Name: dissection and mounting of drosophila pupal eye discs
Uploaded: 2014-11-09T15:00:00.000+00:00
Duration: 7 min 48 s
Description: Watch this Scientific Journal Video about Dissection and Mounting of Drosophila Pupal Eye Discs at JoVE.com

We appreciate and would like to thank the Howard Hughes Medical Institute for the HHMI Professor award to U.B. which made this project possible. We thank the college at the University of California, Los Angeles for providing facilities and teaching infrastructure support for this work. The work was also supported with funding from Midwestern University and a generous donation from the Charity Fidelity Gift Fund. We thank John VandenBrooks for comments on the manuscript and Krista Pearman for her technical assistance.

Dieses Verfahren ist einen 2-tägigen Verfahren. Tag 1 (2 HR + Sezieren Zeit) 1. Pupal Eye Disc Dissection <ol><li> Wählen Sie eine Puppe für die Präparation. HINWEIS: Das Alter der Puppe zu seziert werden durch die experimentellen Bedürfnisse ermittelt werden. Wenn jedoch die Prüfung der Zellmorphologie, wird dies häufig bei 42 h nach Pupariumbildung (APF) bei 25 ° C, die das Alter der in der Video gezeigt Puppen durchgeführt wird. Sammeln weißen Puppen (als 0 hr APF) mit einem angefeuchteten Pinsel und ordnen sie in chronologischer Reihenfolge (basierend auf Sammelzeit) in einer neuen Fliege Essen Fläschchen bei 25 ° C gehalten. Präparieren Sie die im Alter von Puppen in diesem gleichen chronologischen Reihenfolge, um die Zeitunterschiede zwischen Puppen so nah wie möglich zu halten. </li><li> Übertragen Sie die Puppe in einen Tropfen Phosphat-gepufferte Salzlösung (PBS, pH 7,4, siehe Materialien für Rezept) auf einem Silikon Seziersaal Platte. </li><li> Halten Sie die Spitze der Puppenhülle mit einer Pinzette und Pierce und öffnen Sie die lower Teil der Puppenhülle mit einem Paar von scharfen Pinzette. Entfernen Sie die Puppe von der neu gegründeten Loch Fall. </li><li> Einen Diagonalschnitt mit einer Schere, mid-Thorax, in der Puppe. Entfernen Sie den Fremdkörper oder überweisen Sie den Puppenkopf zu einem neuen Tropfen PBS. Sauber mit der Pipette Blasrohr (siehe Materialien und Abbildung 1 für Details) und zusätzliche PBS nach Bedarf. Achten Sie darauf, um Austrocknung zu vermeiden. </li><li> Sicher zu greifen die Besten der Puppenkopf Kutikula mit einer Pinzette. Mit der Spitze des Blasrohr, schieben auf der Puppenhülle, um das Gehirn und anderen Geweben, einschließlich der Augenscheiben, aus der Puppenhülle zu extrudieren. Achten Sie darauf, um zu vermeiden, greifen jedes Gewebe innerhalb der Nagelhaut. </li><li> Mit dem Blasrohr mit PBS gefüllt, blasen das Fett Körpergewebe und andere Verunreinigungen aus dem Kopf Fall klar zu trennen und identifizieren die Gehirn und Auge-Discs. HINWEIS: Halten Sie das Gehirn an den Puppenkopf Gehäuse angebracht wird es der Forscher auf die Verwendung erad Fall als &quot;Griff&quot; für zukünftige Manipulation des Gewebes, ohne die Augenscheiben oder Gehirn. Jedoch ist es üblich, das Gehirn und Augen Scheiben vom Kopfgehäuse zu entfernen und dies ist nicht schädlich für das Gesamtverfahren und Ergebnisse. Es dauert oft mehrere Versuche von Treib und schob die Augenscheiben und Gehirn aus dem Puppenkopf Fall und so, dass sie sich deutlich von umliegenden Gewebe erfolgreich zu extrahieren. Die saubere und getrennte man die Augenscheiben aus dem umgebenden Gewebe zu helfen zu verhindern äußerem Gewebe von Befestigung an der Augenscheiben. </li><li> Nach Dissektion, sofort das Gewebe übertragen, um einem 3 gut Glasschale auf Eis mit 0,5 ml Fix-Lösung (siehe Materialien). Float das Gewebe auf der Oberfläche der Fixier-Lösung für einige Minuten (typischerweise der Zeit, die für die nächste Reihe von Augen Scheiben zu zerlegen). HINWEIS: Ist das nicht das erste Auge Disk in dieser Runde der Dissektion, completel seziert werdeny tauchen die vorherige Augen Disc in den Fix-Lösung. Wenn für besondere Färbeverfahren (zB reaktive Sauerstoffspezies, Lysosomen, etc.) muss dies vor der Fixierung durchgeführt werden. Um dies zu tun, vor der Fortsetzung mit dem entsprechenden Färbungsprotokoll speichern die seziert Augenscheiben in eiskaltem PBS. </li><li> Nach Präparation und Untertauchen aller Augenscheiben seziert, bewegen Sie den 3 gut Glasschale auf einer Wippe und Rock bei Raumtemperatur für 30 min. </li></ol> 2. Immunfärbung <ol><li> Entfernen Sie vorsichtig das Fix-Lösung und 0,5 ml PBS mit 0,3% Triton X-100 (0,3% PBT, siehe Materialien) und waschen Sie das Gewebe auf einer Wippe für 10 min. Wiederholen Sie diesen Schritt noch dreimal für insgesamt vier Waschvorgängen (je 10 min). HINWEIS: Die sorgfältige Entfernung der Lösung aus der Quelle unter einem Präpariermikroskop zu Gewebeschäden oder Verlust zu verhindern, durchgeführt werden. Die Lösung kann vorsichtig mit einer Pipette angesaugt werden; bevorzugen wir eine fein Tipped, Einmaltransferpipette, die die Öffnung abgeflacht ist, um die Wahrscheinlichkeit zu verringern Absaugen Gewebe hatte. </li><li> Am Ende des 4. waschen, entfernen Sie die Wasch- und 0,5 ml Blocklösung (siehe Materialien), um den Brunnen und Inkubation für 30 min. Hinweis: Dieser Zeitrahmen kann nach Ihren Bedürfnissen erweitert werden. </li><li> Am Ende der 30 Minuten Blockzeit, Pipette 10 ul Primärantikörperlösung (in Block-Lösung verdünnt) in die entsprechende Anzahl von Vertiefungen in einer Mikrotiterplatte. HINWEIS: Eine Vertiefung einer Mikrotiterplatte wird über 4 Paar Augenscheiben zum Puppenhülle angebracht oder 8 isolierte Paare von Augenscheiben mit Köpfchen befestigt zubringen. Die primären Antikörper in diesem Protokoll für die repräsentative Daten verwendet werden, sind Anti-Phosphotyrosin (1: 500 Verdünnung) und Anti-Cut (1: 200 Verdünnung). </li><li> Übertragen Sie die sezierten Gewebes in die Mikrotiterplatte Wells mit dem primären Antikörper-Lösung aus der 3 gut Glasschale gefüllt. </li><li> Ortder Mikrotiterplatte auf einem befeuchteten Papiertuch in einem leeren Pipettenspitze Box (oder andere ähnliche Behälter) und bei 4 ° C über Nacht (um eine Austrocknung zu verhindern). </li></ol> Tag 2 (4 h + Montagezeit) <ol start="6"><li> Übertragen Sie das Gewebe aus der Mikrotiterplatte auf ein neues 3 gut Glasschale mit 0,3% PBT (≈0.5 ml) und waschen Sie das Gewebe für 10 Minuten auf der Wippe. Wiederholen Sie diesen Schritt noch dreimal für insgesamt vier Waschvorgängen (je 10 min) </li><li> Entfernen Sie die letzten Wasch und 0,5 ml Block-Lösung zum Brunnen. Block für 30 min. Hinweis: Dieser Zeitrahmen kann nach Ihren Bedürfnissen erweitert werden. </li><li> Entfernen Sie die Block-Lösung und 0,5 ml des Sekundärantikörperlösung zum Brunnen (von fluoreszenzmarkierten entsprechend Ihrer primären Antikörper und Fluoreszenzwellenlänge, in Block-Lösung verdünnt Antikörper hergestellt) und zum Schutz der 3 gut Glasschale von Licht mit einem Deckel oder Aluminium Folie und Inkubation für mindestens 2 Stunden bei RaumTemperatur. HINWEIS: Alle nachfolgenden Schritte sollten in ähnlicher Weise von Lichtexposition geschützt werden. Diese Inkubation kann über Nacht bei 4 ° C erweitert werden, in einem Setup ähnlich wie der primäre Antikörper Inkubation, aber es kann auch in einem 3 gut Glasschale mit Labor-Film oder eine Mikrotiterplatte, wenn Antikörper Erhaltung gewünscht abgedeckt durchgeführt werden. </li><li> Am Ende der Antikörper-Inkubation, entfernen Sie die Sekundärantikörperlösung und 0,5 ml von 0,3% PBT zum Brunnen und Wäsche auf der Wippe für 10 min. Wiederholen Sie diesen Schritt für insgesamt 4 Wäschen (jeweils 10 Minuten). HINWEIS: Wenn DAPI-Färbung gewünscht wird, vor dem Start der ersten Wäsche, fügen 0,5 ul DAPI-Stammlösung (siehe Materialien) zu den 500 ul PBT, die für die erste Wäsche verwendet wird (1: 1000 Verdünnung). </li></ol> 3. Montage <ol><li> Am Ende der letzten Waschung montieren anten / Augenscheiben auf einem Objektträger mit 70% Glycerin oder PBS als Medium, um die br entfernenain und anderen unerwünschten Gewebe in einem Pool auf einer Seite der Folie. </li><li> Das Gewebe vorsichtig bewegen Sie mit hydrostatischen Druck zwischen den Zangen, oder sehr vorsichtig mit einem Zangenspitze, und richten Sie die Augenscheiben in einer Kolonne in Richtung der Mitte des Schlittens. </li><li> Entfernen Sie alle Fremd Glycerin oder PBS aus der ganzen Gewebe (mit Dochtwirkung eines Laborwischtuch oder die Zange arbeiten für diese). </li><li> Legen Sie eine kleine Menge (≈10 ul) Montagemedium entlang einer Seite des Auges Disc Spalte und setzen Sie ein Deckglas auf das Gewebe, um so die Montagemedium auf das Auge Scheiben verteilen. </li><li> Lassen Rutsche sit für 1 bis 2 min, damit das Gewebe / Montagemedium vollständig auszubreiten. </li><li> Wischen Sie zusätzliche Montagemedium weg von den Rändern des Deckglases und verschließen Sie diese mit klarem Nagellack. </li><li> Erfassen Fluoreszenzbilder der Augenscheiben mittels konfokaler Mikroskopie. </li></ol>

