The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.
This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.
The zebrafish (Danio rerio) has emerged as a powerful animal model for the study of vertebrate development, disease, behavior, and in-drug screening1-3. Zebrafish embryos can be obtained in large numbers from a single crossing. Fertilization and development occurs ex utero and the optically clear embryos develop rapidly. Critical developmental events occur during the first 48 hr post-fertilization (hpf). This includes the appearance of organ primordia and the initiation of cytodifferentiation. Knockdown or over-expression of proteins can be achieved through the microinjection of embryos with antisense morpholino (MO) oligonucleotides or mRNA respectively at the one or two cell stage (0.75 hpf).
The WISH of zebrafish embryos facilitates the study of the spatio-temporal expression of specific genes of interest. Application of the WISH technique following microinjection of embryos with mRNA or MO to over-express or knockdown specific protein levels reveals differential regulation of other genes.
Changes in gene expression patterns can be correlated with phenotypic changes and reveal the function of target genes during early organogenesis. Since probes can be prepared and stored in advance, the ISH technique can be applied to reveal gene expression patterns for at least 20 genes at a time using six-well plates and custom made baskets.
1. Whole-mount In Situ Hybridization of Zebrafish Embryos
1.1 Preparation of Embryos
1.2 Synthesis of Digoxygenin-labeled RNA Probes
Reagent | Volume |
Template DNA | 100 ng |
PCR Buffer (10x) | 5 μl |
dNTPs (10 mM) | 2 μl |
Primers (10 mM) | 1 μM each |
Add water to a final volume of | 49.7 μl |
Taq Polymerase (5 unit/μl) | 0.3 μl |
Reaction Volume | 50 μl |
Table 1. PCR amplification reaction.
Reagent | Chemically Competent Cells | Electro Competent Cells |
Fresh PCR reaction product | 1-4 μl | 1-4 μl |
Salt Solution | 1 μl | |
Diluted Salt Solution | 1 μl | |
Water | Up to total volume of 5 μl | Up to total volume of 5 μl |
TOPO vector | 1 μl | 1 μl |
Final Volume | 6 μl | 6 μl |
Table 2. TOPO cloning reaction.
Reagent | Volume |
NTP labeling mixture (10x) | 2 μl |
Transcription buffer (10x) | 2 μl |
Protector RNase inhibitor | 1 μl |
RNA Polymerase SP6 or | |
RNA Polymerase T7 | 2 μl |
Final Volume | 20 μl |
Table 3. RNA labeling reaction.
1.3 Whole-mount In Situ Hybridization
Embryonic Stage (hours post fertilization) | Digestion Period (Proteinase K) |
Up to 6 | 15 sec |
6-12 | 30 sec |
12-18 | 3 min |
24 | 12-15 min |
48 | 25-30 min |
72 | 40 min |
96-120 | 50 min |
2. Double In Situ Hybridization
Using the protocol with 50 embryos per basket (per gene/per experimental condition) the expression pattern of that gene can be achieved in one experiment. Almost all the embryos show similar expression patterns for a particular gene. Representative examples of the in situ hybridization staining are shown in Figures 3-6.
Both the sense and anti-sense riboprobes were synthesized from the cDNAs corresponding to PC5.1, PC5.26, SCL/tal-17, gata-18,9, flk/ kdrl10, shh11, dlx212, fkd7/foxa113, insulin14,15, and trypsin16 have been constructed by amplifying a segment of the full-length mRNA using Taq DNA polymerase, and cloned into pCRII-TOPO (Invitrogen). The authenticity of individual amplicons was confirmed by sequencing. After verification of the clone orientation, corresponding antisense riboprobes were synthesized using the protocols17 with modifications18,19 as described here using either the Sp6 or T7 RNA polymerases. Steps involved in whole-mount in situ hybridization are illustrated briefly in Figure 2. Purple staining observed after hybridization with riboprobe of interest represents both the abundance and the sites of expression of particular gene. For instance PC5.1 shows very discrete expression within the anterior and posterior part of otic vesicle and lateral line primordium at 24hpf and is strongly localized within the anterior and posterior lateral line neuromasts by 72 hpf (Figure 3). In contrast PC5.2 shows ubiquitous expression within the CNS with distinct regionalization within the somites, otic vesicle and pronephric duct and is highly expressed within the liver and intestine at 96 hpf (Figure 4). Absence of gene expression when using respective sense riboprobes serves as a negative control. (Figures 3b and 4b). Expression analyses of blood markers showed staining of SCL/tal-1 within the bilateral cranial cells and in the intermediate cell mass (ICM). Although the staining for gata-1 is also seen within the ICM the expression pattern is different. The expression of flk/ kdrl was observed within the hindbrain, main and intersegmental vessels and in the ICM (Figure 5). The staining for shh was observed in the notochord (Figure 5). The expression of dlx2 at 34 hpf is localized in the telencephalon, diencephalon, hypothalamus and cranial ectomesenchymal arches and the fkd7/foxa1 expression at 24 hpf is seen mainly in the floor plate and hypochord. Expression patterns of the pancreatic markers showed insulin staining in the endocrine pancreas and trypsin in the exocrine pancreas (Figure 6). These results showing expression pattern with high resolution represents the success of the outlined protocol for the detection of the expression of both high and low abundant genes. For further clarity images can be taken using confocal microscopy after mounting the embryos on a microscopic slide20. Some of the examples of the results obtained when the experiment was undertaken under sub-optimal conditions during optimization of the protocol are shown in Figure 7. High background signal with poor shh expression was noticed under inadequate permeabilization and hybridization between 55-60 °C.
