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

Diese Arbeit wurde gefördert von National Science Foundation gewährt IOS-1256565 und IOS-1557638 an AWS. Wir danken Patricia Wittkopp und drei anonymen Rezensenten für ihre hilfreiche Kommentare zu einer früheren Version dieses Artikels.

1. Probenmontage HINWEIS: Tote Fliegen in 70% Ethanol in Wasser vor der Bildgebung. <ol><li> Gießen Sie 10 ml 1,25% Agar in kochendem Wasser in einer 60 mm x 15 mm Petrischale aufgelöst und lassen Sie es einstellen. </li><li> Unter einem Sektionsmikroskop verwenden Sie ein Paar Feinpinzetten, um eine ~ 20 mm ong, 2 mm breite, 1 mm tiefe Rille in der Oberfläche des Gels zu machen. Mit feinen Zangen, eingebettet die ventrale Seite einer erwachsenen Fliege in die Nut, wobei die dorsale Sei...

Diese Methodik ermöglicht die präzise, ​​schnelle und wiederholbare Erfassung von Pigmentierungsdaten in quantitativer Form, die für mehrere nachgeschaltete Analysen geeignet ist. Die Methode wurde verwendet, um Daten über die Wirkung der Temperatur auf die Bauchpigmentierung in einer isogenen Linie von Fliegen zu erwerben. Allerdings könnte die Methodik in Forward-Genetics-Studien verwendet werden, um Gene zu identifizieren, die den Pigmentierungsunterschieden zwischen Individuen, Populationen oder Spezies zug...

