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>

<ol>
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N., 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=Mutations+affecting+the+cardiovascular+system+and+other+internal+organs+in+zebrafish.">Mutations affecting the cardiovascular system and other internal organs in zebrafish.</a> Development. 123, 293-302 (1996).</li><li>Bakkers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+to+study+cardiac+development+and+human+cardiac+disease.">Zebrafish as a model to study cardiac development and human cardiac disease.</a> Cardiovasc Res. 91, 279-288 (2011).</li><li>Jowett, T., Lettice, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+in+situ+hybridizations+on+zebrafish+embryos+using+a+mixture+of+digoxigenin-+and+fluorescein-labelled+probes.">Whole-mount in situ hybridizations on zebrafish embryos using a mixture of digoxigenin- and fluorescein-labelled probes.</a> Trends Genet. 10, 73-74 (1994).</li><li>Gibson, U. E., Heid, C. A., Williams, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+real+time+quantitative+RT-PCR.">A novel method for real time quantitative RT-PCR.</a> Genome Res. 6, 995-1001 (1996).</li><li>Epstein, C. B., Butow, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+technology+-+enhanced+versatility,+persistent+challenge.">Microarray technology - enhanced versatility, persistent challenge.</a> Curr Opin Biotechnol. 11, 36-41 (2000).</li><li>Qian, X., Ba, Y., Zhuang, Q., Zhong, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq+Technology+and+Its+Application+in+Fish+Transcriptomics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+hearts+from+zebrafish+embryos.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transmission+of+a+myocardium-specific+GFP+transgene+reveals+critical+regulatory+elements+in+the+cardiac+myosin+light+chain+2+promoter+of+zebrafish.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Veerkamp, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unilateral+dampening+of+Bmp+activity+by+nodal+generates+cardiac+left-right+asymmetry).">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/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. C., Pisat, N., Johnson, S. L., Dougherty, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+translating+ribosome+affinity+purification+for+zebrafish.">Development of translating ribosome affinity purification for zebrafish.</a> Genesis. 51, 187-192 (2013).</li></ol>

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 within the petri dish while strictly excluding other tissues. Third, sample quality critically depends on limiting RNA degradation which can occur upon disruption of the embryos; this is achieved by using cell culture medium to avoid cell death and by keeping the samples on ice. RNA degradation is stopped once the hearts have been transferred into RNAlater or Trizol. That hearts are beating in the petri dish powerfully demonstrates that the cardiac tissue is physiologically normal after dissection.
We recommend starting with at least 300 hearts (i.e. around 500 embryos), from which enough RNA can safely be precipitated (together with&#160; 5-10 &#181;g glycogen). However, we cannot exclude than fewer hearts would suffice for small-scale experiments. It is simply important to have sufficient RNA to determine the concentration, purity and integrity of the sample. Please, note that RNA contents can vary depending on the developmental stage and on the genetic background of the embryo (e.g. in the case of mutants with abnormal hearts). Hence, the minimal amount of embryos required for an experiment would have to be determined in each individual case.
There are few technical differences between this protocol and an earlier protocol by Burns and MacRae, and we don&#8217;t think that there is a significant difference in efficiency or speed between the two protocols. However, we have introduced improvements: Most importantly, we suggest the use of RNAlater stabilization solution. This is important for two reasons: first, to collect high quality RNA for further experiments such as RT-qPCR by reducing RNA degradation to a minimum, as RNAlater immediately inactivates RNases and stabilizes RNA within tissues or cells. Second, RNAlater is crucial for allowing the collection of hearts on different days, which is important when the number of embryos is a limiting factor. It also allows pooling of the hearts which may be necessary to obtain a sufficient amount of starting material for efficient isolation of high-quality RNA with Trizol. Transferring the hearts directly into Trizol would bypass the use of RNAlater, however, due to health hazards, this step would have to be performed under a chemical hood, which cannot be assumed to be located in the direct vicinity of the fluorescence microscope for all experimenters.
We found that the heart dissection works well with embryos between 20-72 hpf [12; unpublished data for 72 hpf]. However, the extraction procedure becomes more difficult with increasing embryonic age and length: additional and more forceful pipetting is required to successfully introduce the embryos into the long tip and to separate the hearts from other embryonic tissues. As a result, more embryonic debris is present within the sample preparation (see Figure 1C, C&#8217;) and the experimenter has to be more careful in sorting the hearts in the petri dish.
The heart dissection protocol presented here allows an analysis of the entire cardiac expression profile of different cell types, including myocardium and endocardium. Further separation of cardiac cell types could be achieved by using endocardial- and myocardial-specific-reporter lines [e.g. Tg(myl7:GFP)twu34 and Tg(kdrl:mCherry)is5]9,17 for heart extraction combined with FACS sorting. In contrast to the Translating Ribosome Affinity Purification (TRAP) method18,19, which allows tissue-specific transcriptional analysis without tissue dissection, our approach is not limited to actively-translated mRNAs but also includes not-actively-translated mRNAs or noncoding RNAs. An alternative application of the heart dissection protocol is to analyze protein expression within hearts: protein levels can be assessed by Western blot and protein localization can be analyzed by immunohistochemistry, as the anatomy of the heart is preserved during the dissection procedure.
In summary, we present here a detailed protocol for dissecting functional/beating hearts from hundreds of embryos in a short time, which can be used for obtaining heart-specific RNA (or protein) samples of high purity for further analyses.

