Name: upright imaging of drosophila egg chambers
Uploaded: 2015-03-13T14:00:00.000+00:00
Duration: 12 min 29 s
Description: Watch this Scientific Journal Video about Upright Imaging of Drosophila Egg Chambers at JoVE.com

We appreciate assistance from members of the fly community, particularly Dr. Denise Montell, Dr. Lynn Cooley, and Dr. Pernille Rorth, for reagents. We thank Flybase, the Bloomington Drosophila Stock Center, and Developmental Studies Hybridoma Bank for information and providing fly stocks and antibodies, respectively. LM is supported by the Department of Education&#160;Grant, Graduate Assistance in the Areas of National Need (GAANN) training fellowship (P200A120017) and by a NIGMS Initiative for Maximizing Student Development Grant (2 R25-GM55036). A portion of the microscopy work was supported by NSF MRI grant DBI-0722569 and the Keith R. Porter Core Imaging Facility. Research was supported in part by a NSF CAREER Award (1054422) and a Basil O&#8217;Connor Starter Scholar Award from the March of Dimes, both awarded to MSG.

1. Präparation Ovariolen <ol><li> Übertragen Sie etwa fünfzehn 2-4 Tage alte weibliche Fliegen und ein paar Männer auf eine frische fliegen Essen Fläschchen mit Mehr Trockenhefe. Verwenden Baumwolle als Plugin für die Fläschchen, und fügen Sie ein paar Tropfen Wasser auf die Baumwolle, die Luftfeuchtigkeit hoch zu halten. </li><li> Ort Vial in einem 25 ° C Inkubator für 14-16 h, um die Anzahl der Stufe 8-10 Eikammern maximieren. Hinweis: Die Inkubationszeit variiert je nach Temperatur und gewünschte Bühne. </li><li> Bereiten Dissektion Medien, 0,1 M KPO 4 und NP40 Lösungen für den nächsten Tag erforderlich (siehe Materialien). </li><li> Anesthetize die Fliegen mit CO 2, und legen Sie unter einem Dissektionsmikroskop. Pipette einige Tropfen Dissektion Medien in jeden Hohlraum in der Glas Depression Rutsche. </li><li> Halten Zange in jeder Hand in einem ca. 30 ° Winkel zur Tischplatte und orientieren eine weibliche Fliege mit den Flügeln nach unten. Mit Ihrer nicht-dominanten Hand, nehmen Sie den weiblichen mit einer Pinzette, greifen sie an der einnterior des Bauches, in der Nähe des Brustkorbs, und tauchen sie in die Dissektion Medien in der Vertiefung Schieber (1A Einschub). </li><li> Mit der anderen Pinzette, fassen Sie das Exoskelett an der ventralen posterioren des Bauches und durch durchbohren. Besorgen Sie sich die Eierstöcke am hinteren Ende, und ziehen Sie sie aus dem Bauch und legen Sie die frische Medien auf der anderen Seite der Mulde Rutsche. (1A kleines Bild). Vermeiden Auseinanderziehen des Darms. Entsorgen Sie die Karkasse. </li><li> Wiederholen Sie mit den anderen weiblichen Fliegen bis alle Eierstöcke seziert werden. Entsorgen Sie die Männer. </li><li> Mit einer Pinzette, halten Sie ein Eierstock am hinteren Ende (in der Nähe der größten Ei Kammern), und mit der anderen Hand mit einem Pinzette zu einem der vordersten Ei Kammern (Germarium) einklemmen. (1A). </li><li> Langsam und stetig, ziehen eine Ovariole Kette aus dem Eierstock Scheide. Weiterhin individuell sezieren Ovariolen bis alle gewünschten Ei Kammern removed. </li><li> Wiederholen Sie die Schritte 1,7-1,8 für alle seziert Eierstöcke. </li><li> Wenn Sie fertig sind, übertragen Ovariolen auf eine 0,6 ml Tube mit einem Kunststofftransferpipette. </li><li> Lassen