Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 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.

Protokół

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 stage to limit variations in gene expression.
3. Dechorionate the embryos manually.

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.

1. 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).
2. 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.
3. 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).
4. 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.
5. Apply the sample onto a 100 μm 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.
6. Apply the flow-through onto a 30 μm 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.
7. 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.
8. 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 older than 24 hpf, the hearts should be beating, indicating that the tissues are still physiologically normal.

3. Isolating mRNA from Dissected Hearts

1. Collect the hearts in the smallest possible volume (e.g. 10 µl) 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.
2. 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; the final 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.
3. 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).
4. Centrifuge the samples at 15,700 x g for 20 min at 4 °C to sediment the hearts.
5. 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.
6. Under a chemical hood, add 0.5 ml Trizol to the tube containing the sedimented hearts and keep on ice.
7. 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.
8. Vortex the 1.5 ml tube containing the hearts in Trizol to disrupt the hearts and incubate for 5 min at room temperature.
9. Under a chemical hood, add 100 µl 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.
10. Centrifuge the sample at 15,700 x g for 15 min at 4 °C and transfer the aqueous phase into a new 1.5 ml tube.
11. Add 5-10 µg glycogen and 250 µl precooled (at -20 °C) isopropanol; mix well and incubate over night at -20 °C.
12. Centrifuge the sample at 15,700 x g for 30 min at 4 °C and discard the supernatant.
13. Wash the pellet with 1 ml 75% ethanol, centrifuge at 15,700 x g for 15 min at 4 °C and discard the supernatant.
14. Let the ethanol evaporate by drying the pellet at room temperature until it becomes white (e.g. for 10-15 min).
15. Add 20 µl RNase-free ddH₂O to the pellet and incubate for 10-15 min at 55 °C to dissolve the RNA; then keep on ice.
16. Assess the RNA concentration and purity by absorbance measurement. Assess the RNA integrity by running the sample on a 1% agarose gel.

Wyniki

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 ...

Dyskusje

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...

Materiały

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