Questo lavoro è stato sostenuto da NIH R01 NS042228 di MCH I FV1000 Olympus confocale è stata acquisita con una strumentazione NIH condiviso S10RR023717 concedere al Dipartimento di Zoologia UW (PI Bement Bill). Erica Andersen, Namrata Asuri, e Matteo Argilla contribuito in maniera uguale a questo articolo.

1. Montaggio di diapositive di iniezione e diapositive di immagini Iniezione di diapositive: <ol><li> Preparare elastomero di silicone Sylgard secondo le istruzioni del produttore. </li><li> Utilizzare il Sylgard a legare tre microscopio vetro standard diapositive insieme da impilare una diapositiva in cima alla intersezione degli altri due diapositive, che sono disposti fianco a fianco ai bordi lunghi. La slitta superiore crea un angolo retto o &quot;muro&quot; tra la parte superiore e scivoli in basso. </li><li> Lasciate Sylgard set durante la notte. Queste diapositive sono riutilizzabili. </li></ol> Imaging diapositiva: <ol><li> Noi utilizziamo i rettangoli in acciaio inox tagliato le stesse dimensioni di un vetrino da microscopio standard di vetro (75mm x 25mm x 1mm), così la camera si inserisce il supporto diapositive sul palco microscopio. Un foro di 1,5 cm è tagliato al centro del rettangolo. </li><li> Fissare a 22 mm 2 coprioggetto al rettangolo di metallo con Sylgard. Usa basta Sylgard per sigillare completamente il coprioggetto. Per l&#39;immagine su un microscopio invertito, gli embrioni verranno montati su questo coprioggetto e ripreso dal basso. </li><li> Lasciate Sylgard set durante la notte. Queste diapositive sono riutilizzabili e puliti con etanolo al 70% tra usi. </li></ol> 2. Preparazione di costrutti di DNA Per esprimere un transgene in maniera tessuto-specifici, il gene di interesse viene clonato dietro una cellula-specifico promotore. Usiamo un elemento cis-regolamentazione del gene pesce zebra neurogenin1 (-3.1ngn1) 2 o il SOX10 gene (-4.9sox10) 3 a guidare l&#39;espressione nei neuroni sensoriali o cellule della cresta neurale, rispettivamente. Per visualizzare la dinamica della membrana cellulare e, esprimiamo citoplasmatica o di membrana localizzate fluorofori. A immagine di F-actina distribuzione, esprimiamo il DNA che codifica per il dominio di omologia calponina utrophin fusa mCherry (UtrCH-mCherry). Il UtrCH-mCherry sonda biosensore consente la visualizzazione di F-actina, senza interferire con l&#39;actina dinamiche 1. Noi generare questi costrutti del DNA utilizzando la Gateway Invitrogen ricombinazione strategia clonazione 4 e vettori forniti nel Tol2kit zebrafish 5. <ol><li> Genera vettore di espressione (s) utilizzando Multisite-Gateway Tecnologia e vettori da Tol2kit zebrafish 5. Plasmidi contengono la spina dorsale Tol2 da pT2KXIGDin. </li><li> Purificare DNA plasmidico con plasmidi QIAfilter Midi Prep Kit (Qiagen Inc.) ed eluire in acqua sterile. DNA non ha bisogno di essere linearizzata per l&#39;iniezione. </li></ol> 3. Iniezione di costrutti di DNA <ol><li> Diluire il DNA ad una concentrazione finale di 10-50 mg / ml di DNA in rosso fenolo 0,2% (per la visualizzazione durante l&#39;iniezione) in acqua. </li><li> Compilare e calibrare ago: Back-riempiono un ago tirato capillare con circa 1 soluzione di DNA microlitri e inserirla nel supporto ago della apparati di iniezione (usiamo un Picospritzer). Usando una pinza sottile, rompere la punta fuori l&#39;ago in modo che l&#39;apertura punta è di circa 10 mm di diametro. Misurare il diametro della goccia in olio minerale con un micrometro oculare. Regolare la durata regolazione della pressione per ottenere una goccia di circa 1 volume nl. </li><li> Raccogliere 1-cellula embrioni in fase embrionale medio E3 (5mm NaCl, KCl 0,17 mm, 0,33 mm di CaCl 2, 0,33 mm di MgSO 4 * 7H 2 O, pH 7,0). </li><li> Trasferire circa 30-60 embrioni alla diapositiva di iniezione e la posizione degli embrioni in un angolo ad angolo retto formano tra la parte superiore e scivoli in basso. Eliminare la maggior parte E3 in modo che gli embrioni sono tenuti alla diapositiva dalla tensione superficiale. </li><li> Utilizzare un perno piegato dissezione di ruotare gli embrioni in modo che il cellulare è contro il muro della diapositiva iniezione. </li><li> Utilizzare un micromanipolatore per guidare l&#39;ago attraverso il corion embrionale, attraverso il tuorlo e nella cella singola dell&#39;embrione. Iniettare 0,5-1 nl soluzione di DNA all&#39;interno della cellula. </li><li> Trasferire gli embrioni in piastre di Petri in E3 e incubare a 28.5 ° C. Se necessario, lo sviluppo può essere rallentato da incubando gli embrioni a bassa temperatura. </li></ol> 4. Embrioni di montaggio per l&#39;imaging <ol><li> Rimuovere il chorions dagli embrioni con una pinza sottile circa un&#39;ora prima di embrioni raggiungono l&#39;età desiderata per l&#39;imaging (circa 14-17 ore dopo la fecondazione per i nostri scopi). </li><li> Selezionare gli embrioni con cellule marcate utilizzando un microscopio da dissezione dotato di fluorescenza. Noi utilizziamo un obiettivo 4x (ingrandimento 40X) su una portata Nikon AZ100 grazie alla sua combinazione di lunga distanza di lavoro e ad alto