect flight muscles (IFMs) arrangement. (Left) represents a lateral view and (Right) represents a cross section of the thorax. The IFMs are composed of the Dorso-longitudinal (DLM) muscles (in red) and the Dorso-ventral (DVM) muscles (in green).
Preservation of tissue structure and the control over dorso-ventral axis orientation of histological sections are critical to ensure proper assessment of muscle cross-sectional area. To preserve muscle structure we used a fixation mixture modified from Tomlinson et al.4 . Moreover, because muscles are internal tissues, the impermeability of Drosophila&#39;s exoskeleton is a problem as fixation mixtures cannot penetrate to the target tissues. To circumvent this problem, we removed the fly head, legs, wings and the last two segments of the abdomen to create holes that allowed the fixation mixture to enter. As part of the fixation protocol we included treatment with osmium tetroxide (OsO4)5, which is extensively used because of its ability to fix fats, including triglycerides. OsO4 preserves most structures extremely well, particularly at the cytological level and at the same time provides contrast to the image. After fixation, Drosophila thoraces were embedded in resin for transversal semi-thin sectioning (1.5 &#181;m). For improved contrast, tissue can be additionally stained with toluidine blue. Images of complete thoraces were taken at 10X and muscle area was quantified by binarizing images (of equal dimensions) and quantifying percentage of pixels corresponding to muscle tissue (black pixels) out of total, with ImageJ software.
Modifications on the tissue preparation and fixation mixtures, as the increase of the concentration of OsO4 and glutaraldehyde solution, introduced in this protocol, allowed unique preservation of muscle tissue. This is because the protocol avoids the degradation and deformation of the tissue, making the posterior analysis of the samples more reliable&#160;even in highly atrophic conditions associated with neuromuscular degenerative diseases such as Myotonic Dystrophy (DM). In its more common form, DM type 1, this rare genetic disorder is brought about by expanded CUG repeats in myotonic dystrophy protein kinase (DMPK) transcripts. Mutant DMPK RNA aggregates form&#160;ribonuclear foci that sequester the Muscleblind-like nuclear RNA-binding proteins (MBNL1-3; Muscleblind (Mbl) in Drosophila)6. We generated a Drosophila model of Myotonic Dystrophy by expressing 250 CTG repeats under the muscular myosin heavy chain promoter (Mhc-Gal4). Model flies were flightless with a typical &#39;up-held wings&#39; phenotype and had serious muscle atrophy in their IFMs (Figure 2B). Previous studies performed in our laboratory have shown that determination of the muscle area of IFMs is a reliable method to quantify the effects of different chemical or genetic modifiers of the muscle atrophy in these model flies7. As an example, overexpression of Mbl isoform C in flies expressing the 250 CTG repeats in the muscle, achieved a rescue of muscle area, as Mbl depletion by sequestration is the triggering factor in DM1 pathogenesis8 (Figure 2C). Muscle area was also rescued after feeding the DM model flies with Abp1, a hexapeptide with proven anti-DM1 activity9 (Figure 2D).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56179/56179fig2.jpg" /> 
Figure 2. Quantification of dorsoventral sections of resin-embedded adult thoraces. (A-D) Indirect flight muscles of Drosophila melanogaster with the indicated relevant genotypes. (A) Control flies (yw). (B) Expression of 250 non-coding CTG repeats in muscle (UAS-CTG(250)x) caused a reduction of muscle area in DLMs in comparison to control flies. (C) This muscle atrophy phenotype was rescued by overexpression of Muscleblind (MblC) (UAS-CTG(250)x UAS-MblC) and (D) feeding the model flies with the hexapeptide Abp1 (UAS-CTG(250)x Abp1). In all images the dorsal side is on top. Transgenes were driven to muscle using a Myosin heavy-chain promoter (Mhc)-Gal4. (E) Quantification of percentage of muscle area relative to the control flies&#160;confirmed that the differences were significant. The histogram shows means &#177; S.E.M. **p&#60;0.01 and *p&#60;0.05 (Student&#180;s t-test). Scale bar: 200 &#181;m. <a href="//cloudflare.jove.com/files/ftp_upload/56179/56179fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The method here reported will be of interest to researchers focusing on muscle development, maintenance and aging, disease pathology and drug testing as it provides reliable information about how muscle tissue responds to both endogenous and external factors.

