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

Эта работа была профинансирована Национальным научным фондом и предоставила IOS-1256565 и IOS-1557638 AWS. Мы благодарим Патрицию Виткопп и трех анонимных рецензентов за их полезные комментарии к более ранней версии этого документа.

1. Монтаж образцов ПРИМЕЧАНИЕ. Хранить мертвые мухи в 70% этаноле в воде до получения изображений. <ol><li> Залить 10 мл 1,25% агара, растворенного в кипящей воде в чашке Петри 60 мм х 15 мм, и дать ему установить. </li><li> Под рассекающим микроскопом используйте пару тонкопленочных щип...

Эта методика позволяет получить точное, быстрое и повторяемое получение данных о пигментации в количественной форме, подходящей для нескольких последующих анализов. Этот метод был использован для получения данных о влиянии температуры на брюшную пигментацию в изогенной линии мух. Од...

Пигментация показывает огромные фенотипические различия между видами, популяциями и индивидуумами и даже внутри индивидуумов во время онтогенеза 1 , 2 , 3 , 4 , 5 , 6 . Несмотря на многочисленные исследования пигментации у самых разных животных, пигментация, пожалуй, лучше всего изучалась у Drosophila melanogaster , где полная сила молекулярной генетики использовалась для выяснения механизмов развития и физиологии, которые регулируют пигментацию и как эти механизмы развиваются 1 , 6 . Многое известно о генах, которые регулируют биохимический синтез пигментов в D. melanogaster 7 , 8 и генах, которые контролируют временную и пространственную диСтроение этого биосинтеза 9 , 10 , 11 , 12 , 13 . Кроме того, генетическое картирование выявило генетические локусы, лежащие в основе внутри- и межвидовых различий в пигментации у D. melanogaster 14 , 15 , 16 , 17 . Также были изучены взаимосвязи между пигментацией и плейотропными чертами, такими как поведение 18 , 19 и иммунитет 19 , 20 , а также адаптивное значение рисунков пигментации 15 , 21 , 22 . Таким образом, пигментация в D. melanogaster появилась как мощный, но простой mДля разработки и эволюции сложных фенотипов. Пигментация у взрослых D. melanogaster характеризуется различными формами меланизации по всему телу, особенно на крыльях, спинной грудной клетке и животе. Это пигментация каждой кутикулярной пластины (тергита) на дорзальном животе, однако, которая получила наибольшее внимание для исследования. Значительные различия в этой пигментации ( рис. 1А- Ф) обусловлены как генетическими 17 , 23, так и экологическими 24 , 25 факторами. Кутикула брюшного тергита состоит из передних и задних отделов развития ( рис. 1G ), каждая из которых может быть дополнительно разделена в зависимости от пигментации и орнамента 26 . Передний отдел включает шесть кутикулы(A1-a6), а задний отсек включает три (p1-p3) ( рисунок 1G ). Из них кутикула p1, p2 и a1 обычно складывают под тергитом в невытянутых брюшных полотнах, так что они скрыты. Надежная видимая кутикула характеризуется полосой тяжелой пигментации, здесь называемой «пигментной лентой», состоящей из типов кутикулы a4 (волосатая с умеренными щетинками) и a5 (волосатая с большими щетинками), с задним краем полосы Более интенсивно пигментированный, чем передний край ( рис. 1G ). Перед этой полосой находится область слегка пигментированной волосатой кутикулы, которая имеет щетину назад (а3), но не вперед (а2). Изменение пигментации между мухами наблюдается как в интенсивности пигментации, так и в ширине пигментной полосы. В целом, самые большие изменения в большинстве задних сегментов (брюшные сегменты 5, 6 и 7) и ниже в более передних сегментах (брюшное сечениеПункты 3 и 4) 24 . Кроме того, в пигментации D. melanogaster