Il lavoro è stato sostenuto da un finanziamento di base del Medical Research Council. Siamo grati a Craig Nicol per la preparazione di disegni tecnici. Siamo grati a Matthew Pearson e Paul Perry per il loro sostegno imaging.

Tutto il lavoro animale è stato eseguito in conformità con le linee guida istituzionali su licenza dalla UK Home Office (numero di licenza del progetto PPL 60/3785 e 60/4424). 1. Preparazione <ol><li> Etichettare le melanoblasts della pelle di sviluppo combinando una linea di topi che esprimono Cre-ricombinasi appropriata con una linea di topi reporter fluorescente. Tyr :: CREA, Tyr :: CREB 8 e Wnt1 :: Cre 14 sono stati utilizzati con successo come linee Cre-esprimono e R26YFPR 9 come linea giornalista. </li><li> Preparare il terreno di coltura in una cappa a flusso laminare; integrare Dulbecco Modified Eagle Medium (DMEM) con (1% v / v penicillina / streptomicina), 10% v / v di siero di vitello fetale e 0,584 g / L di L-glutammina. </li><li> L&#39;apparato di immagini dal vivo è descritto nella Figura 1 Sterilizzare base della camera di imaging strofinando giù con il 70% di etanolo. Sterilizzare gli inserti, clip, immagini e O-ring immergendo overnight in etanolo al 70%. Utilizzare un coperchio sterile da una piastra da 6 pozzetti come il coperchio della camera. </li><li> La superficie inferiore della camera di imaging costituito da una membrana lummox trattenuto da un O-ring (Figura 1). Inizia con un gas piatto lummox permeabile da 35 mm. Posizionare il piatto sopra la camera di imaging e tagliare fuori la membrana permeabile ai gas con una sola lama di rasoio taglio. Spingere l&#39;O-ring sulla membrana per fissarlo alla camera di imaging. </li><li> Le clip di imaging (6 in totale) morsetto pelle embrionale in posizione (Figura 1) prima dell&#39;uso devono essere riempiti con 1% di agarosio. Preparare una soluzione al 2% di agarosio con dH 2 O sterile nell&#39;apparecchiatura a microonde e raffreddamento a 60 ° C. Miscela 1:1 con 10 ml di terreno di coltura (vedere il passo 2) preriscaldato a 60 ° C. Stare i 6 clip di imaging in un 6-pozzetti sterile. Gocciolare la soluzione di agarosio 1% al centro di ogni clip con una multa pastette e lasciare raffreddare. Aggiungere 1 ml di PBS sterile a freddo a ciascun pozzetto per impedire l&#39;unagarose secchi. Le clip imaging possono essere conservati per diverse ore in PBS. </li><li> Per effettuare la delicata separazione di pelle embrionale e manipolare il tessuto sezionato utilizzare il &#39;metodo bastone capelli&#39; come descritto in Kashiwagi et al. 3 Invece di utilizzare un bastoncino di bambù kebab spiedo può essere utilizzato. Fai una piccola fessura nella parte piatta della spiedino con una singola lama di rasoio taglio e inserire uno spazzolino a setole morbide. Tagliare lo spiedo in modo che sia circa 15 cm di lunghezza. </li><li> Utilizzare un microscopio confocale con una camera ambientale completo o inscenare camera superiore che fornisce 5% di CO 2 nell&#39;aria. Preriscaldare palco microscopio a 37 ° C per almeno 1 ora prima di imaging. Questo aiuta a prevenire la deriva del campione causata dall&#39;espansione dei componenti dello stadio come si riscaldano. </li><li> Preparare un gruppo multi-posizione utilizzando il software di controllo del microscopio in modo che si può centrare rapidamente e facilmente al centro di ogni cultura impostare un sperimentazionet. </li></ol> 2. Procedura <ol><li> Mate i topi e designare il primo coitum giorno dopo giorno come E0.5. Al giorno E14.5, abbattere la femmina per dislocazione cervicale ed esporre la cavità del corpo attraverso una piccola incisione mediana, smontando la pelle verso la testa e la coda e poi