Ovariolen setzen sich am Boden der Röhre, dann nach Fixierung und Färbung Schritten fortfahren. </li></ol> 2. Immunfluoreszenz (IF) Färbung von Ovariolen <ol><li> Dissektion Medium vorsichtig aus Ovariolen mit einer Pipette. Achten Sie darauf, keine Ovariolen oder Eikammern entfernen. Lassen Sie ca. 50 ul Ovariolen und Dissektion Medien. </li><li> In 50 ul von 16% Paraformaldehyd und 150 ul 0,1 M KPO 4 Puffer in einem Rohr, und fügen Lösung Eikammern, sie zu beheben. Inkubation unter Schütteln für 10 min. ACHTUNG: Para ist giftig. </li><li> Entfernen Fixiermittel und spülen Sie zweimal mit 0,5 ml NP40 Waschpuffer. </li><li> Zweimal für 15 Minuten jeweils bei RT waschen. </li><li> Siehe Standard-ZF-Protokoll 15 für den nachfolgenden Schritten. <ol><li> Kurz nach der Fixierung, fügenPrimärantikörper an den geeigneten Verdünnungen inkubieren O / N bei 4 ° C. In NP40 mehrmals waschen, dann fügen Sekundärantikörper (1: 200 Verdünnungen). </li><li> Mehrmals waschen in NP40, dann fügen Sie DAPI (1: 1000) für 10 Minuten, dann wiederholen Wäschen. Sobald IF-Protokoll abgeschlossen ist, fügen Sie 200 ul von 50% Glycerin in PBS zu Eikammern und lassen Sie sich bei Raumtemperatur für 2 Std. </li><li> Entfernen Lösung, ersetzen Sie es mit 70% Glycerin in PBS und mit Folie abdecken. Zeigen Probe bei 4 ᵒC bis einbaufertig. Unterdessen mit Schritt 3 fortfahren. </li></ol></li></ol> 3. Geleistete Vorbereitung von Glycerin Jelly Slides <ol><li> Messen Sie mindestens 4 ml Gelee Glycerin mit einem Spatel und füllen ein Glas Zellkulturröhrchen in die Halbmarathonmarke. Schmelzen Glycerin Gelee in einem Wasserbad bei 55 ° C für 30 min. </li><li> Gießen Sie einen kleinen Tropfen Glycerin, etwa 300 &amp; mgr; l aus der Stammlösung auf zwei Objektträgern. Mit einem Gewebe, verbreiten Glycerin in einer dünnen layer über jede der Folien. Dies liefert eine Basis und ermöglicht die einfache Entfernung von Glycerin Gelee in Schritt 5.6. </li><li> Schneiden Sie die Spitze aus einer gefilterten 1.000 ul Pipettenspitze. Verwenden Schnitt Pipettenspitze 1000-1500 ul warmen Glycerin Gelee langsam auf jedem Objektträger übertragen. Achten Sie darauf, um Blasen zu vermeiden. </li><li> Lassen Glycerin Gelee Folien sitzen für 5 Minuten bei Raumtemperatur eingestellt. </li><li> Platzieren Sie jede Glycerin Gelee Folie in 60 mm x 15 mm Petrischale und mit Plastikfolie abdecken. Bei 4 ° CO / N. Optional Speicher Glycerin Gelee Folien für bis zu 5 Tage bei 4 ° C. </li></ol> 4. Montage Egg Chambers <ol><li> Mit Proben aus Schritt 2.5, Pipette mehr 3 ul Tropfen Eikammern in 70% Glycerin in einem Glycerin Gelee Rutsche. Weiter Pipettieren 3 ul Tropfen, bis alle Eier Kammern sind auf dieser Folie. Stellen Sie sicher, jeder Tropfen mindestens zehn Eikammern. Inzwischen Hitze ein Kulturröhrchen aus Glycerin Gelee in einem 55 ° C Wasserbad. </li><li> Stellen Sie das Glycerin Gelee Rutsche mit Ei Kammern unter einem Dissektionsmikroskop. Einstellen der Vergrößerung, so daß nur ein Tropfen Eikammern ist in dem Sichtfeld (1B). </li><li> Mit einer 30-Gauge-Nadel, wie einem Löffel, nehmen Sie die gewünschte Stufe Eikammer. Wenn nötig, separate gewünschten Ei Kammern von einem Ovariole Kette mit Hilfe der Nadel wie ein Messer. </li><li> Um Verunreinigungen, die mit einer Abbildungs ​​stören