P.E. is supported by the Deutsche Forschungsgemeinschaft (DFG) Research Training Group 1373 and the Graduate School of the Technische Universit&#228;t M&#252;nchen (TUM-GS). G.P. was supported by TUM-GS. L.T. is supported by an EMBO fellowship (EMBO ALTF 108-2013). D.P.'s work on zebrafish was supported by the DFG through the Sonderforschungsbereich "Molecular Mechanisms of Neurodegeneration" (SFB 596); the Center for Integrated Protein Sciences (Munich) and the European Community's Seventh Framework Programme (FP7/2007-2013) under Grant agreement no. 200611 (MEMOSAD). He is currently a New York Stem Cell Foundation-Druckenmiller Fellow and was supported by a fellowship from the German Academy of Sciences Leopoldina. L.G. is supported by funding from the DFG through SFB 870 "Assembly and Function of Neuronal Circuits", Project A11. 
We are grateful to Kristina Wullimann for maintaining our fish facility, Yvonne Hufnagel for technical support and Thomas Misgeld for comments on the manuscript. We are grateful to R. K&#246;ster (Technische Universit&#228;t Braunschweig) for providing the M1 Medusa vector (pSKmemmRFP:5xUAS:H2B-CFP:5xUAS:Centrin2-YFP) from which we cloned out Centrin-YFP and S.C. Suzuki and T. Yoshimatsu (University of Washington) for providing the 14xUAS:MA-cerulean cassette which we used to generate the reporter construct to make CentrinFish. We further thank S.C. Suzuki and T. Yoshimatsu (University of Washington) for the Otx2:Gal4 transgenic line, A. Sagasti for the Sensory:Gal4-VP16 construct (UCLA) and M. Nonet (Washington University in St. Louis) for the pCold Heart Tol2 vector. We acknowledge Bettina Schmid, Alexander Hruscha and Christian Haass (German Center for Neurodegenerative Diseases Munich - DZNE) for contributing to the development of MitoFish.

The authors have nothing to disclose.

Qui, dimostriamo la versatilità del sistema di espressione GAL4-UAS ai mitocondri tag fluorescenza, centrosomi e le membrane cellulari del specifici tipi di cellule in vivo in embrioni di zebrafish. Molte proteine ​​di fusione fluorescenti che etichettano altri organelli o strutture subcellulari si possono trovare nella letteratura pubblicata e possono essere ottenuti dal rispettivo laboratorio, fonti commerciali o depositari plasmide non commerciali (ad esempio, Addgene). Per progettare una nuova...

