We thank O. Subach for providing the original H2B-PSmOrange plasmid and our fish facility team for fish care. We are grateful to the Nikon Imaging Center at the University of Heidelberg for access to microscopy equipment and analysis software. We acknowledge the support of the Core Facility Live Cell Imaging Mannheim at the CBTM (DFG INST 91027/10-1 FUGG). This work was supported by the Excellenzcluster CellNetworks, EcTop Spatio-temporal coordination of signaling processes (EcTop 2), University of Heidelberg to C.A.B. and the Medical Faculty Mannheim of the University Heidelberg and the DFG (FOR 1036/2, 298/3-1 and 298/6-1) to M.C.

1. H2B-PSmOrange mRNA in vitro-Transkription und mRNA Purification <ol><li> Linearisieren die H2B-PSmOrange enthält pCS2 + Plasmid, das die NotI-Restriktionsenzym unter Verwendung von nach den Anweisungen des Herstellers. Hinweis: Führen Sie die nächsten Schritte mit geeigneten Schutzmaßnahmen wie Handschuhe und Kittel mRNA Verschmutzung und Verschlechterung zu verhindern. </li><li> Reinige den linearisierten DNA einer PCR Purification Kit oder Phenol-Chloroform-basierte Verfahren nach Herstellerprotokollen. </li><li> Verwenden 1 ug linearisierte Matrizen-DNA für Sp6-mRNA-Transkription nach Herstellerangaben. </li><li> Entfernen DNA durch Zugabe von 1,0 U RNase freie DNaseI für 10-20 min bei 37 ° C. </li><li> Clean up the mRNA eine RNA bereinigen Kit gemäß dem Protokoll des Herstellers. </li><li> Zur Erhöhung der mRNA Reinheit und Konzentration, fallen die mRNA durch Zugabe von 6 ul Natriumacetat (3,0 M, pH 5,2) und 150 &amp; mgr; l 96% Ethanol. Inkubieren Sie die mixed Lösung bei -20 ° C für mindestens 30 min und bis zu 24 Stunden. </li><li> Sammeln Sie die mRNA, indem die Lösung bei 18,407 xg Zentrifugieren für 45 min bei 4 ° C. Entsorgen Sie die Flüssigkeit und resuspendieren die mRNA in 150 ul 70% Ethanol. Mischen und Zentrifugieren der Lösung für weitere 15 min bei 4 ° C. Entsorgen Sie die Flüssigkeit, trocknet das Pellet für 5 Minuten auf Eis und resuspendieren die mRNA in 20 ul ultrareinem RNase freies Wasser. </li><li> Bewerten Sie die Konzentration und Reinheit der mRNA durch photometrische Messung einer 1: 100-mRNA-Verdünnung (1 ul mRNA in 99 &amp; mgr; l ultrareinem RNase freies Wasser). </li><li> Für kurzzeitige Lagerung, halten Sie die mRNA-Lösung bei -20 ° C. Für die langfristige Lagerung, halten Sie die mRNA bei -80 ° C. </li></ol> 2. H2B-PSmOrange mRNA Mikroinjektion in Zebrafischembryonen <ol><li> Richten Sie Paare in der Nacht vor der Injektion Paarung mit tg (foxD3: GFP); tg (FLH: GFP) doppelt transgenen Fisch (oder jede andere GFP transgenen Fische). Lassen Sie den Fisch getrennt durch einen Abstandshalter platzierenzwischen ihnen die Zeit der Paarung zu steuern. </li><li> Entfernen Sie die Abstandshalter am Morgen der Einspritzung und lassen die Fische Kumpel für 20 min. </li><li> Während der Zeit der Paarung, verdünnte die H2B-PSmOrange mRNA zur endgültigen Konzentration von 130 pg / nl (in RNase freies Wasser) und Transfer 6 ul Injektionslösung in einem Mikroinjektion Kapillare eine Micro Trick. Schauen Sie sich die Einspritzmenge mit einem kalibrierten Glasträger. Stellen Sie den Einspritzdruck zu injizieren 2 nl mRNA-Lösung (260 pg). </li><li> Sammeln Sie die Eier in eine sterilisierte Kunststoffpetrischale mit 1x Embryo (E3) Medium. </li><li> Übertragen Sie 20 bis 30 Embryonen zu einem Einspritzschale mit einem Kunststoffpasteurpipette. </li><li> Injizieren 2 nl mRNA Lösung in der Zelle enthalten oder direkt unter der Zelle in das Eigelb an der Ein-Zell-Stadium 11. </li><li> Transfer injizierten Embryonen in eine Petrischale 1x E3-Medium, das Entfernen unbefruchtete oder nicht normal Embryonen und wachsen sie bei 28 ° C zu entwickeln. Hinweis: Lichtschutz of den Embryonen nicht während des ganzen Experiments erforderlich. </li><li> Heben Zebrafischembryonen in 1x E3 Medium und fügen 0,2 mM PTU (1-Phenyl-2 Thioharnstoff) nach gastrulation Pigmentierung zu hemmen. Ändern Sie das Medium zweimal pro Tag, um die Gefahr von bakteriellen Kontaminationen zu minimieren. </li></ol> 3. Embryo Einbetten <ol><li> Wählen Sie das GFP (grün) und PSmOrange (orange / rot) co-exprimierenden Embryonen ein Binokular mit einem