We would like to thank C. Burns, C. McRae for outlining the basic principle of this purification protocol, and F. Priller for initial implementation of this method in our lab. S.A.-S. is supported by a Heisenberg professorship of the Deutsche Forschungsgemeinschaft (DFG). This work was supported by DFG grant SE2016/7-1.

Questo protocollo segue le linee guida per la cura degli animali della legge tedesca e lo stato di Berlino; gestione zebrafish è stato monitorato dalle autorità locali per la protezione degli animali (LAGeSo, Berlin-Brandenburg). 1. Ottenere embrioni di zebrafish per Cuore Estrazione <ol><li> Croce zebrafish giornalista cardiaca come il Tg (myl7: EGFP) twu34 linea transgenica 9 al fine di ottenere embrioni con espressione GFP-specific cuore. </li><li> Mantenere gli embrioni in acqua uovo a 28,5 ° C, fino alla fase embrionale desiderato 10,11. Assicurarsi che la popolazione embrione raccolto è omogeneo sia geneticamente e stadio di sviluppo per limitare le variazioni nell&#39;espressione genica. </li><li> Dechorionate gli embrioni manualmente. </li></ol> 2. Dissezione Zebrafish embrionali Hearts NOTA: Tenere tutte le soluzioni sul ghiaccio. Se possibile, effettuare il lavoro di squadra: una persona analizza i cuori dal embRyos mentre un&#39;altra persona ordina i cuori allo stereomicroscopio a fluorescenza. Questo permette la lavorazione di diverse centinaia di embrioni in poche ore. <ol><li> Trasferire circa 100 embrioni in una provetta da 1,5 ml centrifugazione. Anestetizzare gli embrioni con tricaine (0,16 mg / ml in E3 medio). Procedere volta gli embrioni sono chiaramente anestetizzati (essi non nuotano e sedimenti sul fondo del tubo, ma i loro cuori sono ancora battendo). </li><li> Rimuovere la soluzione / E3 tricaine e lavare gli embrioni una volta con 1 ml di L-15/10% di media FBS e mantenere in ghiaccio. NOTA: Il mezzo FBS L-15/10% è importante mantenere gli organi e le cellule in vita durante l&#39;intera procedura di dissezione fino a quando i cuori sono stati trasferiti in RNAlater o Trizol. </li><li> Aggiungere 1 ml di L-15/10% di media FBS agli embrioni e pipetta su e giù 5-8 volte con un Round Gel Loading Suggerimento fino a quando il tuorlo è completamente distrutto. Pipetta embrioni goccia a goccia sulla superficie della soluzione, in modo che la goccia scoppio egli embrioni vengono delicatamente interrotte. NOTA: Come con vigore e con quale frequenza gli embrioni devono essere pipettati su e giù dipende dallo stadio di sviluppo degli embrioni, cioè più pipettaggio è necessaria a stadi più avanzati (ad esempio a 56 HPF). </li><li> Per il primo lotto di embrioni sezionati, valutare l&#39;integrità degli embrioni e la percentuale di cuore sezionato allo stereomicroscopio, dal momento che alcuni cuori possono rimanere attaccati ai embrioni se pipettati troppo delicatamente. Regolare il modo di pipettaggio per i prossimi turni di dissezione di conseguenza. NOTA: utilizzare punte bassa ritenzione per pipette e tubi microcentrifuga per ridurre al minimo i cuori attaccare alla plastica. </li><li> Applicare il campione attraverso un filtro 100 micron collocato su un tubo da 50 ml centrifugazione; risciacquare la provetta da 1,5 ml con 1 ml di mezzo L-15/10% FBS e quindi applicare questo al filtro per minimizzare la perdita del campione (alcuni cuori potrebbero essere attaccati alle pareti del tubo). Lavare due volte il filtro con 1 ml L-15 / 10% FBS. In questa fase i cuori passano attraverso il filtro e sono raccolti nel tubo 50 ml. </li><li> Applicare il flusso continuo su un filtro da 30 micron collocato su un tubo di centrifugazione 15 ml e risciacquare il filtro una volta con 1 ml L-15/10% FBS media. In questa seconda fase di filtrazione, i cuori sono trattenuti nel filtro e detriti più piccolo è lavato via. </li><li> Ruotare il filtro a testa in giù e lavare i cuori fuori del filtro in un agarosio rivestite Petri 1% applicando tre lavaggi consecutivi con 1 ml L-15/10% FBS. </li><li> Separare manualmente cuori GFP-positive dalla macerie embrionale nonfluorescent (ad esempio le lenti degli occhi), con un paio di pinze allo stereomicroscopio a fluorescenza e concentrarsi al centro del piatto. Raccogliere loro secondo step 3. NOTA: Se gli embrioni sono più vecchi di 24 HPF, il cuore