tagliando il peritoneo sottostante e pealing ritornare a entrambi i lati del corpo. Sezionare l&#39;utero in freddo sterile PBS tenendo saldamente con una pinza e tagliandoli lungo la mesometrium con le forbici chirurgiche. Mantenere l&#39;utero in ghiaccio fino al momento. Per una descrizione dettagliata vedi Shea et al. (2007) 15. </li><li> Sezionare embrioni dall&#39;utero prima tagliando la lunghezza della parete uterina con forbici chirurgiche per rilasciare il decidua e poi sezionare gli embrioni dalla decidua Separandoli con due coppie di pinza sottile. Schermo per l&#39;espressione reporter fluorescente (se necessario). </li><li> Abbattere gli embrioni per decapitazione prima della dissezione. Subsequently rimuovere la coda e posteriori degli arti e poi gli arti anteriori utilizzando una singola lama di rasoio taglio. Conservare il tessuto per la genotipizzazione, se necessario. </li><li> Fare un&#39;incisione nel ventre vicino alla linea mediana in direzione rostro-caudale. Usando i capelli bastoni descritti al punto 1.6, stuzzicare accuratamente via la pelle dal tessuto connettivo sottostante rotazione dell&#39;embrione allo stesso tempo. Non rimuovere la pelle completamente, ma piuttosto lasciarlo attaccato lungo il bordo ventrale più lontana dalla prima incisione. </li><li> È importante non lasciare la pelle diventare avvitato come è poi molto difficile da montare. Per evitare questo, appiattire la pelle usando i bastoni dei capelli (lato dermico up) sulla superficie di una piastra di Petri a secco e poi tagliare dal tronco embrione utilizzando una singola lama di rasoio taglio. Il lato cutanea può essere distinto dal lato epidermica da un&#39;attenta osservazione della giunzione tra il tessuto sezionato e l&#39;embrione prima che la pelle viene rimosso. La superficie del tessuto è sezionatocirca 0,5 mm 2 quando sezionare un embrione E14.5. </li><li> Posizionare una clip di imaging in cima alla pelle in modo che l&#39;agarosio tocchi il lato del tessuto dermico. Lasciare riposare per 60 sec e poi accuratamente provocare gli bordi del tessuto fino ai lati della clip. Invertire la clip e utilizzare i capelli bastoni di appiattire la pelle ed eliminare eventuali bolle d&#39;aria, avendo cura di toccare la superficie del tessuto (nella zona che verrà ripreso) il meno possibile. </li><li> Utilizzare filo di sutura di seta per legare la pelle alla clip di imaging utilizzando due semplici nodi pollice su entrambi i lati del clip di imaging in modo che stringere e tirare la pelle nella scanalatura vicino alla parte superiore della clip. Invertire la clip, spingere all&#39;interno l&#39;inserto di imaging e posto nella base. Riempire l&#39;inserto con ~ 5 ml di terreno di coltura (vedi passo 1.2). Figura 1 illustra il montaggio della camera di imaging. </li><li> Ripetere protocollo per i rimanenti 5 campioni. </li></ol> 3. VivereImaging <ol><li> La camera di imaging ha le stesse dimensioni di una piastra da 6 pozzetti standard. Utilizzare un apposito inserto piastra per montare la camera sul palco del microscopio confocale. </li><li> Un palcoscenico automatizzato è tenuto a immagine culture in parallelo. Definire le sei posizioni di immagine nel software confocale. Le impostazioni esatte dipenderanno dalla capacità del microscopio utilizzato. Tipicamente una piccola z-stack (3-5 z-posizioni per tenere conto di qualsiasi deriva stage) viene utilizzato in ciascuna delle 6 posizioni ed un z-stack viene catturato ogni 2 min per 18-24 ore. Per applicazioni live-immagini è generalmente vantaggioso utilizzare una più ampia impostazione pinhole otticamente ottimale per ridurre la potenza del laser e il numero di z sezioni necessarie. </li></ol>

