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.

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			<td height="40" style="height:40px;width:348px;">Name of Reagent/ Equipment</td>
			<td style="width:279px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:283px;">Comments/Description</td>
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			<td height="96" style="height:96px;width:348px;">0.1 M Potassium phosphate Buffer (KPO4 Buffer)</td>
			<td style="width:279px;"></td>
			<td style="width:119px;"></td>
			<td style="width:283px;">Add 3.1 g of NaH2PO4&#8226;H2O and 10.9 g of Na2HPO4 (anhydrous) to distilled H2O 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 mo at 4&#176;C.</td>
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			<td height="25" style="height:25px;width:348px;">16% Paraformaldehyde aqueous solution, EM grade</td>
			<td style="width:279px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td>methanol-free to preserve GFP fluorescence</td>
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			<td height="20" style="height:20px;width:348px;">30G x 1/2 inch needle&#160;</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">BD305106</td>
			<td>Regular bevel</td>
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			<td height="22" style="height:22px;width:348px;">Active Dry Yeast</td>
			<td style="width:279px;">Genesee Scientific</td>
			<td>62-103</td>
			<td>To fatten female flies</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Bovine Serum Albumin</td>
			<td style="width:279px;">PAA-cell culture company</td>
			<td style="width:119px;">A15-701</td>
			<td>Used in NP40 Wash Buffer</td>
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		<tr height="39">
			<td height="39" style="height:39px;width:348px;">Dumont #5 Forceps</td>
			<td style="width:279px;">Fine Science Tools</td>
			<td style="width:119px;">11295-10</td>
			<td style="width:283px;">Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection</td>
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		<tr height="39">
			<td height="39" style="height:39px;width:348px;">DAPI (4&#8242;,6-Diamidino-2-phenylindole dihydrochloride)</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">D9542</td>
			<td style="width:283px;">5mg/ml stock solution&#160;&#160;</td>
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		<tr height="39">
			<td height="39" style="height:39px;width:348px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:279px;">Life Technologies</td>
			<td style="width:119px;">16140-071</td>
			<td style="width:283px;">Added to supplement dissection medium (10%)&#160;</td>
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			<td height="24" style="height:24px;width:348px;">Glass culture tube</td>
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			<td>47729-576</td>
			<td>14mL</td>
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			<td height="20" style="height:20px;width:348px;">Glass Depression Slides</td>
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			<td>470019-020</td>
			<td>1.2 mm Thick, Double Cavity</td>
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			<td>70% in PBS</td>
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			<td height="39" style="height:39px;width:348px;">IGEPAL CA-630</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">I3021-500ML</td>
			<td style="width:283px;">(interchangable for Nonidet P-40)&#160; Used in NP40 Wash Buffer&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Leica Fluorescent Stereoscope&#160;</td>
			<td style="width:279px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Leica SP5 Confocal Microscope</td>
			<td style="width:279px;">Leica Microsystems</td>
			<td style="width:119px;"></td>
			<td>40x/0.55NA dry objective&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Micro spatula&#160;</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">82027-518</td>
			<td>Stainless steel</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Microknife&#160;</td>
			<td style="width:279px;">Roboz Surgical Instrument Company</td>
			<td style="width:119px;">37-7546</td>
			<td>45&#7506; angle</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Microscope Slide</td>
			<td style="width:279px;">VWR</td>
			<td style="width:119px;">16004-368</td>
			<td>75x25x1 mm</td>
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		<tr height="58">
			<td height="58" style="height:58px;width:348px;">NP40 Wash Buffer</td>
			<td style="width:279px;"></td>
			<td style="width:119px;"></td>
			<td style="width:283px;">50mM TRIS-HCL, 150mM NaCl, 0.5% Ipegal, 1mg/ml BSA, and 0.02% sodium azide</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Penicillin-Streptomycin-Glutamine</td>
			<td style="width:279px;">Life Technologies</td>
			<td>10378-016</td>
			<td>Added to supplement dissection medium (0.6X)</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Petridish- Polysterine, sterile</td>
			<td style="width:279px;">VWR</td>
			<td>82050-548</td>
			<td>60Wx15H mm</td>
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			<td height="20" style="height:20px;width:348px;">Phosphate Buffer Saline</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P3813-10PAK</td>
			<td>10 packs of Powder</td>
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			<td height="20" style="height:20px;width:348px;">Potassium phosphate dibasic</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P3786-500G</td>
			<td>Used in 0.1M KPO4 Buffer&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Potassium phosphate monobasic</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">P9791-500G</td>
			<td>Used in 0.1M KPO4 Buffer&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Schneider&#8217;s Insect Medium</td>
			<td style="width:279px;">Life Technologies</td>
			<td style="width:119px;"> 
			11720018</td>
			<td>With L-glutamine and sodium bicarbonate</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:348px;">Sodium azide</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">S2002-25G</td>
			<td>Used in fluorescent antibody staining</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Sodium chloride</td>
			<td style="width:279px;">Sigma Aldrich</td>
			<td style="width:119px;">S3014-500G</td>
			<td>Used in NP40 Wash Buffer</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">TRIS-HCL</td>
			<td style="width:279px;">IBI Scientific</td>
			<td style="width:119px;">IB70144</td>
			<td>1M TRIS-HCL, pH 7.4</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:348px;">Volocity 3D Image Analysis Software</td>
			<td style="width:279px;">PerkinElmer</td>
			<td style="width:119px;"></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