können, zu vermeiden, übertragen das Ei Kammer zur zweiten Glycerin jelly Schieber mit dem vorderen zugewandt links (1E -1). Wiederholen, bis es mindestens sieben Eikammern reihen sich in einer Spalte (Abbildung 1C). </li><li> Bilden neue Spalten von sieben bis alle gewünschten Eikammern werden dem zweiten Glycerin jelly Träger übertragen. </li><li> Reißen Sie ein kleines Stück Gewebe und Twist. Verwenden Sie diese Option, um das überschüssige PBS-Glycerin von den Ei Kammern durch leichtes Berühren jeden einzelnen zu absorbieren. Achten Sie darauf, Eikammern nicht entfernen. </li><li> Schneiden Sie dieSpitze eines gefilterten 200 ul Pipettenspitze. Verwenden Sie diese Funktion, um 150 ul Glycerin Gelee, um alle Spalten der Eikammern decken zu übertragen. HINWEIS: Die Menge an Glycerin Gelee benötigt, um die Säulen zu decken auf Eikammer Stufen und die Anzahl der Säulen. Menge eingestellt werden kann. </li><li> Lassen montiert Eikammern sitzen für 5 min bei RT, bis die obere Schicht aus Glycerin Gelee vollständig verfestigt (1D). </li><li> Den Objektträger montiert Ei Kammern in einem 60 mm x 15 mm Petrischale und mit Plastikfolie abdecken. Lagerung bei 4 ° CO / N, damit die Glycerin Gelee Schichten (in der dunklen Ort, wenn es Bedenken über Photobleaching) zusammen zu verfestigen. </li></ol> 5. Herstellung von Egg Chambers for Imaging <ol><li> Zeigen Schieber montiert Ei Kammern unter einem Dissektionsmikroskop. Einstellen der Vergrößerung, so daß nur eine Spalte Eikammern ist in dem Sichtfeld. </li><li> Mit einem Winkel von 45 ° Miniatur Skalpell, schneiden Sie ein straight Linie entlang der rechten Seite der ersten Spalte Eikammern (nahe der hinteren Seite der Eikammern). (1D-E) </li><li> Als nächstes wird über der ersten Eierkammer an der Oberseite und unter der letzten Eikammer in der Spalte geschnitten. An diesem Punkt der Spalte Eikammern sollte auf drei Seiten geschnitten werden. </li><li> Verwendung eines microknife, Scheibe entlang der linken Seite der Säule, so nahe wie möglich an der vorderen Seite der Eikammern (1E -3). </li><li> Je 100 ul kaltem 1X PBT über die Schnitt Spalte. </li><li> Verwenden Sie einen abgerundeten Kanten Spachtel, um das überschüssige Glycerin Gelee um drei Seiten des Schnittes Spalte Eikammern und entsorgen zu entfernen, während der Rest auf der Folie. </li><li> Stecken Sie den Spachtel in den Schnitt an der vorderen Seite des Eier Kammern und trennen Sie die Spalte aus der Primär Glycerin Gelee Block. </li><li> Bereiten Proben entweder invertiert (5.8.1) oder stehend (5.8.2) Mikroskopie. <ol><li> Für Inverses Mikroskop: Transfer ter Spalte Eikammern zu einem Deckglas mit vorderen Seite Eikammern nach unten. Die Schritte 5.2-5.8, bis alle Spalten von Glycerin Gelee wurden entfernt und gelagert. Fügen Sie ein paar Tropfen 50% PBS-Glycerin auf jeden Block von Eikammern, um sie vor dem Austrocknen zu schützen. Bild Eikammern direkt auf Deckglas. </li><li> Für aufrechten Mikroskop: Übertragen Sie die Spalte von Ei Kammern auf einen Objektträger mit der vorderen Seite nach oben ein. Die Schritte 5,2-5,9, bis alle Spalten von Glycerin Gelee wurden entfernt und auf Objektträgern montiert. Fügen Sie ein paar Tropfen 50% PBS-Glycerin auf jeden Block von Ei Kammern und Deckblöcke mit einem # 1 ½ Deckglas. </li></ol></li></ol>

