Nous remercions le soutien à la BHR à travers une subvention à la découverte ainsi que d&#39;un outils de recherche et Grant Instrument de recherches en sciences naturelles et en génie du Canada (CRSNG). Nous reconnaissons également H. Oda et le stock de Bloomington drosophile Centre de fournir des stocks génétiques qui ont été utilisés dans des séquences d&#39;imagerie par exemple la vivre.

Préparation <ol><li> Recueillir des embryons sur le standard des plaques d&#39;agar jus de raisin 4. Il est commode d&#39;utiliser un système automatisé de drosophile Egg Collector (flymax scientifique Equipment Ltd.) Utiliser synchrones en scène des collections embryon réduire le nombre de dechorionations qui doivent être effectuées afin d&#39;obtenir un nombre suffisant d&#39;embryons au stade désiré de développement. </li><li> Préparer une diapositive dechorionation en attachant un morceau de scotch double-face à une lame de microscope, retirer la pellicule protectrice de la bande. </li><li> Préparer la chambre de l&#39;imagerie en direct par couper un morceau de tissu pour tenir dans le puits de la chambre. Placez le tissu dans le puits et le mouillé avec de l&#39;eau distillée. </li><li> Mélanger l&#39;huile d&#39;halocarbures (Halocarbon Products Corp) de la série 700 et série 56 de viscosité à un ratio de 1:1. Le mélange peut être stocké et utilisé indéfiniment. </li><li> Placez plusieurs petites gouttes d&#39;huile d&#39;halocarbures sur la surface d&#39;une lamelle (22 x 40 mm, n ° 2). Le volume n&#39;est pas critique mais doit être de l&#39;ordre de ul 20-40. </li></ol> Procédure <ol><li> Avec l&#39;aide d&#39;un stéréomicroscope, recueillir des embryons à partir du raisin Jus-agar en utilisant soit une paire de pinces de bijoutier (n ° 5) ou d&#39;un pinceau très fin. Les embryons ont tendance à coller les unes aux autres et à des conseils de la pince », ils sont facilement collectées en touffes. </li><li> Avec l&#39;aide d&#39;un stéréomicroscope, abaissez doucement la touffes d&#39;embryons qui ont adhéré à la pointe de la pince sur la surface de la bande sur la diapositive dechorionation. </li><li> En utilisant à nouveau le stéréomicroscope, doucement pousser ou un AVC que les embryons avec le côté de la pince, afin de casser la chorion externe cireuse sans rupture de la membrane interne vitelline (l&#39;embryon va éclater, si la membrane vitelline est rompue). </li><li> Une fois le chorion externe a été rompue, taquiner l&#39;embryon à partir du chorion, l&#39;embryon dechorionated tend à adhérer à la surface de la pince. Prenez soin d&#39;éviter de toucher l&#39;embryon dechorionated à la surface de la bande. </li><li> Dès que l&#39;embryon n&#39;est dechorionated, rapidement, il transfert à la baisse précédemment préparé de l&#39;huile d&#39;halocarbures sur la lamelle. Toucher la pointe de la pince transportant l&#39;embryon dechorionated à la surface de la goutte d&#39;huile sur les halocarbures - ce déloge l&#39;embryon de la pince dans l&#39;huile. </li><li> Confirmer le transfert de l&#39;embryon à la goutte d&#39;huile. Cela peut être fait à l&#39;œil nu à condition que vous travaillez sur un fond noir et d&#39;utiliser une source d&#39;illumination par fibre optique fixé à un angle oblique. </li><li> Avant de tenter de dechorionate autre embryon, nettoyer toute l&#39;huile de la pince. Pince enduit d&#39;huile ont moins d&#39;achat sur la surface du chorion, ce qui rend plus difficile dechorionation. </li><li> Embryons sont porteurs Dechorionated dans l&#39;huile d&#39;halocarbures. En utilisant une pince ou d&#39;un pinceau et tout en regardant à travers une loupe binoculaire, poussez l&#39;embryon au fond de la goutte d&#39;huile et l&#39;organiser dans la position désirée. </li><li> Vite inverser la lamelle sur le bien de la chambre de l&#39;imagerie en direct. Confirmez que les embryons sont au repos contre la lamelle dans l&#39;orientation désirée. En raison de leur flottabilité dans l&#39;huile, les embryons seront désormais flotter contre la surface inférieure de la lamelle. Fixer la lamelle de la chambre de l&#39;imagerie en direct avec du ruban adhésif. Aérer la chambre par des trous dans la bande dans la zone où la bande enjambe le fossé entre la fin de la lamelle et la fin du puits de la chambre de l&#39;imagerie en direct. </li><li> Un microscope à fluorescence verticale ou un stéréomicroscope à fluorescence peut maintenant être utilisé à l&#39;image de l&#39;embryon. </li></ol>

