The authors thank David Larson for developing the original protocol for imaging the live Drosophila pupal eye. We thank our colleagues Richard Carthew, Shoichiro Tsukita and Matthew Freeman, who developed the transgenic flies that we combined to generate our live-imaging fly lines. Brittany Baldwin-Hunter gave helpful comments on the manuscript. This work was supported by start-up funds awarded to Ruth Johnson by Wesleyan University.

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+roles+for+Drosophila+Dicer-1+and+Dicer-2+in+the+siRNA/miRNA+silencing+pathways.">Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways.</a> Cell. 117, 69-81 (2004).</li><li>Zitserman, D., Roegiers, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+of+sensory+organ+precursor+cells+in+intact+Drosophila+pupae.">Live-cell imaging of sensory organ precursor cells in intact Drosophila pupae.</a> J. Vis. Exp. (51), 2706 (2011).</li><li>Classen, A. 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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pathway+of+signals+regulating+effector+and+initiator+caspases+in+the+developing+Drosophila+eye.">A pathway of signals regulating effector and initiator caspases in the developing Drosophila eye.</a> Development. 129, 3269-3278 (2002).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+tugging+force+regulates+the+size+of+cell-cell+junctions.">Mechanical tugging force regulates the size of cell-cell junctions.</a> Proc. Natl. Acad. 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The authors declare that they have no competing financial interests.

Описание разработки дикого типа (выше) образует основу для сравнения паттерна событий в РНК-интерференции или избыточной экспрессии генотипов. Сравнение развития живой ткани имеют неоценимое значение при определении, какие именно клеточные поведения регулируются белка. Кроме того, и...

Соединение дрозофилы глаз характеризуется стереотипной расположения его ~ 750 омматидиев разделенных сотовой решетки-вспомогательных пигментных клеток 4,5. Эти пигментные клетки с рисунком с помощью согласованной комбинации событий: местных движений клеток, клеточного роста, изменение формы клеток и апоптоз. Живая визуализация этого эпителия позволяет изучать молекулярные механизмы, лежащие в основе этих событий в физиологически необходимой и невозмущенной трехмерной контексте. В отличие от предыдущих протоколов 6,7, методика описано здесь, включает в себя эффективный способ стабилизации посторонние движение ткани, которая не может быть отсоединен от процесса формирования изображения. Этот способ повышает исследований поведения клеток в развивающемся Drosophila куколки глаз эпителия - ткани, который растет, вращается, и сдвиги в течение визуализации. Кроме того движение стабилизация техника деописано здесь, будет полезным для изучения клеток и в других контекстах, где происходит постороннее движение. Для визуализации границ клеток сетчатки в области Drosophila, трансгенные линии мух, которые были получены выражения Ubi-ДЕ-кадгерин: GFP, а также UAS-α-катенин: GFP под контролем водителя глаз конкретного ГМР-GAL4 1-3. Использование двух GFP-меченый мембранных маркеров позволяет для визуализации границ клеток в нижней интенсивности света. Это сводит к минимуму повреждения тканей и фотообесцвечивание на повторном воздействии высоких энергий длин волн, позволяя увеличенную частоту кадров и продолжительности фильма. Для повышения эффективности RNAi трансгенов, UAS-DCR-2 также включены во второй летучей линии 8. В третьем обновления из предыдущих протоколов, простой визуализации буровой которые легко собран в большинстве лабораторий описано. Этот аппарат устраняет требование, чтобы иметь специализированный изображений RIG генерируется университета &quot;механический цех&quot; или подобный сервис. Это визуализации установка аналогична той, что используется в графических других куколок тканей 9,10. Представленные здесь простой протокол изображения живых клеток, которые могут быть использованы непосредственно оценить морфогенетические события, которые способствуют глаз паттерна от ~ 17 до 42 ч после образования пупария (HR НПФ). В частности, этот протокол позволяет определить последствия изменения экспрессии генов во время развития куколки.

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			<td height="21" style="height:21px;width:263px;">Name of Material/ Equipment</td>
			<td style="width:124px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">GMR-GAL4; UAS-a-cat:GFP, ubi-DE-Cadherin:GFP ; + / SM5-TM6b</td>
			<td>Available on request from authors</td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">UAS-dcr-2; UAS-a-cat:GFP, ubi-DE-Cadherin:GFP; + / SM5-TM6b&#160;</td>
			<td>Available on request from authors</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Scotch double stick tape</td>
			<td style="width:124px;">3M</td>
			<td style="width:119px;">665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Black Sylgard dissection dish</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>Fill glass petri dish to rim with Sylgard (colored black with finely-ground charcoal powder) or Sylgard 170; leave at room temperature for 24-48 h to polymerize</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Sylgard</td>
			<td style="width:124px;">Dow Corning</td>
			<td style="width:119px;">SYLG184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Sylgard (Black)</td>
			<td style="width:124px;">Dow Corning</td>
			<td style="width:119px;">SYLG170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Glass petri dish</td>
			<td style="width:124px;">Corning</td>
			<td style="width:119px;">7220-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">25 x 75 x 1 mm glass microscope slide</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">12-550-413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">22 x 40 mm glass coverslip</td>
			<td style="width:124px;">VWR</td>
			<td>48393-172</td>
			<td></td>
		</tr>
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			<td height="42" style="height:42px;width:263px;">Forceps&#160;</td>
			<td style="width:124px;">Fine Science Tools</td>
			<td style="width:119px;">91150-20</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Whatman 3mm Chromatography Paper</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">05-713-336</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Vaseline</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">19-086-291</td>
			<td>Or purchase at local pharmacy.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">30ml syringe</td>
			<td style="width:124px;">Fisher Scientific</td>
			<td style="width:119px;">S7510-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Adobe Photoshop CS5</td>
			<td style="width:124px;">Adobe</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Leica TCS SP5 DM microscope</td>
			<td style="width:124px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">LAS AF Version 2.6.0.7266 microscope software</td>
			<td style="width:124px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

