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.

This protocol follows the animal care guidelines of the German and Berlin state law; zebrafish handling was monitored by the local authority for animal protection (LaGeSo, Berlin-Brandenburg).
1. Obtaining Zebrafish Embryos for Heart Extraction
<ol>
	<li>Cross cardiac reporter zebrafish such as the Tg(myl7:EGFP)twu34 transgenic line9 in order to obtain embryos with heart-specific GFP expression.</li>
	<li>Maintain the embryos in egg water at 28.5 &#176;C until the desired embryonic stage10,11. Ensure that the collected embryo population is homogeneous both genetically and by developmental stage to limit variations in gene expression.</li>
	<li>Dechorionate the embryos manually.</li>
</ol>
2. Dissecting Zebrafish Embryonic Hearts
NOTE: Keep all the solutions on ice. If possible, do teamwork: one person dissects the hearts from the embryos while another person sorts the hearts under the fluorescence stereomicroscope. This allows the processing of several hundreds of embryos in a few hours.
<ol>
	<li>Transfer about 100 embryos into a 1.5 ml centrifugation tube. Anesthetize the embryos with tricaine (0.16 mg/ml in E3 medium). Proceed once the embryos are clearly anesthetized (they do not swim and sediment to the bottom of the tube, but their hearts are still beating).</li>
	<li>Remove the tricaine/E3 solution and wash the embryos once with 1 ml L-15/10% FBS medium and maintain on ice. 
	NOTE: The L-15/10% FBS medium is important to keep organs and cells alive during the entire dissection procedure until the hearts have been transferred into RNAlater or Trizol.</li>
	<li>Add 1 ml L-15/10% FBS medium to the embryos and pipette up and down 5-8 times with a Round Gel Loading Tip until the yolk is completely disrupted. Pipette the embryos drop-wise onto the surface of the solution, so that the drop will burst and the embryos are gently disrupted. 
	NOTE: How vigorously and how often the embryos have to be pipetted up and down depends on the developmental stage of the embryos, i.e. more pipetting is needed at late stages (e.g. at 56 hpf).</li>
	<li>For the first batch of dissected embryos, assess the integrity of the embryos and the proportion of dissected hearts under a stereomicroscope, since some hearts may remain attached to the embryos if too gently pipetted. Adjust the manner of pipetting for the next rounds of dissection accordingly. 
	NOTE: Use low retention pipette tips and microcentrifuge tubes to minimize hearts sticking to the plastic.</li>
	<li>Apply the sample onto a 100 &#956;m filter placed on a 50 ml centrifugation tube; rinse the 1.5 ml tube with 1 ml L-15/10% FBS medium and then apply this to the filter in order to minimize loss of the sample (some hearts might be sticking to the walls of the tube). Wash the filter twice with 1 ml L-15/10% FBS. In this step the hearts pass through the filter and are collected in the 50 ml tube.</li>
	<li>Apply the flow-through onto a 30 &#956;m filter placed onto a 15 ml centrifugation tube and rinse the filter once with 1 ml L-15/10% FBS medium. In this second filtration step, the hearts are retained in the filter and smaller debris is washed off.</li>
	<li>Turn the filter upside down and flush the hearts out of the filter into a 1% agarose-coated petri dish by applying three consecutive washes with 1 ml L-15/10% FBS.</li>
	<li>Manually separate GFP-positive hearts from the nonfluorescent embryonic debris (e.g. eye lenses) with a pair of forceps under a fluorescence stereomicroscope and concentrate them in the center of the dish. Collect them according to step 3. 
	NOTE: If the embryos are&#160;older than 24 hpf, the hearts should be beating, indicating that the tissues are still physiologically normal.</li>
</ol>
3. Isolating mRNA from Dissected Hearts
<ol>
	<li>Collect the hearts in the smallest possible volume (e.g. 10 &#181;l) and pipette into a 1.5 ml tube containing 0.75 ml RNAlater (on ice). Verify that no hearts remain in the pipette tip by viewing it under a fluorescence stereomicroscope.</li>
	<li>Alternatively, if a chemical hood is available in the immediate vicinity of the fluorescence microscope, transfer the collected hearts into Trizol (CAUTION) under the chemical hood. This circumvents the possible loss of hearts sticking in the pipette tip and also during centrifugation of the hearts (steps 3.4, 3.5 and 3.7). Pool the hearts from several rounds of isolation into the same tube with Trizol and go directly to step 3.8;&#160;the final&#160;volume should not exceed 10% of the original Trizol volume. 
	NOTE: Read the Trizol Material Safety Data Sheet (MSDS) before use. Handle Trizol reagent under a hood and wear recommended Personal Protective Equipment. Avoid contact with skin, eyes and clothing. Remove all sources of ignition.</li>
	<li>Pool the hearts from several rounds of isolation into the same tube with RNAlater (on ice), as the efficiency of RNA isolation improves with the amount of tissue collected. If the total sample volume exceeds 10% of the original RNAlater volume, use an additional tube with 0.75 ml RNAlater for storage (on ice).</li>
	<li>Centrifuge the samples at 15,700 x g for 20 min at 4 &#176;C to sediment the hearts.</li>
	<li>Under a fluorescence stereomicroscope, collect carefully as much supernatant as possible into a 1% agarose-coated petri dish containing 3 ml L-15/10% FBS medium, but do not disturb the sedimented hearts in the bottom of the centrifugation tube. Since up to 15% of the hearts can remain in the supernatant due to the high viscosity of RNAlater, retrieve them as described in step 3.7.</li>
	<li>Under a chemical hood, add 0.5 ml Trizol to the tube containing the sedimented hearts and keep on ice.</li>
	<li>Under the fluorescent microscope, transfer the hearts (from step 3.5) from the 1% agarose-coated dish containing the RNAlater/L-15/10% FBS solution into another 1% agarose-coated dish with 3 ml L-15/ 10% FBS to dilute out the RNAlater. Collect the hearts in the center of the petri dish and transfer them in a small volume into the tube of sedimented hearts in Trizol under a chemical hood.</li>
	<li>Vortex the 1.5 ml tube containing the hearts in Trizol to disrupt the hearts and incubate for 5 min at room temperature.</li>
	<li>Under a chemical hood, add 100 &#181;l chloroform (CAUTION), mix thoroughly and transfer the Trizol/chloroform solution into a 1.5 ml pre-spun PLG tube (centrifuge 30 sec at maximum speed before use). Incubate for 3 min at room temperature. 
	NOTE: Read the chloroform MSDS before use. Handle chloroform reagent under a hood and wear recommended Personal Protective Equipment. Avoid contact with skin, eyes and clothing. Keep away from incompatibles such as metals, alkalis.</li>
	<li>Centrifuge the sample at 15,700 x g for 15 min at 4 &#176;C and transfer the aqueous phase into a new 1.5 ml tube.</li>
	<li>Add 5-10 &#181;g glycogen and 250 &#181;l precooled (at -20 &#176;C) isopropanol; mix well and incubate over night at -20 &#176;C.</li>
	<li>Centrifuge the sample at 15,700 x g for 30 min at 4 &#176;C and discard the supernatant.</li>
	<li>Wash the pellet with 1 ml 75% ethanol, centrifuge at 15,700 x g for 15 min at 4 &#176;C and discard the supernatant.</li>
	<li>Let the ethanol evaporate by drying the pellet at room temperature until it becomes white (e.g. for 10-15 min).</li>
	<li>Add 20 &#181;l RNase-free ddH2O to the pellet and incubate for 10-15 min at 55 &#176;C to dissolve the RNA; then keep on ice.</li>
	<li>Assess the RNA concentration and purity by absorbance measurement. Assess the RNA integrity by running the sample on a 1% agarose gel.</li>
</ol>