dovrebbe essere battono, indicando che i tessuti sono ancora fisiologicamente normale. </li></ol> 3. Isolamento mRNA da dissezioneted Hearts <ol><li> Raccogliere i cuori del minor volume possibile (ad esempio, 10 mL) e trasferirli in una provetta da 1,5 ml contenente 0,75 ml RNAlater (su ghiaccio). Verificare che nessun cuore rimane nella punta della pipetta visualizzando sotto uno stereomicroscopio a fluorescenza. </li><li> In alternativa, se una cappa chimica è disponibile nelle immediate vicinanze del microscopio a fluorescenza, trasferire i cuori raccolti in Trizol (ATTENZIONE) sotto il cofano chimica. Questo aggira la possibile perdita di cuori che spuntano nella punta della pipetta e anche durante la centrifugazione del cuore (i passaggi 3.4, 3.5 e 3.7). Pool i cuori di diversi cicli di isolamento nello stesso tubo con Trizol e passare direttamente al punto 3.8; il volume finale non deve superare il 10% del volume originale Trizol. NOTA: Leggere il Trizol Scheda di sicurezza (MSDS) prima dell&#39;uso. Maneggiare Trizol reagente sotto una cappa e usura raccomandato dispositivi di protezione individuale. Evitare il contatto con la pelle, gli occhi e clothing. Rimuovere tutte le fonti di accensione. </li><li> Pool cuori di diversi cicli di isolamento nello stesso tubo con RNAlater (su ghiaccio), l&#39;efficienza di isolamento dell&#39;RNA migliora con la quantità di tessuto raccolto. Se il volume totale del campione supera il 10% del volume RNAlater originale, utilizzare un tubo supplementare con 0,75 ml RNAlater per la memorizzazione (su ghiaccio). </li><li> Centrifugare i campioni a 15.700 xg per 20 min a 4 ° C per sedimentare il cuore. </li><li> Sotto un stereomicroscopio a fluorescenza, raccogliere il più accuratamente possibile surnatante in una capsula petri agarosio rivestite 1% contenente 3 ml L-15/10% FBS mezzo, ma non disturbare i cuori sedimentate sul fondo del tubo di centrifugazione. Poiché fino al 15% dei cuori può rimanere nel supernatante causa dell&#39;elevata viscosità di RNAlater, recuperali come descritto al punto 3.7. </li><li> Sotto una cappa chimica, aggiungere 0,5 ml Trizol per il tubo contenente i cuori sedimentati e tenere in ghiaccio. </li><li> Sotto la microsco fluorescentepe, trasferire i cuori (dal punto 3.5) dal piatto agarosio 1% rivestite contenenti la soluzione FBS RNAlater / L-15/10% in un altro piatto 1% agarosio rivestite con 3 ml L-15/10% FBS per diluire il RNAlater. Raccogliere il cuore nel centro della capsula di Petri e trasferirli in un piccolo volume nel tubo di cuori sedimentati in Trizol sotto cappa chimica. </li><li> Vortex la provetta da 1,5 ml contenente i cuori in Trizol perturbare il cuore e incubare per 5 minuti a temperatura ambiente. </li><li> Sotto una cappa chimica, aggiungere 100 ml di cloroformio (ATTENZIONE), mescolare bene e trasferire la soluzione / cloroformio Trizol in un 1,5 ml pre-filata tubo PLG (centrifuga 30 sec alla massima velocità prima dell&#39;uso). Incubare per 3 minuti a temperatura ambiente. NOTA: Leggere le MSDS cloroformio prima dell&#39;uso. Manipolare il reagente cloroformio sotto una cappa e usura raccomandato dispositivi di protezione individuale. Evitare il contatto con la pelle, gli occhi e gli indumenti. Tenere lontano dai materiali incompatibili come i metalli, alcali. </li><li> Centrifugoge il campione a 15.700 xg per 15 min a 4 ° C e trasferire la fase acquosa in una nuova provetta da 1,5 ml. </li><li> Aggiungere 5-10 mg glicogeno e 250 microlitri preraffreddata (a -20 ° C) isopropanolo; mescolare bene e incubare per tutta la notte a -20 ° C. </li><li> Centrifugare il campione a 15.700 xg per 30 min a 4 ° C e scartare il surnatante. </li><li> Lavare il pellet con 1 ml di etanolo al 75%, centrifugare a 15.700 xg per 15 min a 4 ° C e scartare il surnatante. </li><li> Lasciare evaporare etanolo essiccando il pellet a temperatura ambiente fino a quando diventa bianco (ad esempio per 10-15 min). </li><li> Aggiungere 20 microlitri RNasi-free DDH 2 O al pellet e incubare per 10-15 min a 55 ° C per sciogliere il RNA; poi tenere su ghiaccio. </li><li> Valutare la concentrazione di RNA e la purezza mediante misurazione assorbanza. Valutare l&#39;integrità dell&#39;RNA eseguendo il campione su un gel di agarosio 1%. </li></ol>