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 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

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 were GFP labeled. Some 500 embryos (56 hpf) were transferred into 1.5 ml tubes (approx. 100 embryos in each) (Fig. 1A1, 1B). Each tube was processed as described in the Protocol section (step 2) and in Figure 1A. Hearts were released by pipetting up and down several times (Fig. 1A2) and then applied onto a 100 &#956;m filter (Fig. 1A3). Large pieces of embryonic tissue were retained in this filter; this fraction was retrieved and put in Trizol for RNA extraction to obtain an &#8220;embryo without heart&#8221; RNA sample (Fig. 1A3). The flow-through containing hearts was then applied onto a 30 &#956;m filter (Fig. 1A4), which retained the hearts and tissue debris of similar size. GFP-positive hearts were flushed out of the filter into an agarose-coated petri dish (Fig. 1A5, 1C), quickly gathered in the middle of the dish under a fluorescent stereomicroscope and transferred into 0.75 ml RNAlater (Fig. 1A6). Within this tube with RNAlater, we pooled hearts derived from 5 dissection rounds (approx. 300 hearts from 500 embryos representing an extraction efficiency of about 60%). A comparison of the heart samples prior to sorting from the debris from embryos at 56 hpf (Fig. 1C) versus 36 hpf (Fig. 1C&#8217;) is shown in Figure 1. At 56 hpf, the disruption of embryos yielded more embryonic debris (such as lenses, arrowhead, Fig. 1C) than at 36 hpf following the 30 &#956;m filtration step (Fig. 1C&#8217;).
To determine the purity of the 56 hpf &#8220;heart&#8221; sample, we compared the expression levels of cardiac versus extra-cardiac transcripts in &#8220;heart&#8221; and &#8220;embryo without heart&#8221; samples by RT-qPCR. To this end, we extracted mRNA from those two samples and assessed RNA quality on an agarose gel (Figure 2A) and quantity by absorbance measurement (Table 1) as described in the protocol section (step 3). The approx. 300 hearts yielded 660 ng RNA. Next, cDNA was synthesized using M-MLV Reverse transcriptase RNase H(-) and random hexamers according to Manufacturer&#180;s instructions, and RT-qPCR experiments were performed as described12. Relative gene expression levels were calculated for different tissue-specific markers such as myosin light polypeptide 7 [myl7, a myocardial marker]9, kinase insert domain receptor like [kdrl, an endothelial/endocardial marker]13, hemoglobin beta embryonic-1.1 [hbbe1.1, an erythrocyte marker]14, crystallin-alpha A [cryaa, an eye lens marker]15, and glial fibrillary acidic protein [gfap, a central nervous system marker]16 (Figure 2B). The zebrafish eukaryotic translation initiation factor 1B [eif1b]12 was used as an internal reference gene (see Table 2 for GenBank ID numbers, primer sequences, PCR product sizes and tissue specificity for each gene). As expected, myl7 was highly enriched in the &#8220;heart&#8221; sample as compared to the &#8220;embryo without heart&#8221; sample (Fig. 2B). The endothelial cell marker kdrl was found