Fluoreszenz-Lampe und angemessene Emissionsfiltern. Übertragen Sie die positiven Embryonen in eine sterile Petrischale mit 1x E3 Medium. </li><li> Dechorionate Embryonen unter einem Stereomikroskop Pinzette 12. </li><li> Transfer eines Embryos in ein 1,5 ml Röhrchen mit 1,0 ml vorgewärmten 1,0% niedrig schmelzender Agarose (LMA) in ultrareinem Wasser hergestellt, indem ein Cut-Spitze Pipette (P200) verwendet. Hinweis: Wärmen Sie die LMA bis 80 ° C und Abkühlung für 3-5 Minuten bei Raumtemperatur, bevor der Embryo Einbettung. </li><li> Übertragen Sie die Embryos in 150 ul LMA in einemKammerdeckgläser und passen Sie die Ausrichtung des Embryos, zum Beispiel Rückenseite nach unten in das Experiment für den invertierten konfokalen Laser-Scanning-Mikroskop A1R + (oder einem anderen geeigneten Mikroskop) beschrieben, eine feine Spitze aus Kunststoff verwenden. Wenn die LMA polymerisiert wird, füllen Sie die Kammer mit Fisch Wasser, das 0,2 mM PTU und 0,02% Ethyl-3-aminobenzoat Methansulfonat (Tricaine) für die Anästhesie. </li><li> Bevor die Probe Bildgebung, warten Sie 15 Minuten, um die Wirkung von Tricaine zu ermitteln. Die Anästhesie verhindert Embryo Bewegungen, die ein Stereomikroskop mit Hellfeldbeleuchtung überwacht werden kann. Anmerkung: Die Kammern zweimal wiederverwendet werden kann. </li></ol> 4. PSmOrange Photokonversion <ol><li> Legen Sie die Probe unter dem konfokalen Mikroskop und scannen die Probe um den Bereich zu identifizieren, um photoconvert mit 488 nm und 561 nm Laser zur Abbildung GFP und PSmOrange sind. Hinweis: Mit der umgekehrten konfokalen Laser-Scanning-Mikroskop A1R + auf dem umgekehrten stage TiEcontrolled durch das Mikroskop Imaging-Software und ausgestattet mit 488 nm, 561 nm und 640 nm Laser für die Photokonversion und Bildgebung. Jedoch ist die Anwendung sicher nicht auf diese beschränkt Mikroskop solange die Laserlinien zur Umwandlung und Bild verfügbar sind. <ol><li> Setzen Sie den 20X Luft Objektiv ein (NA: 0,75; WD: 1,0 mm; FOV: 0,64 x 0,64 mm). </li><li> Verwenden Sie die folgenden Einstellungen des Mikroskops die PSmOrange Protein vor Photokonversion zu erkennen: 561-nm-Laser, 0,74 mW bei der Fokusebene über dem Ziel gemessen. Hinweis: Diese auf diesem System bis zu 40% entspricht (in der Fokusebene messen, ist der einzige Weg, um die Wirkleistung zu bestimmen). Stellen Sie die Laserleistung nach experimentellen Anforderungen wie die Effizienz der H2B-PSmOrange Injektionen variieren. </li><li> In Zoomfaktoren den Bereich von Interesse zu markieren. Höhere Vergrößerungen reduzieren Bildaufnahmezeit und damit Phototoxizität. </li></ol></li><li> Erwerben Sie eine Z-Stack für den Aufbaus von Interesse unter Verwendung von 488 nm und 561 nm Laser im sequenziellen Modus (Zeilenmodus 1-&gt; 4). Befestigen Sie die z-Schritt zwischen 1,0 und 2,0 um. Rauh und Scan-Frequenz kann verwendet werden, um Bildqualität zu optimieren. </li><li> Scannen Sie die Probe und wählen Sie die Region of Interest (ROI) Werkzeug, um den Bereich zu markieren, um photoconvert. Befestigen Sie den ROI als Stimulationsbereich. Wählen Sie das Foto Aktivierung / Bleaching-Modul, aktivieren Sie die 488-nm-Laser durch das entsprechende Kontrollkästchen aktivieren und stellen Sie die 488-nm-Laser bis 80% (1 mW Laserleistung auf dem Ziel gemessen). Stellen Sie die Scan-Geschwindigkeit bis 0,5 sec / Frame. </li><li> Öffnen Sie die Photo Dienstprogramm (ND Stimulation) und die Photo Einstellungen angeben. <ol><li> Klicken Sie auf den Befehl &quot;Hinzufügen&quot; die Photo Protokoll anzugeben. </li><li> Stellen Sie &quot;Phase&quot; ein in der &quot;Erwerb / Stimulation&quot; -Menü auf &quot;Übernahme&quot; und geben Sie die Anzahl der Bilder vor Photo erworben werden in der &quot;Loops&quot; -Menü. </li><li> Stellen Sie &quot;Phase&quot; 2 auf &quot;St.imulation &quot;und die Anzahl der Stimulationsereignisse geben durchgeführt werden. </li><li> Stellen Sie &quot;Phase&quot; 3 wie für &quot;Phase&quot; 1. Optional beschrieben, legen Sie eine Warte &quot;Phase&quot; nach der &quot;Phase&quot; 2 durch die Zeit vor der letzten Runde der Bildaufnahme zu warten eingeben. </li><li> Sobald alle Parameter eingestellt werden, gelten die Stimulation Einstellung und die