Patrick Cafferty est soutenu par une bourse de recherche postdoctorale de la Société canadienne de la sclérose.

Partie 1: Pré-montage expérimental. <ol><li> Une semaine à l&#39;avance, compagnon mouches pour générer larve qui expriment la GFP sous le contrôle d&#39;un promoteur spécifique gliales. Pour notre expérience, nous visualisés GFP taggés avec une séquence de localisation nucléaire exprimé dans les cellules gliales en utilisant les inversions de polarité (repo) promoteur (3, 4). </li><li> Préparer la couverture glisse au moins un jour à l&#39;avance par trempage 18 mm de diamètre glisse couvercle pendant 10 minutes dans 1% de poly-L-lysine solution et l&#39;air sec durant la nuit. </li><li> Le jour avant l&#39;expérience, nettoyer une chambre de culture Chamlide magnétiques par trempage des composants nuit dans l&#39;éthanol à 70%. </li><li> Le jour de l&#39;expérience, de préparer un milieu de culture par l&#39;ajout de sérum bovin fœtal, la pénicilline-streptomycine solution, et l&#39;insuline pour 10 ml de milieu de Schneider insectes en utilisant une technique aseptique. Les concentrations de travail sont: 1X sérum fœtal bovin; 100U/ml pénicilline; streptomycine 0.1mg/ml, et 0,2 mg / ml d&#39;insuline dans le milieu des insectes Schneider (modifié d&#39;après (5)). </li><li> Autoriser la chambre de culture à l&#39;air sec dans une hotte de culture cellulaire. Laver l&#39;éthanol résiduel de la chambre de culture par un rinçage avec un milieu de culture préparé. </li></ol> Partie 2: Dissection de la drosophile oeil-cerveau complexe. <ol><li> Sélectionnez une larve du troisième stade larvaire errance du côté d&#39;un flacon de volée et placer dans une goutte de milieu de culture frais sur une boîte de Pétri sur la glace pendant plusieurs minutes. Chilling la larve est recommandé de ralentir les contractions péristaltiques du corps pour faciliter la dissection. </li><li> Placer la larve réfrigérée dans une goutte de milieu de culture sur un plat recouvert Sylgard sous un microscope de dissection. </li><li> Sous le microscope de dissection, utiliser une paire de pinces fines Dumont de saisir fermement la larve d&#39;environ un tiers de la distance entre l&#39;extrémité postérieure. Attendez jusqu&#39;à ce que la larve extrude ses crochets la bouche de l&#39;extrémité antérieure. Saisir la bouche des crochets avec une deuxième paire de forceps lors de l&#39;extension complète. Pour disséquer la larve, tirez lentement les deux paires de forceps dans des directions opposées. Le complexe oeil-cerveau, avec les glandes salivaires, des corps gras, et les tissus imaginaux, va se détacher du corps larvaire. Ne tirez pas sur la bouche des crochets trop rapidement sinon ils vont déchirer du complexe oeil-cerveau. </li><li> Coupez les glandes salivaires, des corps gras, et les disques imaginaux de l&#39;aide de ciseaux clipper ultra-fines. Les deux hémisphères du cerveau et des yeux disques sera attaché à la corde nerveuse ventrale et crochets bouche. </li><li> Placez un feuillet de 18 mm couvercle sur une lame de verre. Laisser un bord de la lamelle pendait au bord de la diapositive et pouvoir ensuite ramasser les lamelle. Ajouter une goutte de milieu de culture sur le bordereau de 18 mm de couverture. Transfert du complexe oeil-cerveau dans le milieu de culture en saisissant les crochets la bouche avec une pince. Ne pas saisir le complexe de l&#39;œil-cerveau directement avec la pince ou le tissu sera endommagé. </li><li> Avec des ciseaux de dissection fine, couper la bouche des crochets du complexe oeil-cerveau et le jeter. Retrait des crochets bouche est importante pour l&#39;imagerie vivre comme les crochets buccaux continuera de se contracter dans le milieu de culture des tissus provoquant de se déplacer pendant la microscopie. </li><li> Pour visualiser la migration gliales dans le disque imaginal des yeux, couper soigneusement la tige optique, le tissu mince qui relie le cerveau et le disque yeux et pousser ou vous débarrasser du cerveau (voir figure 1A). Pour visualiser la GLIA dans le pédoncule optique, laissez le cerveau, la tige optique, et le disque imaginal des yeux intacts (voir figure 1E). </li><li> Ramassez la lamelle à l&#39;aide de pinces et de dessiner un cercle au-dessous autour du tissu d&#39;intérêt avec un marqueur permanent. Ce cercle vous