Name: optical cross-sectional muscle area determination of drosophila melanogaster adult indirect flight muscles
Uploaded: 2018-03-31T13:00:00
Description: Watch this Scientific Journal Video about Optical Cross-Sectional Muscle Area Determination of Drosophila Melanogaster Adult Indirect Flight Muscles at JoVE.com

Forfatterne vil gerne takke medlemmerne af translationel genomforskning gruppe og Kathryn J Hanson for feed-back og forbedringerne på denne protokol. Dette projekt blev udført med forskning tilskud SAF2015-64500-R, som omfatter europæiske regionale Udviklingsfond, tildeles Odins af Ministerio de Economia y Competitividad.

1. fiksering og harpiks indlejring
<ol><li>Bedøver fluer med kuldioxid (CO2) eller via hypotermi ved hjælp af en isblok. Bruge en saks og dissekere mikroskop (med lav forstørrelse at se hele flue) til at fjerne benene, vingerne, hoved og den terminale del af maven til at fremme udbredelsen af fiksativ. Omhyggelig håndtering af kroppe er påkrævet på dette trin for at undgå deformering af brystkassen. Bemærk: Start med mindst 12 fluer per genotype at sikre et tilstrækkeligt antal korrekt forarbejd...

<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>

Det er blevet påvist, at Drosophila melanogaster er en nyttig model til at studere menneskets neuromuskulære sygdomme7,10,11, herunder født Dystrophies, som er karakteriseret ved fremkomsten af muskelatrofi. Protokollen præsenteres her er et nyttigt værktøj for kvantificering muskel degeneration forårsaget af debut eller progression af en bestemt sygdom i en flyve model. For eksempel, er det også muligt at følg...