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	<li>Hudson, A. M., Cooley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+oogenesis.">Methods for studying oogenesis.</a> Methods. 68 (1), 207-217 (2014).</li><li>Bastock, R., St Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+oogenesis.">Drosophila oogenesis.</a> Curr Biol. 18 (23), R1082-R1087 (2008).</li><li>Wu, X., Tanwar, P. S., Raftery, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+follicle+cells:+morphogenesis+in+an+eggshell.">Drosophila follicle cells: morphogenesis in an eggshell.</a> Semin Cell Dev Biol. 19 (3), 271-282 (2008).</li><li>Bilder, D., Haigo, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+morphogenetic+repertoire:+perspectives+from+the+Drosophila+egg.">Expanding the morphogenetic repertoire: perspectives from the Drosophila egg.</a> Dev Cell. 22 (1), 12-23 (2012).</li><li>Montell, D. J., Yoon, W. H., Starz-Gaiano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group+choreography:+mechanisms+orchestrating+the+collective+movement+of+border+cells.">Group choreography: mechanisms orchestrating the collective movement of border cells.</a> Nat Rev Mol Cell Biol. 13 (10), 631-645 (2012).</li><li>Spradling, A. C., Bate, M., Marinez-Arias, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+genetics+of+oogenesis.">Developmental genetics of oogenesis.</a> The Development of Drosophila melanogaster. , 1-70 (1993).</li><li>Montell, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Border-cell+migration:+the+race+is+on.">Border-cell migration: the race is on.</a> Nat Rev Mol Cell Biol. 4 (1), 13-24 (2003).</li><li>Silver, D., Geisbrecht, E., Montell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+JAK/STAT+signaling+throughout+border+cell+migration+in+Drosophila.">Requirement for JAK/STAT signaling throughout border cell migration in Drosophila.</a> Development. 132 (15), 3483-3492 (2005).</li><li>Ghiglione, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+cytokine+receptor+Domeless+controls+border+cell+migration+and+epithelial+polarization+during+oogenesis.">The Drosophila cytokine receptor Domeless controls border cell migration and epithelial polarization during oogenesis.</a> Development. 129 (23), 5437-5447 (2002).</li><li>Denef, N., Sch&#252;pbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning:+JAK-STAT+signalling+in+the+Drosophila+follicular+epithelium.">Patterning: JAK-STAT signalling in the Drosophila follicular epithelium.</a> Curr Biol. 13 (10), R388-R390 (2003).</li><li>Beccari, S., Teixeira, L., R&#248;rth, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+JAK/STAT+pathway+is+required+for+border+cell+migration+during+Drosophila+oogenesis.">The JAK/STAT pathway is required for border cell migration during Drosophila oogenesis.</a> Mech Dev. 111 (1-2), 115-123 (2002).</li><li>Montell, D., Rorth, P., Spradling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=slow+border+cells,+a+locus+required+for+a+developmentally+regulated+cell+migration+during+oogenesis,+encodes+Drosophila+C/EBP.">slow border cells, a locus required for a developmentally regulated cell migration during oogenesis, encodes Drosophila C/EBP.</a> Cell. 71 (1), 51-62 (1992).</li><li>Xi, R., McGregor, J., Harrison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gradient+of+JAK+pathway+activity+patterns+the+anterior-posterior+axis+of+the+follicular+epithelium.">A gradient of JAK pathway activity patterns the anterior-posterior axis of the follicular epithelium.