<table><tbody><tr><td>Image-J software</td><td>National Institutes of Health</td><td></td><td>https://imagej.nih.gov/ij/</td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td>Leica UC6</td><td></td></tr><tr><td>Microscope</td><td>Leica</td><td>Leica MZ6</td><td>Bright field technique.</td></tr><tr><td>Razor blades</td><td>Electron Microscopy Sciences</td><td>71970</td><td>Several alternative providers exist.</td></tr><tr><td>Scissors</td><td>World Precision World</td><td>14003</td><td>Several alternative providers exist.</td></tr><tr><td>Embedding molds</td><td>Electron Microscopy Sciences</td><td>70900</td><td>Several alternative providers exist.</td></tr><tr><td>Glutaraldehyde</td><td>Fluka (Sigma)</td><td>49624</td><td>Toxic.</td></tr><tr><td>OsO&lt;sub&gt;4&lt;/sub&gt;</td><td>Polyscience</td><td>0972A</td><td>Extremely toxic.</td></tr><tr><td>Propylene oxide</td><td>Sigma Aldrich</td><td>82320-250ML</td><td>Extremely toxic.</td></tr><tr><td>resin (Durcupan)</td><td>Sigma Aldrich</td><td>44611-44614</td><td>Carcinogenic when it is unpolymerized.</td></tr><tr><td>Toluidine blue</td><td>Panreac</td><td>251176</td><td>Toxic.</td></tr><tr><td>Mountant Medium (DPX)</td><td>Sigma Aldrich</td><td>44581</td><td>Dangerous.</td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>P6148-500G</td><td>Harmful.</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Panreac</td><td>122507</td><td>0.2 M dilution.</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Panreac</td><td>121677</td><td>0.2 M dilution.</td></tr><tr><td>Borax</td><td>Panreac</td><td>3052</td><td>Toxic.</td></tr></tbody></table>

optical cross-sectional muscle area determination of drosophila melanogaster adult indirect flight muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight muscles (IFMs) of flies, specifically the dorso-longitudinal muscles (DLM), as the experimental subject to measure the atrophic phenotype brought about by different genetic causes. In this protocol, we describe how to embed fly thorax muscles for semi thin sectioning, how to obtain a good contrast between muscle and the surrounding tissue, and how to process optical microscope images for semiautomatic acquisition of quantifiable data and analysis. We describe three specific applications of the methodological pipeline. First, we show how the method can be applied to quantify muscle degeneration in a myotonic dystrophy fly model; second, measurement of muscle cross-sectional area can help to identify genes that either promote or prevent muscle atrophy and/or muscle degeneration; third, this protocol can be applied to determine whether a candidate compound is able to significantly modify a given atrophic phenotype induced by a disease-causing mutation or by an environmental trigger.

To quantify whether the overexpression of MblC or the administration of Abp1 had any effect in the atrophic phenotype of the Myotonic Dystrophy fly model we focused on the DLMs, which are part of IFMs (Figure 1). We determined that the model flies, which express 250 non-coding CTG repeats throughout the musculature driven by the Myosin heavy-chain promoter (Mhc)-Gal4, had a 50% reduction of muscle area compared to control flies. In contrast, co-expre...

Watch this Scientific Journal Video about Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles at JoVE.com

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

We report a method to quantify muscle area, which is an indirect method to determine muscle mass in Drosophila adults. We demonstrate the application of our methodology by analyzing the indirect flight muscles in a Drosophila model of Myotonic Dystrophy disease.

