Name: quantifying abdominal pigmentation in drosophila melanogaster
Uploaded: 2017-06-01T12:30:00
Description: Watch this Scientific Journal Video about Quantifying Abdominal Pigmentation in Drosophila melanogaster at JoVE.com

Dit werk werd gefinancierd door de National Science Foundation, verleent IOS-1256565 en IOS-1557638 aan AWS. We bedanken Patricia Wittkopp en drie anonieme recensenten voor hun nuttige opmerkingen over een eerdere versie van dit artikel.

1. Proefmontage OPMERKING: Bewaar dood vliegen in 70% ethanol in water voor afbeelden. <ol><li> Giet 10 ml 1,25% agar opgelost in kokend water in een 60 mm x 15 mm Petri-schotel en laat het instellen. </li><li> Gebruik onder een dissectiemicroscoop een paar fijnpuntenpincen om een ​​~ 20 mm ong, 2 mm brede, 1 mm diepe groef in het oppervlak van de gel te maken. Gebruik fijne pincet, installeer de ventrale zijde van een volwassen vlieg in de groef, met de dorsale kant van de vlieg die b...

Deze methodologie zorgt voor de precieze, snelle en herhaalbare verwerving van pigmentatiegegevens in een kwantitatieve vorm die geschikt is voor meerdere downstream analyses. De methode is gebruikt om gegevens over het effect van temperatuur op buikpigmentatie in een isogene lijn vliegen te verwerven. De methodologie kan echter gebruikt worden in voorwaartse genetische studies om genen die onderliggende pigmentatieverschillen tussen individuen, populaties of soorten, of omgekeerde genetische studies zijn, te identifice...