существует половой диморфизм, причем самцы обычно имеют полностью пигментированные пятый и шестой брюшные тергиты ( рис. 4C ). В большинстве исследований пигментации брюшной полости у D. melanogaster пигментация рассматривалась как категориальный или ординальный признак, при этом образец качественно измерялся 27 , 28 , 29 или полуколичественно по шкале 14 , 15 , 16 , 17 , 24 , 30 , 31 , 32 , 33 , 34 , 35, 36 , 37 . Эти методы неизбежно страдают от недостатка точности, и поскольку они полагаются на субъективную оценку пигментации, трудно сравнивать данные по исследованиям. Несколько авторов определили пространственные размеры пигментации 38 , 39 , интенсивность пигментации определенного типа кутикулы 23 , 25 , 39 , 40 или среднюю интенсивность пигментации по всему тергиту брюшной полости в целом 41 , 42 , 43 . Тем не менее, эти методы количественной оценки не измеряют как интенсивность, так и пространственное распределение брюшной пигментации одновременно и, следовательно, не фиксируют нюансов о том, как пигментация изменяется в зависимости от abdОминальный тергит. Кроме того, некоторые из этих способов 38 , 41 , 42 , 43 количественного определения требуют вскрытия и монтажа брюшной кутикулы. Это требует много времени и разрушает образец, что делает его недоступным для дополнительного морфологического анализа. По мере углубления понимания развития и развития абдоминальной пигментации потребуются более сложные инструменты для быстрого и точного измерения как пространственного распределения, так и интенсивности пигментации. Общая цель этого метода заключается в использовании цифрового анализа изображений для получения реплицируемой и более точной меры брюшной пигментации в D. melanogaster . Методология включает три этапа. Во-первых, взрослая муха неразрушающе монтируется и берется цифровое изображение дорзального живота. Во-вторых, используя макрос ImageJ, пользовательОпределяет переднюю заднюю полосу пикселей, которая простирается от передней части кутикулы a2 до задней части кукурузы a5 (зеленая коробка, рисунок 1G ) как на третьем, так и на четвертом брюшных сегментах. Среднее значение пикселя по ширине этой полосы затем извлекается вдоль ее длинной оси, создавая профиль, который фиксирует пространственное распределение и интенсивность пигментации, когда он изменяется от передней к задней части тергита. В-третьих, сценарий R используется для описания профиля пигментации математически с использованием кубического сплайна. Сценарий R затем использует сплайн и его первую и вторую производные для извлечения ширины кутикулы a2-a5, ширины полосы пигмента и максимального и минимального уровней пигментации. Таким образом, этот метод количественно определяет как пространственные характеристики, так и глубину пигментации брюшной полости. Эта методика количественно определяет пигментацию третьего и четвертого тергитов брюшной полости,Которые были в центре внимания многочисленных предыдущих исследований 1 , 15 , 23 , 24 , 25 , 28 , 33 , 39 , 42 , либо исключительно, либо в сочетании с более поздними тергитами. Хотя и менее переменный, чем пятый и шестой тергиты брюшной полости, третий и четвертый тергиты не полностью пигментированы у самцов, поэтому этот протокол может применяться как для мужчин, так и для женщин. Тем не менее, как показано здесь, протокол может использоваться для измерения пигментации в пятом и шестом брюшных тергитах у женщин. Кроме того, незначительные модификации сценариев, используемых для извлечения характеристик профиля пигментации, должны позволять использовать этот метод для количественной оценки изменения пигментации в широком спектре другихорганизмы.