<ol>
	<li>Kiehart, D. P., Galbraith, C. G., Edwards, K. A., Rickoll, W. L., Montague, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+forces+contribute+to+cell+sheet+morphogenesis+for+dorsal+closure+in+Drosophila.">Multiple forces contribute to cell sheet morphogenesis for dorsal closure in Drosophila.</a> J. Cell Biol. 149, 471-490 (2000).</li><li>Schock, F., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+processes+associated+with+germ+band+retraction+in.">Cellular processes associated with germ band retraction in.</a> , 248-2429 (2002).</li><li>Reed, B. H., Wilk, R., Schock, F., Lipshitz, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin-dependent+apposition+of+Drosophila+extraembryonic+membranes+promotes+morphogenesis+and+prevents+anoikis.">Integrin-dependent apposition of Drosophila extraembryonic membranes promotes morphogenesis and prevents anoikis.</a> Curr. Biol. 14, 372-380 (2004).</li><li>Wieschaus, E. p., Nusslein-Volhard, C., Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila: a practical approach. , 199-227 (1986).</li></ol>

Nous décrivons une nouvelle méthode de préparation des embryons de drosophile pour l&#39;analyse de l&#39;imagerie en direct, que nous appelons le protocole goutte suspendue. Malheureusement, il n&#39;est pas possible d&#39;utiliser le protocole goutte suspendue si l&#39;on travaille avec un microscope inversé. Dans ce cas, la technique sandwich (comme décrit ci-dessus dans le résumé) doivent être utilisées et la compression des embryons reste un sujet de préoccupation. Dans les ex...

L&#39;équipement standard et les matériaux nécessaires à la préparation des embryons pour vivre d&#39;imagerie utilisant le protocole goutte suspendue comprennent les éléments suivants: des lames de microscope, lamelles (22 x 40 mm, n ° 2), ruban adhésif double face, des pinces de bijoutier (style Dumont n ° 5 ), huile d&#39;halocarbures série 56 et série 700 (Halocarbon Products Corp), un couteau ou une lame de rasoir seule pointe, papier de soie, des ciseaux, de l&#39;eau distillée, ruban à usage général, et une pipette apte à délivrer ul 20-40. Le protocole d&#39;imagerie vivons exige également une chambre de sur-mesure imagerie en temps réel. Il est préparé par une réduction de 5 mm en polycarbonate pense feuille de plastique pour les dimensions d&#39;une lame de microscope standard (75 x 25 mm) et en utilisant un rotor pour créer une dépression profonde 3mm dans la glissière (20 X 55 mm). L&#39;accès à un stéréomicroscope (loupe binoculaire) et une source d&#39;illumination par fibre optique est également requise.

the preparation of drosophila embryos for live-imaging using the hanging drop protocol