Photo laufen. Hinweis: Die Laserleistung, die Frequenz des Scannens und der Anzahl der Wiederholungen für jede Runde von Photo variieren und unterscheiden sich von Embryos Embryos. Eine Einstellung zu beginnen, ist in Tabelle 1 gezeigt. </li></ol></li><li> Erwerben Sie eine endgültige Z-Stapel mit 488 nm, 561 nm und 640 nm Laser, wie oben die Einstellungen zu übernehmen. Stellen Sie die 640-nm-Laser die konvertierte PSmOrange mit hoher Laserleistung (bis zu 4,5 mW) zu visualisieren. </li></ol> 5. Demontieren Embryo aus LMA <ol><li> Entfernen Sie den Embryo aus dem Mikroskop enthaltenden Kammer. Entfernen Sie vorsichtig den Embryo aus der Agarose usingen Zange. </li><li> Bringen Sie den Embryo in eine neue sterilisierte Kunststoffpetrischale oder in eine sterilisierte 6-Well-Platte mit 1x E3 und 0,2 mM PTU. Inkubieren des Embryos bei 28 ° C, bis die gewünschte Stufe der Entwicklung. </li></ol> 6. Die Analyse der Fate of photokonvertiertem PSmOrange Protein exprimierenden Zellen <ol><li> Re-einbetten den Embryo als 3.3 und 3.4 unter den Punkten beschrieben. </li><li> Legen Sie die Probe unter dem konfokalen Mikroskop und scannen die Probe zu photokonvertiertem Zellen (640 nm) in der GFP transgenen Struktur von Interesse (488 nm) zu identifizieren. </li><li> Erwerben Sie eine Z-Stapel Abdecken der Strukturen von Interesse unter Verwendung von 488 nm, 561 nm und 640 nm Laser im sequenziellen Modus (Zeilenmodus 1-&gt; 4). Befestigen Sie die z-Schritt zwischen 1,0 und 2,0 um. Rauh und Scan-Frequenz kann verwendet werden, um Bildqualität zu optimieren. </li><li> Verwenden Sie eine der folgenden Methoden, um Zellen zu identifizieren, die Co-express GFP und die photokonvertiertem Protein. <ol><li> Maß automatische Fidschi BildJ Makro 3 <ol><li> Convolve der Originalstapel die &quot;Weichzeichnen&quot; Plugin (→ Bearbeiten → Filter → Weichzeichnen) und subtrahieren die glatten Stapel von der ursprünglichen Verwendung der &quot;Bild Rechner&quot; Plugin (→ Prozess → Bildrechner) verwenden. Verwenden Sie diesen Schritt, um die Strukturen von Interesse zu visualisieren und zur Verringerung der Rechenzeit für die Analyse. </li><li> Übernehmen bestimmte Schwellenwerte zu den grünen und dunkelroten Kanäle, um die photokonvertiertem Zellen zu markieren (→ Bild → → Stellen Threshold). Verwenden Sie den &quot;Analyze Particles&quot; Werkzeug, um die Schwelle Bereiche mit einem passenden Rahmen zu erkennen (→ Analysieren → Analysieren Partikel). </li><li> Öffnen Sie die &quot;Bild Rechner&quot; Plugin und zeigen die überlappenden ROIs im grünen und dunkelroten Kanal in gelb (→ Prozess → Bildrechner). </li></ol></li><li> Microscope Imaging-Software für 3D-Datenauswertung <ol><li> Zeigen Sie den Stapel in3D die Show Volumen Ansichtsoption (→ 3D-Visualisierung Menü → Show Volume anzeigen) verwenden. Verwenden Sie die grafische Schnittstelle, um die grünen, roten und dunkelrotes Kanäle, die Helligkeit, den Kontrast und die Ernte der 3D-Stapel zu wählen Sie die photokonvertiertem Bereich (Abbildung 1) zu markieren. Hinweis: Ähnliche Verfahren werden die Daten sind in den meisten der gemeinsamen Software zur Bildanalyse zu analysieren. </li></ol></li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+intracellular+protein+movements+using+photoswitchable+fluorescent+proteins+PS-CFP2+and+Dendra2.">Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2.</a> Nature Protocols. 2, 2024-2032 (2007).</li><li>Subach, F. V., 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=Photoactivatable+mCherry+for+high-resolution+two-color+fluorescence+microscopy.">Photoactivatable mCherry for high-resolution two-color fluorescence microscopy.</a> Nature Methods. 6, 153-159 (2009).</li><li>Patterson, G. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), e1115 (2009).</li><li>Concha, M. L., 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=Local+tissue+interactions+across+the+dorsal+midline+of+the+forebrain+establish+CNS+laterality.">Local tissue interactions across the dorsal midline of the forebrain establish CNS laterality.</a> Neuron. 39, 423-438 (2003).</li><li>Auer, T. O., Duroure, K., Concordet, J. 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The authors have nothing to disclose.

