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. dissezione ovarioli <ol><li> Trasferire una quindicina di 2-4 giorni di età mosche femmine e alcuni maschi di una nuova mosca alimento flacone con aggiunta di lievito secco attivo. Utilizzare il cotone come una presa per i flaconi, e aggiungere qualche goccia di acqua per il cotone per mantenere l&#39;umidità alta. </li><li> Luogo flacone in un incubatore a 25 ° C per 14-16 ore per massimizzare il numero di stage 8-10 camere uovo. NOTA: Il tempo di incubazione varia a seconda della temperatura e la fase desiderata. </li><li> Preparare i media dissezione, 0.1M KPO 4 e NP40 soluzioni come necessario per il giorno successivo (vedi Materiali). </li><li> Anestetizzare linea con CO 2, e mettere sotto un microscopio dissezione. Dispensare alcune gocce di media dissezione in ogni cavità nella diapositiva depressione vetro. </li><li> Tenere pinze in ogni mano ad un angolo di circa 30 ° rispetto al piano del tavolo e orientare una mosca femmina con le ali giù. Con la mano non dominante, prendere la femmina usando pinze, afferrando il suo al dinterior dell&#39;addome, vicino al torace, e la immergere nei media dissezione nella depressione slitta (Figura 1A riquadro). </li><li> Con l&#39;altra coppia di pinze, afferrare l&#39;esoscheletro al posteriore ventrale dell&#39;addome e perforare attraverso. Afferrare le ovaie alla estremità posteriore, quindi tirare fuori dell&#39;addome e posto in media fresca sull&#39;altro lato della slitta depressione. (Figura 1A riquadro). Evitare di tirare a parte l&#39;intestino. Eliminare la carcassa. </li><li> Ripetere con gli altri mosche femminili fino a quando tutte le ovaie vengono sezionati. Eliminare i maschi. </li><li> Con una coppia di pinze, tenere un&#39;ovaia alla fine posteriore (vicino più grandi camere d&#39;uovo), e con l&#39;altra mano usare un paio di pinze per pizzicare una delle camere più uova anteriori (germarium). (Figura 1A). </li><li> Lentamente e costantemente, tirare una catena ovariole dalla guaina dell&#39;ovaio. Continuare a sezionare fuori ovarioli individualmente fino a quando tutte le camere uovo desiderati sono rdell&#39;uso e rimossi. </li><li> Ripetere i passaggi 1,7-1,8 per tutti ovaie sezionati. </li><li> Una volta completato, il trasferimento ovarioli ad un tubo 0,6 ml con una pipetta di trasferimento di plastica. </li><li> Lasciate ovarioli depositano sul fondo della provetta, quindi procedere al fissaggio e colorazione passi. </li></ol> 2. immunofluorescenza (IF) La colorazione di ovarioli <ol><li> Rimuovere con attenzione i media dissezione da ovarioli con una pipetta. Assicurarsi di non rimuovere eventuali ovarioli o camere di uova. Lasciare circa 50 ml di ovarioli e media dissezione. </li><li> Aggiungere 50 ml di 16% paraformaldeide a 150 ml di 0,1 M KPO 4 Buffer in un tubo, e aggiungere la soluzione alle camere uovo di correggerli. Incubare mentre a dondolo per 10 min. ATTENZIONE: paraformaldeide è tossico. </li><li> Rimuovere fissativo e lavare due volte con 0,5 ml di tampone di lavaggio NP40. </li><li> Lavare due volte per 15 minuti ciascuno a RT. </li><li> Fare riferimento alla norma se il protocollo 15 per passi successivi. <ol><li> In breve, dopo la fissazione, aggiungereanticorpi primari alle diluizioni appropriati e incubare O / N a 4 ° C. Lavare in NP40 più volte, quindi aggiungere anticorpi secondari (1: 200) diluizioni. </li><li> Lavare più volte in NP40, quindi aggiungere DAPI (1: 1.000) per 10 minuti, quindi ripetere lavaggi. Una volta se il protocollo è completo, aggiungere 200 ml di glicerolo al 50% in PBS per camere d&#39;uovo e lasciate riposare a temperatura ambiente per 2 ore. </li><li> Rimuovere la soluzione, sostituirlo con il 70% glicerolo in PBS, e coprire con un foglio. Posizionare campione a 4 ᵒC fino al momento per il montaggio. Nel frattempo, continuare con il passo 3. </li></ol></li></ol> 3. preparazione preliminare di diapositive gelatina glicerina <ol><li> Misurare almeno 4 ml di glicerina gelatina con una spatola e riempire un tubo di coltura cellulare di vetro per il marchio a metà strada. Sciogliere gelatina glicerina in un bagno di acqua a 55 ° C per 30 min. </li><li> Versare una piccola goccia di glicerolo, circa 300 ml, dalla soluzione madre su due vetrini da microscopio. Utilizzando un tessuto, diffuso glicerolo in un sottile layer su ciascuna delle diapositive. Ciò fornisce una base e consente una facile rimozione di gelatina glicerina al punto 5.6. </li><li> Tagliare la punta fuori di un filtrato puntale 1.000 ml. Usa taglio puntale per trasferire 1000-1500 ml di caldo gelatina glicerina lentamente ad ogni vetrino da microscopio. Fare attenzione per evitare le bolle. </li><li> Lasciate diapositive gelatina glicerina riposare per 5 minuti a temperatura ambiente per