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.

Cette procédure est une procédure de deux jours. Jour 1 (2 h + temps de dissection) Dissection 1. pupes yeux disque <ol><li> Sélectionnez une nymphe pour la dissection. NOTE: L&#39;âge de la nymphe à être disséqué sera déterminé par les besoins expérimentaux. Toutefois, si l&#39;examen de la morphologie des cellules, cela est souvent effectué à 42 h après la formation de pupe (CSA) à 25 ° C, ce qui est l&#39;âge de l&#39;pupes montré dans la vidéo. Recueillir pupes blanc (considéré comme de l&#39;APF 0 h) avec un pinceau mouillé et les disposer dans l&#39;ordre chronologique (en fonction du temps de collecte) dans un nouveau flacon de nourriture à la mouche maintenue à 25 ° C. Disséquer les nymphes ans dans ce même ordre chronologique afin de maintenir les différences de temps entre les nymphes aussi près que possible. </li><li> Transférer la nymphe dans une goutte de solution saline tamponnée au phosphate (PBS, pH 7,4, voir matériaux pour la recette) sur une plaque de silicone de dissection. </li><li> Tenez le haut de la coque de nymphose avec une pince et percer et ouvrir la lpartie de tournesol de la pupe avec une paire de pinces coupantes. Retirer la nymphe de l&#39;affaire dans le trou nouvellement formé. </li><li> Faire une coupe en diagonale avec des ciseaux, mi-thorax, à la nymphe. Retirer les débris ou transférer la tête de nymphe à une nouvelle baisse de PBS. Nettoyer avec le tube pipette de ventilateur (voir Figure 1 et matériaux pour plus de détails) et ajouter d&#39;autres PBS selon les besoins. Assurez-vous d&#39;éviter la déshydratation. </li><li> La poignée fermement le haut de la tête cuticule nymphale avec une paire de pinces. Avec la pointe du tube de ventilateur, poussez doucement sur la coque de nymphose à extruder le cerveau et d&#39;autres tissus, y compris les disques de l&#39;oeil, sur la coque de nymphose. Soyez sûr d&#39;éviter l&#39;accaparement tout tissu dans la cuticule. </li><li> Utilisation du tube de soufflage remplie de PBS, soufflez doucement le tissu de corps gras et d&#39;autres débris sur le cas de la tête de séparer clairement et identifier les disques du cerveau et des yeux. REMARQUE: Garder le cerveau fixé au boîtier de la tête de pupe permettra au chercheur d&#39;utiliser l&#39;ilannonce cas comme une &quot;poignée&quot; de manipulation avenir du tissu sans endommager les disques des yeux ou le cerveau. Cependant, il est courant de déloger le cerveau et les yeux de disques le cas de la tête et ce ne nuit pas à l&#39;ensemble de la procédure et les résultats. Il faut souvent plusieurs tentatives de souffler et pousser à extraire avec succès les disques œil et le cerveau de l&#39;intérieur de la tête pupe et afin qu&#39;ils soient clairement séparés du tissu environnant. Le nettoyeur et un plus séparé peuvent obtenir les disques d&#39;oeil du tissu environnant aidera à prévenir les tissus étrangers de fixation sur les disques de l&#39;oeil. </li><li> Après dissection, transférer immédiatement le tissu dans un plat en verre de 3 bien sur la glace avec 0,5 ml de solution Fix (voir matériaux). Flotter le tissu sur la surface de la solution Fix pendant quelques minutes (généralement le temps qu&#39;il faut pour disséquer le prochain jeu de disques de l&#39;œil). REMARQUE: Si ce ne sont pas le premier disque d&#39;oeil à être disséqué dans cette ronde de dissection, Completely immerger le disque de l&#39;œil précédente dans la solution Fix. Si l&#39;exécution des procédures de coloration spéciales (par exemple, les espèces réactives de l&#39;oxygène, les lysosomes, etc.) cela doit être fait avant la fixation. Pour ce faire, stocker les disques oculaires disséqués dans du PBS glacé avant de continuer avec le protocole de coloration appropriée. </li><li> Après dissection et l&#39;immersion de tous les disques de l&#39;oeil étant disséqué, déplacer le plat en verre de 3 à bien un rocker et le rock à la température ambiante pendant 30 min. </li></ol> 2. Immunocoloration <ol><li> Retirez délicatement la solution Fix et ajouter 0,5 ml de PBS avec 0,3% de Triton X-100 (0,3% PBT, voir matériaux) et laver le tissu sur une bascule pour 10 min. Répétez cette étape trois fois pour un total de 4 lavages (10 minutes chacun). REMARQUE: élimination soigneuse de la solution dans le puits peut être réalisée sous un microscope à dissection pour prévenir les lésions tissulaires ou de la perte. La solution peut être aspiré