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+migratory+pathway+of+neural+crest+cells+into+the+skin+of+mouse+embryos.">The migratory pathway of neural crest cells into the skin of mouse embryos.</a> Developmental Biology. 34 (1), 39-46 (1973).</li><li>Kashiwagi, M., Huh, N., Turksen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+Culture+of+Developing+Mouse+Skin+and+Its+Application+for+Molecular+Mechanistic+Studies+of+Morphogenesis.">Organ Culture of Developing Mouse Skin and Its Application for Molecular Mechanistic Studies of Morphogenesis.</a> Epidermal Cells: Methods and Protocols. 289, 39-45 (2005).</li><li>Kashiwagi, M., Kuroki, T., Huh, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+inhibition+of+hair+follicle+formation+by+epidermal+growth+factor+in+an+organ+culture+of+developing+mouse+skin.">Specific inhibition of hair follicle formation by epidermal growth factor in an organ culture of developing mouse skin.</a> Developmental Biology. 189 (1), 22-32 (1997).</li><li>Van Der Wel, L. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MGF+(KIT+ligand)+is+a+chemokinetic+factor+for+melanoblast+migration+into+hair+follicles.">MGF (KIT ligand) is a chemokinetic factor for melanoblast migration into hair follicles.</a> Developmental Biology. 225 (2), 424-436 (2000).</li><li>Delmas, V., Martinozzi, S., Bourgeois, Y., Holzenberger, M., Larue, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre-mediated+recombination+in+the+skin+melanocyte+lineage.">Cre-mediated recombination in the skin melanocyte lineage.</a> Genesis. 36 (2), 73-80 (2003).</li><li>Srinivas, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre+reporter+strains+produced+by+targeted+insertion+of+EYFP+and+ECFP+into+the+ROSA26+locus.">Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus.</a> BMC Developmental Biology. 1 (1), (2001).</li><li>Li, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1+Drives+Melanoblast+Organization+during+Mouse+Development+by+Orchestrating+Pseudopod.">Rac1 Drives Melanoblast Organization during Mouse Development by Orchestrating Pseudopod.</a> Driven Motility and Cell-Cycle Progression. 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Descriviamo un metodo per la cultura pelle embrionale che è particolarità suscettibili di imaging per il live-cell su microscopi confocale invertiti. Il metodo include i recenti miglioramenti che permettono di 6 culture di essere ripreso in parallelo e rimuove la dipendenza matrigel e membrane Nuclepore del metodo originale 6. La differenza fondamentale tecnico da tecniche simili è l&#39;uso di una membrana permeabile ai gas lummox stabilire un&#39;interfaccia liquido aria e anche agire come un coprioggett...