impostare. </li><li> Collocare ogni diapositiva gelatina glicerina in 60 mm × 15 mm piatto di Petri e coprire con pellicola trasparente. Posto a 4 ° CO / N. Facoltativamente, negozio di glicerina scivoli gelatina per un massimo di 5 giorni a 4 ° C. </li></ol> 4. Montaggio Egg Chambers <ol><li> Utilizzando campioni dal punto 2.5, pipette alcune gocce 3 microlitri delle camere uovo nel 70% glicerolo attraverso uno glicerina gelatina diapositiva. Continuare pipettaggio 3 gocce microlitri fino a quando tutte le camere di uova sono in questa diapositiva. Assicurarsi che ogni goccia contiene almeno dieci camere d&#39;uovo. Nel frattempo, scaldare un tubo cultura di gelatina glicerina in un bagno di 55 ° C Acque. </li><li> Posizionare il vetrino gelatina glicerina con le camere d&#39;uovo al microscopio dissezione. Regolare ingrandimento in modo che solo una goccia di camere uovo è nel campo di vista (Figura 1B). </li><li> Utilizzando un ago calibro 30 come un cucchiaio, prendere la camera stadio di uovo desiderato. Quando necessario, camere uovo desiderati separati da una catena ovariole utilizzando l&#39;ago come un coltello. </li><li> Per evitare detriti che possono interferire con le immagini, trasferire la camera delle uova alla seconda diapositiva gelatina glicerina, con la parte anteriore rivolta verso sinistra (Figura 1E -1). Ripetere finché ci sono almeno sette camere uovo allineati in una colonna (Figura 1C). </li><li> Formare nuove colonne di sette finché tutte le camere uovo desiderati vengono trasferiti alla seconda slitta gelatina glicerina. </li><li> Strappare un piccolo pezzo di tessuto e di torsione. Usare questo per assorbire l&#39;eccesso di PBS-Glicerolo dalle camere di uova da sfiorando ciascuno. Fare attenzione a non rimuovere le camere di uova. </li><li> Tagliare ilpunta di un filtrato puntale 200 microlitri. Utilizzare questo per trasferire 150 ml di glicerina gelatina per coprire tutte le colonne delle camere uovo. NOTA: La quantità di glicerina gelatina necessario per coprire le colonne si riferiscono al fasi camera d&#39;uovo e il numero di colonne. Importo può essere regolata. </li><li> Lasciate che le camere d&#39;uovo montati riposare per 5 minuti a temperatura ambiente fino a quando lo strato superiore della gelatina glicerina è completamente solidificato (Figura 1D). </li><li> Luogo di diapositive delle camere uovo montati in un piatto di 60 mm × 15 millimetri di Petri e coprire con pellicola trasparente. Conservare a 4 ° CO / N per consentire gli strati gelatina glicerina per solidificare insieme (posto al buio se non vi è preoccupazione per photobleaching). </li></ol> 5. Preparazione di uova Chambers for Imaging <ol><li> Mettere diapositive delle camere uovo montati sotto un microscopio dissezione. Regolare ingrandimento in modo che solo una colonna di camere uovo è nel campo visivo. </li><li> Utilizzando un angolo di 45 ° bisturi in miniatura, tagliare un straight linea lungo il lato destro della prima colonna di camere d&#39;uovo (vicino al lato posteriore delle camere d&#39;uovo). (Figura 1D-E) </li><li> Quindi, tagliare sopra la prima camera uovo in alto e in basso l&#39;ultima camera di uovo in colonna. A questo punto la colonna di camere uovo deve essere tagliato su tre lati. </li><li> Utilizzando un microknife, fetta lungo il lato sinistro della colonna, il più vicino possibile al lato anteriore delle camere d&#39;uovo (Figura 1E -3). </li><li> Dispensare 100 ml di freddo 1X PBT sulla colonna di taglio. </li><li> Utilizzare una spatola rotonda taglio per rimuovere la gelatina glicerina in eccesso intorno a tre lati della colonna taglio delle camere uovo e scartare, lasciando il resto della diapositiva. </li><li> Inserire la spatola nella taglio effettuato sul lato anteriore della camere d&#39;uovo e separare la colonna dal blocco gelatina glicerina primaria. </li><li> Preparare i campioni per entrambi invertiti (5.8.1) o verticale (5.8.2) microscopia. <ol><li> Per invertito Microscopio: t Transferegli colonna camere uovo per un coprioggetto con il lato anteriore delle camere uovo rivolto verso il basso. Ripetere i passaggi 5,2-5,8 fino a quando tutte le colonne di gelatina glicerina sono stati rimossi e montato. Aggiungere un paio di gocce di 50% PBS-glicerolo su ogni blocco di camere d&#39;uovo per evitare che si secchi. Camere di uova Immagine direttamente sul coprioggetto. </li><li> Per microscopio verticale: Trasferire la colonna di camere uovo di un vetrino da microscopio con il lato anteriore rivolto verso l&#39;alto. Ripetere i passaggi 5,2-5,9 fino a quando tutte le colonne di gelatina glicerina sono stati rimossi e montati su vetrini da microscopio. Aggiungere un paio di gocce di 50% PBS-glicerolo su ogni blocco di camere d&#39;uovo e blocchi di copertura con un # 1 ½ coprioggetto. </li></ol></li></ol>

