Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Summary

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.

Abstract

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.

Introduction

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 mutants^1,2 and the zebrafish is now a widely recognized model organism to study heart diseases

Protocol

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

1. Cross cardiac reporter zebrafish such as the Tg(myl7:EGFP)^twu34 transgenic line⁹ in order to obtain embryos with heart-specific GFP expression.
2. Maintain the embryos in egg water at 28.5 °C until the desired embryonic stage^10,11. Ensure that the collected embryo population is homogeneous both genetically and by developmental....

Results

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 .......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment for raising fish and collecting eggs			see the Zebrafish Book11 for details
Fluorescence stereomicroscope
Refrigerated Microcentrifuge
UV-Spectrophotometer			e.g. Thermo Scientific Nanodrop 2000
Nucleic acid electrophoresis chamber
Petri dishes 4 cm Ø, coated with 1% agarose in E3 medium
Micropipettes and tips (P20, P100, P1000)
1.5 ml centrifugation tubes
15 ml and 50 ml centrifugation tubes
Pair of Dumont #5 forceps
ExactaCruz™ Round Gel Loading Tips in Sterile Rack, 1-200μl	Santa Cruz	sc-201732
100 μm filter (BD Falcon 100 mm Cell Strainer)	BD Biosciences	352360
30 μm filter (Pre-Separation Filters-30 µm)	Miltenyi Biotec	130-041-407
Phase lock gel, heavy, 1.5 ml tubes	Prime	2302810
Egg water medium			60 μg/ml Instant Ocean Sea Salts in ddH2O, 0.00001% (w/v) Methylene Blue
E3 medium			5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4
Tricaine (3-amino benzoic acidethylester)	Sigma-Aldrich	A-5040	4 mg/ml Tricaine stock solution, pH 7
1% agarose (in E3 medium)
1% agarose gel (in TBE buffer)
Leibovitz´s L-15 medium	Gibco	21083-027
FBS (Fetal Bovine Serum)	Sigma	F4135
RNAlater	Ambion	AM7020
Trizol	Ambion	1559606
Glycogen	Invitrogen	10814-010	20 µg/µl in RNase-free water
chloroform
isopropanol
75% ethanol (in DEPC-ddH2O)
Nuclease-free water or sterilized DEPC treated ddH2O
Nucleic acid loading buffer
TBE (Tris/Borate/EDTA) buffer for electrophoresis