aideront à localiser les tissus pour la microscopie à la partie 4. </li></ol> Partie 3: Le montage du disque imaginal des yeux dans une chambre de culture magnétique. <ol><li> En utilisant une pince, saisir le bord pendaison de la lamelle. Transférer la lamelle à la plaque de fond de la chambre Chamlide magnétiques sans déranger les tissus en culture. </li><li> Placez le joint torique silicone sur le corps principal de la chambre de Chamlide. Installer le corps principal sur le dessus de la plaque de fond. </li><li> Ajouter lentement milieu de culture à la chambre. Ne pas ajouter le milieu de culture trop vite ou le tissu d&#39;intérêt sera perturbé. Ne pas remplir complètement la chambre de permettre l&#39;échange de gaz sous le couvercle. </li><li> Placez délicatement le couvercle invisible sur le dessus de la chambre de Chamlide. </li></ol> Partie 4: Visualisation de la migration des cellules gliales dans le disque oculaire. <ol><li> Placez la chambre de culture sur la scène d&#39;un microscope confocal ou fluorescence. Localisez l&#39;échantillon en utilisant le cercle sur la lamelle comme une référence sur le faible grossissement. </li><li> Focus sur l&#39;échantillon en utilisant une lentille 40x. Autoriser le tissu à régler sur la lamelle. Capture d&#39;images du disque optique oculaire ou tige tous les 10-15min en 3-4 heures. Le tissu peut se contracter et de sortir de discussion. Mise au point manuelle du tissu avant la capture d&#39;image permettra d&#39;alléger èmeest le problème. </li></ol> Partie 5: Les résultats représentatifs: Effectuée correctement, notre protocole nous a permis de collecter une série d&#39;images de la GFP-taggés cellules gliales qui ont migré de la tige optique dans le disque imaginal des yeux (figure 1 BD). Alors que l&#39;imagerie en direct pour une période de 60 minutes était suffisant pour observer les changements dans les positions des noyaux gliales dans un disque de type sauvage oeil imaginale, les noyaux gliales dans un mutant d&#39;un gène nécessaire à la migration des cellules gliales complètement échoué à la sortie de la tige optique (flèches Figure 1 FH). Nous avons cultivé oeil-cerveau complexes pour des périodes aussi longues que 240 minutes avant d&#39;observer une détérioration du tissu cultivé. Après les 240 minutes de temps-point, nous commençons à observer des cellules GFP-positives dans le milieu de culture environnante cultivée oeil-cerveau complexes (flèche figure 2 C). En outre GFP vont s&#39;accumuler dans les endroits diffuse dans les tissus suggérant une rupture de l&#39;intégrité des tissus. <img src="/files/ftp_upload/1155/figure1.png" alt="" width="589" height="241" /> Figure 1: Imagerie en direct de la GFP-noyaux marqués gliales dans le type sauvage et mutants de développement de systèmes visuels. A, E) Les images prises à l&#39;aide de contraste d&#39;interférence différentiel (DIC) microscope de type sauvage et mutant cultivé oeil-imaginaux disques (ED). Dans le type sauvage, les cellules gliales sont nés dans et migrent de la tige optique (OS) dans l&#39;œil-disque. Le cerveau a été enlevé pour faciliter l&#39;aplatissement et l&#39;imagerie du disque imaginal des yeux. BD) microscopie à fluorescence du disque oeil de type sauvage cultivée imaginale en (A) à 0, 30 et 60 points de temps minutieux révèle GFP-noyaux marqués gliales migrer à l&#39;intérieur du disque oeil. FH) microscopie à fluorescence du mutant oeil-cerveau complexe dans (E) à 0, 30 et 60 points de temps minutieux démontre un décrochage de la GFP-noyaux marqués gliales au sein de la tige optique (flèche). Le cerveau (BR) a été laissée attachée à la tige optiques pour faciliter l&#39;imagerie des cellules gliales dans le pédoncule. <img src="/files/ftp_upload/1155/Figure_2.png" alt="" width="600" height="156" /> Figure 2: Imagerie en direct de la GFP-taggés membranes gliales dans l&#39;œil d&#39;un disque de type sauvage imaginale. Nous avons réussi cultivés oeil-cerveau complexes pour 240 minutes de périodes. Tissus cultivés plus de 240 minutes commence à se décomposer. Le milieu de culture entourant le disque imaginal des yeux (ED), la tige optique (OS), et le lobe du cerveau (BR) à 0 (A) et 150 minutes (B), comparativement à 300 la minute de temps-point (C), est libre de la GFP de cellules positives, indiqué par une flèche.

