Gli autori ringraziano Christa Haase, Doris Weber e Brigitte Korte per l&#39;assistenza tecnica. Il lavoro in laboratorio Weidinger è sostenuto da finanziamenti della Deutsche Forschungsgemeinschaft WE 4223 / 3-1, WE 4223 / 4-1 e dalla Deutsche Gesellschaft für Kardiologie tramite un Oskar-Lapp-Stipendium e Klaus-Georg-und-Sigrid- Hengstberger-Forschungsstipendium.

<ol>
	<li>Fang, Y., 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=Translational+profiling+of+cardiomyocytes+identifies+an+early+Jak1/Stat3+injury+response+required+for+zebrafish+heart+regeneration.">Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (33), 13416-13421 (2013).</li><li>Tryon, R. C., Johnson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+analysis+of+kit+ligand+a+functional+expression+reveals+lineage-specific+competence+to+promote+melanocyte+rescue+in+the+mutant+regenerating+caudal+fin.">Clonal analysis of kit ligand a functional expression reveals lineage-specific competence to promote melanocyte rescue in the mutant regenerating caudal fin.</a> PloS one. 9 (7), e102317 (2014).</li><li>Wehner, D., 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=Wnt/beta-catenin+signaling+defines+organizing+centers+that+orchestrate+growth+and+differentiation+of+the+regenerating+zebrafish+caudal+fin.">Wnt/beta-catenin signaling defines organizing centers that orchestrate growth and differentiation of the regenerating zebrafish caudal fin.</a> Cell Rep. 6 (3), 467-481 (2014).</li><li>Huang, C. J., 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=Conditional+expression+of+a+myocardium-specific+transgene+in+zebrafish+transgenic+lines.">Conditional expression of a myocardium-specific transgene in zebrafish transgenic lines.</a> Dev Dyn. 233 (4), 1294-1303 (2005).</li><li>Knopf, F., 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=Dually+inducible+TetON+systems+for+tissue-specific+conditional+gene+expression+in+zebrafish.">Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (46), 19933-19938 (2010).</li><li>Provost, E., Rhee, J., Leach, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+2A+peptides+allow+expression+of+multiple+proteins+from+a+single+ORF+in+transgenic+zebrafish+embryos.">Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos.</a> Genesis. 45 (10), 625-629 (2007).</li><li>Suster, M. L., Abe, G., Schouw, A., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transposon-mediated+BAC+transgenesis+in+zebrafish.">Transposon-mediated BAC transgenesis in zebrafish.</a> Nat Protoc. 6 (12), 1998-2021 (2011).</li><li>Bussmann, J., Schulte-Merker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+BAC+selection+for+tol2-mediated+transgenesis+in+zebrafish.">Rapid BAC selection for tol2-mediated transgenesis in zebrafish.</a> Development. 138 (19), 4327-4332 (2011).</li><li>Yuan, S., Sun, Z. <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+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. (27), (2009).</li><li>Kim, J. H., 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=High+cleavage+efficiency+of+a+2A+peptide+derived+from+porcine+teschovirus-1+in+human+cell+lines,+zebrafish+and+mice.">High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.</a> PloS one. 6 (4), e18556 (2011).</li><li>Matthews, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anesthesia+and+euthanasia+in+zebrafish.">Anesthesia and euthanasia in zebrafish.</a> ILAR journal / National Research Council, Institute of Laboratory Animal Resources. 53 (2), 192-204 (2012).</li><li>Hyde, D. R., Godwin, A. R., Thummel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+of+morpholinos+into+the+regenerating+adult+zebrafish+tail+fin.">In vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin.</a> J Vis Exp. (61), (2012).</li><li>Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+isolation+of+PCR-ready+genomic+DNA+from+zebrafish+tissues.">Method for isolation of PCR-ready genomic DNA from zebrafish tissues.</a> Biotechniques. 43 (5), 610 (2007).</li><li>Thermes, 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=I-SceI+meganuclease+mediates+highly+efficient+transgenesis+in+fish.">I-SceI meganuclease mediates highly efficient transgenesis in fish.</a> Mech Dev. 118 (1-2), 91-98 (2002).</li><li>Suster, M. L., Kikuta, H., Urasaki, A., Asakawa, K., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+in+zebrafish+with+the+tol2+transposon+system.">Transgenesis in zebrafish with the tol2 transposon system.</a> Methods Mol Biol. , 56141-56163 (2009).</li><li>Grabher, C., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meganuclease+and+transposon+mediated+transgenesis+in+medaka.">Meganuclease and transposon mediated transgenesis in medaka.</a> Genome Biol. 8, (2007).</li></ol>

