Name: heart dissection in larval, juvenile and adult zebrafish, danio rerio
Uploaded: 2011-09-30T12:00:00
Description: Watch this Scientific Journal Video about Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio at JoVE.com

Thanks to Marie Birne, Areti Tsiola, Jaymie Estevez and Arelys Uribe. This work was supported in part by CUNY Research Foundation and Howard Hughes Medical Institute Grant Summer Program for Undergraduate Students (SPUR) and NIH RO3 41702-00-01. Research was also supported by Queens College and some of the experiments were done on equipment from the Core Facility for Imaging, Cellular and Molecular Biology at Queens College.

Zebrafish were raised using standard protocols13 in the Queens College Animal Facility at 28&deg;C. Individual fish pairs or group matings should be preformed and the resulting embryos cleaned and stored in a 28&deg;C incubator. Fish health should be examined daily. At 5-day post fertilization (dpf), fish should be separated into tanks at a 10-15 fish density and entered into the fish facility nursery. For the protocol presented here zebrafish were collected at larva through adult stages: 15, 30, 45, 60, 90, 1...

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These methods for dissection of the zebrafish heart of post-embryonic fish allows for the complete removal of an undamaged heart. While this technique is straightforward and provides guides for external cuts that will prevent damaging the heart, a steady hand is essential for smaller fish.
A key step during the dissection is to identify the silvery pericardium as the heart is directly within this sac. Visualizing the pericardium provides an important marker for finding and ultimately removing ...

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 <tr>
 <th>Material</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>Paraformaldehyde </td>
 <td>EMD</td>
 <td>PX0055 </td>
 <td>.04g/mL in PBS</td>
 </tr>
 <tr>
 <td>Forceps #55 </td>
 <td>FST</td>
 <td>11295-51</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro-scissors </td>
 <td>FST</td>
 <td>15005-08</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pin holder </td>
 <td>FST </td>
 <td>26016-12</td>
 <td>&nbsp;</td>
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 <td>Micro-needles </td>
 <td>FST </td>
 <td>10130-10</td>
 <td>&nbsp;</td>
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 <td>Nutrient Agar </td>
 <td>BBL</td>
 <td>11472</td>
 <td>23g/L in distilled H2O</td>
 </tr> 
 <tr>
 <td>Petri dishes</td>
 <td>BD Falcon</td>
 <td>351029</td>
 <td>&nbsp;</td>
 </tr>
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 <td>PCR 8-tube strips </td>
 <td>USA Scientific</td>
 <td>1402-2500</td>
 <td>&nbsp;</td>
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 <td>Digital Caliper</td>
 <td>Fisher Scientific</td>
 <td>14-648-17</td>
 <td>&nbsp;</td>
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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.

Watch this Scientific Journal Video about Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio at JoVE.com

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.

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 ...

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