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			<td>Dumont #5 Biology Forceps</td>
			<td>FST</td>
			<td>11252-30</td>
			<td></td>
		</tr>
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			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>5040</td>
			<td></td>
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			<td>Dissecting Scope</td>
			<td>Leica</td>
			<td>MZ16FA</td>
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			<td>Leica</td>
			<td>MDG41</td>
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			<td>Leica</td>
			<td>DFC280</td>
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			<td>Gooseneck Cold Light Source</td>
			<td>Schott</td>
			<td>ACE 1</td>
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			<td>Image Acquisition Control Software</td>
			<td>Micro-Manager v1.3.20</td>
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			<td>https://micro-manager.org/</td>
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			<td>Image Analysis Software</td>
			<td>ImageJ</td>
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			<td>R 3.3.2</td>
			<td></td>
			<td>https://www.r-project.org/</td>
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			<td>LED</td>
			<td>Thor Labs</td>
			<td>LEDWE-15</td>
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			<td>Multimeter</td>
			<td>Fluke</td>
			<td>Fluke 75 Series II</td>
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			<td>60 x 15 mm Petri dish</td>
			<td>Celltreat Scientific Products</td>
			<td>229663</td>
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			<td>Stage micrometer</td>
			<td>Klarman Rulings, Inc.</td>
			<td>KR-867</td>
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quantifying abdominal pigmentation in drosophila melanogaster

Пигментация - это морфологически простая, но очень переменная черта, которая часто имеет адаптивное значение. Он широко использовался как модель для понимания развития и эволюции морфологических фенотипов. Пигментация брюшной полости у Drosophila melanogaster особенно полезна, что позволяет исследователям идентифицировать локусы, лежащие в основе межвидовых и внутривидовых изменений морфологии. До сих пор, однако, брюшная пигментация D. melanogaster в значительной степени анализировалась качественно, за счет скоринга, а не количественно, что ограничивает формы статистического анализа, которые могут быть применены к данным пигментации. Эта работа описывает новую методологию, которая позволяет количественно оценить различные аспекты картины брюшной пигментации у взрослого D. melanogaster . Протокол включает в себя монтаж образцов, захват изображений, извлечение данных и анализ. Все программное обеспечение, используемое для макросъемки и анализа изображенийНаписанный для анализа изображений с открытым исходным кодом. Преимуществом этого подхода является способность точно измерять пигментные черты с использованием методики, которая очень воспроизводима в разных системах визуализации. Хотя эта методика использовалась для измерения вариаций в моделях пигментации трогала у взрослого D. melanogaster , эта методология является гибкой и широко применимой к образцам пигментации в множестве различных организмов.

Протокол использовался для изучения влияния температуры выращивания на брюшную пигментацию. Предыдущие исследования показали, что увеличение температуры развития приводит к уменьшению распространения брюшной пигментации у нескольких видов дрозофилы , включа...

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

Quantifying Abdominal Pigmentation in Drosophila melanogaster

В этой работе представлен метод быстрого и точного количественного определения абдоминальной пигментации Drosophila melanogaster с использованием цифрового анализа изображений . Этот метод упрощает процедуры регистрации фенотипов и анализа данных и включает в себя установку образца, сбор изображений, извлечение значений пикселей и измерение признаков.

Количественное определение пигментации брюшной полости в<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.

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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.

В Ситу маркировки репликации митохондриальной ДНК в Drosophila взрослых яичников с помощью EDU Окрашивание

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.

Высоким содержанием жиров питание и высокая пропускная способность Triacylglyceride Assay в 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.

Измерение уровня осуществления в Drosophila melanogaster , с использованием системы количественной оценки вращающихся упражнения (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 ...

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: Интервал подготовки протокола для нежно индуцированной упражнения в 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).

Очистка низким обильные клеток в зрительной системе дрозофилы

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.

Анализ для количественной протеино-РНК Связывания в бактериях

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.

В Vivo Функциональное исследование связанных с болезнями редких человеческих вариантов с использованием дрозофилы

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.

В Vivo Вперед генетический экран для выявления новых нейропротекторных генов в Drosophila меланогастер

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.

Робуст Полимераза Цепная реакция на основе Ассаем для количественной цитозин-гуанин-гуанин тринуклеотид повторяет в хрупкой X умственной отсталости-1 Гена

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.