<ol>
	<li>Ready, D. F., Hanson, T. E., Benzer, S. <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+retina,+a+neurocrystalline+lattice.">Development of the Drosophila retina, a neurocrystalline lattice.</a> Dev Biol. 53 (2), 217-240 (1976).</li><li>Wolff, T., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beginning+of+pattern+formation+in+the+Drosophila+compound+eye:+the+morphogenetic+furrow+and+the+second+mitotic+wave.">The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave.</a> Development. 113 (3), 841-850 (1991).</li><li>Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+the+Drosophila+eye.">Pattern formation in the Drosophila eye.</a> Curr Opin Genet Dev. 17 (4), 309-313 (2007).</li><li>Bao, S., Cagan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+adhesion+mediated+by+Hibris+and+Roughest+regulates+morphogenesis+and+patterning+in+the+Drosophila+eye.">Preferential adhesion mediated by Hibris and Roughest regulates morphogenesis and patterning in the Drosophila eye.</a> Dev cell. 8 (6), 925-935 (2005).</li><li>Grzeschik, N. A., Knust, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IrreC/rst-mediated+cell+sorting+during+Drosophila+pupal+eye+development+depends+on+proper+localisation+of+DE-cadherin.">IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin.</a> Development. 132 (9), 2035-2045 (2005).</li><li>Sprecher, S. G., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switch+of+rhodopsin+expression+in+terminally+differentiated+Drosophila+sensory+neurons.">Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons.</a> Nature. 454 (7203), 533-537 (2008).</li><li>Zuker, C. S., Cowman, A. F., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+structure+of+a+rhodopsin+gene+from+D.+melanogaster.">Isolation and structure of a rhodopsin gene from D. melanogaster.</a> Cell. 40 (4), 851-858 (1985).</li><li>Voas, M. G., Rebay, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+integration+during+development:+insights+from+the+Drosophila+eye.">Signal integration during development: insights from the Drosophila eye.</a> Dev Dyn. 229 (1), 162-175 (2004).</li><li>Hsiao, H. 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=Dissection+and+immunohistochemistry+of+larval,+pupal+and+adult+Drosophila+retinas.">Dissection and immunohistochemistry of larval, pupal and adult Drosophila retinas.</a> J Vis Exp. , 4347 (2012).</li><li>Call, G. B., 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=Genomewide+clonal+analysis+of+lethal+mutations+in+the+Drosophila+melanogaster+eye:+comparison+of+the+X+chromosome+and+autosomes.">Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes.</a> Genetics. 177 (2), 689-697 (2007).</li><li>Chen, J., 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=Discovery-based+science+education:+functional+genomic+dissection+in+Drosophila+by+undergraduate+researchers.">Discovery-based science education: functional genomic dissection in Drosophila by undergraduate researchers.</a> PLoS Biol. 3 (2), 59 (2005).</li><li>Wolff, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+techniques+for+pupal+and+larval+Drosophila+eyes.">Dissection techniques for pupal and larval Drosophila eyes.</a> CSH Protoc. 2007, (2007).</li><li>Williamson, W. R., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+developing+and+adult+Drosophila+brains+and+retinae+for+live+imaging.">Preparation of developing and adult Drosophila brains and retinae for live imaging.</a> J Vis Exp. 37, 1936 (2010).</li><li>Xu, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117 (4), 1223-1237 (1993).</li><li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4+system+in+Drosophila:+a+fly+geneticist's+Swiss+army+knife.">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. 34 (1-2), 1-15 (2002).</li></ol>

