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. Dissection de ovarioles <ol><li> Transférer environ quinze 2-4 jours d&#39;âge mouches femelles et quelques hommes d&#39;une mouche des aliments frais flacon avec de la levure sèche active ajoutée. Utilisez le coton comme une prise pour les flacons, et ajouter quelques gouttes d&#39;eau au coton pour maintenir l&#39;humidité élevée. </li><li> Lieu flacon dans un incubateur à 25 ° pour 14-16 h pour maximiser le nombre de l&#39;étape 8-10 chambres d&#39;œufs. NOTE: Le temps d&#39;incubation varie selon la température et stade désiré. </li><li> Préparer les médias de dissection, 0,1 M KPO 4 et NP40 solutions au besoin pour le lendemain (voir Matériaux). </li><li> Anesthésier vol utilisant le CO 2, et le placer sous un microscope de dissection. Pipet quelques gouttes de médias de dissection dans chaque cavité dans la glissière de la dépression de verre. </li><li> Tenez pince dans chaque main à un angle d&#39;environ 30 ° vers le haut de table et orienter une mouche femelle avec les ailes vers le bas. Avec votre main non dominante, ramasser la femelle aide d&#39;une pince, son saisissant à la unenterior de l&#39;abdomen, près du thorax, et son plonger dans les médias de dissection dans la dépression coulisse (figure 1A encadré). </li><li> Avec l&#39;autre paire de pinces, saisir l&#39;exosquelette à la partie postérieure de l&#39;abdomen ventral et percer. Prenez les ovaires à l&#39;extrémité postérieure, puis de les sortir de l&#39;abdomen et le placer dans les médias frais de l&#39;autre côté de la glissière de la dépression. (Figure 1A encadré). Évitez de tirer en dehors de l&#39;intestin. Jeter la carcasse. </li><li> Répéter l&#39;opération avec les autres mouches femelles jusqu&#39;à ce que toutes les ovaires sont disséqués. Jeter les mâles. </li><li> Avec une paire de pinces, maintenez un ovaire à l&#39;extrémité postérieure (près des plus grandes salles d&#39;œufs), et avec l&#39;autre main utiliser une paire de pinces pour pincer l&#39;une des chambres les plus antérieures d&#39;œufs (de germarium). (Figure 1A). </li><li> Lentement mais sûrement, tirer une chaîne ovariole hors de la gaine ovaire. Continuer à disséquer ovarioles individuellement jusqu&#39;à ce que toutes les chambres d&#39;œufs souhaités sont removed. </li><li> Répétez les étapes 1.7 à 1.8 pour toutes les ovaires disséqués. </li><li> Une fois terminé, transférer ovarioles dans un tube de 0,6 ml à l&#39;aide d&#39;une pipette de transfert en plastique. </li><li> Laissez ovarioles se déposent au fond du tube, puis passez à fixation et de coloration étapes. </li></ol> 2. immunofluorescence (IF) Coloration des ovarioles <ol><li> Retirez délicatement les médias de dissection de ovarioles avec une pipette. Veillez à ne pas supprimer tous les ovarioles ou chambres d&#39;œufs. Laissez environ 50 pi de ovarioles et les médias de dissection. </li><li> Ajouter 50 ul de 16% de paraformaldéhyde à 150 ul de 0,1 M KPO 4 tampon dans un tube, et ajouter la solution à des chambres d&#39;oeuf pour les corriger. Incuber tout bascule pour 10 min. ATTENTION: paraformaldéhyde est toxique. </li><li> Retirer fixateur et rincer deux fois avec 0,5 ml de tampon de lavage de NP40. </li><li> Laver deux fois pendant 15 min chacune à la température ambiante. </li><li> Reportez-vous à la norme si le protocole 15 pour les étapes