Imaging in vivo fornisce la visualizzazione diretta dei comportamenti cellulari nel contesto più fisiologico. La trasparenza di embrioni di zebrafish, il loro sviluppo rapido ed esterna e una ricca gamma di strumenti genetici che consentono l&#39;etichettatura fluorescente hanno contribuito al crescente utilizzo della microscopia in vivo per chiarire la dinamica degli eventi chiave dello sviluppo. Studi di imaging di sviluppo del sistema nervoso in zebrafish hanno ad esempio notevolmente ampliato la nostra conoscenza del comportamento delle cellule progenitrici neurali e il destino della loro progenie compreso il loro successiva migrazione, differenziazione e integrazione del circuito 1-8. Il palcoscenico è ora impostata per indagare le dinamiche subcellulari alla base di tali comportamenti cellulari. Infatti, zebrafish sono già sfruttate come strumenti per in vivo biologia cellulare. Ora è possibile visualizzare i mitocondri 9-11, centrosomi 2,8,12-14, Golgi 15, Il citoscheletro microtubuli 4 e actina 16, endosomi 17 e componenti della membrana apicale complesso 1,18, tra le altre strutture subcellulari in embrioni di zebrafish in vivo. Finora, gran parte di ciò che si conosce la funzione di questi organelli viene da studiare il loro comportamento in cellule in coltura. Mentre gli studi in vitro hanno dato un enorme comprensione della biologia delle cellule, cellule in coltura non rappresentano appieno la complessità della situazione in vivo e quindi non riflettono necessariamente la funzione e le dinamiche di organelli subcellulari in vivo. Embrioni di zebrafish offrono una valida alternativa in vivo per esaminare le dinamiche subcellulari. Come vertebrati, zebrafish possiede molti organi (ad esempio, retina neurale) che sono omologhi a quelli trovati in specie di mammiferi. Inoltre, gli embrioni di zebrafish sono sempre più utilizzati per modellare malattie umane 19,20 </sup&gt;, compresi quelli relativi alla funzione centrosomica (ad esempio, microcefalia 21 e amaurosi 22 congenita di Leber) e per la funzione mitocondriale (ad esempio, il morbo di Parkinson 23, tauopatie 10,24 e sindrome di Barth 25). In vivo l&#39;imaging a livello cellulare e subcellulare in questi casi consenta una migliore comprensione della biologia cellulare alla base di questi stati patologici. L&#39;obiettivo generale dei metodi descritti qui è quello di fornire una guida completa per indagare organelli e altre strutture subcellulari in embrioni di zebrafish utilizzando in microscopia ottica vivo. L&#39;intero flusso di lavoro coinvolti nella visualizzazione e il monitoraggio delle strutture subcellulari in vivo è descritto - da approcci genetici di etichettatura, a generare transitoriamente esprimere e pesce transgenico stabile, e, infine, per l&#39;imaging con grande campo e microscopia confocale. Mentre ciascuno di questi procedures è utilizzato da numerosi laboratori di zebrafish, i protocolli descritti sono ottimizzati e semplificato per indagare le dinamiche di strutture subcellulari. Due aspetti specifici del lavoro descritto qui meritano menzione: In primo luogo, l&#39;uso del sistema di espressione GAL4-UAS in più configurazioni per geneticamente organelli etichette in specifici tipi di cellule. In secondo luogo, un confronto diretto di tutto il campo e microscopia confocale a strutture subcellulari di immagini in vivo. Le attuali strategie geneticamente organelli etichette e altre strutture subcellulari in zebrafish o fare uso di capped mRNA 1,4,8 o costrutti di DNA a base dove elementi promotori di auto direttamente l&#39;espressione di proteine ​​di fusione 9,14,15. Trascritto in vitro ricoperto risultati di RNA a rapida e ampia espressione, che non è tessuto-specifica però. Inoltre, i livelli di espressione diminuiscono nel tempo come RNA capped viene diluito o degradato. Pertanto, l&#39;uso di RNA basatocostruisce per esaminare le dinamiche organelli in fasi successive di sviluppo è limitata (in genere fino a 3 giorni dopo la fecondazione). Queste limitazioni possono essere superate usando costrutti di DNA, dove il controllo spaziale e temporale di espressione è determinato da elementi promotori specifici. Quando costrutti di DNA a base sono utilizzati nel contesto del sistema GAL4-UAS significativi miglioramenti a livello di espressione del transgene si osservano 26,27. In questo sistema di espressione bilaterale, di tipo a cella specifici elementi promotori guidano l&#39;espressione di un attivatore trascrizionale GAL4, mentre geni reporter vengono clonati a valle della sequenza attivante a monte GAL4-binding (UAS). Combinando UAS giornalisti con opportuni driver GAL4, espressione può essere limitata a specifici tipi di cellule, eludendo la necessità di clonare geni reporter dietro vari promotori ogni volta che un pattern di espressione specifici si desidera. Inoltre, l&#39;espressione di geni multipli UAS giornalista può essereguidato da un unico attivatore GAL4. Il sistema GAL4-UAS fornisce quindi un approccio genetico versatile e flessibile per l&#39;etichettatura subcellulare. Wide-field e microscopi confocale sono i cavalli di lavoro della maggior parte dei laboratori. sistemi a grande campo tipicamente utilizzano una lampada ad arco come sorgente luminosa e rilevare la luce emessa con una camera sensibile che è posto al termine del percorso della luce. Questa modalità di imaging è in genere limitata a campioni sottili come fuori-di luce fuoco oscura informazioni di messa a fuoco in campioni più spessi. Microscopi confocali differiscono dai sistemi largo campo dal fatto che essi sono costruiti per favorire segnali provenienti dal piano focale rispetto a quelli che hanno origine fuori fuoco (cioè, &quot;sezionamento ottico&quot;) 28. Per ottenere sezionamento ottico un foro è inserito nel percorso di emissione in grado coniugato alla sorgente di luce puntiforme. I laser sono utilizzati come sorgenti di luce ed i segnali vengono rilevati con tubi fotomoltiplicatori (PMT). In pratica, un laserfascio viene strisciato sul campione punto per punto e l&#39;emissione di fluorescenza ad ogni punto (pixel) viene rilevata dal PMT. Qui immagine le stesse strutture subcellulari in embrioni di zebrafish vivere utilizzando sia a livello di campo e di microscopia confocale per fornire un confronto diretto di entrambe le modalità di microscopia. L&#39;obiettivo di fondo di fornire tali confronti è quello di offrire le linee guida per la scelta della tecnica di microscopia più appropriata per la questione specifica a portata di mano. Utilizzando gli approcci descritti qui dimostriamo GAL4-UAS basato etichettatura genetica dei mitocondri e dei centrosomi. Questi organelli vengono esposte in diversi tipi di cellule del sistema nervoso e in cellule muscolari con largo campo e microscopia confocale a dimostrare l&#39;idoneità di ogni modalità di imaging. I metodi descritti qui possono essere facilmente adattate per indagare altri organelli e strutture subcellulari nell&#39;embrione zebrafish vivente.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose (2-hydroxyethylagarose)</td>