The authors have nothing to disclose.

While it appears that the process is simple and easy to perform, in reality, this technique requires a great deal of practice to master. Routinely, we start students off by learning to dissect and mount third instar eye discs12, which are much easier to work with. This practice helps to develop an appropriate dissection position of the arms, hands and fingers13 so that manipulation of the forceps under the dissecting microscope is stable, easy and experienced. In essence, the practice period shou...

Drosophila melanogaster: Die Felder der Entwicklungs- und Zellbiologie wurden stark von der Modellorganismus beeinflusst. Bei diesem Modell haben Studien des Auges Disc viel Wissen über Signalisierung, Zellbiologie und anderen Bereichen beigetragen. Die späten dritten Larvenstadium Auge Disc wurde ausgiebig untersucht und ist ein leistungsfähiges Modell zu nutzen, da es eine Momentaufnahme aus einer Reihe von Entwicklungsperioden, jede mit ihrer eigenen einzigartigen Signalmoleküle und Prozesse, wie der morphogenetischen Furche fortschreitet über das Auge Disc 8. Es besteht jedoch ein Bedarf für unser Verständnis von Entwicklungsprozessen weiter ausbauen in den Puppenentwicklungsphase. Zwar gab es auf der Puppenaugenscheibe 3-7 wurden Studien, unser Wissen nicht nähern die Breite der Arbeit, die auf dem dritten Larvenaugenscheibe durchgeführt worden ist. Dies liegt zum Teil an die größere Schwierigkeit der Zerlegung des Puppenaugen Disc. Daher ist eine Präsentation derrichtige Methode der Präparation konnte erheblich erweitern Forschung in diesem Bereich. Zwar gibt es Stufen innerhalb Puppenaugenscheibe Entwicklung, die leicht zerlegt werden, insbesondere um die Mitte des Puppenperiode, sind andere Zeiträume viel schwieriger zu sezieren. Das Protokoll stellt ein Verfahren zum Präparieren Puppenaugenscheiben, die universell für alle Puppenentwicklungszeitrahmen verwendet werden kann. Dieses Protokoll kann als eine Alternative zu einem anderen Protokoll 9, die eine einfachere und schnellere Methode zur Augenscheiben von den Zeitpunkten, zu sezieren midpupal zeigt verwendet werden. Dieses Protokoll wurde ursprünglich gefilmt und für die Ausbildung fortgeschrittene Studierende in der UCLA Undergraduate Research Consortium in Functional Genomics (URCFG) 10,11 in der Technik der Puppenaugen Dissektion entwickelt. Viele Studenten waren in der Lage, um dieses Video und Verfahren zu nutzen, um diese anspruchsvolle Technik zu erlernen.