Pigmentatie toont een enorme fenotypische variatie tussen soorten, populaties en individuen, en zelfs bij individuen tijdens ontogenie 1 , 2 , 3 , 4 , 5 , 6 . Hoewel er veel studies zijn over pigmentatie in een grote verscheidenheid aan dieren, is pigmentatie wellicht het beste bestudeerd in Drosophila melanogaster , waar de volledige kracht van de moleculaire genetica is gebruikt om de ontwikkelings- en fysiologische mechanismen die pigmentatie regelen en hoe deze mechanismen evolueren 1 toe te lichten , 6 . Er is veel bekend over de genen die de biochemische synthese van pigmenten regelen in D. melanogaster 7 , 8 en de genen die de temporale en ruimtelijke diVerdeling van deze biosynthese 9 , 10 , 11 , 12 , 13 . Bovendien heeft genetische mapping de genetische loci onderliggende intra- en interspecifieke verschillen in pigmentatie in D. melanogaster 14 , 15 , 16 , 17 geïdentificeerd . De relaties tussen pigmentatie en pleiotropische eigenschappen, zoals gedrag 18 , 19 en immuniteit 19 , 20 , zijn ook onderzocht, evenals de adaptieve betekenis van pigmentatiepatronen 15 , 21 , 22 . Als zodanig is pigmentatie in D. melanogaster ontstaan ​​als een krachtige maar simpele mOdel voor de ontwikkeling en evolutie van complexe fenotypes. Pigmentatie in volwassen D. melanogaster wordt gekenmerkt door verschillende melanisatiepatronen over het lichaam, met name op de vleugels en dorsale thorax en buik. Het is de pigmentatie van elke cutikulaire plaat (tergiet) op de dorsale buik, maar dat heeft de meeste onderzoeks aandacht gekregen. Er is aanzienlijke variatie in deze pigmentatie ( Figuur 1A -F ), door beide genetische 17 , 23 en milieu 24 , 25 factoren. De kutikula van een buiktergiet bestaat uit voorste en achterste ontwikkelingscompartimenten ( Figuur 1G ), die elk verder onderverdeeld kunnen worden, afhankelijk van pigmentatie en versiering 26 . Het voorste compartiment bevat zes kutikulaTypen (a1-a6), en het achterste compartiment omvat drie (p1-p3) ( figuur 1G ). Van deze worden de p1-, p2- en a1-kutikula typisch gevouwen onder de tergiet in onuitgespreide abdomenen, zodat ze verborgen zijn. De betrouwbare zichtbare kutikula wordt gekenmerkt door een band met zware pigmentatie, hierna aangeduid als een pigmentband, bestaande uit kutikeltypes a4 (harig met matige borstels) en a5 (harig met grote borstels), met de achterste rand van de band Meer intensief gepigmenteerd dan de voorste rand ( figuur 1G ). Voorkant van deze band is een gebied van licht gepigmenteerde harige kutikula, die achterste achterkant (a3) ​​maar niet anterior (a2) heeft. Variatie in pigmentatie tussen vliegen wordt waargenomen in zowel de intensiteit van pigmentatie als in de breedte van de pigmentband. In het algemeen is variatie het grootste in de meest achterste segmenten (abdominale segmenten 5, 6 en 7) en is lager in de meer anterior segmenten (buikzeeGents 3 en 4) 24 . Verder is er een seksuele dimorfie in D. melanogaster pigmentatie, waarbij mannen meestal volledig gepigmenteerde vijfde en zesde buiktergieten hebben ( Figuur 4C ). In de meeste studies van buikpigmentatie in D. melanogaster is pigmentatie behandeld als een categorische of ordinale eigenschap, waarbij het patroon kwalitatief 27 , 28 , 29 of semi-kwantitatief wordt gemeten op een schaal 14 , 15 , 16 , 17 , 24 , 30 31 , 32 , 33 , 34 , 3536 , 37 . Deze methoden hebben onvermijdelijk een gebrek aan precisie, en omdat ze vertrouwen op de subjectieve beoordeling van pigmentatie, is het moeilijk om de gegevens over studies te vergelijken. Verscheidene auteurs hebben de ruimtelijke dimensies van pigmentatie 38 , 39 gekwantificeerd, de intensiteit van pigmentatie van een bepaald kutikeltype 23 , 25 , 39 , 40 of de gemiddelde intensiteit van pigmentatie over de buiktergiet als geheel 41 , 42 , 43 . Desalniettemin meet deze kwantificatiewerkwijzen echter niet de intensiteit en de ruimtelijke verdeling van buikpigmentatie tegelijkertijd en derhalve niet de nuances van hoe pigmentatie varieert over de abdOminale tergiet Verder vereisen verscheidene van deze kwantificatiewerkwijzen 38 , 41 , 42 , 43 de dissectie en montage van de buikspieren. Dit is zowel tijdrovend en vernietigt het monster, waardoor het niet meer beschikbaar is voor aanvullende morfologische analyses. Aangezien het begrip van de ontwikkeling en evolutie van buikpigmentatie verdiept, zullen meer geavanceerde hulpmiddelen nodig zijn om snel en nauwkeurig zowel de ruimtelijke verdeling als de intensiteit van pigmentatie te meten. Het algemene doel van deze methode is het gebruik van digitale beeldanalyse om een ​​replicabele en nauwkeuriger maatregel van de buikpigmentatie in D. melanogaster te verkrijgen . De methodologie omvat drie fasen. Ten eerste is de volwassen vlieg niet destructief gemonteerd en wordt een digitaal beeld van de dorsale buik genomen. Ten tweede, met behulp van een ImageJ macro, de gebruikerDefinieert een anterior-posterior strook pixels die zich uitstrekken van de voorkant van de a2-cuticle naar de achterkant van de a5-cuticle (groene doos, figuur 1G ) op zowel de derde als vierde buikspijkers. De gemiddelde pixelwaarde over de breedte van deze strip wordt vervolgens langs zijn lange as geëxtraheerd, waardoor een profiel wordt verkregen dat de ruimtelijke verdeling en intensiteit van pigmentatie vastlegt als het van de voorste naar de achterkant van de tergiet verandert. Ten derde wordt een R-script gebruikt om het pigmentatieprofiel wiskundig te beschrijven met behulp van een kubieke spline. Het R-script gebruikt dan de spline en zijn eerste en tweede afgeleide om de breedte van de a2-a5-kutikula, de breedte van de pigmentband en de maximale en minimumpigmenten van pigmentatie te extraheren. De methode kent dus zowel de ruimtelijke eigenschappen als de diepte van de buikpigmentatie. Deze methodologie kwantificeert de pigmentatie van de derde en vierde abdominale tergieten,Die de focus zijn geweest van tal van eerdere studies 1 , 15 , 23 , 24 , 25 , 28 , 33 , 39 , 42 , ofwel uitsluitend of in combinatie met meer posterior tergieten. Hoewel minder variabel dan de vijfde en zesde abdominale tergieten, zijn de derde en vierde tergieten niet helemaal gepigmenteerd bij mannen, dus dit protocol kan zowel bij mannen als vrouwen worden toegepast. Desalniettemin, zoals hier getoond, kan het protocol worden gebruikt om pigmentatie in de vijfde en zesde buiktergieten bij vrouwen te meten. Bovendien moeten kleine wijzigingen van de scripts die gebruikt worden om de eigenschappen van het pigmentatieprofiel te extraheren, de methode toestaan ​​om de variatie in pigmentatie in een grote verscheidenheid aan andere te kwantificerenorganismen.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dumont #5 Biology Forceps</td>
			<td>FST</td>
			<td>11252-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scope</td>
			<td>Leica</td>
			<td>MZ16FA</td>
			<td></td>
		</tr>
		<tr>
			<td>Base</td>
			<td>Leica</td>
			<td>MDG41</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Leica</td>
			<td>DFC280</td>
			<td></td>
		</tr>
		<tr>
			<td>Gooseneck Cold Light Source</td>
			<td>Schott</td>
			<td>ACE 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Acquisition Control Software</td>
			<td>Micro-Manager v1.3.20</td>
			<td></td>
			<td>https://micro-manager.org/</td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td>ImageJ</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Data Analysis Software</td>
			<td>R 3.3.2</td>
			<td></td>
			<td>https://www.r-project.org/</td>
		</tr>
		<tr>
			<td>LED</td>
			<td>Thor Labs</td>
			<td>LEDWE-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Multimeter</td>
			<td>Fluke</td>
			<td>Fluke 75 Series II</td>
			<td></td>
		</tr>
		<tr>
			<td>60 x 15 mm Petri dish</td>
			<td>Celltreat Scientific Products</td>
			<td>229663</td>
			<td></td>
		</tr>
		<tr>
			<td>Stage micrometer</td>
			<td>Klarman Rulings, Inc.</td>
			<td>KR-867</td>
			<td></td>
		</tr>
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</table>