live-imaging of the drosophila pupal eye

Врожденные процессы развития Drosophila куколки могут смещаться и искажать глаз эпителий способами, которые делают индивидуальное поведение клеток трудно отследить во время съемки живых клеток. Эти процессы включают в себя: сетчатки вращение, рост клеток и организменном движение. Кроме того, нарушения в топологии эпителия, в том числе тонких ударов и складки часто вводят в куколки получают для работы с изображениями, сделать это сложно приобрести в фокусе изображения более чем на несколько омматидиев в одном фокальной плоскости. Рабочий процесс описанный здесь исправляет эти вопросы, что позволяет легко анализировать клеточных процессов во время Drosophila развития куколки глаз. Соответственно, в постановке куколки расположены в буровой изображений, которые можно легко собрать в большинстве лабораторий убиквитин-ДЕ-кадгерин:. GFP и ГМР-GAL4 управляемом БАС-α-катенин: GFP используются для визуализации границ клеток в эпителии глаз 1 -3. После деконволюцииприменяется для флуоресцентных изображений, снятых при нескольких фокальных плоскостях, максимальный выступ изображения создаются для каждого момента времени и улучшено с использованием редактирования изображений программное обеспечение. Выравнивание алгоритмы используются для быстрого стабилизации лишнего движения, что делает индивидуальное поведение клеток легче отслеживать.

Live-визуализация куколки глаза успешная стратегия для наблюдения клеток поведения, которые способствуют нанесению нейроэпителия. Роль специфических белков может быть легко оценена путем экспрессии трансгенов, которые изменяют уровни белка в процессе разработки глаз. Для этого GMR-GAL...

Watch this Scientific Journal Video about Live-imaging of the Drosophila Pupal Eye at JoVE.com

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Live-визуализация<em&gt; Drosophila</em&gt; Куколки глаз

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

Research

JoVE Journal

Developmental Biology

Live-imaging of the Drosophila Pupal Eye

9.3K Views.  Wesleyan University. This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

Video: Live-imaging of the Drosophila Pupal Eye

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

Долгосрочный Живая съемка<em&gt; Drosophila</em&gt; Диски глаз

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells named wing imaginal discs formed during embryonic development. In the larval stages the imaginal discs grow, increasing its number of cells and forming monolayered epithelial&#160;structures. Inside the pupal case, the imaginal discs bud out and fold into bilayers along a line that becomes the future margin of the wing. During this process, the longitudinal primodia veins originate vein cells on the prospective dorsal and ventral surfaces of the wing. During the pupal stage the stripes of vein cells of each surface communicate in order to generate tight tubes; at the same time, the cross-veins begin their formation.
With the help of appropriate molecular markers, it is possible to identify the major elements composing the wing during its development. For this reason, the ability to accurately detect transcripts or proteins in this structure is critical for studying their abundance and localization related to the development process of the wing.
The procedure described here focuses on manipulating pupal wings, providing detailed instructions on how to dissect the wing during the pupal stage. The dissection of pupal tissue is more difficult to perform than their counterparts in third instar larvae. This is why this approach was developed, to obtain rapid and efficient high quality samples. Details of how to immunostain and mount these wing samples, to allow the visualization of proteins or cell components, are provided in the protocol. With little expertise it is possible to collect 8-10 high quality pupal wings in a short amount of time.

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

The ability to accurately detect transcripts or proteins in Drosophila tissues is critical for studying their abundance and localization related to the development process. Here is the description of a straightforward procedure to dissect pupal wings. These wings can be used as samples in several techniques (immunohistochemistry, PCR assay, etc.).

Быстрый и эффективный метод для того чтобы рассечь куколки крылья дрозофилы подходит для Immunodetections или анализы ПЦР

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Биоинжиниринг гуманизированных микроуровней костного мозга в мышах и их визуализация с помощью Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Живая визуализация мышей

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

Рассечение и пятнать дрозофилы куколки яичников

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

Рассечение дрозофилы куколки сетчатки для иммуногистохимии, Западный анализ и РНК изоляции

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

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

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

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

Live Imaging и анализ мышечных сокращений в дрозофилы эмбриона

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

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

Размораживание, культивирования и Cryopreserving дрозофила клеточных линий

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Изображение и анализ ориентации тканей и динамики роста в развивающихся дрозофилы Эпителия во время Pupal Этапы

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

Подготовка Drosophila Ларваль и Pupal Тесты для анализа клеточного отдела в прямом эфире, Intact Ткани

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...