<ol>
	<li>Aigouy, B., Lepelletier, L., Giangrande, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+chain+migration+requires+pioneer+cells.">Glial chain migration requires pioneer cells.</a> J. Neurosci. 28, 11635-11641 (2008).</li><li>Silies, M., Yuva, Y., Engelen, D., Aho, A., Stork, T., Klambt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+migration+in+the+eye+disc.">Glial cell migration in the eye disc.</a> J. Neurosci. 27, 13130-13139 (2007).</li><li>Brand, A. H., 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=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>Sepp, K. J., Auld, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+lacZ+enhancer+trap+lines+to+GAL4+lines+using+targeted+transposition+in+Drosophila+melanogaster.">Conversion of lacZ enhancer trap lines to GAL4 lines using targeted transposition in Drosophila melanogaster.</a> Genetics. 151, 1093-1101 (1999).</li><li>Gibson, M. C., Patel, A. B., Nagpal, R., 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=The+emergence+of+geometric+order+in+proliferating+metazoan+epitheia.">The emergence of geometric order in proliferating metazoan epitheia.</a> Nature. 442, 1038-1041 (2006).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+for+Drosophila+neural+development.">Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.</a> Trends Neurosci. 24, 251-254 (2001).</li></ol>

The authors have nothing to disclose.

Dans ce protocole, nous décrivons l&#39;observation de la migration des cellules gliales dans le disque imaginal des yeux en utilisant la microscopie vivre. Dans notre exemple de type sauvage (figure 1 après JC), nous avons utilisé un marqueur GFP nucléaires d&#39;observer le mouvement des cellules gliales dans le disque les yeux au cours d&#39;une heure. Dans un mutant d&#39;un gène candidat requis pour la migration des cellules gliales actuellement à l&#39;étude dans notre laboratoire, nous avons obser...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Poly-L-lysine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P8920</td>
<td>&nbsp;</td>
<tr>
<td>Schneider's Insect Media</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0146</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P4458</td>
<td>&nbsp;</td>
<tr>
<td>Insulin solution from bovine pancreas</td>
<td>Reagent</td>
<td>Sigma</td>
<td>I0516</td>
<td>&nbsp;</td>
<tr>
<td>Chamlide Magnetic chamber</td>
<td>Tool</td>
<td>Live cell Instrument</td>
<td>CM-R-10</td>
<td>35 mm dish type chamber for 18 mm coverslip</td>
</tr>
<tr>
<td>Ultra fine clipper scissors</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>15200-00</td>
<td>&nbsp;</td>
<tr>
<td>Dumont #5 forceps</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>11251-20</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>Microscope</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>Any fluorescent imaging system that has the necessary filters and excitation for GFP can be used. </td>
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live imaging of glial cell migration in the drosophila eye imaginal disc