quantifying abdominal pigmentation in drosophila melanogaster

Pigmentatie is een morfologisch eenvoudig maar zeer variabel kenmerk dat vaak adaptieve betekenis heeft. Het heeft uitgebreid gediend als model om de ontwikkeling en evolutie van morfologische fenotypes te begrijpen. Abdominale pigmentatie in Drosophila melanogaster is bijzonder handig, zodat onderzoekers de loci kunnen identificeren die onderling en intraspecifieke variaties in morfologie liggen. Tot op heden is echter D. melanogaster buikpigmentatie grotendeels kwalitatief geanalyseerd, door middel van scoren, in plaats van kwantitatief, welke de vormen van statistische analyse beperken die op pigmentatiegegevens kan worden toegepast. Dit werk beschrijft een nieuwe methodologie die het mogelijk maakt om verschillende aspecten van het abdominale pigmentatiepatroon van volwassen D. melanogaster te kwantificeren . Het protocol bevat voorbeeldmontage, beeldopname, data-extractie en analyse. Alle software die wordt gebruikt voor macro-opnames en analysemogelijkhedenGeschreven voor open-source beeldanalyse. Het voordeel van deze aanpak is het vermogen om pigmentatiekenmerken nauwkeurig te meten met behulp van een methodologie die zeer reproducteerbaar is over verschillende beeldvormingssystemen. Hoewel de techniek is gebruikt om variatie in de tergal pigmentatiepatronen van volwassen D. melanogaster te meten , is de methodologie flexibel en breed toepasbaar op pigmentatiepatronen in veel verschillende organismen.