<table><tbody><tr><td>Phosphate-buffered saline (PBS, pH 7.4)</td><td></td><td></td><td>80 g NaCl, 2g&amp;nbsp; KCl, 14.4 g Na&lt;sub&gt;2&lt;/sub&gt;HPO4, 2.4 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, Bring volume to 1 &amp;#956;l, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1x PBS with autoclaved ddH&lt;sub&gt;2&lt;/sub&gt;O before using.</td></tr><tr><td>Triton X-100</td><td>Promega</td><td>H5142</td><td>Caution: Irritant! Wear gloves.</td></tr><tr><td>0.3% PBT</td><td></td><td></td><td>1.5 ml of Triton X-100, 500 ml 1X PBS.</td></tr><tr><td>37% formaldehyde solution</td><td>Fisher Scientific</td><td>F75P1GAL</td><td>Caution: Toxic, probable human carcinogen! Wear gloves.&amp;nbsp;</td></tr><tr><td>Fix Solution</td><td></td><td></td><td>(&amp;asymp;4% Formaldehyde in PBS) 50 &amp;mu;l of 37% Formaldehyde, 450 &amp;mu;l 1x PBS, make fresh before use</td></tr><tr><td>Normal goat serum</td><td>Rockland antibodies &amp;amp; assays</td><td>B304</td><td>Aliquot in 1 ml volumes and store at -80 &amp;#176;C</td></tr><tr><td>Block Solution</td><td></td><td></td><td>10% NGS in PBT.&amp;nbsp; This can be made and stored at 4 &amp;deg;C for a few days prior to use.</td></tr><tr><td>DAPI stock solution</td><td>Life Technologies</td><td>D3571</td><td>For coutnerstaining nuclei. Prepare a 1 mg/ml solution with ddH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>VectaShield Mounting Medium</td><td>Vector Labs</td><td>H-1000</td><td>Mounting medium</td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516</td><td>For mounting. Prepare 70% dilution with ddH&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>Equipment</td><td></td><td></td><td></td></tr><tr><td>Nutating mixer</td><td>VWR</td><td>82007-202</td><td>Used to rock tissue in 3 well glass dish</td></tr><tr><td>SylGard 182 Silicone Elastomer Kit</td><td>Krayden</td><td>NC9897184</td><td>Used to make silicone dissection dish</td></tr><tr><td>Silicone dissecting dish</td><td></td><td></td><td>Mix Sylgard elastomer kit (above) according to directions gently (to avoid bubbles). Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37 &amp;deg;C incubator.</td></tr><tr><td>3 well glass dish</td><td>Corning</td><td>7220-85</td><td>The 3 well variety of these are no longer available, this is the 9 well product.</td></tr><tr><td>72 well microwell minitray</td><td>Nunc</td><td>438733</td><td></td></tr><tr><td>Sharp forceps (Dumont #55)</td><td>Fine Science Tools</td><td>11255-20</td><td></td></tr><tr><td>Vannas-type Micro Scissors, Straight, 5mm blade</td><td>Ted Pella</td><td>1346</td><td></td></tr><tr><td>100 mm Borosilicate glass capillaries</td><td>World Precision Instruments</td><td>1B100-4</td><td>Pull with needle puller to make fine point tip that allows a small stream of PBS to flow.</td></tr><tr><td>Disposable Transfer Pipets, Fine Tip</td><td>Samco Scientific</td><td>231</td><td></td></tr><tr><td>Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches</td><td></td><td></td><td></td></tr><tr><td>PVC tubing (1/8 x 3/16 x 1/32)</td><td>Nalgene</td><td>8000-0010</td><td>Use these with pulled needle to assemble the blower tube as shown in Figure 2.</td></tr><tr><td>Tygon Silicone tubing (3/32 x 5/32 x 1/32)</td><td>Saint Gobain Performance Plastics</td><td>ABW00004</td><td></td></tr><tr><td>Tygon Silicone tubing (1/32 x 3/32 x 1/32)</td><td>Saint Gobain Performance Plastics</td><td>ABW00001</td><td></td></tr></tbody></table>

