Patrick Cafferty è supportato da una borsa di studio post-dottorato dalla Multiple Sclerosis Society of Canada.

Parte 1: Pre-set-up sperimentale. <ol><li> Una settimana di anticipo, compagno vola a generare larva che esprimono GFP sotto il controllo di un gliali specifico promotore. Per il nostro esperimento abbiamo visualizzato GFP taggati con una sequenza di localizzazione nucleare espressi nelle cellule gliali con la polarità invertita (pronti contro termine) promotore (3, 4). </li><li> Preparare copertura scivola almeno un giorno di anticipo da una immersione 18 millimetri coprioggetto tondo per 10 minuti in 1% di poli-L-lisina soluzione e aria secca durante la notte. </li><li> Il giorno prima dell&#39;esperimento, pulire una camera Chamlide cultura magnetico immergendo i componenti durante la notte nel 70% di etanolo. </li><li> Il giorno dell&#39;esperimento, preparare un terreno di coltura con l&#39;aggiunta di siero fetale bovino, penicillina-streptomicina soluzione, e l&#39;insulina a 10 ml di terreno insetti Schneider con tecnica asettica. Le concentrazioni di lavoro sono: 1X siero fetale bovino; 100U/ml penicillina, streptomicina 0.1mg/ml e 0,2 mg / ml di insulina nel medio insetti Schneider (modificato da (5)). </li><li> Lasciare che la camera di cultura ad asciugare in una cappa di coltura cellulare. Lavare etanolo residua dalla camera di cultura lavando con terreno di coltura preparato. </li></ol> Parte 2: Preparazione del occhio-cervello complesso Drosophila. <ol><li> Selezionare un terzo instar larva errante dal lato di una fiala mosca e posto in una goccia di terreno di coltura fresco su una piastra di Petri in ghiaccio per diversi minuti. Agghiacciante la larva si consiglia di rallentare le contrazioni peristaltiche del corpo per facilitare la dissezione. </li><li> Posizionare la larva raffreddato in una goccia di terreno di coltura su un piatto rivestito Sylgard al microscopio di dissezione. </li><li> Sotto il microscopio dissezione, usare un paio di pinze Dumont fine per afferrare saldamente la larva circa un terzo della distanza dalla fine posteriore. Attendere che la larva estrude la sua bocca ganci a partire dalla fine anteriore. Afferrare la bocca ganci con un secondo paio di pinze sulla piena estensione. A sezionare la larva, tirare lentamente le due coppie di pinze in direzioni opposte. L&#39;occhio-cervello complesso, insieme con le ghiandole salivari, corpi grassi, e il tessuto immaginale, tirerà fuori dal corpo larvale. Non tirare la bocca ganci troppo in fretta altrimenti si strappano l&#39;occhio-cervello complesso. </li><li> Troncare ghiandole salivari, corpi grassi, e dischi immaginale ultra-sottile forbici clipper. I due emisferi del cervello e degli occhi-dischi sarà attaccato al cordone nervoso ventrale e ganci bocca. </li><li> Inserire un vetrino 18 mm su un vetrino. Lasciare un bordo del vetrino appeso fuori dal bordo della diapositiva in modo da raccogliere dopo il vetrino. Aggiungere una goccia di terreno di coltura sul slittamento 18 millimetri di copertura. Trasferire la occhio-cervello complesso nel terreno di coltura afferrando i ganci bocca con una pinza. Non cogliere l&#39;occhio-cervello complesso direttamente con il forcipe o il tessuto sarà danneggiato. </li><li> Dissezione con forbici bene, tagliare la bocca ganci da occhio-cervello complesso e scartare. La rimozione dei ganci bocca è importante per l&#39;imaging vivere come i ganci bocca continuerà a contrarsi nel terreno di coltura che causa il tessuto di muoversi durante la microscopia. </li><li> Per visualizzare la migrazione gliale all&#39;interno del disco occhio immaginale, accuratamente tagliato il gambo ottica, il tessuto sottile che collega il cervello e il disco occhi e allontanare o scartare il cervello (vedi figura 1A). Per visualizzare glia all&#39;interno del fusto ottica, lasciare il cervello, gambo di ottica, e il disco immaginale occhio intatto (vedi figura 1E). </li><li> Sollevare il coprioggetto con pinze e disegnare un cerchio intorno sotto il tessuto di interesse con un pennarello indelebile. Questo cerchio aiuterà a localizzare il tessuto