</a> Dev Cell. 4 (2), 167-177 (2003).</li><li>Toomre, D., Bewersdorf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+wave+of+cellular+imaging.">A new wave of cellular imaging.</a> Annu Rev Cell Dev Biol. 26, 285-314 (2010).</li><li>McDonald, J., Pinheiro, E., Kadlec, L., Schupbach, T., Montell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+EGFR+ligands+participate+in+guiding+migrating+border+cells.">Multiple EGFR ligands participate in guiding migrating border cells.</a> Dev Biol. 296 (1), 94-103 (2006).</li><li>Van de Bor, V., Zimniak, G., C&#233;r&#233;zo, D., Schaub, S., Noselli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+localisation+of+cytokine+mRNA+is+essential+for+JAK/STAT+activation+during+cell+invasiveness.">Asymmetric localisation of cytokine mRNA is essential for JAK/STAT activation during cell invasiveness.</a> Development. 138 (7), 1383-1393 (2011).</li><li>Hayashi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glypicans+regulate+JAK/STAT+signaling+and+distribution+of+the+Unpaired+morphogen.">Glypicans regulate JAK/STAT signaling and distribution of the Unpaired morphogen.</a> Development. 139 (22), 4162-4171 (2012).</li><li>Airoldi, S. J., McLean, P. F., Shimada, Y., Cooley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intercellular+protein+movement+in+syncytial+Drosophila+follicle+cells.">Intercellular protein movement in syncytial Drosophila follicle cells.</a> J Cell Sci. 124 (Pt 23), 4077-4086 (2011).</li><li>R&#248;rth, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+gain-of-function+genetics+in+Drosophila).">Systematic gain-of-function genetics in Drosophila).</a> Development. 125 (6), 1049-1057 (1998).</li><li>Lee, T., Lee, A., 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=Development+of+the+Drosophila+mushroom+bodies:+sequential+generation+of+three+distinct+types+of+neurons+from+a+neuroblast.">Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast.</a> Development. 126 (18), 4065-4076 (1999).</li><li>Shimada, Y., Burn, K. M., Niwa, R., Cooley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+response+of+protein+localization+and+microtubule+organization+to+nutrient+stress+during+Drosophila+early+oogenesis.">Reversible response of protein localization and microtubule organization to nutrient stress during Drosophila early oogenesis.</a> Dev Biol. 355 (2), 250-262 (2011).</li><li>Qui&#241;ones-Coello, A. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+strategies+for+protein+trapping+in+Drosophila.">Exploring strategies for protein trapping in Drosophila.</a> Genetics. 175 (3), 1089-1104 (2007).</li><li>Buszczak, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+carnegie+protein+trap+library:+a+versatile+tool+for+Drosophila+developmental+studies.">The carnegie protein trap library: a versatile tool for Drosophila developmental studies.</a> Genetics. 175 (3), 1505-1531 (2007).</li><li>Belu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upright+imaging+of+Drosophila+embryos.">Upright imaging of Drosophila embryos.</a> J Vis Exp. (43), (2010).</li><li>Witzberger, M. M., Fitzpatrick, J. A., Crowley, J. C., Minden, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=End-on+imaging:+a+new+perspective+on+dorsoventral+development+in+Drosophila+embryos.">End-on imaging: a new perspective on dorsoventral development in Drosophila embryos.</a> Dev Dyn. 237 (11), 3252-3259 (2008).</li></ol>