Figure 1. Preparation and use of Eppendorf-nylon mesh baskets to hold staged embryos. One well can hold six Eppendorf-nylon mesh baskets. The image shows the 6-well sterile plates containing six Eppendorf-nylon mesh baskets used for successive dilutions of methanol in PBS.
Figure 2. Diagram showing the steps involved in the whole-mount in situ hybridization technique. After fixing staged embryos can be stored in methanol at -20 °C until used. Probes can be synthesized in advance and kept at -80 °C. Storing probes in small aliquots is advisable if they are to used over long periods. In general whole-mount in situ hybridization experiments can be completed in 3 or 4 days. The staining reaction for weakly expressed genes will take up to 16 hr at room temperature or longer when the staining reaction is undertaken at 4 °C Click here to view larger figure.
Figure 3. The expression of PC5.1 by whole-mount in situ hybridization analyses was carried out using 24 hpf and 72 hpf staged embryos. (a) Lateral view of 24 hpf embryos show very discrete expression of PC5.1 within the anterior and posterior part of the otic vesicle and lateral line primordium using PC5.1 antisense riboprobe. At 72 hpf specific staining was observed within the anterior and posterior lateral line neuromasts. (b) Absence of gene expression using PC5.1 sense riboprobe serves as a negative control. Click here to view larger figure.
Figure 4. At 24 hpf and 96 hpf whole-mount in situ hybridization analyses of PC5.2 was carried out using PC5.2 anti-sense and sense riboprobes. (a) Lateral view of 24 hpf embryos show ubiquitous expression within the CNS, somites otic vesicle and pronephric duct. Specific staining in the liver and gut is seen at 96 hpf using PC5.2 antisense riboprobe. (b) Absence of gene expression using PC5.2 sense riboprobe serves as a negative control. Click here to view larger figure.
Figure 5. Whole-mount in situ hybridization analyses at 24 hpf using SCL/tal-1 riboprobe showed staining within the bilateral cranial cells and in the intermediate cell mass (ICM); gata-1 riboprobe staining is seen in the ICM; the flk/ kdrl expression within the hindbrain, main, and intersegmental vessels and in the ICM. The staining for shh was observed in the notochord.
Figure 6. Whole-mount in situ hybridization analyses at 34 hpf using dlx2 riboprobe revealed expression of dlx2 in the telencephalon, diencephalon, hypothalamus, and cranial ectomesenchymal arches; the fkd7/foxa1 expression at 24 hpf is seen mainly in the floor plate and hypochord; insulin staining is observed in the endocrine pancreas and trypsin expression in the exocrine pancreas.
Figure 7. Adequate permeabilization and appropriate hybridization temperature are critical for clear in situ hybridization signals. Whole-mount in situ hybridization analyses at 24 hpf and 48 hpf using an shh riboprobe revealed expression within the notochord and floorplate without background signal when embryos are permeabilized by proteinase K digestion for 12-15 min and hybridized at 70 °C. Incomplete expression patterns and high background signals were observed when the embryos were subjected to inadequate digestion (5 min proteinase K treatment) or hybridized at low temperature (60 °C). Click here to view larger figure.
We have developed improved methods to visualize RNA with high resolution. The in situ hybridization procedures are carried out using simple custom-made baskets with porous bottoms (Figure 1). Embryos are processed using RNase-free solutions in 6-well plates at room temperature under sterile conditions.
To segregate embryos baskets are made from different colored Eppendorf tubes. Baskets with or without a rim are used for easy orientation and to identify the embryos within the basket as corresponding to a particular probe.
Prehybridization at 65 °C and hybridization at 70 °C with 50% deionized formamide was found to be particularly important to obtain high resolution signals. In addition incubating embryos twice with alkaline Tris buffer for 15 min post PBST washes after incubation with anti-DIG antibody and prior to incubation in the presence of BCIP and NBT facilitates the appearance of high quality clear signals. In our hands we experienced that the permeabilization and fixing of embryos for the optimal time and hybridization at 70 °C were very critical in obtaining clear signals. Inadequate permeabilization produced high background signals. Extended permeabilization or inadequate fixing resulted in the degradation of embryos whereas extended fixing inhibited signal detection. Hybridization between 55-60 °C also produced high background signals (Figure 7).
The sense probe will detect a signal for the AS transcript if the gene of interest encodes antisense (AS) transcripts. After the staining reaction is stopped placing embryos in methanol for 30 min or longer converts the signal to a true purple color, and removes non-specific staining. Fixed embryos can be stored in the dark in stop solution at 4 °C for several months.