per microscopia nella parte 4. </li></ol> Parte 3: Montaggio del disco immaginale occhio in una camera di cultura magnetico. <ol><li> Utilizzando pinze, afferrare il bordo impiccagione del vetrino. Trasferire il vetrino per la piastra inferiore della camera di Chamlide magnetico senza disturbare il tessuto in coltura. </li><li> Mettere il silicone O-ring sul corpo principale della camera Chamlide. Installare il corpo principale sopra la piastra inferiore. </li><li> Aggiungere lentamente terreno di coltura alla camera. Non aggiungere il terreno di coltura troppo in fretta o il tessuto di interesse saranno disturbati. Non riempire completamente la camera per consentire lo scambio di gas sotto il coperchio. </li><li> Posizionare con delicatezza il coperchio invisibile sulla parte superiore della camera di Chamlide. </li></ol> Parte 4: Visualizzazione di migrazione di cellule gliali nel disco occhi. <ol><li> Posizionare la camera di cultura sul palcoscenico di un microscopio confocale o fluorescenza. Individuare l&#39;esempio utilizzando il cerchio sul vetrino come riferimento il basso ingrandimento. </li><li> Focus sul campione utilizzando un obiettivo 40x. Lasciare che il tessuto al depositarsi sui coprioggetti. Catturare le immagini del disco ottico dell&#39;occhio o perseguitare ogni 10-15min in 3-4 ore. Il tessuto può contrarsi e spostarsi fuori fuoco. Messa a fuoco manuale il tessuto prima di acquisizione delle immagini allevierà °è un problema. </li></ol> Parte 5: risultati Rappresentante: Eseguita correttamente, il nostro protocollo ci ha permesso di raccogliere una serie di immagini di GFP-tagged cellule gliali che migrarono dal gambo ottica nel disco occhio immaginale (Figura 1 BD). Mentre immagini dal vivo per un periodo di 60 minuti è stato sufficiente a osservare i cambiamenti nelle posizioni dei nuclei gliali in un disco selvaggio occhio tipo immaginale, nuclei gliali in un mutante di un gene necessario per la migrazione delle cellule gliali completamente non è riuscito a uscire dal gambo di ottica (frecce Figura 1 FH). Abbiamo colto occhio-cervello complessi per periodi fino a 240 minuti prima di osservare il deterioramento del tessuto coltura. In seguito i 240 minuti di tempo-punto si comincia a osservare le cellule GFP positive nel terreno di coltura circostante colto occhio-cervello complessi (freccia Figura 2 C). Inoltre in GFP si accumula nei punti diffuso in tutto il tessuto che suggerisce una rottura dell&#39;integrità dei tessuti. <img src="/files/ftp_upload/1155/figure1.png" alt="" width="589" height="241" /> Figura 1: immagini dal vivo di GFP-tagged nuclei gliali del tipo di sviluppo selvaggio e mutante sistemi visivi. A, E) delle immagini scattate con contrasto interferenziale differenziale (DIC) microscopio di colture di tipo selvatico e mutante occhio-immaginale dischi (ED). Nel tipo selvatico, le cellule gliali sono nato e migrano dal gambo ottica (OS) nell&#39;occhio-disco. Il cervello è stata rimossa per facilitare l&#39;appiattimento e l&#39;imaging del disco occhio immaginale. BD) microscopia a fluorescenza del tipo selvatico disco occhio colto immaginale in (A) a 0, 30 e 60 punti tempo minuscolo rivela GFP-tagged nuclei gliali migrare all&#39;interno del disco occhio. FH) microscopia a fluorescenza del mutante occhio-cervello complesso (E) a 0, 30 e 60 punti dimostra un tempo minuscolo stallo di GFP-tagged nuclei gliali entro il gambo ottica (freccia). Il cervello (BR) è stato lasciato attaccato al peduncolo ottico per facilitare l&#39;imaging della glia all&#39;interno del gambo. <img src="/files/ftp_upload/1155/Figure_2.png" alt="" width="600" height="156" /> Figura 2: immagini dal vivo di GFP-tagged membrane gliali in un disco selvaggio occhio tipo immaginale. Abbiamo colto con successo occhio-cervello complessi per 240 minuti a periodi. Tessuto colto più di 240 minuti inizia a crollare. Il terreno di coltura che circonda il disco immaginale occhio (ED), pedinare ottica (OS), e del lobo cerebrale (BR) a 0 (A) e 150 minuti (B), rispetto al minuto 300 time-point (C), è libero di cellule GFP-positive, indicato da una freccia.