Transgene Embryonen fluoreszierenden Reporter trägt grundlegend zu verstehen, die embryonale Entwicklung geholfen. Es besteht jedoch immer noch die wesentliche Notwendigkeit für Promotoren die spezifische Visualisierung von besonderen Strukturen zu erleichtern. In ihrer Abwesenheit, verlassen sich die Forscher auf Techniken wie die Photokonversion von fluoreszierenden Proteinen über den Ursprung und die Entwicklung ihrer Struktur von Interesse zu erfahren. Dies wiederum ist eine wesentliche Voraussetzung, die molekul...

Exponentially improving imaging techniques allow following developmental processes over time periods of up to about four consecutive days3. In zebrafish and many other animal model systems, specific cells, tissues, axonal or vascular structures are marked by transgenic green or sometimes red or yellow fluorescent proteins to facilitate visualization. However, in most transgenic lines the transgene is not specifically expressed in the cells of interest but also additional structures, which hinders the precise tracking of for instance single cells or groups of cells.
Fluorescent photoconvertible proteins are well suited for cell tracking during embryonic development. The prerequisites for the application of such proteins are a long-lived nature, a well-separated emission range upon conversion and bright fluorescence. Available photoconvertible proteins comprise those that change their emission range upon conversion such as Kaede4, KiGR5, mEos26, PS-CFP2 or Dendra27 and others which are only fluorescent when photoactivated (PAmCherry8, PAGFP9 or PATagRFP10). Their applications to track cells in existing FP-transgenic animals are however limited as they often involve a green fluorescent state or do not fulfill all of the above criteria. Only recently, Subach and colleagues reported the PSmOrange protein, which changes its emission from orange/red to far red upon photoconversion and was successfully applied in cells in culture and cultured cells injected into mice2.
To investigate the protein's suitability for cell tracking in a living embryo, we generated an expression construct for the microinjection of nuclear-tagged H2B-PSmOrange into zebrafish embryos. We find that the protein fulfills all prerequisites for successful cell tracking in GFP transgenic backgrounds during the first 4 (and possibly more) days of zebrafish embryonic development. During this time, most of the cell migratory events are completed in fish making the PSmOrange system an excellent addition to the zebrafish toolkit.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PCR Purification Kit</td>
			<td style="width:233px;">Qiagen</td>
			<td align="right" style="width:119px;">28104</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mMESSAGE mMACHINE SP6 Transcription kit</td>
			<td>Ambion</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy MiniElute Cleanup kit</td>
			<td>Qiagen</td>
			<td align="right">74204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic Pasteur</td>
			<td>alpha laboratories</td>
			<td>LW4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Original H2B-PSmOrange Plasmid</td>
			<td>Addgene</td>
			<td>31920</td>
			<td>The plasmid described in the paper is available in the Carl lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FemtoJet Microinjector</td>
			<td>Eppendorf</td>
			<td>5247 000.013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps (5 Inox)</td>
			<td>NeoLab</td>
			<td>2-1633</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab-Tek II Chambered #1.5 German Coverglass System&#160;</td>
			<td>Nunc</td>
			<td align="right">155382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon A1R+</td>
			<td>Nikon GmbH Germany</td>
			<td>No Number</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon PLAN Apo &#955; 20x air objective&#160;</td>
			<td>Nikon GmbH Germany</td>
			<td>No Number</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">NIS Elements AR Software (v. 4.30.02)</td>
			<td>Nikon GmbH Germany/Laboratory Imaging</td>
			<td>No Number</td>
			<td></td>
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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.

