Spatial and Temporal Expression of the Zebrafish Genome by Large-Scale In Situ Hybridization Screening

doi:10.1016/S0091-679X(04)77027-2

Methods in Cell Biology

Volume 77, 2004, Pages 505-519

https://doi.org/10.1016/S0091-679X(04)77027-2 Get rights and content

Publisher Summary

This chapter focuses on spatial and temporal expression of the zebrafish genome by large-scale in situ hybridization screening. Wholemount in situ hybridization is a method widely used to describe the expression patterns of developmentally regulated genes. Use of a highly sensitive in situ hybridization assay allows for reliable visualization of the gene expression, including the genes expressed at low levels. The chapter describes a technique that employs in vitro synthesized RNA tagged with either digoxigenin (DIG) or fluoresceinuridine-5´-triphosphate (UTP) to determine gene expression patterns in wholemount embryos. Following hybridization, the transcript is visualized immunohistochemically, using an antidigoxygenin (or antifluorescein) antibody conjugated to alkaline phosphatase, the substrate of which is chromogenic. The in situ hybridization technique described here is important for defining synexpression groups. Synexpression analysis can reveal that a group of genes share temporal and spatial expression patterns, suggesting that they might be controlled by the same signaling pathways. Over a 6-year period, researchers have analyzed more than 17,000 cDNAs and identified 4600 spatially restricted expression patterns. Because of redundancy (33% established by comparison with the genome sequence), this corresponds to about 3000 different genes. Descriptions of more than 1000 gene expression patterns have been released to the public through ZFIN in the gene expression section.

Section snippets

Introduction and Goals

Whole-mount in situ hybridization is a method widely used to describe the expression patterns of developmentally regulated genes. Use of a highly sensitive in situ hybridization assay allows for reliable visualization of gene expression, including genes expressed at low levels. Here we describe a technique that employs in vitro synthesized RNA tagged with either digoxigenin (DIG) or fluorescein uridine-5′-triphosphate (UTP) to determine gene expression patterns in whole-mount embryos. Following

Isolation and Preparation of the DNA Template

This method is used to prepare large amounts of antisense RNA probes and is divided into two steps: preparation of DNA and synthesis of antisense RNA probe.

For preparation of the DNA template, 5 μg of DNA is linearized in a 2-h digestion, using the appropriate restriction enzyme under appropriate conditions of salt and temperature. Care must be taken to ensure that the direction of insert of interest is known to allow for the correct production of the antisense and sense probes. Once the

Preparation of Embryos

Eggs are collected from single mating pairs about 1 h after laying. They are cleaned and unfertilized eggs are discarded. Embryos are allowed to develop in regular fish water until the end of gastrulation. For embryos older than 24 h, to prevent pigmentation, fish water is replaced at the end of gastrulation (10 hpf) by a 0.0045% solution of 1-phenyl-2-thiourea (Sigma, Cat. No. P-7629) in 1× Danieau's medium (58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO₄, 0.6 mM Ca(NO₃), 2, 5 mM HEPES, pH 7.6).

Chorions

Reagents and Buffers for In Situ Hybridization

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10× PBS (Dulbecco, Sigma, Cat. No. D-5652).
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MeOH.
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Tween 20 (Sigma, Cat. No. P-1379).
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Proteinase K (Boehringer, Cat. No. 1000 144).
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Anti-DIG antibody-alkaline phosphatase Fab fragment (Boehringer, Cat. No. 1 093 274).
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BSA fraction V, protease free (Sigma, Cat. No. A-3294).
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Formamide: high-purity grade (Sigma or Carlo Erba, Cat. No. 452286), deionized by adding and stirring slowly twice for 15 min each with 10 g⧸l Serdolit MB-3 (Serva, Cat. No. 40721). The solution is filtered to remove the resin and

In Situ Hybridization Protocol

This protocol is adapted from Thisse 1993, Thisse 2001, Thisse 1998).

Preparation of Probes

Two different antisense RNA probes are used, one labeled with DIG 11-UTP and the second with fluorescein 12-UTP. The protocol for synthesis with fluorescein 12-UTP is identical to that used for the DIG 11-UTP described previously, except for the transcription mix. This fluorescein-labeled probe is kept in the dark as much as possible during the in situ hybridization steps. Incubate for 2 h at 37 °C 1 μg linearized DNA with the following:

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2 μl 100 mM DTT.
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1.3 μl NTP mix (16.4 μl ATP 100 mmol⧸l,

Recording Results

Labeled embryos are mounted in 100% glycerol. Because of the photosensitivity of the yolk cell, embryos at early developmental stages are first treated for 5 min in an acidic buffer (either PBS, pH < 3.5, or glycine buffer, pH 2.2). This treatment prevents photoreactivity of the yolk proteins, and even under intense light the yolk cell remains unstained. However, this acidic treatment affects embryo morphology. Therefore, although very convenient at early developmental stages (when only a few

Concluding Remarks

Over a 6-year period, we have analyzed more than 17,000 cDNAs and identified 4600 spatially restricted expression patterns. Because of redundancy (33% established by comparison with the genome sequence) this corresponds to about 3000 different genes. Descriptions of more than 1000 gene expression patterns have been released to the public through ZFIN (http:⧸⧸zfin.org, mirror sites in France at http:⧸⧸www.igbmc.u-strasbg.fr and in Japan at http:⧸⧸:www.grs.nig.ac.jp:6060) in the gene expression

Acknowledgements

We thank S. Geschier for maintenance of the fish and M. Fürthauer, A. Agathon, S. Obrecht-Pflumio, and B. Loppin for their contribution at different steps of the analysis. This work was supported by funds from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Hôpital Universitaire de Strasbourg, and the National Institutes of Health (R01 RR15402).

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