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<li>Gibson, U. E., Heid, C. A., Williams, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+method+for+real+time+quantitative+RT-PCR%5BTitle%5D)+AND+%22Genome+Res%22%5BJournal%5D)">A novel method for real time quantitative RT-PCR.</a> Genome Res. 6, 995-1001 (1996).</li>
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<li>Qian, X., Ba, Y., Zhuang, Q., Zhong, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA-Seq+Technology+and+Its+Application+in+Fish+Transcriptomics%5BTitle%5D)+AND+%22OMICS%22%5BJournal%5D)">RNA-Seq Technology and Its Application in Fish Transcriptomics.</a> OMICS. 18, 98-110 (2014).</li>
<li>Burns, C. G., MacRae, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+of+hearts+from+zebrafish+embryos%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Purification of hearts from zebrafish embryos.</a> Biotechniques. 40, 274-278 (2006).</li>
<li>Huang, C. J., Tu, C. T., Hsiao, C. D., Hsieh, F. J., Tsai, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Germ-line+transmission+of+a+myocardium-specific+GFP+transgene+reveals+critical+regulatory+elements+in+the+cardiac+myosin+light+chain+2+promoter+of+zebrafish%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish.</a> Dev Dyn. 228, 30-40 (2003).</li>
<li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stages+of+embryonic+development+of+the+zebrafish%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li>
<li>Westerfield, M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWesterfield%2C+M+%22The+zebrafish+book.+A+guide+for+the+laboratory+use+of+zebrafish+%28Danio+rerio%29%22">The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio)</a>. , 4th, University of Oregon Press. Eugene. (2000).</li>
<li>Veerkamp, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unilateral+dampening+of+Bmp+activity+by+nodal+generates+cardiac+left-right+asymmetry%29%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Unilateral dampening of Bmp activity by nodal generates cardiac left-right asymmetry).</a> Dev Cell. 24, 660-667 (2013).</li>
<li>Jin, S. W., Beis, D., Mitchell, T., Chen, J. N., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+and+molecular+analyses+of+vascular+tube+and+lumen+formation+in+zebrafish%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+embryonic+globin+genes+of+the+zebrafish%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">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/pubmed?term=((Developmental+expression+patterns+of+the+zebrafish+small+heat+shock+proteins%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">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/pubmed?term=((gfap+and+nestin+reporter+lines+reveal+characteristics+of+neural+progenitors+in+the+adult+zebrafish+brain%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">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/pubmed?term=((Moesin1+and+Ve-cadherin+are+required+in+endothelial+cells+during+in+vivo+tubulogenesis%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Moesin1 and Ve-cadherin are required in endothelial cells during in vivo tubulogenesis.</a> Development. 137, 3119-3128 (2010).</li>
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The authors have nothing to disclose.