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The authors have no competing financial interests.

Qui si descrive un metodo per montare e immagine piccola, sviluppando camere uovo da una prospettiva dall&#39;estremità. Tecniche comuni per le camere di imaging di uova sono ottimizzati per le viste laterali e soprattutto permettono una visualizzazione precisa delle cellule del follicolo medio-laterale quando colorati con anticorpi fluorescenti. L&#39;uso di Z-stack o 3-D ricostruzioni aiuti in visualizzazione di più piani focali, ma è ancora insufficiente per la risoluzione sub-cellulare dei poli delle camere uovo ...

Studio di Drosophila melanogaster ha fornito grandi intuizioni nella regolazione genetica di una vasta gamma di fenomeni. In particolare, non vi è stata un&#39;ampia ricerca sullo sviluppo delle uova, perché ovogenesi fornisce un modo trattabili per indagare molti diversi processi di sviluppo, tra cui patterning tessuti, i cambiamenti di polarità cellulare, la commutazione del ciclo cellulare, e la regolazione traslazionale 1,2,3,4. Un importante evento morfogenica durante oogenesi è la specificazione, l&#39;acquisizione di motilità e la migrazione di un insieme di cellule chiamate cellule di confine (recensito in 5). Poiché la migrazione delle cellule è una caratteristica chiave della morfogenesi animali, e perché la regolazione genetica di questo processo è ben conservato, meccanismi di determinati in mosche sono suscettibili di essere importante in altri contesti. Quindi, stiamo studiando il controllo molecolare della migrazione delle cellule di confine. Per i nostri studi, abbiamo sviluppato un nuovo metodo per osservare i poli anteriori di sviluppo delle uova,dove sorgono le cellule di confine, per esaminare come si sviluppano nel dettaglio. All&#39;interno l&#39;ovaio, esistono camere uovo a vari stadi di sviluppo lungo le catene di chiamate ovarioli, che sono racchiusi in un sottile guaina (Figura 1A). Ogni camera di uovo andrà a formare un uovo. Durante oogenesi, diversi tipi di cellule devono sviluppare in modo coordinato. Il D. Camera melanogaster uovo è composto da 16 celle germinali, tra cui un ovocita, circondato da un singolo strato epitelio follicolare 6. Un piccolo numero di cellule specializzate, chiamate cellule polari, sorgono a anteriore e posteriore poli dell&#39;epitelio. Le cellule di frontiera 6-8 originano nell&#39;epitelio anteriore della camera di uovo, indotta dalle cellule polari 5,7. A metà oogenesi (fase 9), le cellule di confine si staccano dai loro vicini e migrano tra le cellule nutrici per raggiungere l&#39;ovocita al posteriore della camera di uovo 5,7. Questo movimento deve essere realizzatomentre le cellule di confine rimangono in un cluster circostante due celle polari non mobili, rendendo questo tipo di migrazione cellulare collettiva. La migrazione di successo del cluster cella di bordo assicura il corretto sviluppo del micropilo del guscio d&#39;uovo, che è necessaria per la fecondazione. Le cellule polari anteriori istruiscono il destino delle cellule di confine attivando una cascata di trasduzione del segnale. Cella polare secernono una citochina, non accoppiato (UPD), che si lega ad un recettore transmembrana, Domeless (DOME), sulla vicina cellule del follicolo durante fase 8 dello sviluppo dell&#39;ovocita 8,9. Il legame di UPD provoca Janus tirosin chinasi (JAK) di fosforilare il trasduttore di segnale e attivatore di trascrizione (STAT) 8,10,11. STAT sposta quindi al nucleo di attivare la trascrizione. Le cellule di confine lenti (SLBO) è un fattore di trascrizione che è un bersaglio trascrizionale diretta di STAT ed è anche necessario per la migrazione delle cellule di confine 12. Viste laterali delle camere uovo indicano tattività di STAT cappello è regolata in un gradiente attraverso l&#39;epitelio anteriori 8,11,13. Cellule del follicolo vicini alle cellule polari hanno i più alti livelli di attivazione STAT, quindi diventano cellule di frontiera e invadono il tessuto germinale adiacente. Per capire come le cellule di confine sono specificati all&#39;interno e si staccano dal epitelio, abbiamo bisogno di osservare come il tessuto è organizzato. Se consideriamo le camere di uova da una prospettiva antero-on, ci aspetteremmo simmetria radiale