soigneusement avec une pipette; nous préférons une amende-tipped, jetable pipette de transfert qui a eu l&#39;ouverture aplatie pour réduire le risque de tissus aspiration. </li><li> A la fin de la 4ème lavage, retirer le lavage et ajouter 0,5 ml de solution de bloc (voir matériaux) pour le bien et incuber pendant 30 min. NOTE: Ce délai peut être prolongé en fonction de vos besoins. </li><li> A la fin du temps de blocage de 30 min, la pipette 10 ul de la solution d&#39;anticorps primaire (dilué dans une solution Block) dans le nombre approprié de puits dans une plaque de microtitration. REMARQUE: Un puits d&#39;une microplaque pourra accueillir environ 4 paires de disques oculaires attachés à la coque de nymphose ou 8 paires isolées de disques oculaires avec des cerveaux connectés. Les anticorps primaires utilisés dans ce protocole pour les données représentatives sont antiphosphotyrosine (dilution 1: 500) et anti-Cut (dilution 1: 200). </li><li> Transférer le tissu disséqué dans les puits de plaques de microtitration remplies de la solution d&#39;anticorps primaire de la boîte de verre de 3 puits. </li><li> Lieula microplaque sur une serviette en papier mouillée (pour prévenir la déshydratation) dans une boîte vide de la pipette de pointe (ou d&#39;autres contenants similaires) et conserver à 4 ° C pendant la nuit. </li></ol> Jour 2 (4 h + temps de montage) <ol start="6"><li> Transférer le tissu de la microplaque à un nouveau plat en verre de 3 et de 0,3% de PBT (≈0.5 ml) et laver le tissu pendant 10 min sur la bascule. Répétez cette étape trois fois pour un total de 4 lavages (10 min chacun) </li><li> Retirez le dernier lavage et ajouter 0,5 ml de solution de blocs pour le bien. Bloquer pendant 30 min. NOTE: Ce délai peut être prolongé en fonction de vos besoins. </li><li> Retirer la solution de bloc et ajouter 0,5 ml de la solution d&#39;anticorps secondaire (fait à partir d&#39;anticorps marqués par fluorescence correspondant à votre anticorps primaire et longueur d&#39;onde fluorescente, dilué dans une solution Block) pour le bien et protéger le plat en verre de 3 et de la lumière avec une couverture ou aluminium déjouer et incuber pendant au moins 2 h à la salletempérature. NOTE: Toutes les étapes suivantes devraient également être protégés contre l&#39;exposition de la lumière. Cette incubation peut être prolongée pour une nuit à 4 ° C, dans une configuration similaire à l&#39;incubation d&#39;anticorps primaire, mais il peut également être effectuée dans un récipient en verre 3 de puits recouvert d&#39;un film de laboratoire, ou une plaque de microtitration, si la conservation de l&#39;anticorps est souhaitée. </li><li> À la fin de l&#39;incubation d&#39;anticorps, enlever la solution d&#39;anticorps secondaire et ajouter 0,5 ml de 0,3% PBT pour le bien et laver sur la bascule pour 10 min. Répétez cette étape pour un total de 4 lavages (10 min chacun). NOTE: Si la coloration DAPI est souhaité, avant de commencer le premier lavage, ajouter 0,5 pi de solution DAPI des stocks (voir matériaux) pour les 500 pi de PBT qui seront utilisés pour le premier lavage (1: 1000 dilution). </li></ol> 3. Montage <ol><li> A la fin de la dernière lavage, monter les disques yeux des antennes / sur une diapositive en utilisant 70% de glycérol ou PBS comme un moyen de supprimer le largeain et d&#39;autres tissus non désirés dans une piscine d&#39;un côté de la lame. </li><li> Déplacez doucement le tissu en utilisant la pression hydrostatique entre les forceps, ou très doucement avec une pointe forceps, et aligner les disques d&#39;oeil dans une colonne vers le centre de la diapositive. </li><li> Retirer tous glycérol étrangère ou PBS à travers le tissu (en utilisant effet de mèche d&#39;un laboratoire lingette ou les forceps travailler pour cela). </li><li> Placer une petite quantité (≈10 pi) de milieu de montage sur un côté de la colonne de disque de l&#39;oeil et placer une lamelle couvre-objet sur le tissu de façon à répartir le milieu de montage sur les disques de l&#39;oeil. </li><li> Laissez diapositive sit pendant 1 à 2 minutes pour permettre le / milieu de montage de tissu pour étaler complètement. </li><li> Essuyez tout support de montage de congé supplémentaire autour des bords de la lamelle et le sceller avec du vernis à ongles transparent. </li><li> Capturer des images fluorescentes des disques de l&#39;oeil à l&#39;aide de la microscopie confocale. </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...

