Patrick Cafferty wird durch ein Postdoc-Stipendium von der Multiple Sclerosis Society of Canada unterstützt.

Teil 1: Pre-Versuchsanordnung. <ol><li> Eine Woche im Voraus, fliegt Kumpel zur Larve zu generieren, die ausdrückliche GFP unter der Kontrolle eines Glia-spezifischen Promotors. Für unser Experiment haben wir visualisiert GFP mit einem Kernlokalisierungssequenz in Gliazellen mit umgekehrter Polarität (Repo)-Promotor (3, 4) zum Ausdruck markiert. </li><li> Bereiten Deckgläser mindestens einen Tag im Voraus durch Einweichen 18 mm runde Deckgläser für 10 Minuten in 1% Poly-L-Lysin-Lösung und in der Luft über Nacht trocknen. </li><li> Am Tag vor dem Experiment, sauber ein Chamlide magnetischen Kultur Kammer durch Einweichen über Nacht Komponenten in 70% Ethanol. </li><li> Am Tag des Experiments, bereiten Kulturmedium durch Zugabe von fötalem Rinderserum, Penicillin-Streptomycin-Lösung und Insulin zu 10 ml Insektenmedium Schneider unter aseptischen Bedingungen. Die Arbeitsgruppe Konzentrationen sind: 1X Rinderfötenserum; 100U/ml Penicillin; 0.1mg/ml Streptomycin und 0,2 mg / ml Insulin in Insektenmedium Schneider (modifiziert nach (5)). </li><li> Lassen Sie die Kultur Kammer an der Luft in einer Zellkultur Haube trocknen. Abwaschen Restethanol aus dem Kulturraum durch Spülen mit vorbereiteten Kulturmedium. </li></ol> Teil 2: Präparation des Drosophila Auge-Gehirn-Komplex. <ol><li> Wählen Sie einen dritten Larvenstadium wandernde Larven von der Seite eine Fliege Fläschchen und in einen Tropfen gekühlt Kulturmedium auf eine Petrischale auf Eis für mehrere Minuten. Chilling die Larve wird empfohlen, peristaltische Kontraktionen Körper für eine einfache Zerlegung langsam. </li><li> Legen Sie die gekühlte Larve in einen Tropfen Kulturmedium auf einem Sylgard beschichtet Gericht unter einem Dissektionsmikroskop. </li><li> Unter dem Dissektionsmikroskop, verwenden Sie ein Paar Dumont feinen Pinzette fest zu greifen die Larve etwa ein Drittel des Weges aus dem hinteren Ende. Warten Sie, bis die Larve dringt in ihren Mund Haken aus dem vorderen Ende. Fassen Sie den Mund Haken mit einer zweiten Pinzette auf volle Streckung. Um sezieren die Larve, langsam ziehen die zwei Pinzetten in entgegengesetzte Richtungen. Die Auge-Gehirn-Komplex, zusammen mit Speicheldrüsen, Fettkörper und imaginalen Gewebe, wird wegziehen aus dem larvalen Körper. Ziehen Sie nicht den Mund Haken zu schnell sonst werden sie abreißen des Auge-Gehirn-Komplex. </li><li> Trim entfernt Speicheldrüsen, Fettkörper und Imaginalscheiben mit ultrafeinen clipper Schere. Die beiden Hemisphären des Gehirns und der Augen-Discs, die Bauchmark und Mund Haken befestigt werden. </li><li> Ein 18 mm Deckglas auf einen Objektträger. Lassen Sie einen Rand des Deckglases hängen über den Rand der Folie, um später abholen Deckglas. Fügen Sie einen Tropfen Kulturmedium auf die 18 mm Deckglas. Übertragen Sie die Auge-Gehirn-Komplex in das Kulturmedium durch Erfassen des Mundhaken mit einer Pinzette. Greifen Sie nicht die Auge-Gehirn-Komplex direkt mit der Pinzette oder das Gewebe beschädigt wird. </li><li> Mit feinen Sezierung Schere, schneiden Sie den Mund Haken aus dem Auge-Hirn-komplex und zu verwerfen. Entfernung von der Mündung Haken ist für Live-Bildgebung wichtig wie die Mundhaken wird weiterhin in das Kulturmedium wodurch das Gewebe während der Mikroskopie zu bewegen Vertrag. </li><li> Um glialen Migration innerhalb des Auges Imaginalscheibe visualisieren, sorgfältig schneiden die optische Stengel, die dünnen Gewebe, Gehirn und Auge Scheibe verbindet und wegzuschieben oder zu verwerfen das Gehirn (siehe Abbildung 1A). Zur Visualisierung Glia innerhalb der optischen Stiel, verlassen das Gehirn, Optik Stiel und Auge Imaginalscheibe intakt (siehe Abbildung 1E). </li><li> Pick-up das Deckglas mit einer Pinzette und ziehen Sie einen Kreis unter der ganzen Gewebe von Interesse mit einem Permanent-Marker. Dieser Kreis wird bei der Ortung des Gewebes für die