The authors have no competing financial interests.

Hier beschreiben wir eine Methode zu montieren und Bild klein, entwickelt Eikammern von einer End-on-Perspektive. Gemeinsame Methoden zur Bildgebung Ei Kammern für Seitenansichten optimiert und vor allem ermöglichen eine präzise Visualisierung von mediolaterale Follikelzellen, wenn mit fluoreszierenden Antikörpern gefärbt. Die Verwendung von Z-Stapeln oder 3-D-Rekonstruktion Hilfsmittel in Anzeigen mehrerer Brennebenen, aber ist immer noch für subzelluläre Auflösung der Pole des elliptischen Eikammern (2...

Studium der Drosophila melanogaster hat große Einblicke in die genetische Regulation einer Vielzahl von Phänomenen ist. Insbesondere gab es umfangreiche Forschung auf Ei-Entwicklung, da Oogenese eine tractable Weg zu vielen verschiedenen Entwicklungsprozessen, einschließlich Gewebe Strukturieren Zellpolaritätswechsel, Zellzyklus-Schalt und translationale Regulation 1,2,3,4 untersuchen. Ein wichtiger morphogenic Ereignis während der Oogenese ist die Spezifikation, den Erwerb von Motilität und Migration aus einem Satz von Zellen, die sogenannten Randzellen (in 5 überprüft). Da Zellmigration ist ein Schlüsselmerkmal von tierischen Morphogenese und weil die genetische Regulation dieses Prozesses ist gut konserviert, sind wahrscheinlich auch in anderen Zusammenhängen wichtig Mechanismen in Fliegen bestimmt. So untersuchen wir die molekularen Kontrolle der Grenzzellmigration. Für unsere Untersuchungen haben wir eine neue Methode, um die vorderen Pole der sich entwickelnden Eier Beobachtung entwickelt,denen Grenz Zellen entstehen, zu untersuchen, wie sie im Detail zu entwickeln. Innerhalb der Eierstöcke, Eikammern bei verschiedenen Entwicklungsstadien existieren entlang Ketten genannt Ovariolen, die in einer dünnen Hülle (1A) umhüllt sind. Jedes Ei Kammer wird weitergehen, um ein Ei zu bilden. Während der Oogenese, müssen mehrere unterschiedliche Zelltypen in einer koordinierten Art und Weise zu entwickeln. Die D. melanogaster Eikammer aus 16 Keimlinien-Zellen, einschließlich einer Eizelle von einem einlagigen Follikelepithels 6 umgeben. Eine kleine Anzahl von spezialisierten Zellen, die so genannte polare Zellen, entstehen an den vorderen und hinteren Pole des Epithels. Die 6-8 enden Zellen stammen im anterioren Epithel der Eierkammer, von den Polarzellen 5,7 induziert. Mitte der Oogenese (Stufe 9), die Grenzzellen lösen sich von ihren Nachbarn und wandern zwischen den Nährzellen, die Eizelle an der hinteren der Eikammer 5,7 zu erreichen. Diese Bewegung muss erreicht werden,während die Grenzzellen in einer Cluster-Umgebung zwei unbewegliche Polarzellen, so dass dies eine Art kollektive Zellmigration. Erfolgreiche Migration der Grenzzellverband sorgt für die richtige Entwicklung der Mikropyle der Eierschale, die für die Befruchtung notwendig ist. Die vorderen polare Zellen anweisen Grenzzellschicksals durch die Aktivierung eines Signalübertragungskaskade. Polar Zellen eine Cytokin, ungepaarte (UPD), die zu einer Transmembranrezeptor, Domeless (DOME) bindet, auf benachbarte Follikelzellen in Stufe 8 von Oozytenentwicklung 8,9. Die Bindung von UDP verursacht Janus Tyrosin-Kinase (JAK), um das Signal Transducer und Aktivator der Transkription (STAT) 8,10,11 phosphorylieren. STAT bewegt sich dann in den Kern, um die Transkription zu aktivieren. Langsam Border Cells (SLBO) ist ein Transkriptionsfaktor, der eine direkte transkription Ziel von STAT ist und wird auch für die Grenzzellmigration 12 erforderlich. Seitenansichten Eikammern zeigen that STAT-Aktivität wird in einem Gradienten über die vordere Epithel 8,11,13 geregelt. Follikelzellen nächsten zu den polaren Zellen die höchste aktiviert STAT, damit sie sich Randzellen und dringen in den benachbarten Keimbahngewebes. Um zu verstehen, wie die Grenzzellen innerhalb festgelegter und nehmen Sie aus dem Epithel, müssen wir beobachten, wie sich das Gewebe organisiert. Wenn wir sehen Eikammern von einer anterioren-on Sicht würden wir radiale Symmetrie der STAT-Aktivität in den Follikelzellen rund um die Polarzellen erwarten. Eine End-Ansicht würde auch genauer Unterschiede von Membranproteinen und Zell-Zell-Interfaces zeigen vor und während der Ablösung als Vergleich die Zellen in unterschiedlichen Brennebenen. Da Eikammern sind länglich und miteinander durch Stiel Zellen angebracht, auf Objektträger nieder sie seitlich, so dass es schwierig ist, den vorderen Architektur zu beobachten. So viele Informationen über die Zellen, an den Polen des Eies Kammerwurde von Seitenansichten abgeleitet. Obwohl einige Informationen durch algorithmische 3-D-Rekonstruktionen von optischen Schnitten, Lichtstreuung, Photobleaching und schlechtere Auflösungsgrenzen in der Z-Achse erhalten werden diese Angaben weniger detailliert und zuverlässig in der Abwesenheit von teuren Techniken wie superauflösende Mikroskopie 14 . Andere Arten von Abschnitt basierten Bildgebung (beispielsweise Elektronenmikroskopie oder Mikrotom Schnitte) erfordert umfangreiche Manipulation von Geweben, einschließlich Entwässerung, was die Wahrscheinlichkeit für Artefakte. So eine neue Methode zur Bild D. entwickelten wir melanogaster Eikammern während aufrecht. Diese Methode hat sich bereits bei der Aufklärung, wie bewegliche Zellen sind vom Schicksal bestimmt (siehe Repräsentative Ergebnisse und Manning et al, im Berichts) bewährt und dürfte im weiteren Sinne wertvoll in Studien anderer Aspekte der Oogenese sein.