Les domaines de la biologie du développement et de la cellule ont été fortement touchés par l&#39;organisme modèle: Drosophila melanogaster. Dans ce modèle, les études du disque de l&#39;œil ont contribué beaucoup de connaissances sur la signalisation, biologie cellulaire et d&#39;autres domaines. Le troisième disque stade de l&#39;œil larvaire fin a été largement étudié et est un modèle puissant à utiliser, car il donne un aperçu d&#39;une série de périodes de développement, chacun avec ses propres molécules et des processus de signalisation unique, comme le sillon morphogénétique progresse à travers le disque de l&#39;oeil 8. Cependant, il est nécessaire d&#39;étendre notre compréhension des processus de développement dans la phase de chrysalide de développement. Bien qu&#39;il y ait eu des études sur le disque d&#39;œil pupe 3-7, notre connaissance ne se rapproche pas de l&#39;ampleur du travail qui a été effectué sur le troisième disque stade de l&#39;œil. Cela est dû, en partie, à la plus grande difficulté à disséquer le disque d&#39;oeil chrysalide. En conséquence, une présentation de labonne méthode de dissection pourrait grandement augmenter la recherche dans ce domaine. Bien qu&#39;il existe des étapes dans le développement du disque de l&#39;œil des pupes qui sont facilement disséqués, en particulier autour de la période mi-pupe, d&#39;autres périodes sont beaucoup plus difficiles à disséquer. Ce protocole représente une méthode pour disséquer disques oculaires de nymphose qui peuvent être universellement utilisés pour tous les délais de développement des pupes. Ce protocole peut être utilisé comme une alternative à un autre protocole que 9 montre un procédé plus facile et plus rapide à disséquer les disques de l&#39;oeil à partir des points de temps midpupal. Ce protocole a été filmé et mis au point pour la formation des étudiants avancés de premier cycle dans le cycle UCLA Consortium de recherche en génomique fonctionnelle (URCFG) 10,11 dans la technique de dissection de l&#39;oeil chrysalide. Beaucoup d&#39;étudiants de premier cycle ont pu utiliser cette vidéo et méthode pour apprendre cette technique difficile.

<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.

A titre d&#39;exemple de l&#39;utilisation de ce protocole, des résultats illustrant midpupal (42 heures APF à 25 ° C) des disques de l&#39;oeil immunocolorées avec des anticorps différents sont présentés dans la figure 2. En utilisant un anticorps dirigé contre les résidus de phosphotyrosine, la membrane des cellules peut être observée (figure 2A). Ceci peut être utilisé pour identifier l&#39;agencement régulier de cellules dans l&#39;œil ommatidial nymphal suivant les procédés de mise...

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

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

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

Research

JoVE Journal

Biology

Dissection and Mounting of Drosophila Pupal Eye Discs

12.1K vues.  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.

Vidéo: 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-imagerie de la<em&gt; Drosophila</em&gt; Nymphe yeux

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

Recherche

Biologie du développement

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

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

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

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.

À long terme d&#39;imagerie en direct de<em&gt; drosophile</em&gt; Disc Eye

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.

Imagerie de la migration en direct des cellules gliales dans le disque Eye drosophile Imaginal

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

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.

Dissection et immunohistochimique de larve, de nymphe et adulte<em&gt; Drosophila</em&gt; Rétines

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

Neurosciences

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;

Dissection de la rétine de pupes de drosophile pour immunohistochimie, analyse occidentale et isolement d’ARN

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.

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

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

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.

Préparation des adultes Drosophile Yeux pour la coupe mince et analyse microscopique

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.

Dissection et Immunocoloration Imaginal disques de<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.

Dissection du complexe œil-cerveau à partir de pupes de mouches : une méthode pour isoler le tissu rétinien

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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