<ol>
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Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+analyses+of+vascular+tube+and+lumen+formation+in+zebrafish.">Cellular and molecular analyses of vascular tube and lumen formation in zebrafish.</a> Development. 132, 5199-5209 (2005).</li><li>Brownlie, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+embryonic+globin+genes+of+the+zebrafish.">Characterization of embryonic globin genes of the zebrafish.</a> Dev Biol. 255, 48-61 (2003).</li><li>Marvin, M., 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=Developmental+expression+patterns+of+the+zebrafish+small+heat+shock+proteins.">Developmental expression patterns of the zebrafish small heat shock proteins.</a> Dev Dyn. 237, 454-463 (2008).</li><li>Lam, C. S., Marz, M., Strahle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gfap+and+nestin+reporter+lines+reveal+characteristics+of+neural+progenitors+in+the+adult+zebrafish+brain.">gfap and nestin reporter lines reveal characteristics of neural progenitors in the adult zebrafish brain.</a> Dev Dyn. 238, 475-486 (2009).</li><li>Wang, 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=Moesin1+and+Ve-cadherin+are+required+in+endothelial+cells+during+in+vivo+tubulogenesis.">Moesin1 and Ve-cadherin are required in endothelial cells during in vivo tubulogenesis.</a> Development. 137, 3119-3128 (2010).</li><li>Heiman, M., 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=A+translational+profiling+approach+for+the+molecular+characterization+of+CNS+cell+types.">A translational profiling approach for the molecular characterization of CNS cell types.</a> Cell. 135, 738-748 (2008).</li><li>Tryon, R. 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The authors have nothing to disclose.

Questo protocollo permette il rapido arricchimento di zebrafish tessuto cardiaco embrionale per analisi dell&#39;espressione genica. La quantità e la qualità del campione RNA cardio-specifica dipende molto pochi passi fondamentali: in primo luogo, la quantità di campione viene notevolmente migliorata se la perdita di cuori è impedito ad ogni passo del protocollo, poiché la purificazione dell&#39;RNA funziona solo con sufficiente materiale di partenza. In secondo luogo, la purezza del campione, che dipende interamen...