ingrandimento. </li><li> Inserire un embrione in una provetta per microcentrifuga con circa 50 microlitri E3 contenente 0,2% Tricaine anestetico (concentrazione 10X). </li><li> Aggiungere 500 microlitri 1% agarosio a basso punto di fusione nella E3 con 10 mM HEPES (mantenuta a 42 ° C), diluendo il Tricaine ad una concentrazione finale di 0,02%. </li><li> Trasferire immediatamente l&#39;embrione in una goccia di agarosio alcoprioggetto della diapositiva immagini utilizzando una pipetta di vetro ampio foro. </li><li> Prima della agarosio indurisce, la posizione l&#39;embrione così la regione con le cellule marcato è rivolto verso il basso contro il coprioggetto fondo (verso il basso dorsale per le cellule della cresta neurale e dorsolaterale giù per spinale i neuroni sensoriali). </li><li> Assemblare camera di imaging: Coat un lato di un anello di plastica (2 cm di diametro esterno, 3 mm di altezza) con grasso al silicone vuoto e apporre la diapositiva di imaging in metallo. Cappotto l&#39;altro lato del ring con grasso. </li><li> Riempire la camera con E3 HEPES contenente 10 mM e 0,02% Tricaine. Chiudere la parte superiore della camera apponendo a mm 22 2 coprioggetto alla superficie superiore unta dell&#39;anello. </li></ol> 5. Imaging 4D di comportamenti cella Olympus IX81 FV1000 con microscopio confocale I dettagli della acquisizione delle immagini dipende dalle specifiche del sistema microscopio. Qui si descrive il time-lapse processo di acquisizione per il microscopio confocale FV1000 Olympus. Vedi precedente protocollo di Giove per time-lapse acquisizione con la Zeiss LSM 510 sistema confocale 6. <ol><li> Posizionare la camera di imaging nel supporto diapositive sul palco microscopio e mettere a fuoco l&#39;embrione utilizzando un obiettivo 10X o 20X con luce trasmessa. </li><li> Passare a un obiettivo 60X (NA di 1,2 o superiore) e concentrarsi sulla cella etichettata con epifluorescenza. Noi utilizzare un olio di immersione (UPLSAPO 60xO NA 1,35) o immersione in acqua (UPLSAPO 60xW NA 1,20) lente. </li><li> Nel software FV (Ver. 1.7c), fare clic su XY ripetere per avviare la scansione laser. </li><li> Regolare le impostazioni di acquisizione e controllo di acquisizione delle immagini (uscita laser, velocità di scansione, HV, guadagno, e livelli di offset) per ottimizzare il segnale mantenendo potenza del laser al minimo (25% o inferiore) per evitare danni ai tessuti e ridurre photobleaching. </li><li> Di acquisire una serie Z, impostare i limiti superiore e inferiore della Z-Stack, insieme a passo. Assicurarsi che l&#39;icona della profondità è impegnato in modo che l&#39;icona Sweep XY XY e Z ha evidenziato. Avviare l&#39;acquisizione facendo clic sull&#39;icona Sweep XYZ. </li><li> Di acquisire una serie di time-lapse, impostare l&#39;intervallo di tempo e numero di punti di tempo sotto la voce TimeScan nella finestra delle impostazioni di acquisizione. Clicca l&#39;icona Ora nella finestra di controllo Image Acquisition modo che l&#39;icona Sweep XY ha XY (Z) e T evidenziato. Avviare l&#39;acquisizione cliccando il (Z) XY icona Sweep T. </li><li> In alternativa, il TimeController può essere usata per impostare un time-lapse. Questa programmazione è utile quando si generano grandi insiemi di dati, in quanto non supera l&#39;allocazione di memoria. In dispositivo, aprire la finestra TimeController. Creare un nuovo Task Imaging. La finestra dei parametri immagine si aprirà. Fare clic sul pulsante Stato FV per aggiornare le impostazioni di acquisizione. Assicurarsi Salva ed Elimina sono controllati sotto la voce Termina. Impostare il nome del file di output e fare clic su OK per salvare le impostazioni e chiudere la finestra. Utilizzare la funzione Loop per impostare l&#39;intervallo di tempo desiderato e il numero di punti di tempo. Clicca Ready e Start per iniziare l&#39;acquisizione. La pausa e Z-shift funzioni possono essere utilizzate per riorientare mentre imaging. </li></ol> 6. Rappresentante Risultati Le figure ed i film rappresentano esempi di neuroni sensoriali e cellule della cresta neurale etichettati con membrana mirati fluorofori o la sonda biosensore actina. <img src="/files/ftp_upload/1726/1726fig1.jpg" alt="Figura 1" /> Figura 1 immagini confocale (z-proiezioni) di un Tg (ngn1: GFP-CAAX). Embrione transgenico iniettato con 25 pg ngn1: mCherry-CAAX DNA. Il mCherry-CAAX etichette un neurone rosso (A) in uno sfondo con tutte le ROHON-Beard neuroni sensoriali etichettati verde (B). C) immagine a video dei canali sia verde e rosso. Barra di scala = 50 micron. <img src="/files/ftp_upload/1726/1726fig2.jpg" alt="Figura 2" /> Figura 2 confocale z-proiezioni di neuroni negli embrioni iniettati con ~ 20 pg ngn1:.. MCherry-UtrCH DNA A) ROHON-Beard neuroni sensoriali in 16,5 embrione HPF. F-actina segnale è forte in coni di crescita (punte di freccia) e sporgenze lungo gli assoni di recente formazione. B) Neuron in 20 embrione hpf mostrando forti segnali di F-actina in coni di crescita di ramificati assoni periferici (punte di freccia) e più debole segnale di centrale più matura assoni (frecce). Barra di scala = 50 micron.