suivantes. <ol><li> Brièvement, après fixation, ajouteranticorps primaires aux dilutions appropriées et incuber O / N à 4 ° C. Laver à NP40 à plusieurs reprises, puis ajouter des anticorps secondaires (1: 200 dilutions). </li><li> Lavez plusieurs fois dans NP40, puis ajouter DAPI (1: 1000) pendant 10 min, puis répétez lavages. Une fois que si le protocole est complète, ajouter 200 ul de 50% de glycérol dans du PBS pour chambres d&#39;oeufs et laisser reposer à température ambiante pendant 2 h. </li><li> Retirer la solution, le remplacer par 70% de glycérol dans du PBS, et couvrir de papier d&#39;aluminium. Placez échantillon à 4 ᵒC jusqu&#39;au moment pour le montage. Pendant ce temps, passez à l&#39;étape 3. </li></ol></li></ol> 3. Préparation préalable de glycérine Jelly Diapositives <ol><li> Mesurer au moins 4 ml de glycérine gelée aide d&#39;une spatule et remplir un tube de culture de cellules de verre pour la marque à mi-chemin. Faire fondre la gelée de glycérine dans un bain d&#39;eau à 55 ° C pendant 30 min. </li><li> Verser une petite goutte de glycérol, environ 300 pi, de la solution de réserve sur deux lames de microscope. Utiliser un tissu, répartis glycérol dans une fine layer sur chacune des diapositives. Ceci fournit une base et permet de retirer facilement la glycérine gelée à l&#39;étape 5.6. </li><li> Coupez la pointe de d&#39;une pointe de pipette 1000 pi filtré. Utilisez copier pointe de la pipette pour transférer 1000-1500 ul de la glycérine chaude gelée lentement à chaque lame de microscope. Prenez soin d&#39;éviter les bulles. </li><li> Laissez diapositives glycérine gelée reposer pendant 5 min à température ambiante à définir. </li><li> Placez chaque diapositive glycérine gelée dans 60 mm × 15 mm plat de Pétri et couvrir d&#39;une pellicule plastique. Lieu à 4 ° CO / N. Eventuellement, magasin glycérine diapositives de gelée jusqu&#39;à 5 jours à 4 ° C. </li></ol> 4. Montage Chambers oeufs <ol><li> En utilisant des échantillons de l&#39;étape 2.5, pipette quelques gouttes 3 pi de chambres d&#39;œufs dans 70% de glycérol dans une diapositive de la gelée de glycérine. Continuer pipetage 3 gouttes ul jusqu&#39;à ce que toutes les chambres d&#39;oeufs sont sur cette diapositive. Assurez-vous que chaque goutte contient au moins dix chambres d&#39;œufs. Pendant ce temps, chauffer un tube de culture de glycérine gelée dans un bain-marie à 55 °. </li><li> Placer la lame de gelée de glycérine avec des chambres d&#39;oeuf sous un microscope à dissection. Ajuster le grossissement de telle sorte que seule une goutte de chambres d&#39;oeuf est dans le champ de vision (figure 1B). </li><li> En utilisant une aiguille de calibre 30 comme une cuillère, ramasser la chambre d&#39;œufs de stade désiré. Si nécessaire, les chambres d&#39;œufs souhaités séparée d&#39;une chaîne de ovariole utilisant l&#39;aiguille comme un couteau. </li><li> Pour éviter les débris qui peuvent interférer avec l&#39;imagerie, transférer la chambre de l&#39;œuf à la deuxième diapositive glycérine gelée, à la face antérieure gauche (figure 1E -1). Répéter jusqu&#39;à ce que il ya au moins sept chambres d&#39;oeuf alignés dans une colonne (figure 1C). </li><li> Former de nouvelles colonnes de sept jusqu&#39;à ce que toutes les chambres d&#39;oeuf désirées sont transférées à la seconde glissière de glycérine gelée. </li><li> Déchirez un petit morceau de tissu