Zebrafish (Danio rerio) è ampiamente usato in biologia dello sviluppo per studiare organogenesi in vivo per la sua veloce, trasparente e extrauterina sviluppo embrionale, combinato con dimensioni ridotte e la disponibilità di linee giornalista transgeniche con espressione tessuto-specifica di proteine ​​fluorescenti. Questo piccolo vertebrato è particolarmente adatto per studiare lo sviluppo cuore perché l&#39;ossigenazione del primo embrione zebrafish non si basa sul battito cardiaco e il flusso di sangue; queste caratteristiche hanno permesso la caratterizzazione di un gran numero di mutanti cardiovascolari 1,2 e il pesce zebra è ora un organismo modello ampiamente riconosciuto per lo studio delle malattie del cuore 3. Per studiare l&#39;espressione del gene durante lo sviluppo embrionale, le trascrizioni sono comunemente analizzati con tutto il montaggio in situ (WISH) 4, RT-qPCR 5, 6 microarrays, o generazione sequenziamento next (RNA-Seq) 7. MentreWISH consente un&#39;analisi spazio-temporale di espressione genica nell&#39;intero embrione, livelli di trascrizione sono solitamente valutati mediante RT-qPCR, microarrays o approcci RNA-Seq. Tuttavia, questi metodi richiedono arricchimento tessuto specifico per profili di espressione genica. Poiché il cuore embrionale zebrafish rappresenta una piccola frazione dell&#39;intero dell&#39;embrione, studi trascrittoma durante lo sviluppo cardiaco richiedono un protocollo per la dissezione e arricchimento di cuori. Inoltre, per ottenere dati fisiologicamente rilevanti, è importante mantenere il tessuto cardiaco completamente funzionale fino estrazione dell&#39;RNA. Qui, descriviamo un protocollo per isolare rapidamente fisiologicamente normale e battere i cuori di centinaia di embrioni di zebrafish per ottenere in modo efficiente campioni di RNA di alta qualità per ulteriori analisi. Il presente metodo è basato sul protocollo riportato da Burns e MacRae 2006 8. Per arricchimento del tessuto cardiaco, entrambi i metodi utilizzano una linea giornalista infarto transgenicae sfruttare le proprietà di adesione differenziale di zebrafish cuori embrionali rispetto ad altri tessuti. In breve, pipettando molti embrioni su e giù attraverso un puntale stretto, cuori sono simultaneamente rilasciati dai corpi embrionali e successivamente separati dai detriti embrionale in due fasi di filtrazione rapidi; cuori fluorescente vengono poi ordinati manualmente da residui restanti e raccolti per ulteriori elaborazioni.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr >
			<td >Equipment for raising fish and collecting eggs&#160;</td>
			<td></td>
			<td></td>
			<td>see the Zebrafish Book11 for details</td>
		</tr>
		<tr >
			<td >Fluorescence stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Refrigerated Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >UV-Spectrophotometer</td>
			<td></td>
			<td></td>
			<td>eg. Thermo Scientific Nanodrop 2000</td>
		</tr>
		<tr >
			<td >Nucleic acid electrophoresis chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Petri dishes 4cm &#216;, coated with 1% agarose in E3 medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipettes and tips (P20, P100, P1000)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1,5ml centrifugation tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >15ml and 50ml centrifugation tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Pair of Dumont #5 forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >ExactaCruz&#8482; Round Gel Loading Tips in Sterile Rack, 1-200&#956;l&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-201732</td>
			<td></td>
		</tr>
		<tr >
			<td >100 &#956;m filter (BD Falcon 100 mm Cell Strainer)</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr >
			<td >30 &#956;m filter (Pre-Separation Filters-30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr >
			<td >Phase lock gel, heavy, 1,5ml tubes&#160;</td>
			<td>Prime</td>
			<td>2302810</td>
			<td></td>
		</tr>
		<tr >
			<td >Egg water medium</td>
			<td></td>
			<td></td>
			<td>60&#956;g/ml Instant Ocean Sea Salts in ddH2O, 0.00001% (w/v) Methylene Blue</td>
		</tr>
		<tr >
			<td >E3 medium&#160;</td>
			<td></td>
			<td></td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4</td>
		</tr>
		<tr >
			<td >Tricaine (3-amino benzoic acidethylester)</td>
			<td>Sigma-Aldrich</td>
			<td>A-5040</td>
			<td>4mg/ml Tricaine stock solution, pH 7</td>
		</tr>
		<tr >
			<td >1% agarose (in E3 medium)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1% agarose gel (in TBE buffer)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Leibovitz&#180;s L-15 medium&#160;</td>
			<td>Gibco</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr >
			<td >FBS (Fetal Bovine Serum)</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr >
			<td >RNAlater</td>
			<td>Ambion</td>
			<td>AM7020</td>
			<td></td>
		</tr>
		<tr >
			<td >Trizol</td>
			<td>Ambion</td>
			<td>1559606</td>
			<td></td>
		</tr>
		<tr >
			<td >Glycogen&#160;</td>
			<td>Invitrogen</td>
			<td>10814-010</td>
			<td>20 &#181;g/&#181;L in RNase-free water</td>
		</tr>
		<tr >
			<td >chloroform</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >isopropanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >75% ethanol (in DEPC-ddH2O)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Nuclease-free water or sterilized DEPC treated ddH2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Nucleic acid loading buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >TBE (Tris/Borate/EDTA) buffer for electrophoresis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