			<td>Sigma-Aldrich</td>
			<td>A4018-10G</td>
			<td>Low-gelling temperature Type VII</td>
		</tr>
		<tr>
			<td>Block heater</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Thermomixer compact</td>
		</tr>
		<tr>
			<td>Ca(NO3)2 Calcium nitrate hydrate, 99.996%&#160;</td>
			<td>Aldrich</td>
			<td>202967-50g</td>
			<td>To prepare 30x Danieau&#39;s</td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Qimaging</td>
			<td>Retiga Exi Fast 1394</td>
			<td></td>
		</tr>
		<tr>
			<td>Ceramic Coated Dumont #5 Forceps</td>
			<td>Dumont - Fine Science Tools</td>
			<td>11252-50</td>
			<td>#5 Forceps</td>
		</tr>
		<tr>
			<td>Confocal laser-scanning microscope</td>
			<td>Olympus</td>
			<td>FV1000 Fluoview</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture dish heater</td>
			<td>Warner Instrument Corporation</td>
			<td>DH-35</td>
			<td>Heating ring</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfate salt</td>
			<td>Fluka Analytical</td>
			<td>A5040-100G</td>
			<td>Tricaine (anesthetic)</td>
		</tr>
		<tr>
			<td>Fluorescence dissecting microscope</td>
			<td>Leica</td>
			<td>M205 FA</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneClean kit</td>
			<td>MP Biomedicals</td>
			<td>111001200</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Culture Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-14-C</td>
			<td>35mm petri dish, 14mm microwell, No. 0 coverglass</td>
		</tr>
		<tr>
			<td>Glass needles</td>
			<td>World Precision Instruments Inc.&#160;</td>
			<td>TW100F-4</td>
			<td>For microinjections</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H3375-250g</td>
			<td>To prepare 30x Danieau&#39;s</td>
		</tr>
		<tr>
			<td>High vacuum grease</td>
			<td>Dow Corning&#160;</td>
			<td>DCC000001242 150g</td>
			<td>Silicon dioxide grease</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Scientific</td>
			<td>Heraeus</td>
			<td>To maintain zebrafish embryos at 28.5&#8304; C</td>
		</tr>
		<tr>
			<td>KCl 99%</td>
			<td>Sigma-Aldrich</td>
			<td>S7643-5kg</td>
			<td>To prepare 30x Danieau&#39;s</td>
		</tr>
		<tr>
			<td>MgSO4.7H2O &#160;&#160;Magnesium sulfate heptahydrate 98+% A.C.S reagent</td>
			<td>Sigma-Aldrich</td>
			<td>230291-500g</td>
			<td>To prepare 30x Danieau&#39;s</td>
		</tr>
		<tr>
			<td>Microinjector&#160;</td>
			<td>Eppendorf</td>
			<td></td>
			<td>FemtoJet</td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td>0.5-20uL</td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Maerzhaeuser Wetzlar</td>
			<td>MM33 Rechts/00-42-101-0000/M3301R</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette holder</td>
			<td>Intracel</td>
			<td>P/N 50-00XX-130-1</td>
			<td></td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE SP6 Transcription Kit</td>
			<td>Ambion</td>
			<td>AM1340</td>
			<td>To transcribe PCS-Transposase</td>
		</tr>
		<tr>
			<td>NaCl BioXtra &#62;99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-1kg</td>
			<td>To prepare 30x Danieau&#39;s</td>
		</tr>
		<tr>
			<td>Nanophotometer</td>
			<td></td>
			<td></td>
			<td>To measure DNA/RNA concentration</td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>Flaming/Brown</td>
		</tr>
		<tr>
			<td>NIR Apo 40x/0.80W&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td>Water-dipping-cone objective</td>
		</tr>
		<tr>
			<td>N-Phenylthiourea Grade I, approx. 98%</td>
			<td>Sigma</td>
			<td>P7629-10G</td>
			<td>PTU (prevents pigmentation)</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Sarstedt AG&#160;</td>
			<td>821472</td>
			<td>92 x 16mm&#160;</td>
		</tr>
		<tr>
			<td>Plastic molds&#160;</td>
			<td>Adaptive Science Tools</td>
			<td>TU-1</td>
			<td>For microinjections</td>
		</tr>
		<tr>
			<td>Plexiglas cover-with a hole</td>
			<td>Custom-made</td>
			<td></td>
			<td>The hole in the Plexiglas cover should be 3 mm larger than the diameter of the water-dipping-cone objective</td>
		</tr>
		<tr>
			<td>Tea-strainer (Plastic)</td>
			<td></td>
			<td></td>
			<td>To collect zebrafish eggs</td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Warner Instrument Corporation</td>
			<td>TC-344B</td>
			<td>Dual Automatic Temperature Controller</td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Sarstedt AG&#160;</td>
			<td>86.1171</td>
			<td>3.5mL plastic transfer pipettes</td>
		</tr>
		<tr>
			<td>UMPlanFI 100x/1.00W</td>
			<td>Olympus</td>
			<td></td>
			<td>Water-dipping-cone objective</td>
		</tr>
		<tr>
			<td>UMPlanFLN 20x/0.50W&#160;</td>
			<td>Olympus</td>
			<td></td>
			<td>Water-dipping-cone objective</td>
		</tr>
		<tr>
			<td>Widefield microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>PTU (50x Stock)</td>
			<td></td>
			<td></td>
			<td>Dissolve 76 mg PTU in 50 ml distilled water 
			Stir vigorously at room temperature&#160; 
			Store at -20 oC in 1 ml aliquots 
			Use at 1x working solution</td>
		</tr>
		<tr>
			<td>Tricaine (20x Stock)</td>
			<td></td>
			<td></td>
			<td>Dissolve 200 mg Tricaine in 48 ml distilled water 
			Add 2 ml 1M Tris base (pH9) 
			Adjust to pH 7&#160; 
			Store at -20 oC in 1 ml aliquots 
			Use at 1x working solution</td>
		</tr>
		<tr>
			<td>Danieau&#39;s Solution (30x Stock)</td>
			<td></td>
			<td></td>
			<td>1740 mM&#160; NaCl&#160; 
			&#60;21 mM&#160;&#160;&#160;&#160;&#160; KCl&#160; 
			12 mM&#160; &#160;&#160;&#160;&#160;MgSO4.7H2O&#160; 
			18 mM&#160;&#160;&#160;&#160;&#160; Ca (NO3)2 
			150 mM&#160;&#160;&#160; HEPES buffer&#160; 
			Distilled water upto 1 L&#160; 
			Store at 4 oC&#160; 
			Use at 0.3x working solution</td>
		</tr>
	</tbody>
</table>