et la torsion. Utilisez cette option pour absorber l&#39;excès de PBS-glycérol des chambres d&#39;œufs en effleurant chacun. Prenez soin de ne pas supprimer les chambres d&#39;œufs. </li><li> Coupez lepointe d&#39;une pointe de pipette 200 pi filtré. Utilisez cette option pour transférer 150 pi de glycérine gelée pour couvrir toutes les colonnes de chambres d&#39;œufs. REMARQUE: La quantité de glycérine gelée nécessaire pour couvrir les colonnes est basé sur les étapes de la chambre d&#39;œufs et le nombre de colonnes. Montant peut être ajustée. </li><li> Laissez chambres d&#39;œufs montés reposer pendant 5 min à température ambiante jusqu&#39;à ce que la couche supérieure de la glycérine gelée est complètement solidifié (figure 1D). </li><li> Placer la lame de chambres d&#39;œufs montés dans une boîte de 60 mm × 15 mm de Pétri et couvrir d&#39;une pellicule plastique. Conserver à 4 ° CO / N pour permettre aux couches de gelée de glycérine pour solidifier ensemble (lieu dans l&#39;obscurité se il est préoccupé par photoblanchiment). </li></ol> 5. Préparation des Chambres oeufs for Imaging <ol><li> Placer les lames de chambres d&#39;oeufs montés sous un microscope à dissection. Ajuster le grossissement de telle sorte que seulement une colonne de chambres d&#39;oeuf est dans le champ de vision. </li><li> L&#39;utilisation d&#39;un angle de 45 ° scalpel miniature, couper un Straight ligne le long du côté droit de la première colonne de chambres d&#39;oeuf (à proximité de la face postérieure des chambres d&#39;oeuf). (Figure 1D-E) </li><li> Ensuite, couper au-dessus de la première chambre d&#39;oeuf en haut et en dessous de la dernière chambre de l&#39;oeuf dans la colonne. A ce stade, la colonne de chambres d&#39;oeuf doit être coupée sur trois côtés. </li><li> Utilisation d&#39;un microknife, tranche le long du côté gauche de la colonne, aussi près que possible de la face antérieure des chambres d&#39;oeufs (Figure 1E) -3. </li><li> Introduire à la pipette 100 pi de froid 1X PBT sur la colonne de coupe. </li><li> Utilisez une spatule à bords arrondis pour éliminer l&#39;excès de glycérine gelée autour de trois côtés de la colonne de chambres d&#39;œufs et jetez la coupe, en laissant le reste de la diapositive. </li><li> Insérer la spatule dans la coupe faite à la face antérieure de chambres d&#39;oeuf et séparer la colonne du bloc de glycérine gelée primaire. </li><li> Préparer les échantillons pour les deux inversées (5.8.1) ou verticale (5.8.2) microscopie. <ol><li> Pour microscope inversé: t Transfertil colonne de chambres d&#39;oeufs à une lamelle avec le côté antérieur de chambres d&#39;œufs vers le bas. Répéter les étapes 5.2 à 5.8 jusqu&#39;à ce que toutes les colonnes de la glycérine gelée ont été enlevés et monté. Ajouter quelques gouttes de 50% de PBS-glycérol sur chaque bloc de chambres d&#39;oeuf pour les empêcher de se dessécher. Chambres image d&#39;œufs directement sur la lamelle. </li><li> Pour microscope droit: Transférer la colonne de chambres d&#39;oeuf à une lame de microscope avec le côté antérieur vers le haut. Répéter les étapes 5.2 à 5.9 jusqu&#39;à ce que toutes les colonnes de la glycérine gelée ont été retirés et montés sur des lames de microscope. Ajouter quelques gouttes de 50% de PBS-glycérol sur chaque bloc de chambres d&#39;oeufs et des blocs de couverture avec un ½ lamelle # 1. </li></ol></li></ol>