Thorax af frugtflue indeholder to forskellige klasser af flyvningen muskler, som er funktionelt, fysiologisk og anatomisk adskilte. Disse muskler er: indirekte flyvning musklerne (IFM), som er sammensat af dorso-langsgående (DLM) og dorso-ventrale (DVM) muskler (figur 1) og synkrone flyvningen kontrollere musklerne1,2. Disse muskler skabe sammen den forhøjede mekaniske strøm, der kræves for flyvningen. Størrelse, distribution og rostro-caudale disponeringen af IFMs tillader en nem orientering for tværgående skæring3 (figur 2A). Derfor har vi valgt disse muskler til at studere muskelatrofi i Drosophila melanogaster.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56179/56179fig1.jpg"> Figur 1. Diagram af thorax af frugtflue viser de indirekte flyvning muskler (IFMs) arrangement. (Venstre) repræsenterer en lateral udsigt og (til højre) repræsenterer et tværsnit af brystkassen. IFMs er sammensat af Dorso-langsgående (DLM) muskler (i rødt) og Dorso-ventrale (DVM) muskler (i grønt).
Bevarelse af væv struktur og kontrol over dorso-ventrale akse orientering af histologiske sektioner er afgørende for at sikre korrekt vurdering af muskel tværsnitsareal. At bevare muskelstruktur vi brugte en fiksering blanding ændret fra Tomlinson et al. 4 . Desuden, fordi musklerne er indre væv, uigennemtrængelighed af Drosophilaexoskeleton er et problem som fiksering blandinger ikke kan trænge igennem til målvæv. For at omgå dette problem, vi har fjernet den flyve hoved, ben, vinger og de sidste to segmenter af maven til at skabe huller, der tillod fiksering blandingen ind. Som en del af fiksering protokollen vi inkluderet behandling med osmium dinitrogentetraoxid (OsO4)5, som er flittigt brugt på grund af dens evne til at løse fedtstoffer, herunder triglycerider. OsO4 bevarer de fleste strukturer ekstremt godt, især på den cytologiske niveau og samtidig står i kontrast til billedet. Efter fiksering, var Drosophila thoraces indkapslet i harpiks for tværgående semi-tynd skæring (1,5 µm). For forbedret kontrast, kan væv desuden farves med toluidin blå. Billeder af komplet thoraces blev taget på 10 X og muskel område var kvantificeres ved binarizing billeder (af samme dimensioner) og kvantificere procentdel af pixels svarende til muskelvæv (sort pixel) af i alt, med ImageJ software.
Ændringer på væv forberedelse og fiksering blandinger, som forøgelse af koncentrationen af OsO4 og glutaraldehyd løsning, indført i denne protokol, tilladt unikke bevarelse af muskelvæv. Dette skyldes, at protokollen undgår forringelse og deformation af væv, gøre den posteriore analyse af prøverne mere pålidelige selv under meget atrofisk forhold forbundet med neuromuskulære degenerative sygdomme såsom født dystrofi (DM). I sin mere almindelige form, DM type 1, er denne sjældne genetiske lidelse forårsaget af udvidet LBC gentagelser i født dystrofi protein kinase (DMPK) udskrifter. Mutant DMPK RNA aggregater form ribonuclear foci, at udskille Muscleblind-lignende nukleare RNA-bindende proteiner (MBNL1-3; Muscleblind (Mbl) i Drosophila)6. Vi genereret en Drosophila model af født dystrofi udtrykke 250 CTG gentager muskuløs myosin heavy chain promotor (Mhc-Gal4). Model fluer var flightless med en typisk 'op-holdt vinger' fænotype og havde alvorlig muskel atrofi i deres IFMs (figur 2B). Tidligere undersøgelser udført i vores laboratorium har vist, at bestemmelsen af muskel arealet af IFMs er en pålidelig metode til at kvantificere virkningerne af forskellige kemiske eller genetisk modificering af muskelatrofi i disse model flyver7. Som forbillede opnåede overekspression af Mbl isoform C i fluer udtrykker de 250 CTG gentager i musklen, en redning af muskel område, som Mbl udtynding af binding er den udløsende faktor i DM1 patogenese8 (figur 2 c). Muskel område blev også reddet efter fodring DM model flyver med Abp1, en hexapeptide med dokumenteret anti-DM1 aktivitet9 (figur 2D).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56179/56179fig2.jpg"> Figur 2. Kvantificering af dorsoventral dele af harpiks-embedded voksne thoraces. (A-D) indirekte flyvningen muskler af Drosophila melanogaster med de anførte relevante genotyper. A kontrol flyver (yw). (B) udtryk for 250 ikke-kodende CTG gentager i muskel (UAS-CTG(250)x) forårsaget en reduktion af muskel areal i DLMs i forhold til kontrol fluer. (C) denne muskel atrofi fænotype blev reddet af overekspression af Muscleblind (MblC) (UAS-CTG (250) x UAS-MblC) og (D) fodring model flyver med hexapeptide Abp1 (UAS-CTG (250) x Abp1). I alle billeder er den dorsale side på toppen. Transgener blev drevet til muskel ved hjælp af en Myosin heavy chain promotor (Mhc)-Gal4. (E) kvantificering af procentdel af muskel område i forhold til kontrol fluer bekræftet, at forskellene var betydelig. Histogrammet viser middel ± S.E.M. **p< 0,01 og * p < 0,05 (Student´s t-test). Skalalinjen: 200 µm. <a href="//cloudflare.jove.com/files/ftp_upload/56179/56179fig2large.jpg" target="_blank">venligst klik her for at se en større version af dette tal.</a>
Metoden her rapporteret vil være af interesse for forskere med fokus på muskel udvikling, vedligeholdelse og aldring, sygdom patologi og test af lægemidler, da det giver pålidelige oplysninger om, hvordan muskelvæv reagerer på både endogene og eksterne faktorer.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>OsO4</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>Na2HPO4</td>
			<td>Panreac</td>
			<td>122507</td>
			<td>0.2 M dilution.</td>
		</tr>
		<tr>
			<td>NaH2PO4</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

Muskelmasse spilde, er kendt som muskelatrofi, en fælles fænotype i Drosophila modeller af neuromuskulære sygdomme. Vi har brugt de indirekte flyvning muskler (IFMs) af fluer, specielt de dorso-langsgående muskler (DLM), som den eksperimentelle under foranstaltning atrofisk fænotype som følge af forskellige genetiske årsager. I denne protokol, vi beskriver hvor hen til indkapsle flyve thorax muskler for semi tynde skæring, hvordan du kan få en god kontrast mellem muskel og det omgivende væv og sådan behandle optisk mikroskop billeder for semiautomatisk erhvervelse af kvantificerbare data og analyse. Vi beskriver tre specifikke anvendelser af metodologiske rørledningen. Først, vi viser, hvordan metoden kan anvendes til at kvantificere muskel degeneration i født dystrofi flyve model. andet, måling af muskel tværsnitsareal kan hjælpe med at identificere gener, der enten fremmer eller forhindrer muskelatrofi og/eller muskel degeneration; for det tredje, denne protokol kan anvendes til at afgøre, om en kandidat sammensatte er købedygtig væsentligt ændre en given atrofisk fænotype induceret af en sygdomsfremkaldende mutationeller af en miljømæssige udløser.