Het protocol werd gebruikt om het effect van de opslagtemperatuur op buikpigmentatie te onderzoeken. Uitgaande onderzoeken hebben aangetoond dat een toename van de ontwikkelingstemperatuur resulteert in een afname in de verspreiding van buikpigmentatie in verschillende soorten Drosophila , waaronder D. melanogaster 30 , 32 . Specifiek, in buiktergieten 3 en 4 neemt de mate van pigmentatie (breedte van de pigment...

Watch this Scientific Journal Video about Quantifying Abdominal Pigmentation in Drosophila melanogaster at JoVE.com

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Dit werk biedt een methode om de abdominale pigmentatie van Drosophila melanogaster snel en nauwkeurig te kwantificeren met behulp van digitale beeldanalyse . Deze methode stroomlijnt de procedures tussen fenotype-acquisitie en data-analyse en omvat voorbeeldmontage, beeldverzameling, pixelwaarde-extractie en eigenschappen van eigenschappen.

Kwantificering van buikpigmentatie in<em&gt; Drosophila melanogaster</em

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

The overall goal of this methodology is to rapidly quantify abdominal pigmentation in the fruit fly Drosophila melanogaster. This method can help answer key questions in the field of evolutionary biology such as identifying the genetic loci that underlie in turn in trust-specific variation morphology and understanding the development and evolution of morphological phenotypes. The main advantage of this technique is that it allows users to non-destructively measure various aspects of abdominal pigmentation including the spread and depth of pigmentation.Though this method can provide insight into Drosophila melanogaster's abdominal pigmentation, it can also be applied to other Drosophila species and easily adapted to measure pigmentation patterns in other insects and animal taxa. Prior to imaging, the flies can be stored in 70%ethanol to maintain their pigmentation. Visualization requires a mounting pad.Load a 60 millimeter Petri dish with about 10 milliliters of 1.25%agar in water. Under a microscope, use fine forceps to dig a 20 millimeter long, two millimeter wide, one millimeter deep trench into the gel. Several trenches can be made in one dish.Then embed flies into the trench with the surface for analysis sitting just above the gel surface. In this case, the dorsal side is of interest. Now, fill the dish with 70%ethanol until the flies are completely submerged.This reduces reflections off the cuticle that can obscure the pigmentation pattern. Now, start the imaging software and digital camera connected to the microscope. For a light source, cold light from a double gooseneck device is ample.In the image analysis software, set the image type to 8-bit. Then in the image capture software, toggle the Live setting to open a view window of the camera. Set the image type to Grayscale.Now, set the light source to its maximum intensity and position the lights about 120 millimeters from the stage. Set the microscope to 60 times magnification so the field of view is three millimeters wide. Then place the two millimeter stage micrometer into the field of view and focus on it.Returning to the software, adjust the exposure time to view the micrometer using the field found in the exposure box. Then set the scale. Open the straight line tool and draw the length of two millimeters along the stage micrometer.Then input that length under the Set Scale option. The window should then display the scale in pixels per micron. Take note of the scale and OK the adjustments.Now, return to the image capture software and use the Stop and Snap commands to capture an image of the stage micrometer. Then return to the image analysis software and save the image as a Tiff file. Now, focus on the dorsal midline of a mounted fly.The midline should be positioned straight up for the best imaging results. Push aside the wings and any other obtruding structures and adjust the lighting to best illuminate the cuticle. Next, set the exposure in the image capture software using the pixel value histogram as a guide to capture the full pixel range of the image without saturation, thereby maximizing contrast.Retain this exposure and lighting throughout the experiment. Now, before capturing an image, ensure that the abdominal segments of interest are in view. Then capture an image and save the image with the sample ID number in the filename.Use the format described in the written protocol. This analysis makes use of a macro provided by the authors. The image series to analysis should all be contained within the same file folder.Now, start the macro and follow the prompted commands. First, identify the location of the stored files. Then identify the location for where the analysis data should be stored.Next, input the number of characters that were used in the filenames so the software reads the image series correctly. The next prompt is for the width of the region of interest. Here, input the number of pixels along the anterior-posterior strip that needs to be analyzed.Now, at the prompt, make a choice to analyze the first image of the series. The other options are to analyze the next image in the series or to exit the analysis. The line tool gets automatically selected.Use it to identify the midline on the image by drawing a line along it in the anterior to posterior direction. Next, follow the macro prompts to identify the posterior edge of Tergite4 using the line tool. The line tool is made available to draw the line from the posterior midline edge to the right lateral edge so that the center of the line is just posterior to the posterior edge of the pigment band.Then define the anterior edge of Tergite4 using the line tool. Draw a line from the anterior midline edge to the right lateral edge. The middle of the line defined by a white square should sit at the anterior edge of the lightly pigmented cuticle.With this information, the macro now generates a pigmentation profile. However, the profile might be inaccurate if bristles or other blemishes cross the strip along which the profile is measured. To correct for this, select the Live preview on the histogram window and adjust the line such that the profile is smooth.For the image analysis strip to accurately capture the various aspects of pigmentation, it is vital to identify an analysis region that will have minimal noise caused by image or specimen irregularities. Now proceed to analyze Tergite3 in the same image and then move on to the next image. The described procedure was used to explore a known change in abdominal pigmentation caused by rearing temperature.The third and fourth tergites were examined several times under various lightings and exposures. Various aspects of the pigmentation were explored. And specifically, it was found that the pigmentation bands were wider when flies were reared at cooler temperatures.The calculated values were then compared to general subjective measures made on the same flies by five different observers. In the assessment of 45 female flies, the average observed ranking of the width of the fourth abdominal pigmentation band was tightly correlated with the macro's automated measurement. The same pigmentation band was also manually measured using imaging software.These results were also strongly correlated with measurements generated using the macro. The software was then tested to measure pigmentation in female ebony mutants which are well-known to have increased pigmentation in the third and fourth pigment bands. The method was also used to measure the fifth and sixth abdominal bands of wild type females.The automated technique always provided useful measurements of the pigment density and band size. After watching this video, you should have a good understanding of how to quickly and precisely quantify abdominal pigmentation in Drosophila. Mastering the technique mostly requires the depth use of forceps.Once mastered, the imaging portion of this technique can be done at a rate of 50 specimens per hour. Extracting the pigmentation measures from these images can be done at a pace of 70 images per hour.