<table><tbody><tr><td>0.1 M Potassium phosphate Buffer (KPO&lt;sub&gt;4&lt;/sub&gt; Buffer)</td><td></td><td></td><td>Add 3.1 g of NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;H&lt;sub&gt;2&lt;/sub&gt;O and 10.9 g of Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (anhydrous) to distilled H&lt;sub&gt;2&lt;/sub&gt;O to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 month at 4&amp;deg;C.</td></tr><tr><td>16% Paraformaldehyde aqueous solution, EM grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td>methanol-free to preserve GFP fluorescence</td></tr><tr><td>30G x 1/2 inch needle&amp;nbsp;</td><td>VWR</td><td>BD305106</td><td>Regular bevel</td></tr><tr><td>Active Dry Yeast</td><td>Genesee Scientific</td><td>62-103</td><td>To fatten female flies</td></tr><tr><td>Bovine Serum Albumin</td><td>PAA-cell culture company</td><td>A15-701</td><td>Used in NP40 Wash Buffer</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11295-10</td><td>Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection</td></tr><tr><td>DAPI (4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride)</td><td>Sigma Aldrich</td><td>D9542</td><td>5 mg/ml stock solution&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16140-071</td><td>Added to supplement dissection medium (10%)&amp;nbsp;</td></tr><tr><td>Glass culture tube</td><td>VWR</td><td>47729-576</td><td>14 ml</td></tr><tr><td>Glass Depression Slides</td><td>VWR</td><td>470019-020</td><td>1.2 mm Thick, Double Cavity</td></tr><tr><td>Glycerin Jelly&amp;nbsp;</td><td>Electron Microsocpy Sciences</td><td>17998-10</td><td>Mounting media</td></tr><tr><td>Glycerol</td><td>IBI Scientific</td><td>IB15760</td><td>70% in PBS</td></tr><tr><td>IGEPAL CA-630</td><td>Sigma Aldrich</td><td>I3021-500ML</td><td>(interchangable for Nonidet P-40)&amp;nbsp; Used in NP40 Wash Buffer&amp;nbsp;</td></tr><tr><td>Leica Fluorescent Stereoscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Leica SP5 Confocal Microscope</td><td>Leica Microsystems</td><td></td><td>40x/0.55NA dry objective&amp;nbsp;</td></tr><tr><td>Micro spatula&amp;nbsp;</td><td>VWR</td><td>82027-518</td><td>Stainless steel</td></tr><tr><td>Microscope Slide</td><td>VWR</td><td>16004-368</td><td>75x25x1 mm</td></tr><tr><td>NP40 Wash Buffer</td><td></td><td></td><td>50 mM TRIS-HCl, 150 mM NaCl, 0.5% Ipegal, 1 mg/ml BSA, and 0.02% sodium azide</td></tr><tr><td>Penicillin-Streptomycin-Glutamine</td><td>Life Technologies</td><td>10378-016</td><td>Added to supplement dissection medium (0.6X)</td></tr><tr><td>Petridish- Polysterine, sterile</td><td>VWR</td><td>82050-548</td><td>60 W x 15 H mm</td></tr><tr><td>Phosphate Buffer Saline</td><td>Sigma Aldrich</td><td>P3813-10PAK</td><td>10 packs of Powder</td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma Aldrich</td><td>P3786-500G</td><td>Used in 0.1 M KPO&lt;sub&gt;4&lt;/sub&gt; Buffer&amp;nbsp;</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P9791-500G</td><td>Used in 0.1 M KPO&lt;sub&gt;4&lt;/sub&gt; Buffer&amp;nbsp;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Schneider&amp;rsquo;s Insect Medium</td><td>Life Technologies</td><td>&lt;br/&gt; 11720018</td><td>With L-glutamine and sodium bicarbonate</td></tr><tr><td>Sodium azide</td><td>Sigma Aldrich</td><td>S2002-25G</td><td>Used in fluorescent antibody staining</td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>S3014-500G</td><td>Used in NP40 Wash Buffer</td></tr><tr><td>TRIS-HCl</td><td>IBI Scientific</td><td>IB70144</td><td>1 M TRIS-HCl, pH 7.4</td></tr><tr><td>Volocity 3D Image Analysis Software</td><td>PerkinElmer</td><td></td><td>For processing confocal Z-stacks</td></tr></tbody></table>