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.

Als Beispiel für die Verwendung dieses Protokolls, Ergebnisse darstellt midpupal (42 Std APF bei 25 ° C) Augenscheiben mit unterschiedlichen Antikörpern immungefärbt sind in Abbildung 2 Durch Verwendung eines gegen Phosphotyrosinreste gerichteten Antikörper präsentiert, kann die Membran der Zellen (Abbildung 2A). Dies kann verwendet werden, um die regelmäßige Anordnung der Ommatidien Zellen im Puppenauge identifizieren folgenden endgültigen Strukturierungsprozessen, die vor der midpupal Stufe a...

Watch this Scientific Journal Video about Dissection and Mounting of Drosophila Pupal Eye Discs at JoVE.com

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 ...

dissection-and-mounting-of-drosophila-pupal-eye-discs

Research

JoVE Journal

Biology

Dissection and Mounting of Drosophila Pupal Eye Discs

12.1K Aufrufe.  University of California at Los Angeles (UCLA). 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.

Serie: Dissection and Mounting of Drosophila Pupal Eye Discs

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

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

Biologie

Long-term Live Imaging of Drosophila Eye Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have been used to study many biological processes, such as cell proliferation, differentiation, growth, migration, apoptosis, competition, cell-cell signaling, and compartmental boundary formation. However, methods for the long-term ex vivo culture and live imaging of the imaginal discs have not been satisfactory, despite many efforts. Recently, we developed a method for the long-term ex vivo culture and live imaging of imaginal discs for up to 18 h. In addition to using a high insulin concentration in the culture medium, a low-melting agarose was also used to embed the disc to prevent it from drifting during the imaging period. This report uses the eye-antennal discs as an example. Photoreceptor R3/4-specific m&#948;0.5-Ga4 expression was followed to demonstrate that photoreceptor differentiation and ommatidial rotation can be observed during a 10 h live imaging period. This is a detailed protocol describing this simple method.

Long-term Live Imaging of Drosophila Eye Disc

This protocol describes a detailed method for the long-term ex vivo culture and live imaging of a Drosophila imaginal disc. It demonstrates photoreceptor differentiation and ommatidial rotation within the 10 h period of live imaging of the eye disc. The protocol is simple and does not require expensive setup.

Langfristige Live-Imaging von<em&gt; Drosophila</em&gt; Augen Disc

Live imaging provides the ability to continuously track dynamic cellular and developmental processes in real time. Drosophila larval imaginal discs have ...

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

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

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

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 ...

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 2. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 3 4. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes5: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s 6 7 8.
Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc9 and initiates the assembly of undifferentiated cells into ommatidia10-11. The 'founder cell' R8 is specified first and recruits R1-6 and then R7 12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes 15, including rhabdomere formation, synaptogenesis and eventually rh expression.
In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Dissection and Immunohistochemistry of Larval, Pupal and Adult Drosophila Retinas

The Drosophila retina is a crystal-like lattice composed of a small number of cell types that are generated in a stereotyped manner 1. Its amenability to sophisticated genetic analysis allows the study of complex developmental programs. This protocol describes dissections and immunohistochemistry of retinas at three discrete developmental stages, with a focus on photoreceptor differentiation.