quantifying-abdominal-pigmentation-in-drosophila-melanogaster

Research

JoVE Journal

Genetics

Quantifying Abdominal Pigmentation in Drosophila melanogaster

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

In Situ Labeling van mitochondriaal DNA-replicatie in Drosophila volwassen Eierstokken van Edu kleuring

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

Hoog vet dieet voeding en High Throughput triglyceride test bij Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

Meten van oefening niveaus bij Drosophila melanogaster met behulp van de roterende oefening kwantificering (systeemvereisten)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and lowered activity levels. These multifactorial diseases arise from a combination of genetic, environmental, and behavioral factors. One such complex disease is Metabolic Syndrome (MetS), which is a cluster of metabolic disorders, including hypertension, hyperglycemia, and abdominal obesity. Exercise and dietary intervention are the primary treatments recommended by doctors to mitigate obesity and its subsequent metabolic diseases. Exercise intervention, in particular aerobic interval training, stimulates favorable changes in the common risk factors for Type 2 Diabetes Mellitus (T2DM), Cardiovascular Disease (CVD), and other conditions. With the influx of evidence describing the therapeutic effect exercise has on metabolic health, establishing a system that models exercise in a controlled setting provides a valuable tool for assessing the effects of exercise in an experimental context. Drosophila melanogaster is a great tool for investigating the physiological and molecular changes that result from exercise intervention. The flies have short lifespans and similar mechanisms of metabolizing nutrients when compared to humans. To induce exercise in Drosophila, we developed a machine called the TreadWheel, which utilizes the fly&#39;s innate, negative geotaxis tendency to gently induce climbing. This enables researchers to perform experiments on large cohorts of genetically diverse flies to better understand the genotype-by-environment interactions underlying the effects of exercise on metabolic health.