The advantages and disadvantages of WISH
WISH is an efficient technique to characterize both the temporal and spatial expression of target genes during embryogenesis and larval stages. This protocol also permits visualization of RNA expression of two genes or co-localization of RNA and protein expression at the same time using appropriate modifications depending upon the experimental objectives. This high resolution protocol can be used to detect RNA expression in many tissues and cell types. It can be used for the detection of very low abundant RNA species with minimal background signals. However, to visualize accurate expression of genes within interior compartments of tissues requires in situ hybridization using tissue sections.
The authors have nothing to disclose.
Taq Polymerase | Invitrogen | 10342053 | |
TopoTA Cloning Kit- Dual Promoter pCRII-TOPO | Invitrogen | K4650-01 | |
HQ Mini Plasmid Purification Kit | Invitrogen | K2100-01 | |
Agarose | Roche | 11 685 660 001 | |
Not1 and HindIII Restriction Enzymes | Invitrogen | 15441-025, 15207-012 | |
Buffer Saturated Phenol, ultrapure | Invitrogen | 15513039 | Store at 4 °C. Eyes, skin, and respiratory tract irritant and suspected carcinogen. Care should be taken while handling. |
Chloroform | Fisher | C607-1 | Toxic and suspected carcinogen. Work under fume hood. |
RNase-free DNAse I | Roche | 4716728001 | Prepare small aliquots and store at -20°C. |
DIG-RNA Labeling Mix with Sp6, T7 and T3 RNA Polymerase | Roche | 11277073910 | |
RNase Inhibitor | Roche | 10777-019 | |
3.0 M Sodium Acetate (pH 5.5) | Fisher | 50-751-7355 | |
100% Ethanol | Commercial alcohols | ||
Diethyl Pyrocarbonate (DEPC) | Sigma | 40718-25ML | DEPC is an eye, skin, and respiratory irritant. Avoid contact with skin and eyes. Use safety glasses, gloves, and mask. |
NaOH | Fisher | SS255-1 | |
EDTA 0.5 M (pH 8.0) | Fisher | 50-751-7404 | |
Instant Ocean Salt | Aquarium Systems | N/A | |
Phosphate Buffered Saline (PBS) | Fisher | FL-03-0900 | |
Paraformaldehyde (PFA) | Sigma | P6148-1KG | Prepare and store at -20°C as 40 ml aliquots for later use. PFA is toxic. Use safety glasses, gloves, and dust mask. |
Phenylthiocarbomide (PTC) | Sigma | P7629-25G | PTC is highly toxic. Use safety glasses, gloves, and dust mask. |
Methanol | Fisher | A947-4 | |
Dumont (Watchmaker’s) Forceps pattern no. 5 | Fine Science Tools | ||
Six-well Cell Culture Cluster | Sarstedt | 83.1839 | |
Baskets are made of nylon mesh and Eppendorf tubes | In-house preparation | To make the baskets, cut Eppendorf tubes (1.5 ml) with or without rims to remove the conical end. Cut nylon mesh into small pieces corresponding to the size of the cut ends of the Eppendorf tubes. Place tubes with the nylon mesh covering the cut end on an electrical hot plate until both the tube and nylon mesh stick together (carry out in fume hood). Cut off the excess mesh from the baskets and store them in 100% methanol until used. | |
Polyoxyethylenesorbitan Monolaurate (Tween 20) | Sigma | P1379-500ML | |
Proteinase K | Fermentas | EO0491 | |
Acetic Anhydride | Sigma | 242845 | |
Triethanolamine | Sigma | 90279-100ML | |
Formamide, high purity grade | Sigma | F9037 | |
AG501-X8 Resin | Bio Rad | 142-6424 | |
Citric Acid monohydrate | Sigma | C0706-500G | |
Heparin Sodium Salt | Sigma | H3393-25KU | |
Saline-sodium Citrate Buffer (SSC) | Sigma | S6639 | |
tRNA from bakers yeast type X | Sigma | R5636-1ML | |
NaCl | Fisher | S640-500 | |
Tris-HCl | Fisher | BP153-1 | |
MgCl2 | Fisher | S25403 | |
Levamisole Hydrochloride | Sigma | 31742 | |
N,N-Dimethylformamide anhydrous (DMF) |
Sigma | 227056 | Irritant, toxic, combustible, and suspected teratogen. Handle with proper safety attire including gloves and goggles. |
5-Bromo-4-chloro-3-indolyl phosphate (BCIP) | Sigma | N6639 | Protect this solution from light. |
Nitroblue tetrazolium (NBT) | Sigma | 840W | Protect this solution from light. |
Albumin from Bovine Serum, purified fraction V (BSA) | Sigma | A8806 | |
Sheep Anti-digoxigenin-AP Fab Fragments | Roche | 1093274910 | |
Glycerol | Sigma | G5516-500ML | |
96-well cell culture plates | Sarstedt | 83.1835 | |
Tg(mnx1:GFP)ml2/Tg(hb9:GFP)ml2 | ZIRC | Tg(mnx1:GFP)ml2 | |
Instant Ocean | Aquarium Systems | N/A |