Mikroskopie in Teil 4 zu unterstützen. </li></ol> Teil 3: Anschluss des Auges Imaginalscheibe in einem magnetischen Kultur Kammer. <ol><li> Mit einer Pinzette fassen Sie den hängenden Rand des Deckglases. Übertragen Sie die Deckglas auf die Bodenplatte des Chamlide magnetische Kammer, ohne die Gewebe in der Kultur. </li><li> Setzen Sie den Silikon-O-Ring auf den Hauptteil der Chamlide Kammer. Installieren Sie den Hauptteil auf der Bodenplatte. </li><li> Langsam Kulturmedium in die Kammer. Fügen Sie nicht das Kulturmedium zu schnell oder das Gewebe von Interesse gestört werden. Nicht vollständig füllen die Kammer für den Gasaustausch unter dem Deckmantel ermöglichen. </li><li> Sanft statt der unsichtbaren Abdeckung auf der Oberseite des Chamlide Kammer. </li></ol> Teil 4: Visualisierung der Migration Gliazellen im Auge CD. <ol><li> Legen Sie die Kultur Kammer auf der Bühne eines konfokalen oder Fluoreszenzmikroskop. Suchen Sie das Beispiel mithilfe der Kreis auf dem Deckglas als Referenz auf geringer Vergrößerung. </li><li> Konzentrieren Sie sich auf der Probe mit einem 40x Objektiv. Lassen Sie das Gewebe auf das Deckglas zu begleichen. Machen Sie Bilder des Auges Disc oder optischen Stiel jede 10-15min über 3-4 Stunden. Das Gewebe kann zucken und bewegen out of focus. Manuelles Fokussieren des Gewebes vor der Aufnahme von Bildern wird zu lindern thist Problem. </li></ol> Teil 5: Repräsentative Ergebnisse: Korrekt ausgeführt, erlaubt unser Protokoll wir eine Reihe von Bildern von GFP-markierten Gliazellen, die von der Optik Stiel ins Auge Imaginalscheibe (Abbildung 1 BD) migriert zu sammeln. Während Live-Bildgebung für eine 60-Minuten-Zeitraum war ausreichend, um Veränderungen in den Positionen der Glia-Kerne in einem Wildtyp-Auge Imaginalscheibe beobachten Gliazellen Kerne in einer Mutante für ein Gen, die für Gliazellen Migration völlig versäumt, die Optik Stiel (Pfeile Ausfahrt Abbildung 1 FH). Wir haben kultivierten Auge-Hirn-Komplexe für die Zeiträume, solange 240 Minuten vor Beobachtung Verschlechterung der gezüchtetes Gewebe. Nach dem 240-Minuten-Punkt fangen wir an, GFP-positiven Zellen in das Kulturmedium umliegenden kultivierten Auge-Gehirn-Komplexe (Pfeil Abbildung 2 C) zu beobachten. Darüber hinaus GFP wird in diffuse Flecken in den Geweben was auf einen Abbau im Gewebe Integrität zu akkumulieren. <img src="/files/ftp_upload/1155/figure1.png" alt="" width="589" height="241" /> Abbildung 1: Live Darstellung von GFP-markierten Gliazellen Kerne in den Entwicklungsländern Wildtyp und Mutante visuellen Systemen. A, E) aufgenommene Bilder mit Differential-Interferenz-Kontrast (DIC) Mikroskopie von kultivierten Wildtyp und Mutante eye-Imaginalscheiben (ED). Im Wildtyp sind Gliazellen in geboren und wandern von der Optik Stiel (OS) in das Auge-Disc. Das Gehirn wurde entfernt, um Abflachung und Bildgebung des Auges Imaginalscheibe erleichtern. BD) Fluoreszenzmikroskopie des Wildtyp-kultivierten Auge Imaginalscheibe in (A) bei 0, zeigt 30, und 60 Minuten Zeitpunkten GFP-markierten Gliazellen Kerne innerhalb des Auges Scheibe wandern. FH) Fluoreszenzmikroskopie des mutierten Auge-Gehirn-Komplex in (E) bei 0, zeigt, 30 und 60 Minuten Zeit, die Punkte a Abwürgen des GFP-markierten Gliazellen Kerne innerhalb der optischen Stiel (Pfeil). Das Gehirn (BR) wurde links an der Optik Stengel Bildgebung von Glia innerhalb der Stiel zu erleichtern. <img src="/files/ftp_upload/1155/Figure_2.png" alt="" width="600" height="156" /> Abbildung 2: Live Darstellung von GFP-markierten Gliazellen Membranen in einem Wildtyp-Auge Imaginalscheibe. Wir haben erfolgreich Auge-Hirn-Komplexe für die 240-Minuten-Perioden kultiviert. Tissue kultivierten länger als 240 Minuten beginnt zu bröckeln. Das Kulturmedium rund um das Auge Imaginalscheibe (ED), Optik Stiel (OS), und das Gehirn Lappen (BR) auf 0 (A) und 150 Minuten (B), im Vergleich zu den 300 Minuten Zeit-Punkt (C), ist frei der GFP-positiven Zellen, die durch einen Pfeil.