At kvantificere om overekspression af MblC eller administration af Abp1 havde nogen effekt i atrofisk Fænotypen af født dystrofi flyve model vi fokuserede på de DLMs, som er en del af IFMs (figur 1). Vi har besluttet, at modellen flyver, som express 250 ikke-kodende CTG gentager hele muskulaturen drevet af Myosin heavy-kæde promoter (Mhc)-Gal4, havde en 50% reduktion af muskel areal i forhold til at styre fluer. Derimod Co udtryk af MblC og udvid...

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

Optisk tværsnit muskel område bestemmelse af Drosophila Melanogaster voksen indirekte flyvning muskler

Vi rapporterer en metode til at kvantificere muskel område, som er en indirekte metode til at bestemme muskelmasse i Drosophila voksne. Vi demonstrere anvendelsen af vores metodik ved at analysere den indirekte flyvning musklerne i Drosophila model af født dystrofi sygdom.

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

The overall goal of this procedure is to quantify the muscle area in the Drosophila adult muscles as a means to indirectly quantify muscle mass and correctly size the generation or muscle atrophy. For this purpose, we focused on dorsal longitudinal muscles because of their size, distribution, and also count on this position, a low and easy orientation for transversal sectioning. One, fixation and resin embedding.The first step of the procedure is to anesthetize the flies with CO2, and, with the help of scissors and forceps, remove the legs, the wings, and the terminal part of the abdomen. Secondly, carefully transfer the carcasses, to avoid the deformation of the thorax, to a one point five milliliter tube on ice, placing a maximum of six individuals per tube containing 200 microliters of solution one. The carcasses cannot remain in this solution for more than 20 minutes.Finally, add 200 microliters of solution two and incubate on ice for 30 minutes. After incubation, remove all of the liquid, add 200 microliters of solution two, and incubate the carcasses on ice for one to two hours. The next step is to remove the solution and dehydrate the samples through a graded ethanol series.Incubate the samples twice in propylene oxide for ten minutes each incubation. Then, replace the propylene oxide with a mixture of 500 microliters resin and 500 microliters propylene oxide and incubate the samples at room temperature overnight. The next day, replace the mixture with 100 percent resin and incubate the samples for four hours.Afterwards, transfer the carcasses to plastic molds containing new resin, making sure you put one carcass in each well of the mold. With a wooden stick, orient the carcasses in the wells and let the resin polymerase overnight in a pasteur oven at 70 degrees Celsius. To discard the resin, bake it at 70 degrees Celsius for 24 hours, because solid, baked resin is not toxic.Two, trimming and sectioning of blocks. With the help of razor blades, trim away the resin and form a trapezoidal shape surrounding the carcass to give the appropriate orientation in the microtome. Check in the microscope that the muscles are perfectly oriented.After this, cut one point five micrometer thick sections with an ultramicrotome, and transfer the sections into water drops over gelatinized slides. Finally, place the slides containing the sections on a heating block at 70 to 80 degrees Celsius until all the water evaporates. Three, staining IFM sections.Cover the sections with toluidine blue for approximately 30 seconds on the heating block. Then, rinse in several changes of water and leave the slides on a warm plate to evaporate the water. Four, mounting IFM sections.Taking the slides, put one or two drops of mountant medium on the sections and put on a long coverslip. Five, acquisition of images and quantification of muscle area. Use ImageJ software to select an area containing only the DLMs.Taking as a control the dimensions of the image of the fly with the biggest muscle area, finerize the images, and delete the areas, or pixels, that do not correspond to the muscles of interest. Quantify the percentage of pixels corresponding to muscle tissue 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.Representative results. We determined that the model flies, which express 250 noncoding CTG repeats throughout the musculature, driven by the myosin heavy chain promoter, MhC Gal four, had a 50 percent reduction of muscle area compared to control flies. In contrast, co expression of muscle blind C and expanded CTG repeats with the same driver strongly suppressed such phenotype and cross sectional muscle area was 70 percent of control flies.Administration of the Abp1 hexapeptide in the nutritive media similarly suppressed the atrophic phenotype and model flies that had taken the compound showed approximately 60 percent of normal muscle area, instead of 50 percent, that is typical of DF1 flies.Conclusions. 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. Finally, this method is also optimized to detect small muscle area differences, or even to quantify the area of different structures such as nephyocytes.Thank you for watching.

Research

JoVE Journal

Biology

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

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

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Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

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Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

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Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

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Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...