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Il zebrafish adulto ha una straordinaria capacità di rigenerare con successo molti organi interni e appendici. Una conoscenza approfondita dei meccanismi molecolari e cellulari coinvolti richiede un&#39;analisi tessuto-specifica delle funzioni dei geni e vie di segnalazione. Verso questo, il sistema di Teton fornisce un efficace strumento per l&#39;espressione genica controllata spatiotemporally in zebrafish embrionali e adulte. I costrutti sistema Teton e metodologie descritte in questo manoscritto sono stati utilizza...

Il pesce zebra è una ben consolidata organismo modello vertebrato per studiare molti aspetti dello sviluppo, fisiologia, patologia, e la rigenerazione. Con la crescente adozione di zebrafish come modello per i processi biologici post-embrionali, strumenti sperimentali per, la manipolazione inducibile tessuto-specifica dell&#39;espressione genica o vie di segnalazione sono diventati sempre più importanti. In particolare, gli studi in organo e rigenerazione appendice in zebrafish adulti hanno sofferto di una mancanza di strumenti per la dissezione dei requisiti spazio-temporali di vie di segnalazione durante questi processi rigenerativi. Attualmente, tre diversi sistemi sono stati utilizzati per realizzare condizionale, espressione tessuto-specifica del gene in rigenerazione organi di zebrafish adulto: il sistema Cre-lox, espressione mosaico di heat-shock transgeni inducibile mediante trasposoni mediata transgenesi somatica, e il sistema di Teton 1 -3. TETON riferisce ad una variante di un tetraciclina-CONTlaminati sistema di attivazione trascrizionale, dove l&#39;espressione viene attivato in presenza di tetraciclina o un suo derivato, ad esempio doxiciclina. Il sistema Cre-lox, come è finora stato utilizzato in pesci adulti, si basa su un ricombinasi Tamoxifen controllato Cre (CreERT2), la cui espressione è spazialmente limitata da elementi regolatori tessuto-specifici. Rimozione Cre-driven di una cassetta di STOP facilita l&#39;espressione del gene di interesse guidata da un promotore che deve essere attiva in tutti i tipi cellulari 1. Creazione Transposon mediata di transgeni somatiche mosaically espresse offre un sistema di espressione del transgene inducibile in linee cellulari individuali. Iniezione di embrioni di zebrafish con trasposone Tol2 che porta un gene di interesse sotto il controllo trascrizionale di un shock termico risultati promotore in individui chimerici tipicamente portano il transgene solo in linee cellulari discreti di un organo rigenerante 2. Mentre entrambi i sistemi permettono condizionale tessuti speespressione genica fica, il sistema Cre-lox non è reversibile, e la strategia di utilizzo di etichettatura clonale basato trasposoni-risente della sua natura stocastica. Così, noi ed altri hanno recentemente adattato sistemi Teton transgenici per l&#39;utilizzo in zebrafish, che facilitano temporalmente e l&#39;espressione genica spazialmente controllata che si aggiunge sintonizzabile e reversibile 3-5. Il sistema di Teton qui utilizzato comprende una linea pilota transgenica (TetActivator), in cui un Doxiciclina (DOX) -inducible attivatore trascrizionale (migliorato transattivatore tetraciclina inverso, irtTA, breve Teta) è sotto il controllo di sequenze regolatorie genomiche tessuto-specifici. In secondo luogo, esso richiede una linea transgenica responder (TetResponder) che ospita un gene di interesse sotto il controllo trascrizionale del gestore tetraciclina (Tet elemento di risposta; tetre) (Figura 1A). Pertanto, l&#39;uso di combinazioni specifiche di linee TetActivator e TetResponder consente condizionale tessuto-specificomanipolazione fic dell&#39;espressione genica. Abbiamo recentemente utilizzato il sistema di Teton a sondare per le funzioni di tessuto-specifici di Wnt segnalazione / beta-catenina nell&#39;adulto rigeneranti coda zebrafish fin 3. Nel protocollo descritto qui si descrive un flusso di lavoro per la messa a punto e l&#39;uso del sistema di Teton in zebrafish, in particolare per gli studi di rigenerazione fin. Questo include le istruzioni dettagliate su come generare linee TetActivator e TetResponder transgeniche stabili e di un protocollo per l&#39;induzione transgene in embrioni e zebrafish adulto. Inoltre, si descrivono tecniche per la verifica dell&#39;espressione genica tessuto-specifica nel rigenerato aletta, compreso un protocollo per la preparazione di criosezioni di