imaging subcellular structures in the living zebrafish embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Qui l&#39;uso di tutto il campo e microscopia confocale a mitocondri immagini e centrosomi in embrioni di zebrafish vivente è direttamente confrontato e contrapposto. A seconda della posizione delle cellule in cui dinamiche organelli devono essere esaminate e la frequenza intrinseca degli specifici eventi subcellulari, generalmente o grande campo o la microscopia confocale è la scelta migliore. Abbiamo ripreso organelli nei neuroni RB situati sulla superficie dell&#39;embrione e in cel...

Watch this Scientific Journal Video about Imaging Subcellular Structures in the Living Zebrafish Embryo at JoVE.com

Imaging Subcellular Structures in the Living Zebrafish Embryo

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

Imaging strutture subcellulari nell&#39;embrione Living Zebrafish

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

imaging-subcellular-structures-in-the-living-zebrafish-embryo

Research

JoVE Journal

Developmental Biology

11.5K Views.  Technische Universität München. Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy. 

Video: Imaging Subcellular Structures in the Living Zebrafish Embryo

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Misurare Protein Stabilità in Living embrioni di zebrafish Usando fluorescenza Decay Dopo Fotoconversione (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Ricerca

Biologia dello sviluppo

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Microperla impianto nel zebrafish

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Analisi delle larve Zebrafish muscolo scheletrico Integrità con Blu di Evans Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Monitoraggio celle in Zebrafish GFP-transgenico Utilizzo del sistema fotoconvertibile PSmOrange

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

Osservando divisione mitotica e Dynamics in un embrione vivo Zebrafish

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Induzione di ipossia in rana vivente e embryos zebrafish

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

Uso di Immunolabeling per analizzare i microtubuli stabili, dinamici e nascenti nell'embrione di pesce zebra

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

AFM e Microrheology nella cella tuorlo embrione di pesce zebra

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Cella singola fotoconversione in Zebrafish intatto vivente

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

Seguendo i movimenti del tessuto Endocardial tramite cella fotoconversione nell'embrione di pesce zebra

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...