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

Summary

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.

Abstract

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews^1-6), however, work examining post-embryonic through adult cardiac development has been limited^7-10. 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 100μm 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.

Protocol

Zebrafish were raised using standard protocols13 in the Queens College Animal Facility at 28°C. Individual fish pairs or group matings should be preformed and the resulting embryos cleaned and stored in a 28°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…

Discussion

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 …

Materials

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