The TreadWheel: Interval Training Protocol for Gently Induced Exercise in Drosophila melanogaster

The TreadWheel uses rotational motion to gently induce exercise in adult Drosophila melanogaster by exploiting flies&#39; innate, negative geotaxis. It allows for analysis of the interactions between exercise and factors, such as genotype, sex, and diet, and their effect on physiological and molecular assays to assess metabolic health.

De tredmolen: Interval Training Protocol voor zachtjes geïnduceerde oefening bij Drosophila melanogaster

The incidence of complex metabolic diseases has increased as a result of a widespread transition towards lifestyles of increased caloric intake and ...

Purification of Low-abundant Cells in the Drosophila Visual System

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

Zuivering van Low-overvloedig cellen in het visuele systeem van Drosophila

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding of an RNA binding protein (RBP) to the initiation region of the mRNA, which interferes with ribosome binding. In the presented method, we utilize this blocking phenomenon to quantify the binding affinity of RBPs to their cognate and non-cognate binding sites. To do this, we insert a test binding site in the initiation region of a reporter mRNA and induce the expression of the test RBP. In the case of RBP-RNA binding, we observed a sigmoidal repression of the reporter expression as a function of RBP concentration. In the case of no-affinity or very low affinity between binding site and RBP, no significant repression was observed. The method is carried out in live bacterial cells, and does not require expensive or sophisticated machinery. It is useful for quantifying and comparing between the binding affinities of different RBPs that are functional in bacteria to a set of designed binding sites. This method may be inappropriate for binding sites with high structural complexity. This is due to the possibility of repression of ribosomal initiation by complex mRNA structure in the absence of RBP, which would result in lower basal reporter gene expression, and thus less-observable reporter repression upon RBP binding.

In this method, we quantify the binding affinity of RNA binding proteins (RBPs) to cognate and non-cognate binding sites using a simple, live, reporter assay in bacterial cells. The assay is based on repression of a reporter gene.

Een test voor het kwantificeren van eiwit-RNA-binding bij bacteriën

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

In vivo functionele studie van ziekten-geassocieerde zeldzame menselijke varianten met behulp van Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

In vivo genetisch scherm naar voren om nieuwe neuroprotectieve genen in Drosophila melanogaster te identificeren

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

A Robust Polymerase Chain Reaction-based Assay for Quantifying Cytosine-guanine-guanine Trinucleotide Repeats in Fragile X Mental Retardation-1 Gene

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; untranslated region (UTR) of the Fragile X mental retardation-1 (FMR1) gene promoter. Conventionally, capillary electrophoresis fragment analysis on a genetic analyzer is used for the sizing of the CGG repeats of FMR1, but additional Southern blot analysis is required for exact measurement when the repeat number is higher than 200. Here, we present an accurate and robust polymerase chain reaction (PCR)-based method for quantification of the CGG repeats of FMR1. The first step of this test is PCR amplification of the repeat sequences in the 5&#8217;UTR of the FMR1 promoter using a Fragile X PCR kit, followed by purification of the PCR products and fragment sizing on a microfluidic capillary electrophoresis instrument, and subsequent interpretation of the number of CGG repeats by referencing standards with known repeats using the analysis software. This PCR-based assay is reproducible and capable of identifying the full range of CGG repeats of FMR1 promoters, including those with a repeat number of more than 200 (classified as full mutation), 55 to 200 (premutation), 46 to 54 (intermediate), and 10 to 45 (normal). It is a cost-effective method that facilitates classification of the FXS and Fragile X-associated disorders with robustness and rapid reporting time.

An accurate and robust polymerase chain reaction-based assay for quantifying cytosine-guanine-guanine trinucleotide repeats in the Fragile X mental retardation-1 gene facilitates molecular diagnosis and screening of Fragile X syndrome and Fragile X-related disorders with shorter turn-around time and investment in equipment.

Een robuuste polymerase kettingreactie op basis van Cytosine-guanine-guanine Trinucleotide herhalingen in fragiele X mentale retardatie-1 gen

Fragile X syndrome (FXS) and associated disorders are caused by expansion of the cytosine-guanine-guanine (CGG) trinucleotide repeat in the 5&#8217; ...

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.