Die Pigmentierung zeigt eine enorme phänotypische Variation zwischen Arten, Populationen und Individuen und sogar innerhalb von Individuen während der Ontogenese 1 , 2 , 3 , 4 , 5 , 6 . Obwohl es in einer Vielzahl von Tieren unzählige Pigmenttests gibt, ist die Pigmentierung am besten in Drosophila melanogaster untersucht worden , wo die volle Kraft der Molekulargenetik verwendet wurde, um die Entwicklungs- und physiologischen Mechanismen zu erforschen, die die Pigmentierung regulieren und wie sich diese Mechanismen entwickeln 1 , 6 Es ist viel über die Gene bekannt, die die biochemische Synthese von Pigmenten in D. melanogaster 7 , 8 und den Genen, die das zeitliche und räumliche di kontrollieren, regulierenVerteilung dieser Biosynthese 9 , 10 , 11 , 12 , 13 . Darüber hinaus hat die genetische Kartierung die genetischen Loci identifiziert, die intra- und interspezifischen Unterschiede in der Pigmentierung in D. melanogaster 14 , 15 , 16 , 17 liegen . Auch die Beziehungen zwischen Pigmentierung und pleiotropen Merkmalen wie Verhalten 18 , 19 und Immunität 19 , 20 wurden ebenso erforscht wie die adaptive Bedeutung der Pigmentierungsmuster 15 , 21 , 22 . Als solches ist die Pigmentierung in D. melanogaster als eine mächtige und dennoch einfache m aufgetauchtOdel für die Entwicklung und Entwicklung von komplexen Phänotypen. Pigmentierung im erwachsenen D. melanogaster zeichnet sich durch deutliche Melanisierungsmuster über den Körper, besonders an den Flügeln und dem dorsalen Thorax und Bauch. Es ist die Pigmentierung jeder cuticular Platte (tergite) auf dem dorsalen Abdomen, jedoch, die die meisten Forschung Aufmerksamkeit erhalten hat. Es gibt eine beträchtliche Variation in dieser Pigmentierung (Abbildung 1A- F ), weil sowohl genetische 17 , 23 und Umwelt 24 , 25 Faktoren. Die Nagelhaut eines abdominalen Tergits besteht aus vorderen und hinteren Entwicklungsfächern (Abbildung 1G ), die jeweils je nach Pigmentierung und Ornamentierung weiter unterteilt werden können. Das vordere Fach umfasst sechs NagelhautTypen (a1-a6), und das hintere Kompartiment enthält drei (p1-p3) (Abbildung 1G ). Von diesen werden die p1, p2 und a1 Nagelhaut typischerweise unter dem Tergit in ungedehnten Abdomen gefaltet, so dass sie verborgen sind. Die zuverlässig sichtbare Nagelhaut zeichnet sich durch eine Bande von schwerer Pigmentierung aus, die hier als &quot;Pigmentband&quot; bezeichnet wird und aus Nagelhauttypen a4 (haarig mit mäßigen Borsten) und a5 (behaart mit großen Borsten) mit dem hinteren Rand des Bandes besteht Intensiver pigmentiert als die vordere Kante (Abbildung 1G ). Anterior zu dieser Band ist eine Region von leicht pigmentierten behaarten Nagelhaut, die Borsten nach hinten (a3) ​​aber nicht anteriorly (a2) hat. Eine Variation der Pigmentierung zwischen Fliegen wird sowohl in der Intensität der Pigmentierung als auch in der Breite des Pigmentbandes beobachtet. Im Allgemeinen ist die Variation am stärksten in den hintersten Segmenten (Bauchsegmente 5, 6 und 7) und ist in den anterioren Segmenten niedriger (abdominal seGionen 3 und 4) 24 . Darüber hinaus gibt es einen sexuellen Dimorphismus in D. melanogaster Pigmentierung, wobei Männer im Allgemeinen vollständig pigmentierte fünfte und sechste Bauch-Tergite (Abbildung 4C ) haben. In den meisten Untersuchungen der Bauchpigmentierung in D. melanogaster wurde die Pigmentierung als kategorisches oder ordinales Merkmal behandelt, wobei das Muster qualitativ 27 , 28 , 29 oder halbquantitativ auf einer Skala 14 , 15 , 16 , 17 , 24 , 30 gemessen wurde , 31 , 32 , 33 , 34 , 35, 36 , 37 . Diese Methoden leiden unweigerlich an einem Mangel an Präzision, und weil sie sich auf die subjektive Bewertung der Pigmentierung verlassen, ist es schwierig, die Daten über Studien zu vergleichen. Mehrere Autoren haben die räumlichen Dimensionen der Pigmentierung 38 , 39 , die Intensität der Pigmentierung eines bestimmten Häutchen Typ 23 , 25 , 39 , 40 oder die durchschnittliche Intensität der Pigmentierung über die Bauch-Tergit als Ganzes 41 , 42 , 43 quantifiziert. Dennoch messen diese Quantifizierungsmethoden nicht sowohl die Intensität als auch die räumliche Verteilung der Bauchpigmentierung gleichzeitig und erfassen daher nicht die Nuancen, wie sich die Pigmentierung über den Abd unterscheidetOminal tergite Weiterhin erfordern mehrere dieser Quantifizierungsverfahren 38 , 41 , 42 , 43 die Dissektion und Montage der Bauchhöhle. Dies ist sowohl zeitaufwändig als auch zerstört die Probe, so dass es für weitere morphologische Analysen nicht verfügbar ist. Als das Verständnis der Entwicklung und Entwicklung der Bauchpigmentierung vertieft, werden anspruchsvollere Werkzeuge zur schnellen und präzisen Messung sowohl der räumlichen Verteilung als auch der Intensität der Pigmentierung erforderlich sein. Das übergeordnete Ziel dieser Methode ist es, die digitale Bildanalyse zu nutzen, um eine replizierbare und präzisere Messung der Bauchpigmentierung in D. melanogaster zu erhalten . Die Methodik umfasst drei Stufen. Zuerst wird die erwachsene Fliege zerstörungsfrei montiert und ein digitales Bild des dorsalen Bauches genommen. Zweitens, mit einem ImageJ-Makro, der BenutzerDefiniert einen anterior-posterioren Streifen von Pixeln, der sich von der vorderen Seite der a2-Nagelhaut bis zum hinteren der a5-Nagelhaut (grüner Kasten, Abbildung 1G ) auf dem dritten und vierten Bauchsegment erstreckt. Der mittlere Pixelwert über die Breite dieses Streifens wird dann entlang seiner langen Achse extrahiert, wodurch ein Profil erzeugt wird, das die räumliche Verteilung und die Intensität der Pigmentierung einfängt, wenn es sich von der vorderen zur hinteren des Tergits ändert. Drittens wird ein R-Skript verwendet, um das Pigmentprofil mathematisch mit einem kubischen Spline zu beschreiben. Das R-Skript verwendet dann das Spline und seine erste und zweite Ableitung, um die Breite der a2-a5-Nagelhaut, die Breite des Pigmentbandes und die maximale und minimale Pigmentierung zu extrahieren. Die Methode quantifiziert daher sowohl die räumlichen Eigenschaften als auch die Tiefe der Bauchpigmentierung. Diese Methodik quantifiziert die Pigmentierung der dritten und vierten Bauch-Tergite,Die im Fokus zahlreicher früherer Studien 1 , 15 , 23 , 24 , 25 , 28 , 33 , 39 , 42 waren , entweder ausschließlich oder in Kombination mit mehr posterioren Tergiten. Obwohl weniger variabel als die fünfte und sechste Bauch-Tergite, die dritte und vierte Tergite sind nicht vollständig pigmentiert bei Männern, so dass dieses Protokoll kann sowohl für Männer und Frauen angewendet werden. Dennoch kann, wie hier gezeigt, das Protokoll verwendet werden, um die Pigmentierung in der fünften und sechsten Bauch-Tergite bei Frauen zu messen. Darüber hinaus sollten kleinere Modifikationen der Skripte, die verwendet werden, um die Eigenschaften des Pigmentierungsprofils zu extrahieren, das Verfahren zur Quantifizierung der Variation der Pigmentierung in einer Vielzahl von anderen verwendenOrganismen