to be moderately enriched in the heart sample, as expected (about 40% of the cardiac cells at 56 hpf are kdrl-expressing endocardial cells) (Fig. 2B). The erythrocyte marker hbbe1.1 was overrepresented in the &#8220;heart&#8221; sample, even though the hearts were still beating after dissection, which should expel red blood cells from the heart (Fig. 2B). In contrast, the expression levels of the CNS and lens markers (gfap and cryaa, respectively) were extremely low in the &#8220;heart&#8221; sample (Fig. 2B). Altogether, we conclude that the heart dissection protocol yields high quality and cardiac-specific RNA from entire zebrafish embryos.
<img alt="Figure 1" src="/files/ftp_upload/52087/52087fig1highres.jpg" /> 
Figure 1. Heart extraction from zebrafish embryos. (A) Scheme depicting the heart extraction procedure. About 100 live embryos are collected in a 1.5 ml tube (A1, B,B&#8217;), anesthetized and then pipetted up and down several times through a narrow pipette tip to release the hearts (A2). The resulting solution containing the embryonic tissues (A2) is then applied onto a 100 &#956;m filter (A3). Embryonic tissues without yolk and hearts, and large embryonic debris remain in the filter (A3). The flow-through is collected in a 50 ml tube (A3) and then applied onto a 30 &#956;m filter (A4). The hearts and small debris are retained in the 30 &#956;m filter and transferred into a small agarose-coated petri dish (A5, C,C&#8217;). EGFP labelled hearts (C,C&#8217;; arrow) are manually sorted from the remaining debris, such as lenses (C, arrowhead), and are collected in RNAlater (A6). This procedure is repeated at least 5 times and all hearts are pooled together for further processing. (B-C&#8217;) Overlay of fluorescence (EGFP in green) and DIC images of live transgenic Tg(myl7:EGFP)twu34 embryos at 56 hpf (B,C) and 36 hpf (B&#8217;,C&#8217;) at two steps of the heart dissection procedure: prior to embryo dissection (B,B&#8217;) and just after flushing from the 30 &#956;m filter, prior to sorting the hearts from the debris in the petri dish (C,C&#8217;). Scale bar: 500 &#956;m.
<img alt="Figure 2" src="/files/ftp_upload/52087/52087fig2highres.jpg" /> 
Figure 2. Analysis of RNA quality and purity by electrophoresis and RT-qPCR, respectively. (A) Total RNA from the &#8220;heart&#8221; sample (2 &#956;l) and from the &#8220;embryo without heart&#8221; sample (1 &#956;l), derived from 56 hpf embryos, resolved on a 1% agarose gel. Prominent S18 and S28 rRNA bands indicate that RNAs are not degraded. (B) Relative expression levels of the tissue specific markers myl7 (myocardium), kdrl (endothelium), hbbe1.1 (erythrocytes), cryaa (lens), and gfap (CNS) as determined by RT-qPCR for the &#8220;heart&#8221; sample normalized to the &#8220;embryo without heart&#8221; sample. The RT-qPCR experiments were performed as technical triplicates and data are given as means &#177; SEM. An unpaired t-test analysis was performed. **** p&#60;0,0001; ** p&#60;0,005. bp, base pair.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>sample ID</td>