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop within the uterus. A newly developed ex vivo upright droplet culture method provides an attractive alternative to studies performed in utero. The ex vivo droplet culture provides the ability to examine and manipulate cellular interactions and diverse signaling pathways through use of various blocking and activating compounds; additionally, the effects of various pharmacological reagents on the development of specific organs can be studied without unwanted side effects of systemic drug delivery in utero. As compared to other in vitro systems, the droplet culture not only allows for the ability to study three-dimensional morphogenesis and cell-cell interactions, which cannot be reproduced in mammalian cell lines, but also requires significantly less reagents than other ex vivo and in vitro protocols. This paper demonstrates proper mouse fetal organ dissection and upright droplet culture techniques, followed by whole organ immunofluorescence to demonstrate the effectiveness of the method. The ex vivo droplet culture method allows the formation of organ architecture comparable to what is observed in vivo and can be utilized to study otherwise difficult-to-study processes due to embryonic lethality in in vivo models. As a model application system, a small-molecule inhibitor will be utilized to probe the role of vascularization in testicular morphogenesis. This ex vivo droplet culture method is expandable to other fetal organ systems, such as lung and potentially others, although each organ must be extensively studied to determine any organ-specific modifications to the protocol. This organ culture system provides flexibility in experimentation with fetal organs, and results obtained using this technique will help researchers gain insights into fetal development.

Using Ex Vivo Upright Droplet Cultures of Whole Fetal Organs to Study Developmental Processes during Mouse Organogenesis

The&#160;ex vivo&#160;upright droplet culture is an alternative to current&#160;in vitro&#160;and&#160;in vivo&#160;experimental techniques. This protocol is easy to perform and requires smaller amounts of reagent, while permitting the ability to manipulate and study fetal vascularization, morphogenesis, and organogenesis.

Utilisation<em&gt; Ex Vivo</em&gt; Cultures Upright gouttelettes de toute fœtus organes pour étudier les processus de développement au cours de la souris Organogenèse

Investigating organogenesis in utero is a technically challenging process in placental mammals due to inaccessibility of reagents to embryos that develop ...

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. ...

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 ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

In Vivo Imagerie de la morphogénèse du Muscle-tendon en pupes de drosophile

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

Analyse de la dynamique de l'actomyosine à l'échelle locale cellulaire et tissulaire à l'aide de films time-lapse de chambres d'oeufs drosophila cultivées

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

Imagerie et analyse en direct des contractions musculaires dans l'embryon de Drosophila

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the number of novel cell lines is expected to increase. The DGRC aims to familiarize researchers with using Drosophila cell lines as an experimental tool to complement and drive their research agenda. Procedures for working with a variety of Drosophila cell lines with distinct characteristics are provided, including protocols for thawing, culturing, and cryopreserving cell lines. Importantly, this publication demonstrates the best practices required to work with Drosophila cell lines to minimize the risk of contaminations from adventitious microorganisms or from other cell lines. Researchers who become familiar with these procedures will be able to delve into the many applications that use Drosophila cultured cells including biochemistry, cell biology and functional genomics.

Thawing, Culturing, and Cryopreserving Drosophila Cell Lines

Drosophila cell lines are important reagents for both fundamental and biomedical research. This article provides protocols for thawing, subculturing, and the cryopreservation of commonly used Drosophila cell lines to assist researchers in incorporating the use of these reagents in their research.

Dégel, cultivant et cryoconservation des lignées de cellules de drosophile

There are currently over 160 distinct Drosophila cell lines distributed by the Drosophila Genomics Resource Center (DGRC). With genome engineering, the ...

Human Egg Maturity Assessment and Its Clinical Application

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the egg&#39;s developmental competence. Presence of the first polar body (PB) together with the meiotic spindle indicates completion of the oocyte maturation and the egg&#39;s readiness for fertilization. In clinical practice, it is customary to assume that all oocytes displaying a PB are mature metaphase (MII) oocytes. However, PB extrusion precedes the formation of the bipolar MII spindle. This asynchrony makes the mere presence of PB an unreliable marker of oocyte maturity. Noninvasive spindle imaging using polarized light microscopy (PLM) allows quick and easy inspection of whether the PB-displaying oocyte actually reassembled a meiotic spindle prior to ICSI. Here, we present a standard protocol to perform human egg maturity assessment in the clinical laboratory. We also show how to optimize the time of ICSI with respect to the oocyte&#39;s developmental stage in order to prevent premature sperm injection of late-maturing oocytes. Using this approach, even immature oocytes extruding PB in vitro can be clinically utilized. Affirmation that MII spindle is present prior to sperm injection and individual adjustment of the time of ICSI is particularly important in poor prognosis in-vitro fertilization (IVF) cycles with a low number of oocytes available for fertilization.

We provide an outline of the clinical protocol for non-invasive assessment of human egg maturity using polarized light microscopy.

Évaluation de la maturité des œufs humains et son application clinique

The optimal timing of intracytoplasmic sperm injection (ICSI) is of a serious concern for fertility programs because untimely sperm entry diminishes the ...

Imaging Dpp Release from a Drosophila Wing Disc

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-&#946; superfamily includes TGF-&#946;, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-&#946; superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.

Imaging Dpp Release from a Drosophila Wing Disc

The timing of exposure to ligands may impact their developmental consequences. Here we show how to image release of a Drosophila bone morphogenetic protein (BMP) called Dpp from cells of the wing disc.