pinne zebrafish adulti. Inoltre, si discute considerazioni per la progettazione del transgene TetActivator, la scelta di un metodo di transgenesi e rilevamento di espressione TetResponder. Quindi, l&#39;obiettivo generale di questo protocollo è quello di servire da modelloprevista per il montaggio di un sistema téton funzionale zebrafish per ottenere l&#39;espressione genica condizionale tessuto-specifico, che può essere applicato a qualsiasi tessuto di interesse. Abbiamo creato un vettore TetActivator consentendo I-SCEI o generazione Tol2 mediata delle linee TetActivator stabili utilizzando sequenze di regolazione genomica brevi (frammenti enhancer; Weidinger laboratorio banca dati plasmide no 1247;. Figura 1B). Questo costrutto contiene una cassetta TetActivator consistente della variante mutante M2 del dominio repressore Tet inverso fuso con il virus Herpes simplex VP16 transattivazione dominio derivato 3F [irtTAM2 (3F)]. Espressione dei TetActivator (TETA) può essere facilmente monitorato dal momento che è co-espressa con il fluoroforo AmCyan dalla stessa griglia di lettura aperta; un peptide P2A media ribosomiale per saltare, che dovrebbe portare la produzione di Teta e AmCyan proteine ​​separate a un rapporto 1: 1 5,6. Il costrutto contiene anche un poylinker 5 &#39;del Tcassette etActivator per facilitare l&#39;inserimento di genomica sequenze regolatrici di interesse utilizzando metodi di clonazione convenzionali. Inoltre, abbiamo creato un costrutto costituito dal cassetto sopra descritto TetActivator più una cassetta di selezione kanamicina (Weidinger database di plasmide laboratorio no 1180;. Figura 1C), che possono essere ricombinati in un cromosoma artificiale batterico (BAC) contenente una grande regione genomica ( di solito nel codone di inizio di un gene la cui espressione modello deve essere imitata dal transgene). Entrambi i costrutti sono disponibili presso il laboratorio Weidinger su richiesta.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Breeding boxes</td>
			<td>Aqua Schwarz</td>
			<td>AquaBox 1</td>
			<td></td>
		</tr>
		<tr>
			<td>Compound fluorescent microscope</td>
			<td>e.g. Leica, Zeiss</td>
			<td>varies with the manufacturer</td>
			<td>to image fluorescent tissue sections</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>e.g. Leica, Zeiss</td>
			<td>varies with the manufacturer</td>
			<td>to image fluorescent tissue sections</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>e.g. Leica, Thermo-Scientific</td>
			<td>varies with the manufacturer</td>
			<td>for cryosectioning</td>
		</tr>
		<tr>
			<td>4&#8217;, 6- diamidino-2-phenylinodole (Dapi)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td>use 1/5000 in PBS 
			for visualization of nuclei</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma-Aldrich</td>
			<td>D9891</td>
			<td>prepare stocks in 50% EtOH at 50 mg/ml (97 mM) 
			for TetResponder induction</td>
		</tr>
		<tr>
			<td>E3 embryo medium&#160;</td>
			<td></td>
			<td></td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2&#903; 2 H2O, 0.33 mM MgSO4&#903;7 H2O, 0.2 &#8240; (w/v) methylene blue, pH 6.5 
			for embryo/larvae husbandry</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>4 % (w/v) paraformaldehyde in PBS, pH 7.5 
			for fixation</td>
		</tr>
		<tr>
			<td>1x Phosphat-buffer saline (PBS)</td>
			<td></td>
			<td></td>
			<td>1.7 mM KH2PO4, 5.2 mM Na2HPO4, 150 mM NaCl, pH 7.5</td>
		</tr>
		<tr>
			<td>1x Phosphat-buffer saline + Tween 20 (PBT)</td>
			<td></td>
			<td></td>
			<td>1x PBS with 0.1 % Tween 20</td>
		</tr>
		<tr>
			<td>Superfrost Ultra Plus adhesion microscope slides</td>
			<td>Thermo Scientific&#160;</td>
			<td>1014356190</td>
			<td>for collection of tissue sections</td>
		</tr>
		<tr>
			<td>Stereo fluorescent microscope</td>
			<td>e.g. Leica, Zeiss</td>
			<td>varies with the manufacturer</td>
			<td>for fluorescence-based genotyping</td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td>e.g. Biorad, Applied Biosystems</td>
			<td>varies with the manufacturer</td>
			<td>for PCR-based genotyping</td>
		</tr>
		<tr>
			<td>Tissue freezing medium (TFM)</td>
			<td>Triangel Biomedical Sciences</td>
			<td>TFM-C</td>
			<td>for embedding of tissue samples</td>
		</tr>
		<tr>
			<td>Tricaine (L-Ethyl-m-amino-benzoate-methane sulfonate/MS-222)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>for anesthesia 
			use at 1 mg/ml in E3 embryo medium</td>
		</tr>
	</tbody>
</table>