Protéine fluorescente verte (GFP) basé timelapse vivons-imagerie est une technique puissante pour étudier la régulation génétique de processus dynamiques tels que la morphogenèse des tissus, adhérence cellule-cellule, ou la mort cellulaire. Embryons de drosophile exprimant la GFP sont facilement imagée en utilisant la microscopie soit stéréoscopique ou confocale. Un but de tout protocole vivons-imagerie est de minimiser les effets néfastes tels que la déshydratation et l&#39;hypoxie. Protocoles antérieurs pour préparer des embryons de drosophile pour live-analyse de l&#39;imagerie ont impliqué plaçant embryons dechorionated dans l&#39;huile d&#39;halocarbures et de les intercaler entre un halocarbure perméables au gaz de membrane et une lamelle<sup&gt; 1-3</sup&gt;. L&#39;introduction de la compression à travers embryons de montage de cette manière représente une complication indésirable pour toute analyse biomécanique basée sur la morphogenèse. Notre méthode, que nous appelons le protocole goutte suspendue, résultats dans une excellente viabilité des embryons lors de l&#39;imagerie en direct et ne nécessite pas que les embryons soient compressées. Brièvement, le protocole goutte suspendue implique le placement d&#39;embryons dans une goutte d&#39;huile sur les halocarbures qui est suspendu à partir d&#39;une lamelle, qui est, à son tour, en position fixe sur une chambre humide. En plus d&#39;offrir l&#39;échange de gaz et prévenir la déshydratation, cet arrangement profite de la flottabilité de l&#39;embryon dans l&#39;huile d&#39;halocarbures pour les empêcher de dériver hors de position lors de l&#39;acquisition timelapse. Cette vidéo décrit en détail la façon de recueillir et de préparer des embryons de drosophile pour l&#39;imagerie en direct en utilisant le protocole goutte suspendue. Ce protocole est adapté pour les embryons d&#39;imagerie utilisant dechorionated stéréomicroscopie ou microscope composé debout fluorescence.

Watch this Scientific Journal Video about The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol at JoVE.com

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Une méthode simple, peu coûteux et efficace de préparer les embryons de drosophile pour live-imagerie analyse est présentée. Notre protocole prévoit l&#39;échange d&#39;humidité et de gaz et ne compresse pas l&#39;embryon de drosophile. Cette méthode est adaptée pour la GFP imagerie basée sur des embryons de drosophile direct en utilisant un stéréomicroscope ou microscope composé debout.

La préparation des Drosophile Les embryons destinés à Live-imagerie utilisant le protocole goutte suspendue

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

the-preparation-drosophila-embryos-for-live-imaging-using-hanging

Research

JoVE Journal

Biology

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

15.0K vues.  University of Waterloo. Une méthode simple, peu coûteux et efficace de préparer les embryons de drosophile pour live-imagerie analyse est présentée. Notre protocole prévoit l'échange d'humidité et de gaz et ne compresse pas l'embryon de drosophile. Cette méthode est adaptée pour la GFP imagerie basée sur des embryons de drosophile direct en utilisant un stéréomicroscope ou microscope composé debout.

Vidéo: The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Préparation des cultures de neurones d&#39;embryons de drosophile Midgastrula scène

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Recherche

Biologie

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Différenciation in vitro de souris embryonnaires souches (MES) Cellules utilisant la méthode de goutte suspendue

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Imagerie de la migration en direct des cellules gliales dans le disque Eye drosophile Imaginal

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

En direct l&#39;imagerie du cerveau de poisson zèbre embryonnaires par microscopie confocale

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Préparation d&#39;embryons pour la microscopie électronique des Drosophile Tube cardiaque embryonnaire

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

En direct de l&#39;imagerie Drosophila melanogaster Migrations hémocytaires embryonnaires

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Préparation de développement et de l&#39;adulte Drosophile Cerveaux et la rétine d&#39;imagerie en temps réel

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Préparation individuelle Drosophila Œuf Chambres pour l&#39;imagerie en direct

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivre imagerie de dédouanement cellulaire par apoptose au cours<em&gt; Drosophila</em&gt; Embryogenèse

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

Le / GFP-FLP Méthode tomate / FRT pour l&#39;imagerie en direct de mosaïque adulte<em&gt; Drosophila</em&gt; cellules photoréceptrices

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...