This protocol allows the rapid enrichment of zebrafish embryonic heart tissue for gene expression analyses. The quantity and quality of the cardiac-specific RNA sample greatly depends on a few crucial steps: first, the quantity of the sample is greatly improved if loss of hearts is prevented at every step of the protocol, since RNA purification will only work with sufficient starting material. Second, the purity of the sample, which depends entirely on the experimenter, is determined by sorting and collecting hearts with...

Zebrafish (Danio rerio) is widely used in developmental biology to study organogenesis in vivo due to its fast, transparent and extrauterine embryonic development, combined with small size and the availability of transgenic reporter lines with tissue-specific expression of fluorescent proteins. This small vertebrate is particularly well suited to study heart development because oxygenation of the early zebrafish embryo does not rely on heart beat and blood flow; these features have allowed the characterization of large numbers of cardiovascular mutants1,2 and the zebrafish is now a widely recognized model organism to study heart diseases3.
To study gene expression during embryonic development, transcripts are commonly analyzed by whole-mount in situ hybridization (WISH)4, RT-qPCR5, microarrays6, or next generation sequencing (RNA-Seq)7. While WISH allows a spatial-temporal analysis of gene expression within the entire embryo, transcript levels are usually assessed by RT-qPCR, microarrays or RNA-Seq approaches. However, these methods require tissue enrichment for specific gene expression profiling.
Since the zebrafish embryonic heart represents a small fraction of the entire embryo, transcriptome studies during cardiac development require a protocol for dissection and enrichment of hearts. In addition, to obtain physiologically relevant data, it is important to maintain the cardiac tissue fully functional until RNA extraction. Here, we describe a protocol for rapidly isolating physiologically normal and beating hearts from hundreds of zebrafish embryos to efficiently obtain high quality RNA samples for further analyses. The present method is based on the protocol reported by Burns and MacRae, 20068. For enrichment of cardiac tissue, both methods use a transgenic myocardial reporter line and take advantage of the differential adhesion properties of zebrafish embryonic hearts versus other tissues. Briefly, by pipetting many embryos up and down through a narrow pipette tip, hearts are simultaneously released from embryonic bodies and subsequently separated from embryonic debris in two rapid filtration steps; fluorescently labelled hearts are then manually sorted from remaining debris and collected for further processing.

<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

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 cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

Here, we describe a representative heart dissection experiment using the zebrafish Tg(myl7:GFP)twu34 transgenic line9, which expresses green fluorescent protein (GFP) exclusively within the myocardium (Fig. 1). We collected both the dissected hearts and the embryos from which they were derived to assess the purity of the heart sample. Briefly, homozygous Tg(myl7:GFP)twu34 zebrafish9 were outcrossed with wild-type so that all embryonic hearts ...

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

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

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

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Developmental Biology

14.4K Views.  Max Delbrück Center for Molecular Medicine.  Cardiale genexpressie profielen te analyseren tijdens zebravis ontwikkeling van het hart, heeft totaal RNA worden geëxtraheerd uit geïsoleerde harten. Hier presenteren we een protocol voor het verzamelen van functionele / kloppende harten door snelle handmatige dissectie van de zebravis embryo's tot cardiale-specifieke mRNA te verkrijgen. 

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.

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

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.

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.

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.

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

Bioengineering

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.

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

Biology

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.

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.

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.

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.

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

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

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