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.

<table><tbody><tr><td>0.1 M Potassium phosphate Buffer (KPO&lt;sub&gt;4&lt;/sub&gt; Buffer)</td><td></td><td></td><td>Add 3.1 g of NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;H&lt;sub&gt;2&lt;/sub&gt;O and 10.9 g of Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (anhydrous) to distilled H&lt;sub&gt;2&lt;/sub&gt;O to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 month at 4&amp;deg;C.</td></tr><tr><td>16% Paraformaldehyde aqueous solution, EM grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td>methanol-free to preserve GFP fluorescence</td></tr><tr><td>30G x 1/2 inch needle&amp;nbsp;</td><td>VWR</td><td>BD305106</td><td>Regular bevel</td></tr><tr><td>Active Dry Yeast</td><td>Genesee Scientific</td><td>62-103</td><td>To fatten female flies</td></tr><tr><td>Bovine Serum Albumin</td><td>PAA-cell culture company</td><td>A15-701</td><td>Used in NP40 Wash Buffer</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11295-10</td><td>Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection</td></tr><tr><td>DAPI (4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride)</td><td>Sigma Aldrich</td><td>D9542</td><td>5 mg/ml stock solution&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16140-071</td><td>Added to supplement dissection medium (10%)&amp;nbsp;</td></tr><tr><td>Glass culture tube</td><td>VWR</td><td>47729-576</td><td>14 ml</td></tr><tr><td>Glass Depression Slides</td><td>VWR</td><td>470019-020</td><td>1.2 mm Thick, Double Cavity</td></tr><tr><td>Glycerin Jelly&amp;nbsp;</td><td>Electron Microsocpy Sciences</td><td>17998-10</td><td>Mounting media</td></tr><tr><td>Glycerol</td><td>IBI Scientific</td><td>IB15760</td><td>70% in PBS</td></tr><tr><td>IGEPAL CA-630</td><td>Sigma Aldrich</td><td>I3021-500ML</td><td>(interchangable for Nonidet P-40)&amp;nbsp; Used in NP40 Wash Buffer&amp;nbsp;</td></tr><tr><td>Leica Fluorescent Stereoscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Leica SP5 Confocal Microscope</td><td>Leica Microsystems</td><td></td><td>40x/0.55NA dry objective&amp;nbsp;</td></tr><tr><td>Micro spatula&amp;nbsp;</td><td>VWR</td><td>82027-518</td><td>Stainless steel</td></tr><tr><td>Microscope Slide</td><td>VWR</td><td>16004-368</td><td>75x25x1 mm</td></tr><tr><td>NP40 Wash Buffer</td><td></td><td></td><td>50 mM TRIS-HCl, 150 mM NaCl, 0.5% Ipegal, 1 mg/ml BSA, and 0.02% sodium azide</td></tr><tr><td>Penicillin-Streptomycin-Glutamine</td><td>Life Technologies</td><td>10378-016</td><td>Added to supplement dissection medium (0.6X)</td></tr><tr><td>Petridish- Polysterine, sterile</td><td>VWR</td><td>82050-548</td><td>60 W x 15 H mm</td></tr><tr><td>Phosphate Buffer Saline</td><td>Sigma Aldrich</td><td>P3813-10PAK</td><td>10 packs of Powder</td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma Aldrich</td><td>P3786-500G</td><td>Used in 0.1 M KPO&lt;sub&gt;4&lt;/sub&gt; Buffer&amp;nbsp;</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P9791-500G</td><td>Used in 0.1 M KPO&lt;sub&gt;4&lt;/sub&gt; Buffer&amp;nbsp;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Schneider&amp;rsquo;s Insect Medium</td><td>Life Technologies</td><td>&lt;br/&gt; 11720018</td><td>With L-glutamine and sodium bicarbonate</td></tr><tr><td>Sodium azide</td><td>Sigma Aldrich</td><td>S2002-25G</td><td>Used in fluorescent antibody staining</td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>S3014-500G</td><td>Used in NP40 Wash Buffer</td></tr><tr><td>TRIS-HCl</td><td>IBI Scientific</td><td>IB70144</td><td>1 M TRIS-HCl, pH 7.4</td></tr><tr><td>Volocity 3D Image Analysis Software</td><td>PerkinElmer</td><td></td><td>For processing confocal Z-stacks</td></tr></tbody></table>

upright imaging of drosophila egg chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

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

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. ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Mesoscopici fluorescenza per Tomografia In-vivo Imaging di sviluppo Drosophila</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Biologia

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

Imaging e analisi dell'orientamento dei tessuti e delle dinamiche di crescita nell'epiteliale della Drosophila in via di sviluppo durante le fasi pupali

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

Imaging di animali interi di Drosophila melanogaster utilizzando la tomografia microincisa

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

Visualizzazione degli effetti dei danni ossidativi sulle camere delle uova Drosophila utilizzando l&apos;imaging dal vivo

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

Migrazione nucleare nell'ovocita di Drosophila

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Imaging verticale di Drosophila Embrioni

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Rilevazione di Anastasis In Vivo di biosensore CaspaseTracker

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

Medicina

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Imaging Upright di

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Capitoli in questo video

Live-imaging del

Calcio Imaging in

Mesoscopici fluorescenza per Tomografia In-vivo Imaging di sviluppo Drosophila

Imaging e analisi dell'orientamento dei tessuti e delle dinamiche di crescita nell'epiteliale della Drosophila in via di sviluppo durante le fasi pupali

Imaging di animali interi di Drosophila melanogaster utilizzando la tomografia microincisa

Visualizzazione degli effetti dei danni ossidativi sulle camere delle uova Drosophila utilizzando l'imaging dal vivo

Migrazione nucleare nell'ovocita di Drosophila

Imaging verticale di Drosophila Embrioni

Rilevazione di Anastasis In Vivo di biosensore CaspaseTracker

Drosophila pupa Addome Immunoistochimica