High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

Summary

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.

Abstract

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.

Introduction

The zebrafish (Danio rerio) has emerged as a powerful animal model for the study of vertebrate development, disease, behavior, and in-drug screening^1-3. 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 mR....

Protocol

1. Whole-mount In Situ Hybridization of Zebrafish Embryos

1.1 Preparation of Embryos

1. Collect embryos at the required developmental stages. Please see Kimmel et al.5 for embryonic stage description.
2. Remove chorions manually using forceps (Dumont Watchmakers Forceps no. 5).
3. Fix embryos in a 4% solution of paraformaldehyde (PFA) made up in PBS (Phosphate Buffered Saline) overnight at 4 °C.
4. Wash embryos with PBS at least 3x for 5 min each at room temperature.
5. Store embryos in 100% methanol (MeOH) at -20 °C until used for future application of techniques including whole....

Results

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-1^.......

Discussion

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 wi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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