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>

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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. 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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><tbody><tr><td>Equipment for raising fish and collecting eggs&amp;nbsp;</td><td></td><td></td><td>see the Zebrafish Book&lt;sup&gt;11&lt;/sup&gt; 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>&lt;em&gt;e.g.&lt;/em&gt; Thermo Scientific Nanodrop 2000</td></tr><tr><td>Nucleic acid electrophoresis chamber</td><td></td><td></td><td></td></tr><tr><td>Petri dishes 4 cm &amp;Oslash;, 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>1.5 ml centrifugation tubes</td><td></td><td></td><td></td></tr><tr><td>15 ml and 50 ml 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&amp;trade; Round Gel Loading Tips in Sterile Rack, 1-200&amp;mu;l&amp;nbsp;</td><td>Santa Cruz</td><td>sc-201732</td><td></td></tr><tr><td>100 &amp;mu;m filter (BD Falcon 100 mm Cell Strainer)</td><td>BD Biosciences</td><td>352360</td><td></td></tr><tr><td>30 &amp;mu;m filter (Pre-Separation Filters-30 &amp;micro;m)</td><td>Miltenyi Biotec</td><td>130-041-407</td><td></td></tr><tr><td>Phase lock gel, heavy, 1.5 ml tubes&amp;nbsp;</td><td>Prime</td><td>2302810</td><td></td></tr><tr><td>Egg water medium</td><td></td><td></td><td>60 &amp;mu;g/ml Instant Ocean Sea Salts in ddH2O, 0.00001% (w/v) Methylene Blue</td></tr><tr><td>E3 medium&amp;nbsp;</td><td></td><td></td><td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 0.33 mM MgSO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Tricaine (3-amino benzoic acidethylester)</td><td>Sigma-Aldrich</td><td>A-5040</td><td>4 mg/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&amp;acute;s L-15 medium&amp;nbsp;</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&amp;nbsp;</td><td>Invitrogen</td><td>10814-010</td><td>20 &amp;micro;g/&amp;micro;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 ...

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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

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

Isolamento e caratterizzazione di singole cellule da embrioni di zebrafish

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Ricerca

Biologia dello sviluppo

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 ...

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

L'analisi dello spostamento della deformazione meccanica miocardiale (DIAMOND) rivela l'eterogeneità segmentale della funzione cardiaca nel pesce zebra embrionale

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression.  ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

Microscopia di fluorescenza di luce-foglio di catturare immagini 4-dimensionale degli effetti di modulazione dello sforzo di taglio sul cuore Zebrafish in via di sviluppo

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

Bioingegneria

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Dissezione di cuore in larvale, Zebrafish giovanile e degli adulti, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Biologia

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Immunocolorazione di Dissected Cuore Zebrafish embrionali

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recupero di Zebrafish adulti cuori per applicazioni ad alta velocità effettiva

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

Microscopia light sheet di dinamiche cardiache veloci in embrioni zebrafish

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid ...

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

Modifiche precise mediate da CRISPR-Cas9 nei cuori di zebrafish

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...

Su larga scala Zebrafish embrionali Cuore dissezione per l'analisi trascrizionale

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