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.

Per stabilire un sistema di Teton funzionale per l&#39;espressione del gene inducibile tessuto-specifica, TetActivator transgenici e linee TetResponder bisogno di essere generato (Figura 1A). Questo si ottiene microinjecting TetActivator (Figura 1B - C) o TetResponder (Figura 1E) costruisce in embrioni di zebrafish e la successiva integrazione della linea germinale. Costrutti TetActivator funzionali possono essere generati sia per clonazione di brevi se...

Watch this Scientific Journal Video about Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration at JoVE.com

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

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

L&#39;utilizzo del sistema di Teton per studiare Meccanismi molecolari della rigenerazione Zebrafish

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

use-teton-system-to-study-molecular-mechanisms-zebrafish

Research

JoVE Journal

Developmental Biology

10.6K Views.  Ulm University. Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

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

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

Ablazione specifiche Hepatocyte in Zebrafish allo Studio biliare-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Ricerca

Biologia dello sviluppo

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 ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Sviluppo di un modello di fegato Fibrotic etanolo-indotta in zebrafish per studiare progenitrici cellulo-mediata epatociti Rigenerazione

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

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 ...

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to any type of injury. Common injury models in these animals remove large amounts of tissue, which damages multiple organs. To overcome this broad tissue damage, we describe here a method to selectively remove a single organ, the pharynx, in the planarian Schmidtea mediterranea. We achieve this by soaking animals in a solution containing the cytochrome oxidase inhibitor sodium azide. Brief exposure to sodium azide causes extrusion of the pharynx from the animal, which we call &#34;chemical amputation.&#34;&#160;Chemical amputation removes the entire pharynx, and generates a small wound where the pharynx attaches to the intestine. After extensive rinsing, all amputated animals regenerate a fully functional pharynx in approximately one week. Stem cells in the rest of the body drive regeneration of the new pharynx. Here, we provide a detailed protocol for chemical amputation, and describe both histological and behavioral methods to assess successful amputation and regeneration.

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

Chimica l'amputazione e la rigenerazione della faringe nella Planaria planarie mediterranea

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Metodi per lo studio della rigenerazione in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Ibridazione dell'intero Monte In Situ negli embrioni di pesce zebra e nella formazione dei tubi, analisi dei prodotti iPSC-EC per studiare il ruolo dell'endoglin nello sviluppo vascolare

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

Esame della rigenerazione muscolare nei modelli zebrafish di malattia muscolare

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

Uso della coltura di organi di tipo Trowell per studiare la regolazione delle cellule staminali dentali

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

Espressione GFP indotta dalla luce in embrioni di zebrafish utilizzando il sistema optogenetico TAEL/C120

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control ...