di attività STAT nelle cellule follicolari che circondano le cellule polari. Un dall&#39;estremità vista sarebbe anche mostrare più accurato differenze di proteine ​​di membrana e interfacce cellula-cellula prima e durante il distacco che confrontando cellule in differenti piani focali. Perché le camere di uova sono oblunghe e attaccato tra loro da cellule gambo, si depositano su vetrini lateralmente, il che rende difficile osservare l&#39;architettura anteriore. Pertanto, molte informazioni sulle cellule ai poli della camera uovo hastato dedotto da viste laterali. Anche se alcune informazioni possono essere ottenute attraverso algoritmici 3-D ricostruzioni di sezioni ottiche, dispersione della luce, photobleaching e limiti più poveri della risoluzione dell&#39;asse Z rendere queste informazioni meno dettagliate e affidabili, in assenza di tecniche costose come super-risoluzione microscopio 14 . Altri tipi di immagini sezione a base (per esempio, microscopia elettronica o microtomo sezionamento) richiedono ampie manipolazione dei tessuti, comprendenti disidratazione, aumentando la probabilità di artefatti. Così, abbiamo sviluppato un nuovo metodo per l&#39;immagine D. camere melanogaster uovo mentre in posizione verticale. Questo metodo è già dimostrato utile nel chiarire come cellule mobili sono fated (vedere risultati rappresentativi e Manning et al, in esame), ed è probabile che sia più ampiamente prezioso negli studi di altri aspetti della oogenesis.

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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>
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			<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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			<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>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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			<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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			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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.

Il metodo di imaging in posizione verticale ha permesso di vedere direttamente l&#39;organizzazione delle cellule dell&#39;epitelio follicolare anteriore in fase 8. indicatore generale per il destino delle cellule del follicolo, il Occhi Assente (EYA) di proteine, così come il marcatore DNA nucleare DAPI, ha mostrato anche espressione in questo campo delle celle, e hanno dimostrato che tutte le cellule potrebbero essere visti con simili intensità di colorazione (Figura 2b &quot;). Proteine ​​regol...

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.

Imaging Upright di<em&gt; Drosophila</em&gt; Chambers Egg

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 Views.  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.

Video: 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-imaging del<em&gt; Drosophila</em&gt; Pupa Eye

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

Ricerca

Biologia dello sviluppo

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.

Utilizzando<em&gt; Ex Vivo</em&gt; Upright Droplet Culture di interi organi fetali per studiare i processi evolutivi durante mouse Organogenesi

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.

Calcio Imaging in<em&gt; Drosophila</em&gt; All&#39;attivazione 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.

A lungo termine Imaging in diretta di<em&gt; Drosophila</em&gt; Disco 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 Formazione immagine del muscolo-tendine morfogenesi in Drosophila pupe

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.

Analisi delle dinamiche di Actomyosin su scale cellulari e tissutali locali utilizzando film time-lapse di camere d'uovo di Drosophila coltivate

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.

Live Imaging e analisi delle contrazioni muscolari nell'embrione di 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.

Scongelamento, coltura e Cryopreserving linee di cellule di Drosophila

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.

Valutazione della maturità dell'uovo umano e la sua applicazione clinica

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.