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Muscle mass wasting, known as muscle atrophy, is a common phenotype in Drosophila models of neuromuscular diseases. We have used the indirect flight ...

optical-cross-sectional-muscle-area-determination-drosophila

Research

JoVE Journal

Biology

9.0K Views.  University of Valencia. Wij rapporteren een methode om te kwantificeren spier gebied, dat een indirecte methode is om spiermassa in Drosophila volwassenen. We laten zien dat de toepassing van onze methodologie door het analyseren van de indirecte vlucht spieren in een Drosophila model van myotone dystrofie ziekte.

Video: Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

Genetics

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Developmental Biology

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

Detection and Quantification of Single mRNA Molecules in Drosophila

Studying Muscle Transcriptional Dynamics at Single-molecule Scales in Drosophila

Skeletal muscles are large syncytia made up of many bundled myofibers that produce forces and enable body motion. Drosophila is a classical model to study muscle biology. The combination of both Drosophila genetics and advanced omics approaches led to the identification of key conserved molecules that regulate muscle morphogenesis and regeneration. However, the transcriptional dynamics of these molecules and the spatial distribution of their messenger RNA within the syncytia cannot be assessed by conventional methods. Here we optimized an existing single-molecule RNA fluorescence in situ hybridization (smFISH) method to enable the detection and quantification of individual mRNA molecules within adult flight muscles and their muscle stem cells. As a proof of concept, we have analyzed the mRNA expression and distribution of two evolutionary conserved transcription factors, Mef2 and Zfh1/Zeb. We show that this method can efficiently detect and quantify single mRNA molecules for both transcripts in the muscle precursor cells, adult muscles, and muscle stem cells.

Author Spotlight: Investigating mRNA Spatial Distribution in Drosophila Muscle Tissue 

Drosophila is a well-established model for studying key molecules that regulate myogenesis. However, current methods are insufficient to determine mRNA transcriptional dynamics and spatial distribution within syncytia. To address this limitation, we optimized an RNA fluorescence in situ hybridization method allowing the detection and quantification of mRNAs at a single-molecule scale.

Skeletal muscles are large syncytia made up of many bundled myofibers that produce forces and enable body motion. Drosophila is a classical model to study ...

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The well characterized larval neuromuscular junction is often used for such studies. However, rapid larval development followed by muscle histolysis and nervous system remodeling during metamorphosis makes this model problematic for the study of slow age-dependent degenerative changes like those occurring in amyotrophic lateral sclerosis. Alternatively, adult flies live for 90 days and the adult leg can be used to study motor neuron changes over the course of adult lifespan using in vivo fluorescent imaging through the cuticle. Here, we describe a leg dissection technique coupled with immunocytochemistry, which allows for the study of molecular changes at the neuromuscular junction of identified adult leg motor neurons. These techniques can be coupled with a myriad of antibodies labeling both pre- and post-synaptic structures. Together these procedures allow a more complete characterization of slow age-dependent changes in adult flies and can be applied across multiple motor neuron disease models.

Dissection and Immunohistochemistry of the Drosophila Adult Leg to Detect Changes at the Neuromuscular Junction for an Identified Motor Neuron

We describe a dissection technique that preserves the architecture of the neuromuscular junction and enables a detailed immunocytochemical study of motor neurons in the adult Drosophila leg.

Drosophila melanogaster represents a genetically tractable model to study neuronal structure and function, and subsequent changes in disease states. The ...