Tradizionalmente pelle embrionale è stato coltivate sezionando dall&#39;embrione mouse e montaggio su Nuclepore membrana di policarbonato. La membrana viene poi collocato sul mezzo di coltura, mantenendo così una interfaccia aria-liquido sulla superficie del tessuto sviluppo 3,4. Questa tecnica è stata utilizzata per saggiare il comportamento melanoblast fissando il tessuto e valutare la distribuzione melanoblast con β-galattosidasi come marcatore 7. Abbiamo sviluppato un metodo che permette melanoblasts live-imaging fluorescente in ex vivo della cultura pelle 6. Qui si descrive il metodo in dettaglio da dissezione, per l&#39;installazione, per vivere confocale e comprende alcuni recenti miglioramenti. Melanoblasts sono i precursori embrionali dei melanociti, le cellule che producono pigmento nei capelli e la pelle. Melanoblasts sorgono nella cresta neurale adiacente al tubo neurale intorno al giorno embrionale 9 (E9) nel topo sviluppo embryo. Successivamente migrano lungo un percorso dorsolaterale tra l&#39;ectoderma e somiti in via di sviluppo. A E12.5 si muovono dal derma all&#39;epidermide dove proliferano e continuano la loro migrazione. Il follicolo capelli modello primario comincia a formarsi nell&#39;epidermide a E14.5 e E15.5 melanoblasts sono la localizzazione di questi follicoli. Per una rassegna di sviluppo melanoblast / melanociti vedere Thomas &amp; Erickson (2008) 1. Per etichettare la popolazione melanoblast abbiamo combinato Tyr :: CREB animali che esprimono Cre-ricombinasi guidata dal promotore del mouse tirosinasi 8 con animali R26YFPR che esprimono la proteina fluorescente gialla (YFP) condizionalmente dal locus Rosa26 9. Descriviamo un metodo per la cultura della pelle e catturare le immagini embrionali utilizzando un microscopio confocale invertito. E &#39;adatta forma il metodo originale descritto in Mort et al. (2010) 6. L&#39;attualemetodo permette l&#39;imaging in un formato da 6 pozzetti e rimuove la dipendenza membrane Nuclepore e matrigel a sostenere la cultura. Invece utilizzando un piccolo blocco di 1% agarosio per stabilizzare la pelle embrionale. Eliminare la dipendenza matrigel è importante soprattutto in situazioni in cui la risposta della pelle embrionale di fattori di crescita solubili è al centro di studio. Il nostro metodo originale è già stato utilizzato per acquisire nuove conoscenze in sviluppo melanoblast 10-13 e ci aspettiamo che i miglioramenti descriviamo qui renderà più potente come una tecnica sperimentale specialmente dove più culture parallele sono un requisito.

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		<col />
		<col />
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM high glucose</td>
			<td style="width:132px;">Biochrom AG</td>
			<td style="width:119px;">F0475</td>
			<td style="width:167px;">without phenol red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">P3032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Streptomycin</td>
			<td style="width:132px;">Sigma</td>
			<td style="width:119px;">S9137</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal calf serum</td>
			<td style="width:132px;">Hyclone</td>
			<td style="width:119px;">SV30160.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamax</td>
			<td style="width:132px;">Gibco</td>
			<td style="width:119px;">35050-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Live imaging chamber</td>
			<td style="width:132px;">Custom made</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lummox dishes</td>
			<td style="width:132px;">Sarstedt</td>
			<td style="width:119px;">94.6077.410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">6-well plate</td>
			<td style="width:132px;">Greiner Bio-One</td>
			<td style="width:119px;">657-160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Single edged razor blade</td>
			<td style="width:132px;">Fisher Scientific</td>
			<td style="width:119px;">1244-3170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:132px;">Biogene</td>
			<td style="width:119px;">300-300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fine pastette</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Kebab skewers</td>
			<td style="width:132px;">Waitrose</td>
			<td style="width:119px;"></td>
			<td>Bamboo BBQ skewers 30cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Toothbrush</td>
			<td style="width:132px;">Generic</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dishes</td>
			<td style="width:132px;">Greiner Bio-One</td>
			<td style="width:119px;">633185</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Suture thread</td>
			<td style="width:132px;">Look</td>
			<td style="width:119px;">SP115</td>
			<td>Black silk suture thread</td>
		</tr>
	</tbody>
</table>

ex vivo culture of mouse embryonic skin and live-imaging of melanoblast migration