Les cellules gliales du vertébrés et invertébrés organismes doivent migrer vers des régions cibles finale afin de ensheath et de soutien des neurones associés. Bien que des progrès récents ont été faits pour décrire la migration en direct des cellules gliales dans l&#39;aile de développement nymphal (1), les études de<em&gt; Drosophile</em&gt; Migration des cellules gliales ont généralement impliqués l&#39;examen des tissus fixés. Analyse microscopique direct des cellules mobiles offre la possibilité d&#39;examiner le comportement cellulaire tout au long du processus migratoire, y compris la détermination du taux de change et en direction de la croissance. Couplé à l&#39;utilisation d&#39;outils génétiques, imagerie en temps réel peut être utilisé pour déterminer les rôles plus précis pour des gènes spécifiques dans le processus de développement. Les travaux antérieurs de Silies<em&gt; Et al.</em&gt; (2) a décrit la migration des cellules gliales en provenance de la tige optique, une structure qui relie l&#39;œil et le cerveau de développement, dans le disque imaginal des yeux dans les tissus fixés. Nous exposons ici un protocole pour examiner la migration en direct des cellules gliales dans le<em&gt; Drosophile</emDisque oeil&gt; imaginale. Nous profitons d&#39;une ligne de drosophile qui exprime la GFP dans glie en développement de suivre la progression des cellules gliales de type sauvage et chez les animaux mutants.

Watch this Scientific Journal Video about Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc at JoVE.com

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

Nous décrivons ici un protocole visant à étudier la migration des cellules gliales dans le développement de l&#39;œil de drosophile en utilisant l&#39;analyse microscopique direct couplé à la GFP taggés cellules gliales.

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-glial-cell-migration-drosophila-eye-imaginal

Research

JoVE Journal

Biology

10.6K vues.  University of British Columbia - UBC. Nous décrivons ici un protocole visant à étudier la migration des cellules gliales dans le développement de l'œil de drosophile en utilisant l'analyse microscopique direct couplé à la GFP taggés cellules gliales.

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

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

Dissection de disques imaginaux de la 3ème Instar larves de drosophile

Recherche

Biologie

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle synapses, we developed a larval tissue preparation that had features of live intact larvae, but also had good optical properties.  This new preparation also allowed for access of perfusates to the synapse.  We used fly larvae, immersed them in artificial hemolymph, and relaxed their normal rhythmic body contractions by chilling them. Next we dissected off the posterior segments of each animal and with a blunt insect pin pushed the mouth parts backward through the body cavity.  This everted the larval body wall, like turning a sock inside-out.  We completed the dissection with ultra-fine dissection scissors and thus exposed the visceral side of the body wall muscles. The glial structures at the NMJ expressed membrane targeted GFP under the control of glial specific promoters. The post-synaptic membrane, the SSR (Subsynaptic Reticula) in muscle expressed synaptically targeted dsRed. We needed to acutely label the motor neuron terminals, the third part of the synapse. To do this we applied primary antibodies to HRP, conjugated to a far-red emitting flurophore.  To test for dye diffusion properties into the perisynaptic space between the motor neuron terminals and the SSR, we applied a solution of large Dextran molecules conjugated to far-red emitting flurophore and collected images.

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

We described structural features of the Glia-neuromuscular synapses in a novel Inside-out tissue preparation of live fly larvae using fluorescent dyes with confocal microscopy. We labeled live neuron terminals with fluorescent primary antibodies to HRP, and also visualized the perisynaptic space with fluorescent Dextrans.

Visualiser le Live Drosophile Gliales-jonction neuromusculaire avec des colorants fluorescents

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

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

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

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

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

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 ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

Un journaliste Roman DP pour faciliter la visualisation du disque Eye Imaginal au Drosophile</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

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 ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Changement de forme d&#39;imagerie cellulaire en vie Drosophile Embryons

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

Vivez l&#39;imagerie des protéines GFP-étiquetés<em&gt; Drosophila</em&gt; Les ovocytes

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

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, ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

Dissection et de montage de<em&gt; Drosophila</em&gt; pupes disques yeux

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...