<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 diesem Protokoll beschreiben wir Beobachtung von Gliazellen Migration in das Auge Imaginalscheibe mit Live-Mikroskopie. In unserem Wildtyp Beispiel (Abbildung 1 AD), verwendeten wir eine nukleare GFP Marker Gliazellen Bewegung in die Augen zu beobachten Disc im Laufe von einer Stunde. In einer Mutante für einen Kandidaten-Gen für Gliazellen Migration derzeit untersucht in unserem Labor erforderlich, beobachteten wir einen Stillstand der Glia-Zellkernen in der Optik Stiel während eines Zeitraums von einer ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<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>
</tr>		</tbody>
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live imaging of glial cell migration in the drosophila eye imaginal disc

Gliazellen der Wirbeltiere und wirbellose Organismen müssen Endziel Regionen wandern, um umhüllen und Unterstützung verbunden Neuronen. Während die jüngsten Fortschritte gemacht worden, um die Live-Migration von Gliazellen in den Entwicklungsländern Puppenstadium Flügel (1), Studium der Beschreibung<em&gt; Drosophila</em&gt; Gliazelle Migration haben in der Regel die Untersuchung von fixiertem Gewebe beteiligt. Live-mikroskopische Analyse von beweglichen Zellen bietet die Möglichkeit, zelluläre Verhalten in der gesamten Migrationsprozess, einschließlich der Bestimmung der Geschwindigkeit und Veränderung in Richtung des Wachstums zu untersuchen. Gepaart mit der Nutzung der genetischen Werkzeugen können Live-Aufnahmen verwendet werden, um genauere Rollen für bestimmte Gene in den Prozess der Entwicklung zu bestimmen. Frühere Arbeiten von Silies<em&gt; Et al.</em&gt; (2) hat die Migration von Glia aus der Optik Stiel, eine Struktur, die den Entwicklungsländern Auge und Gehirn verbindet beschrieben, in das Auge Imaginalscheibe in fixiertem Gewebe. Hier skizzieren wir ein Protokoll für die Prüfung der Live-Migration von Gliazellen in den<em&gt; Drosophila</em&gt; Auge Imaginalscheibe. Wir nutzen eine Drosophila Linie, die GFP zum Ausdruck in der Entwicklung Glia zu Gliazellen Progression in Wildtyp und Mutante in Tieren zu folgen.

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

Hier beschreiben wir ein Protokoll, um die Migration von Gliazellen in den Entwicklungsländern Drosophila Auge mit Live-mikroskopische Analyse mit GFP markierten Gliazellen gepaart untersuchen.

Live-Imaging von Gliazellen Migration in der Drosophila Eye Imaginalscheibe

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 Aufrufe.  University of British Columbia - UBC. Hier beschreiben wir ein Protokoll, um die Migration von Gliazellen in den Entwicklungsländern Drosophila Auge mit Live-mikroskopische Analyse mit GFP markierten Gliazellen gepaart untersuchen.

Serie: 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 der Imaginalscheiben vom 3. Instar Drosophila-Larven

Forschung

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.

Visualisierung der Live- Drosophila Glial-neuromuskulären Synapse mit Fluoreszenzfarbstoffen

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.

Die Herstellung Drosophila Embryonen für Live-Imaging mit dem Hanging-Drop-Protokoll

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.

A Novel RFP Reporter in der Visualisierung des Eye Imaginalscheibe in Aid 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.

Live-Bildgebung Drosophila melanogaster Embryonale Hemocyte Migrationen

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 Zellgröße Veränderung der Lebens- Drosophila Embryos

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.

Live-Imaging von GFP-markierten Proteine ​​in<em&gt; Drosophila</em&gt; Oozyten

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.

Live-Imaging von apoptotischen Ausverkauf während<em&gt; Drosophila</em&gt; Embryogenese

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.

Die Tomate / GFP-FLP / FRT Verfahren zur Live-Imaging von Mosaic Erwachsene<em&gt; Drosophila</em&gt; Sehzellen

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 und Montage<em&gt; Drosophila</em&gt; 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 ...