Melanoblasts sono cresta neurale precursori dei melanociti derivati; le cellule responsabili della produzione del pigmento nella pelle e dei capelli. Melanoblasts migrano attraverso l&#39;epidermide dell&#39;embrione dove successivamente si colonizzano i capelli sviluppare follicoli 1,2. Migrazione neurale delle cellule cresta è ampiamente studiato in vitro, ma in vivo non sono ancora ben sviluppati, soprattutto nei sistemi mammiferi. Una alternativa è quella di utilizzare ex vivo della cultura organotipica 3-6. Cultura di topo pelle embrionale richiede il mantenimento di una interfaccia aria-liquido (ALI) sulla superficie del tessuto 3,6. Alta risoluzione live-imaging topo pelle embrionali è stata ostacolata dalla mancanza di un buon metodo che non solo mantiene questo ALI ma permette anche la cultura di invertire e quindi compatibile con lenti dell&#39;obiettivo a breve distanza di lavoro e microscopi più confocale. Questo articolo descrive i recenti miglioramenti ad un method che utilizza una membrana permeabile ai gas per superare questi problemi e permettere ad alta risoluzione confocale di pelle embrionale in coltura ex vivo 6. Utilizzando un melanoblast specifico Cre-ricombinasi esprimendo linea di topi in combinazione con la linea giornalista R26YFPR siamo in grado di etichettare fluorescente popolazione melanoblast all&#39;interno di queste culture pelle. La tecnica consente live-imaging melanoblasts e osservazione del loro comportamento e le interazioni con il tessuto in cui si sviluppano. Risultati rappresentativi sono inclusi per dimostrare la capacità di vivere-image 6 culture in parallelo.

La Figura 2 mostra i risultati rappresentativi di un esperimento lasso di tempo con la pelle embrionale da Tyr :: CREB x embrioni R26YFPR a E14.5. 6 colture pelle embrionali sono stati ripresi dalla microscopia confocale ogni 2 min per 18 ore. Il pacchetto software di analisi delle immagini ImageJ del freeware è stato utilizzato per analizzare il comportamento dei melanoblasts YFP-etichettati nei 6 film. I melanoblasts erano tracciati automaticamente utilizzando il plugin wrMTrck svil...

Watch this Scientific Journal Video about Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration at JoVE.com

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Descriviamo la dissezione e ex vivo della cultura di topo pelle embrionale. Il sistema di coltura mantiene un&#39;interfaccia aria-liquido attraverso la superficie del tessuto e permette l&#39;imaging su un microscopio invertito. Melanoblasts, un componente della pelle in via di sviluppo, sono fluorescente permettendo loro comportamento da osservare mediante microscopia confocale.

Ex vivo Cultura embrionali di topo della pelle e live-imaging delle Migrazioni Melanoblast

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

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Biology

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

12.5K Views.  University of Edinburgh.  Descriviamo la dissezione e ex vivo della cultura di topo pelle embrionale. Il sistema di coltura mantiene un'interfaccia aria-liquido attraverso la superficie del tessuto e permette l'imaging su un microscopio invertito. Melanoblasts, un componente della pelle in via di sviluppo, sono fluorescente permettendo loro comportamento da osservare mediante microscopia confocale. 

Video: Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

Metodo per la Cultura di Chick primi embrioni ex vivo (Nuova Cultura)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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Biologia

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Vivere Imaging della migrazione cellule gliali nella Eye Disco Drosophila immaginale

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

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Vivere Imaging of the Brain Zebrafish embrionali da microscopia confocale

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Immagini dal vivo di Drosophila melanogaster Migrazioni Hemocyte embrionali

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Imaging in vivo e trattamenti terapeutici in un modello di mouse ortotopico di cancro ovarico

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Isolamento e coltura di cellule dalla Zona nefrogenica del Rene embrionali di topo

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex Cultura Vivo di cellule primarie umane tube di Falloppio epiteliale

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema per<em&gt; Ex vivo</emColtura&gt; del pancreas embrionale

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Metodo di Cultura per il mouse embrionale del cuore

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Mouse fetale Whole Intestino Sistema Cultura per<em&gt; Ex Vivo</em&gt; La manipolazione delle vie di segnalazione e tridimensionale Imaging in diretta di Sviluppo del villo

Ex vivo Cultura embrionali di topo della pelle e live-imaging delle Migrazioni Melanoblast

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