<ol>
	<li>Burkel, B. M., von Dassow, G., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+fluorescent+probes+for+actin+filaments+based+on+the+actin-binding+domain+of+utrophin.">Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin.</a> Cell motility and the cytoskeleton. 64, 822-832 (2007).</li><li>Blader, P., Plessy, C., Strahle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+regulatory+elements+with+spatially+and+temporally+distinct+activities+control+neurogenin1+expression+in+primary+neurons+of+the+zebrafish+embryo.">Multiple regulatory elements with spatially and temporally distinct activities control neurogenin1 expression in primary neurons of the zebrafish embryo.</a> Mech Dev. 120, 211-218 (2003).</li><li>Wada, N., Javidan, Y., Nelson, S., Carney, T. J., Kelsh, R. N., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+is+required+for+cranial+neural+crest+morphogenesis+and+chondrogenesis+at+the+midline+in+the+zebrafish+skull.">Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.</a> Development. 132, 3977-3988 (2005).</li><li>Cheo, D. L., Titus, S. A., Byrd, D. R., Hartley, J. L., Temple, G. F., Brasch, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerted+assembly+and+cloning+of+multiple+DNA+segments+using+in+vitro+site-specific+recombination:+functional+analysis+of+multi-segment+expression+clones.">Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones.</a> Genome Res. 14, 2111-2120 (2004).</li><li>Kwan, K. M., Fujimoto, E., Grabher, C., Mangum, B. D., Hardy, M. E., Campbell, D. S., Parant, J. M., Yost, H. J., Kanki, J. P., Chien, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev Dyn. 236, 3088-3099 (2007).</li><li>O'Brien, G. S., Rieger, S., Martin, S. M., Cavanaugh, A. M., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J Vis Exp. 24, (2009).</li><li>Berndt, J. D., Clay, M. R., Langenberg, T., Halloran, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-kinase+and+myosin+II+affect+dynamic+neural+crest+cell+behaviors+during+epithelial+to+mesenchymal+transition+in+vivo.">Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo.</a> Dev Biol. 324, 236-244 (2008).</li></ol>

La concentrazione ottimale di DNA iniettato può variare a seconda delle dimensioni e la forza del promotore costruire e devono essere determinati empiricamente. L&#39;iniezione di DNA troppo può portare a embrioni non sani con la morte delle cellule esteso, mentre troppo poco si tradurrà in una piccola percentuale di embrioni iniettati esprimere il transgene. Il livello di espressione del DNA è correlata con la potenza del segnale fluoroforo, che varia da cellula a cellula. Mentre l&#39;ordinamento degli embrioni so...