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The authors have no competing financial interests.

Nous décrivons ici une méthode pour monter et petite image, développer chambres d&#39;œufs dans une perspective de fin sur. Techniques communes pour les chambres imagerie d&#39;œufs sont optimisés pour des vues latérales et permettent principalement la visualisation précise des cellules folliculaires médio-latérale lorsqu&#39;ils sont colorés avec des anticorps fluorescents. L&#39;utilisation de piles ou Z-3-D reconstitutions aides à la visualisation de plusieurs plans focaux, mais est encore insuffisante po...

Etude de Drosophila melanogaster a fourni de grandes connaissances dans la régulation génétique d&#39;un large éventail de phénomènes. En particulier, il ya eu des recherches approfondies sur le développement des œufs, parce oogenèse fournit un moyen souple pour enquêter sur de nombreux processus de développement différents, y compris les motifs des tissus, des changements de polarité cellulaire, la commutation du cycle cellulaire, et la réglementation de translation 1,2,3,4. Un événement important au cours de l&#39;ovogenèse morphogénétique est la spécification, l&#39;acquisition de la motilité et la migration d&#39;un ensemble de cellules appelées cellules frontalières (examinés dans cinq). Depuis la migration cellulaire est un élément clé de la morphogénèse des animaux, et parce que la régulation génétique de ce processus est bien conservé, des mécanismes déterminés mouches sont susceptibles d&#39;être importants dans d&#39;autres contextes. Ainsi, nous étudions le contrôle moléculaire de la migration cellulaire de frontière. Pour nos études, nous avons développé une nouvelle méthode pour observer les pôles antérieurs des œufs en développement,où les cellules se posent aux frontières, d&#39;examiner comment elles se développent en détail. Dans l&#39;ovaire, chambres d&#39;œufs à divers stades de développement existent le long des chaînes appelées ovarioles, qui sont entourées d&#39;une gaine mince (figure 1A). Chaque chambre de l&#39;œuf va se poursuivre pour former un œuf. Au cours de l&#39;ovogenèse, plusieurs types de cellules différents doivent se développer de manière coordonnée. Le D. chambre melanogaster d&#39;oeuf se compose de 16 cellules germinales, dont un ovocyte, entouré par une seule couche de l&#39;épithélium folliculaire 6. Un petit nombre de cellules spécialisées, appelées cellules polaires, se pose au antérieures et postérieures pôles de l&#39;épithélium. Les cellules de bordure 6-8 proviennent de l&#39;épithélium antérieur de la chambre d&#39;oeuf, induite par les cellules polaires 5,7. À la mi-ovogenèse (étape 9), les cellules de bordure se détachent de leurs voisins et migrent entre les cellules nourricières pour atteindre l&#39;ovocyte à la partie postérieure de la chambre d&#39;oeuf 5,7. Ce mouvement doit être accomplietandis que les mailles de bord restent dans un cluster polaires entourant deux cellules non-mobiles, ce qui rend ce type de migration cellulaire collective. Migration réussie de la grappe de cellules de la frontière assure le bon développement du micropyle de la coquille d&#39;oeuf, qui est nécessaire pour la fécondation. Les cellules polaires antérieures instruisent le destin des cellules de la frontière en activant une cascade de transduction du signal. Cellules sécrètent une cytokine polaires, non apparié (UPD), qui se lie à un récepteur transmembranaire, Domeless (DOME), sur les cellules de follicules voisin lors de l&#39;étape 8 de développement des ovocytes 8,9. La liaison de UPD provoque Janus tyrosine kinase (JAK) de phosphoryler le transducteur de signaux et activateur de la transcription (STAT) 8,10,11. STAT se déplace ensuite vers le noyau pour activer la transcription. Cellules frontaliers lents (SLBO) est un facteur de transcription qui est une cible transcriptionnelle directe de STAT et est également requis pour la migration des cellules de la frontière 12. Vues latérales de chambres d&#39;œufs indiquent tactivité de STAT chapeau est réglementée dans un gradient à travers l&#39;épithélium antérieure 8,11,13. Cellules folliculaires proches des cellules polaires ont les plus hauts niveaux de activé STAT, ainsi ils deviennent des cellules frontières et envahissent le tissu de la lignée germinale adjacente. Pour comprendre comment les cellules frontières sont spécifiées à l&#39;intérieur et se détachent de l&#39;épithélium, nous avons besoin d&#39;observer comment le tissu est organisé. Si nous considérons chambres d&#39;œufs dans une perspective antérieure sur, nous nous attendrions à symétrie radiale de l&#39;activité de STAT dans les cellules folliculaires qui entourent les cellules polaires. Une vue en bout serait également montrer avec plus de précision les différences de protéines membranaires et des interfaces cellule-cellule avant et pendant le détachement que de comparer les cellules dans différents plans focaux. Parce que les chambres d&#39;oeuf sont oblongues et fixées les unes aux autres par les cellules de la tige, ils se installent sur des lames latéralement, ce qui rend difficile l&#39;observation de l&#39;architecture antérieure. Ainsi, beaucoup d&#39;informations sur les cellules au niveau des pôles de la chambre d&#39;oeuf aété déduit de vues latérales. Bien que certaines informations peut être obtenue par algorithmiques reconstructions 3-D de sections optiques, diffusion de la lumière, de photoblanchiment, et les limites les plus pauvres de la résolution de l&#39;axe Z rendre cette information moins détaillée et fiable en l&#39;absence de techniques coûteuses comme la microscopie de super-résolution 14 . D&#39;autres types de formation d&#39;image sur la base de l&#39;article (par exemple, la microscopie électronique ou microtome découpe) exigent beaucoup de manipulation de tissus, y compris la déshydratation, ce qui augmente la probabilité pour les artefacts. Ainsi, nous avons développé une nouvelle méthode pour l&#39;image D. chambres melanogaster d&#39;œufs tandis que debout. Cette méthode a déjà fait ses preuves utiles à élucider comment les cellules mobiles sont voués (voir résultats représentatifs et Manning et al, en cours d&#39;examen), et est susceptible d&#39;être plus largement utile dans les études d&#39;autres aspects de l&#39;ovogenèse.