<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.

In questo protocollo si descrivono osservazione della migrazione delle cellule gliali nel disco occhio immaginale usando la microscopia dal vivo. Nel nostro esempio wild-type (figura 1 dC), abbiamo utilizzato un marcatore nucleare GFP per osservare il movimento delle cellule gliali nel disco occhio nel corso di un&#39;ora. In un mutante di un gene candidato necessaria per la migrazione delle cellule gliali attualmente allo studio nel nostro laboratorio, abbiamo osservato uno stallo dei nuclei delle cellule glia...

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<td>Poly-L-lysine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P8920</td>
<td>&nbsp;</td>
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<td>Schneider's Insect Media</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S0146</td>
<td>&nbsp;</td>
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<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>
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<td>Ultra fine clipper scissors</td>
<td>Tool</td>
<td>Fine scientific tools</td>
<td>15200-00</td>
<td>&nbsp;</td>
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<td>Dumont #5 forceps</td>
<td>Tool</td>
<td>Fine scientific tools</td>
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<td>&nbsp;</td>
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<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

Cellule gliali di entrambi gli organismi vertebrati e invertebrati devono migrare verso le regioni obiettivo finale per ensheath e supportare i neuroni associati. Anche se i recenti progressi sono stati fatti per descrivere la migrazione live delle cellule gliali nell&#39;ala sviluppo pupale (1), gli studi di<em&gt; Drosophila</emLa migrazione delle cellule&gt; gliali sono in genere impegnati l&#39;esame di tessuto fissato. Vivono analisi al microscopio delle cellule mobili offre la possibilità di esaminare il comportamento cellulare durante tutto il processo migratorio, ivi compresa la determinazione del tasso di cambi di direzione e di crescita. In coppia con l&#39;uso di strumenti genetici, immagini dal vivo possono essere utilizzati per determinare i ruoli più precisi per i geni specifici nel processo di sviluppo. Precedenti lavori Silies<em&gt; Et al.</em&gt; (2) ha descritto la migrazione di cellule gliali provenienti dal gambo ottica, una struttura che collega l&#39;occhio e il cervello in via di sviluppo, nel disco occhio immaginale in tessuto fissato. Qui delineare un protocollo per l&#39;esame della migrazione live delle cellule gliali nella<em&gt; Drosophila</em&gt; Disco occhio immaginale. Ci avvaliamo di una linea di Drosophila che esprime GFP in cellule gliali di sviluppo per seguire la progressione delle cellule gliali di tipo negli animali selvatici e mutanti.

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

Qui descriviamo un protocollo per esaminare la migrazione delle cellule gliali in un occhio lo sviluppo di Drosophila mediante l&#39;analisi microscopica vivono in coppia con tag GFP cellule gliali.

Vivere Imaging della migrazione cellule gliali nella Eye Disco Drosophila immaginale

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

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10.6K Views.  University of British Columbia - UBC. Qui descriviamo un protocollo per esaminare la migrazione delle cellule gliali in un occhio lo sviluppo di Drosophila mediante l'analisi microscopica vivono in coppia con tag GFP cellule gliali.

Video: 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.

Dissezione di dischi immaginale da larve 3 Instar Drosophila

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Biologia

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.

Visualizzazione di Live Drosophila Gliali-giunzione neuromuscolare con tinture fluorescenti

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 preparazione di Drosophila Gli embrioni per Live-Imaging Utilizzando il protocollo goccia

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 Reporter Novel RFP per aiutare nella visualizzazione del Disco Eye immaginale in Drosophila</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.

Immagini dal vivo di Drosophila melanogaster Migrazioni Hemocyte embrionali

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.

Imaging Forma Modifica cella in Living Drosophila Embrioni

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.

Vivo Imaging delle proteine ​​GFP-etichettati in<em&gt; Drosophila</em&gt; Gli oociti

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.

Vivere Imaging di apoptotica delle cellule libera nel corso<em&gt; Drosophila</em&gt; Embriogenesi

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.

Il pomodoro / GFP-FLP / FRT Metodo per Live Imaging del Mosaico adulti<em&gt; Drosophila</em&gt; cellule fotorecettrici

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.

Dissezione e Montaggio di<em&gt; Drosophila</em&gt; pupa Dischi Eye

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