Dissektion und Immunhistochemie von Larven, Puppen und Erwachsenenbildung<em&gt; Drosophila</em&gt; Netzhäute

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including ...

Neurowissenschaften

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

Dissektion der Drosophila Pupal Netzhaut Immunohistochemistry, westlichen Analyse und RNA-Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

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 ...

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a plethora of mutant strains and technical tricks have been developed to allow sophisticated questions to be asked and answered in a reasonable amount of time. Fundamental insight into the interplay of components of all known major signaling pathways has been obtained in forward and reverse genetic Drosophila studies. The fly eye has proven to be exceptionally well suited for mutational analysis, since, under laboratory conditions, flies can survive without functional eyes. Furthermore, the surface of the insect eye is composed of some 800 individual unit eyes (facets or ommatidia) that form a regular, smooth surface when looked at under a dissecting microscope. Thus, it is easy to see whether a mutation might affect eye development or growth by externally looking for the loss of the smooth surface ('rough eye' phenotype; Fig. 1) or overall eye size, respectively (for examples of screens based on external eye morphology see e.g.1). Subsequent detailed analyses of eye phenotypes require fixation, plastic embedding and thin-sectioning of adult eyes.
The Drosophila eye develops from the so-called eye imaginal disc, a bag of epithelial cells that proliferate and differentiate during larval and pupal stages (for review see e.g. 2). Each ommatidium consists of 20 cells, including eight photoreceptors (PR or R-cells; Fig. 2), four lens-secreting cone cells, pigment cells ('hexagon' around R-cell cluster) and a bristle. The photoreceptors of each ommatidium, most easily identified by their light sensitive organelles, the rhabdomeres, are organized in a trapezoid made up of the six "outer" (R1-6) and two "inner" photoreceptors (R7/8; R8 [Fig. 2] is underneath R7 and thus only seen in sections from deeper areas of the eye). The trapezoid of each facet is precisely aligned with those of its neighbors and the overall anteroposterior and dorsoventral axes of the eye (Fig. 3A). In particular, the ommatidia of the dorsal and ventral (black and red arrows, respectively) halves of the eye are mirror images of each other and correspond to two chiral forms established during planar cell polarity signaling (for review see e.g. 3).
The method to generate semi-thin eye sections (such as those presented in Fig. 3) described here is slightly modified from the one originally described by Tomlinson and Ready4. It allows the morphological analysis of all cells except for the transparent cone cells. In addition, the pigment of R-cells (blue arrowheads in Fig. 2 and 3) can be used as a cell-autonomous marker for the genotype of a R-cell, thus genetic requirements of genes in a subset of R-cells can readily be determined5,6.

Preparation of Adult Drosophila Eyes for Thin Sectioning and Microscopic Analysis

A standard approach to prepare adult Drosophila eyes for semi-thin sectioning and light microscopic analysis is presented here. The protocol can be used for gross morphological analysis of eye defects, or with the indicated adjustments can be used to determine genetic requirements of genes in specific cell types of the eye (e.g. clonal analysis of photoreceptors) or for electron microscopic analysis.

Vorbereitung von Adult Drosophila Augen für Thin Schnitte und mikroskopische Untersuchung

Drosophila has long been used as model system to study development, mainly due to the ease with which it is genetically tractable. Over the years, a ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

Präparation und Immunfärbung von Imaginalscheiben aus<em&gt; Drosophila melanogaster</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Dissection of the Eye-Brain Complex from Fly Pupae: A Method to Isolate Retinal Tissue

Source: DeAngelis, M. W., Johnson, R. I. <a href="https://www.jove.com/video/59299/" target="_blank">Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation</a>. J. Vis. Exp.&#160;(2019).
This video describes how to dissect and isolate the eye-brain complex from Drosophila pupae. The featured protocol demonstrates the procedure yielding high-quality tissue, compatible with, for example, immunostaining, as well as, other gene expression experiments.

Dissektion des Auge-Hirn-Komplexes aus Fliegenpuppen: Eine Methode zur Isolierung von Netzhautgewebe

Source: DeAngelis, M. W., Johnson, R. I. Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation. J. Vis. ...

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