upright imaging of drosophila egg chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

Die aufrechte bildgebenden Verfahrens konnten wir direkt die Organisation von Zellen in der vorderen Follikelepithels auf Stufe 8 zu sehen Ein allgemeiner Marker für Follikel Zellschicksal, die Augen Absent (EYA) Protein, sowie die Kern-DNA-Marker DAPI, hat auch Ausdruck in diesem Bereich der Zellen, und zeigten, dass alle Zellen könnten mit ähnlichen Färbungsintensitäten (2B &#39;) zu sehen ist. Proteine, die in Reaktion auf das Cytokin UPD reguliert zeigten jedoch erlichen Muster Expressionsnivea...

Watch this Scientific Journal Video about Upright Imaging of Drosophila Egg Chambers at JoVE.com

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Upright-Imaging von<em&gt; Drosophila</em&gt; Egg Chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

upright-imaging-of-drosophila-egg-chambers

Research

JoVE Journal

Developmental Biology

Upright Imaging of Drosophila Egg Chambers

7.6K Aufrufe.  University of Maryland Baltimore County. The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

Serie: Upright Imaging of Drosophila Egg Chambers

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

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

Live-Bilder aus dem<em&gt; Drosophila</em&gt; Pupal Augen

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

Forschung

Entwicklungsbiologie

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

Imaging Calcium in<em&gt; Drosophila</em&gt; Bei Eiaktivierung

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mesoscopic Fluoreszenz-Tomographie für In-vivo Imaging der Entwicklungsländer Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Biologie

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

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

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

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

Bildgebung und Analyse der Gewebeorientierung und Wachstumsdynamik in der Entwicklung drosophila Epithelia während der Pupal-Stadien

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

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

Ganztierbildgebung von Drosophila melanogaster mittels Mikrocomputed Tomographie

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

Visualisierung der Auswirkungen oxidativer Schäden auf Drosophila-Eierkammern mit Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

Kernmigration in der Drosophila-Eizelle

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Upright Imaging von Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Erkennung von Anastasis In Vivo durch CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Medizin

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Upright-Imaging von

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