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<td>Tricaine (Ethyl 3-aminobenzoate methanesulfonate)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5040-250G</td>
<td>&nbsp;</td>
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<td>Sylgard Silicone Elastomer Kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corporation</td>
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<td>QIAfilter Plasmid Midi Kit</td>
<td>&nbsp;</td>
<td>Qiagen Inc.</td>
<td>12243</td>
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<td>Low melting point agarose</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15517-014</td>
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<td>Picospritzer</td>
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live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos

Il pesce zebra è un modello ideale per i comportamenti delle cellule durante lo sviluppo di imaging in vivo. Embrioni di zebrafish sono fecondati esternamente e quindi facilmente accessibile in tutte le fasi di sviluppo. Inoltre, la loro chiarezza ottica permette l&#39;imaging ad alta risoluzione del cellulare e dinamica molecolare in ambiente naturale dell&#39;embrione intatto. Stiamo utilizzando un approccio dal vivo imaging per analizzare i comportamenti delle cellule durante la migrazione delle cellule neurali cresta e la conseguenza e la guida degli assoni neuronali. Immagini dal vivo è particolarmente utile per comprendere i meccanismi che regolano i processi di motilità cellulare. Per visualizzare i dettagli della motilità cellulare, come l&#39;attività protrusiva e dinamica molecolare, è vantaggioso per etichettare le singole celle. In zebrafish, iniezione di DNA plasmidico produce un transitorio pattern di espressione del mosaico e offre vantaggi distinti rispetto ad altri metodi di etichettatura delle cellule. Per esempio, linee transgeniche spesso etichetta popolazioni di cellule intere, e quindi possono oscurare la visualizzazione delle sporgenze fine (o cambiamenti nella distribuzione molecolare) in una singola cella. Inoltre, l&#39;iniezione di DNA alla cellula uno stadio è meno invasiva e più precisa di iniezioni di colorante nelle fasi successive. Qui si descrive un metodo per l&#39;etichettatura di singoli neuroni di sviluppo o cellule della cresta neurale e di imaging il loro comportamento in vivo. Iniettiamo DNA plasmidico in 1 cellula-embrioni fase, che si traduce in espressione del transgene mosaico. I vettori contengono cellule specifiche per i promotori che l&#39;espressione dell&#39;unità di un gene di interesse in un sottogruppo di neuroni sensoriali o cellule della cresta neurale. Forniamo esempi di cellule marcate con GFP membrana mirati o con una sonda biosensore che consente la visualizzazione di F-actina in cellule viventi 1. Erica Andersen, Namrata Asuri, e Matteo Argilla contribuito in maniera uguale a questo lavoro.

Watch this Scientific Journal Video about Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos at JoVE.com

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

Questo protocollo descrive l&#39;imaging di singoli neuroni o cellule della cresta neurale nel vivere embrioni di zebrafish. Questo metodo è utilizzato per esaminare i comportamenti e la localizzazione cellulare di actina mediante fluorescenza confocale microscopia time-lapse.

Immagini dal vivo della motilità cellulare e citoscheletro dei singoli neuroni e cellule della cresta neurale in embrioni di zebrafish

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

live-imaging-cell-motility-actin-cytoskeleton-individual-neurons

Research

JoVE Journal

Biology

13.3K Views.  University of Wisconsin-Madison. Questo protocollo descrive l'imaging di singoli neuroni o cellule della cresta neurale nel vivere embrioni di zebrafish. Questo metodo è utilizzato per esaminare i comportamenti e la localizzazione cellulare di actina mediante fluorescenza confocale microscopia time-lapse.

Video: Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Topo Epidermal cresta neurale delle cellule staminali (EPI-NCSC) Culture

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Ricerca

Biologia

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Due fotoni assotomia e time-lapse confocale in embrioni di zebrafish vivo

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Imaging cellulare dal vivo di F-actina Dynamics tramite microscopia a fluorescenza Speckle (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Due-Photon-Based fotoattivazione in Live embrioni di zebrafish

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Il trapianto di GFP che esprimono blastomeri per dell&#39;immagine in tempo reale dello sviluppo della retina e del cervello in embrioni di zebrafish Chimerico

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Live-cell imaging e analisi quantitativa della embrionali in cellule epiteliali Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

Studio del citoscheletro di actina in vivo le cellule endoteliali Esprimendo GFP-Actina

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

4D Imaging di aggregazione proteica in cellule vive

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Vivere Imaging di apoptotica delle cellule libera nel corso<em&gt; Drosophila</em&gt; Embriogenesi

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Profiling cellule staminali embrionali umane individuale mediante RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...