Neuroscience

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy (OCM)

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical coherence microscopy (OCM) has been demonstrated to be capable of imaging small animal hearts with high spatial resolution and ultrahigh imaging speed. The high image contrast and noninvasive properties make OCM ideal for performing longitudinal studies without requiring tissue dissections or staining. Drosophila has been widely used as a model organism in cardiac developmental studies due to its high number of orthologous human disease genes, its similarity of molecular mechanisms and genetic pathways with vertebrates, its short life cycle, and its low culture cost. Here, the experimental protocols are described for the preparation of Drosophila and optical imaging of the heartbeat with a custom OCM system throughout the life cycle of the specimen. By following the steps provided in this report, transverse M-mode and 3D OCM images can be acquired to conduct longitudinal studies of the Drosophila cardiac morphology and function. The en face and axial sectional OCM images and the heart rate (HR) and cardiac activity period (CAP) histograms, were also shown to analyze the heart structural changes and to quantify the heart dynamics during Drosophila metamorphosis, combined with the videos constructed with M-mode images to trace cardiac activity intuitively. Due to the genetic similarity between Drosophila and vertebrates, longitudinal study of heart morphology and dynamics in fruit flies could help reveal the origins of human heart diseases. The protocol here would provide an effective method to perform a wide range of studies to understand the mechanisms of cardiac diseases in humans.

Drosophila Preparation and Longitudinal Imaging of Heart Function In Vivo Using Optical Coherence Microscopy OCM

Here, the experimental protocols are described for preparing Drosophila at different developmental stages and performing longitudinal optical imaging of Drosophila heartbeats using a custom optical coherence microscopy (OCM) system. The cardiac morphological and dynamical changes can be quantitatively characterized by analyzing the heart structural and functional parameters from OCM images.

Longitudinal study of the heartbeat in small animals contributes to understanding structural and functional changes during heart development. Optical ...

High-Resolution Imaging and Analysis of Drosophila Eye Defects

A Rapid, Simple Workflow for Quantification of External Adult Drosophila Structures

The Drosophila compound eye is a precisely patterned tissue that has revealed molecular mechanisms and biological processes that drive morphogenesis. It is a simple structure of repeating unit eyes, termed ommatidia, that is used to characterize genetic interactions and gene functions. Mutations that affect eye architecture can be easily detected and analyzed; hence, this system is frequently used in under-resourced institutions. Further phenotypic analysis often includes a Scanning Electron Microscope (SEM) to generate high-magnification images suitable for quantitative analysis. However, SEMs are expensive and require costly reagents; sample preparation spans days; and, often, they need full-time staff for sample preparation and instrument maintenance. This limits their utility at under-resourced institutions or during budgetary austerity. In entomology, the use of high-resolution digital imaging technology is a common practice for the identification and characterization of species. This paper describes a method that combines strategies and allows for high-resolution digital imaging of adult Drosophila structures and quantitative analysis using the open software ImageJ. The workflow is a rapid and student-friendly alternative that remedies the limitations of underfunded and under-resourced research facilities with a cost-effective and rapid approach to quantitative phenotypic analysis.

Here, we present a rapid, low-cost, workflow for high-resolution imaging of adult Drosophila eyes to quantify patterning and growth defects. We describe our protocol for sample preparation by point-mounting, high-resolution image acquisition, and image analysis.

The Drosophila compound eye is a precisely patterned tissue that has revealed molecular mechanisms and biological processes that drive morphogenesis. It ...

C. elegans Muscle Area Measurements: A Standardized Method to Quantify Striated Muscle Morphology

Source: Teoh, J. S. et al. <a href="https://www.jove.com/video/59978/quantitative-approaches-for-studying-cellular-structures-organelle">Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in&#160;Caenorhabditis elegans.</a>&#160;J. Vis. Exp.&#160;(2019).
C. elegans have two types of muscles, single sarcomere and multiple sarcomere, or striated muscles.&#160; This video describes the arrangement of the striated body wall muscles, which are responsible for a worm&#8217;s locomotion and includes a sample protocol to measure muscle morphology using image analysis software.

Source: Teoh, J. S. et al. Quantitative Approaches for Studying Cellular Structures and Organelle Morphology in&#160;Caenorhabditis elegans.&#160;J. Vis. ...

Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles

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