large-scale zebrafish embryonic heart dissection for transcriptional analysis

Il cuore embrionale zebrafish è composto da poche centinaia di cellule, che rappresentano solo una piccola frazione dell&#39;intero dell&#39;embrione. Pertanto, per evitare che il trascrittoma cardiaco venga mascherato dal trascrittoma embrionale globale, è necessario raccogliere un numero sufficiente di cuore per ulteriori analisi. Inoltre, lo sviluppo cardiaco zebrafish procede rapidamente, raccolta cuore e metodi di estrazione di RNA devono essere veloce per garantire l&#39;omogeneità dei campioni. Qui, vi presentiamo un protocollo dissezione manuale rapido per la raccolta funzionali / cuori che battono da embrioni di zebrafish. Questo è un presupposto essenziale per la successiva estrazione di RNA specifico cardiaco-per determinare cardiaco-specifici livelli di espressione genica per analisi trascrittomica, come ad esempio Real time PCR quantitativa (RT-qPCR). Il metodo si basa sulle proprietà adesive differenziali del cuore embrionale zebrafish rispetto ad altri tessuti; questo permette la rapida PHYSeparazione Sical di cardiaca dal tessuto extracardiache da una combinazione di fluidica perturbazione forza di taglio, filtrazione graduale e la raccolta manuale dei transgenici cuori fluorescente.

Qui, descriviamo un esperimento rappresentativo dissezione cuore con il pesce zebra Tg (myl7: GFP) twu34 transgenici linea 9, che esprime la proteina verde fluorescente (GFP) esclusivamente all&#39;interno del miocardio (Fig. 1). Abbiamo raccolto sia il cuore sezionato e gli embrioni da cui sono stati ricavati per valutare la purezza del campione cuore. In breve, omozigote Tg (myl7: GFP) twu34 zebrafish 9 sono stati outcrossed con wild-type in modo che tutti ...

Watch this Scientific Journal Video about Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis at JoVE.com

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Per analizzare i profili di espressione genica cardiaco durante lo sviluppo del cuore zebrafish, RNA totale deve essere estratto dai cuori isolati. Qui, vi presentiamo un protocollo per la raccolta funzionali / cuori che battono per una rapida dissezione manuale da embrioni di zebrafish per ottenere mRNA specifici cardiaco.

Su larga scala Zebrafish embrionali Cuore dissezione per l&#39;analisi trascrizionale

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

large-scale-zebrafish-embryonic-heart-dissection-for-transcriptional

Research

JoVE Journal

Developmental Biology

14.3K Views.  Max Delbrück Center for Molecular Medicine.  Per analizzare i profili di espressione genica cardiaco durante lo sviluppo del cuore zebrafish, RNA totale deve essere estratto dai cuori isolati. Qui, vi presentiamo un protocollo per la raccolta funzionali / cuori che battono per una rapida dissezione manuale da embrioni di zebrafish per ottenere mRNA specifici cardiaco. 

Video: Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

Analisi di Cardiomyocyte Sviluppo usando immunofluorescenza in cuore embrionali di topo

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Ricerca

Biologia dello sviluppo

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain&#39;s macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution EM. Here we describe a universal method for nanotomy applied to investigate the zebrafish larval brain in health and upon non-invasive brain injury.

Su larga scala scansione microscopia elettronica a trasmissione (Nanotomy) di sana e feriti Zebrafish Cervello

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Produzione su larga scala dei cardiomiociti da cellule staminali umane pluripotenti usando un altamente riproducibile piccole molecole-Based Differenziazione protocollo

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

Analisi dei vasi coronarici in sgomberati cuori embrionali

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

La dissezione del larvale Zebrafish gonadica Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Visualizzazione e analisi quantitativa di angiogenesi embrionale in<em&gt; Xenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

Dissezione e cultura del rene embrionale del mouse

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

Microscopia elettronica e correlativo super-risoluzione per risolvere la localizzazione della proteina nella Retina di Zebrafish

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

Nanoparticelle-mediata siRNA silenziamento genico nel cuore di Zebrafish adulto

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Sistema basato su microiniezione per In Vivo l'impianto di cardiomiociti embrionali nell'embrione aviaria

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...