1 veranschaulicht ein Beispiel des PSmOrange Photosystem. Die Zirbeldrüse Komplex ist eine konservierte Struktur im Wirbeltier dorsalen Zwischenhirn. Wie in vielen anderen Wirbeltieren besteht dieser Komplex der Pinealorgan in der Mitte des Zwischenhirns und den linksseitigen parapineal Zellen. Elegant, aber zeitaufwendig Uncaging Experimente zeigten, dass parapineal Zellen 13 in den vorderen Teil des Pinealorgan stammen. In tg (foxD3: GFP); tg (FLH: GFP) tra...

Watch this Scientific Journal Video about Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System at JoVE.com

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

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.

Tracking-Zellen in GFP-transgenen Zebrafisch Mit dem Photokonvertierbare PSmOrange-System

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

tracking-cells-gfp-transgenic-zebrafish-using-photoconvertible

Research

JoVE Journal

Developmental Biology

8.5K Aufrufe.  Heidelberg University. 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.

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

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

Live-Imaging von angeborenen Immun und Präkanzeröse Zell-Interaktionen unter Verwendung eines induzierbaren Gal4 / UAS Expression System in Zebrafisch-Larven Haut

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Forschung

Entwicklungsbiologie

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

Die Nutzung der Teton-System, um molekulare Mechanismen bei Zebrafisch Regeneration Studieren

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

Alginat Encapsulation pluripotenter Stammzellen Mit einer Co-Axial Düsen

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

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.

Analyse von Zebrafisch-Larven Skelettmuskel Integrität mit 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 ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Die Herstellung von pluripotenten Stammzellen aus Maus Fruchtwasserzellen ein Transposon-System verwenden

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

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.

Einzellige Ladungszustand im lebendigen intakten Zebrafisch

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 ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Computergestützte Analyse der Keimbahn Caenorhabditis Elegans , die Verteilung der Kerne, Proteine und das Zytoskelett zu studieren

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In vitro-Generation der Somiten Derivate aus menschlichen induzierten Zellen pluripotenten Stammzellen

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Dreidimensionales Angiogenese-Assay-System mit Co-Culture-Spheroiden, die von endotheliaalen Koloniebildenden Zellen und mesenchymalen Stammzellen gebildet werden

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, proliferation, and differentiation. Time-lapse imagining offers unique insight to the behavior of cells during eye development because it allows us to visualize oculogenesis in vivo. Zebrafish are an excellent model to visualize this process due to their highly conserved vertebrate eye and their ability to develop rapidly and externally while remaining optically transparent. Time-lapse imaging studies of zebrafish eye development are greatly facilitated by use of the transgenic zebrafish line Tg(rx3:GFP). In the developing forebrain, rx3:GFP expression marks the cells of the single eye field, and GFP continues to be expressed as the eye field evaginates to form an optic vesicle, which then invaginates to form an optic cup. High resolution time lapse imaging of rx3:GFP expression, therefore, allows us to track the eye primordium through time as it develops into the retina. Lightsheet microscopy is an ideal method to image ocular morphogenesis over time due to its ability to penetrate thicker samples for fluorescent imaging, minimize photobleaching and phototoxicity, and image at a high speed. Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software. The resulting dataset can provide valuable insights into the process of ocular morphogenesis, as well as perturbations to this process as a result of genetic mutation, exposure to pharmacological agents, or other experimental manipulations.

Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software.&#160;

Visualisierung der Augenmorphogenese mittels Lightsheet-Mikroskopie mittels rx3:GFP Transgener Zebrafische

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, ...