<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>
	</tbody>
</table>

quantifying abdominal pigmentation in drosophila melanogaster

Pigmentierung ist ein morphologisch einfaches, aber sehr variables Merkmal, das oft anpassungsfähige Bedeutung hat. Es hat vielmehr als Modell für das Verständnis der Entwicklung und Entwicklung von morphologischen Phänotypen gedient. Die Bauchpigmentierung in Drosophila melanogaster war besonders nützlich, so dass Forscher die Locus identifizieren können, die inter- und intraspezifischen Variationen in der Morphologie zugrunde liegen. Bisher wurde jedoch die D. melanogaster- Bauchpigmentierung weitgehend qualitativ durch Scoring statt quantitativ untersucht, was die Formen der statistischen Analyse begrenzt, die auf Pigmentationsdaten angewendet werden können. Diese Arbeit beschreibt eine neue Methodik, die die Quantifizierung verschiedener Aspekte des Bauchpigmentierungsmusters des erwachsenen D. melanogaster ermöglicht . Das Protokoll umfasst Probenmontage, Bildaufnahme, Datenextraktion und Analyse. Die gesamte Software, die für die Bildaufnahme und Analyse verwendet wird, verfügt über MakrosGeschrieben für Open-Source-Bildanalyse. Der Vorteil dieses Ansatzes ist die Fähigkeit, Pigmentierungsmerkmale präzise zu messen, wobei eine Methodik verwendet wird, die über verschiedene Abbildungssysteme hoch reproduzierbar ist. Während die Technik verwendet wurde, um die Variation in den tergischen Pigmentierungsmustern des erwachsenen D. melanogaster zu messen , ist die Methodik flexibel und breit auf Pigmentierungsmuster in unzähligen verschiedenen Organismen anwendbar.

Das Protokoll wurde verwendet, um die Wirkung der Aufzuchttemperatur auf die Bauchpigmentierung zu untersuchen. Frühere Studien haben gezeigt, dass eine Erhöhung der Entwicklungstemperatur zu einer Verringerung der Ausbreitung der Bauchpigmentierung bei mehreren Arten von Drosophila führt , einschließlich D. melanogaster 30 , 32 . Speziell bei den Bauch-Tergiten 3 und 4 nimmt das Ausmaß der Pigmentierung (B...

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

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Diese Arbeit präsentiert eine Methode zur schnellen und präzisen Quantifizierung der Bauchpigmentierung von Drosophila melanogaster mittels digitaler Bildanalyse . Diese Methode rationalisiert die Vorgänge zwischen der Phänotyp-Erfassung und der Datenanalyse und umfasst die Probenmontage, die Bilderfassung, die Pixelwert-Extraktion und die Merkmalsmessung.

Quantifizierung der Bauchpigmentierung 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 der mitochondrialen DNA - Replikation in Drosophila Adult Ovarien von EdU Anfärben

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

Forschung

Genetik

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.

Fettreiche Ernährung Fütterung und Hochdurchsatz 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 ...

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.

Übung Messhöhen in Drosophila Melanogaster mit der rotierenden Bewegung Quantifizierung System (Anforderung)

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.

TreadWheel: Intervall Trainingsprotokoll sanft induzierte Bewegung 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 ...

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).

Reinigung des Low-reichlich Zellen im visuellen System von 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.

Ein Assay zur Quantifizierung der Protein-RNA-Bindung in Bakterien

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 Funktionelle Studie von krankheitsassoziierten seltenen menschlichen Varianten mit 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 Forward Genetic Screen zur Identifizierung neuartiger neuroprotektive gene in Drosophila melanogaster

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.

Ein robuster Polymerase-Kettenreaktions-basierter Assay zur Quantifizierung von Cytosin-Guanin-Guanin-Trinukleotid-Wiederholungen in fragilen X Mental Retardation-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.