<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.

La méthode d&#39;imagerie verticale nous a permis de voir directement l&#39;organisation des cellules de l&#39;épithélium folliculaire antérieure à l&#39;étape 8. Un marqueur général pour le destin des cellules du follicule, l&#39;Absent Eyes (EYA) protéine, ainsi que le marqueur d&#39;ADN nucléaire DAPI, a montré même l&#39;expression à travers ce champ de cellules, et ont montré que toutes les cellules ont pu être observés avec des intensités de coloration similaires (figure 2B &quot;).</stron...

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.

Imagerie Montant du<em&gt; Drosophila</em&gt; Chambres d&#39;oeufs

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 vues.  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.

Vidéo: 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-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

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.

Imagerie de calcium dans<em&gt; Drosophila</em&gt; Activation à Egg

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.

Mésoscopique fluorescence tomographie pour In vivo L&#39;imagerie du développement Drosophile</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.

Imagerie et analyse de l’orientation tissulaire et de la dynamique de croissance dans l’épithélie de drosophile en développement pendant les stades pupal

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.

Imagerie d’animaux entiers de Drosophila melanogaster à l’aide de la tomographie microcalculée

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.

Visualisation des effets des dommages oxydatifs sur les chambres d&apos;oeufs Drosophila à l&apos;aide de l&apos;imagerie en direct

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.

Migration nucléaire dans l’ovocyte de la drosophile

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.

Imagerie verticale de<em&gt; Drosophila</em&gt; Embryons

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.

Détection de Anastasis In Vivo par CaspaseTracker biocapteur

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

Médecine

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.

Imagerie Montant du

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Imagerie et analyse de l’orientation tissulaire et de la dynamique de croissance dans l’épithélie de drosophile en développement pendant les stades pupal

Imagerie d’animaux entiers de Drosophila melanogaster à l’aide de la tomographie microcalculée

Visualisation des effets des dommages oxydatifs sur les chambres d'oeufs Drosophila à l'aide de l'imagerie en direct

Migration nucléaire dans l’ovocyte de la drosophile

Imagerie verticale de

Détection de Anastasis In Vivo par CaspaseTracker biocapteur

L'immunohistochimie drosophile Abdomen Nymphe