			<td>ng/&#956;l</td>
			<td>A260</td>
			<td>A280</td>
			<td>260 / 280</td>
			<td>260 / 230</td>
			<td>cursor abs.</td>
			<td>340 raw</td>
		</tr>
		<tr>
			<td>hearts</td>
			<td>32.97</td>
			<td>0.82</td>
			<td>0.41</td>
			<td>2.01</td>
			<td>0.35</td>
			<td>2.355</td>
			<td>0.031</td>
		</tr>
		<tr>
			<td>embryos without hearts</td>
			<td>568.82</td>
			<td>14.22</td>
			<td>7.00</td>
			<td>2.03</td>
			<td>2.04</td>
			<td>6.965</td>
			<td>0.018</td>
		</tr>
	</tbody>
</table>
Table 1. RNA quantity and quality by spectrophotometric measurement. The quantity and quality of the RNA of the &#8220;heart&#8221; and &#8220;embryo without heart&#8221; samples obtained from 56 hpf embryos as measured by spectrophotometry.\
<table border="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>gene</td>
			<td>GenBank #</td>
			<td>primer sequence</td>
			<td>PCR product size (bp)</td>
			<td>Tissue-specific marker</td>
		</tr>
		<tr>
			<td rowspan="2">myl7</td>
			<td rowspan="2">BX248505</td>
			<td>F: 5&#39;-ACAGCAAAGCAGACAGTGAA-3&#39;</td>
			<td rowspan="2">163</td>
			<td rowspan="2">myocardium</td>
		</tr>
		<tr>
			<td>R: 5&#39;-TAACTCCATCCCGGTTCTGA-3&#39;</td>
		</tr>
		<tr>
			<td rowspan="2">kdrl</td>
			<td rowspan="2">CR759732</td>
			<td>F: 5&#39;-ACAACGACACTGGCATCTAC-3&#39;</td>
			<td rowspan="2">170</td>
			<td rowspan="2">endothelium/ endocardium</td>
		</tr>
		<tr>
			<td>R: 5&#39;-TGTTCTACAGGGGACCACAA-3&#39;</td>
		</tr>
		<tr>
			<td rowspan="2">gfap</td>
			<td rowspan="2">BX324157</td>
			<td>F: 5&#39;-AGACAACTTGGCCTCAGAC-3&#39;</td>
			<td rowspan="2">247</td>
			<td rowspan="2">central nervous system</td>
		</tr>
		<tr>
			<td>R: 5&#39;-ATCCACATGAACCTGTTGGG-3&#39;</td>
		</tr>
		<tr>
			<td rowspan="2">cryaa</td>
			<td rowspan="2">BX248514</td>
			<td>F: 5&#39;-ATCCAACACCCTTGGTTCAG-3&#39;</td>
			<td rowspan="2">190</td>
			<td rowspan="2">eye lens</td>
		</tr>
		<tr>
			<td>R: 5&#39;-TCAGACCTCACCTCAGAGAC-3&#39;</td>
		</tr>
		<tr>
			<td rowspan="2">hbbe1.1</td>
			<td rowspan="2">BC095024</td>
			<td>F: 5&#39;-TGGTTGTGTGGACAGACTTCGA-3&#39; 8</td>
			<td rowspan="2">102</td>
			<td rowspan="2">erythrocyte</td>
		</tr>
		<tr>
			<td>R: 5&#39;-CGATAAGACACCTTGCCAGAGC-3&#39; 8</td>
		</tr>
		<tr>
			<td rowspan="2">eif1b</td>
			<td rowspan="2">BX323079</td>
			<td>F: 5&#39;-CAGAACCTCCAGTCCTTTGATC-3&#39; 12</td>
			<td rowspan="2">195</td>
			<td rowspan="2">reference gene</td>
		</tr>
		<tr>
			<td>R: 5&#39;-GCAGGCAAATTTCTTTTTGAAGGC-3&#39; 12</td>
		</tr>
	</tbody>
</table>
Table 2. Primers used for the RT-qPCR experiments. Name, GenBank accession number, sequence, PCR product sizes and tissue-specificity are shown for all genes analyzed.

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

Nanyang Technological University

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Developmental Biology

14.3K Views.  Max Delbrück Center for Molecular Medicine. 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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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