Abstract
Examination of spatial and temporal gene expression pattern is a key step towards understanding gene function. Therefore, in situ hybridization of mRNA is one of the most powerful and widely used techniques in biology. Recent advances allow the reliable and simultaneous detection of mRNA transcripts, or combinations of mRNA and protein, in zebrafish embryos.
Here we describe a standard protocol for visualizing the precise expression pattern of a single transcript or multiple gene products. The procedure employs fixation and permeabilization of embryos, followed by hybridization with tagged antisense riboprobes. Excess probes are then washed and hybrids are detected by enzyme-mediated immunohistochemistry utilizing either chromogenic or fluorescent substrates.
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References
O’Keefe, H.P., Melton, D.A., Ferreiro, B. and Kintner, C. (1991) In situ hybridization. Methods Cell. Biol. 36, 443–463.
Thisse, B., Heyer, V., Lux, A., Alunni, V., Degrave, A., Seiliez, I., Kirchner, J., Parkhill, J. P. and Thisse, C. (2004) Spatial and temporal expression of the zebrafish genome by large-scale in situ hybridization screening. Methods Cell. Biol. 77, 505–519.
Hauptmann, G. (1999) Two-color detection of mRNA transcript localizations in fish and fly embryos using alkaline phosphatase and beta-galactosidase conjugated antibodies. Dev. Genes Evol. 209, 317–321.
Jowett, T. (2001) Double in situ hybridization techniques in zebrafish. Methods 23, 345–358.
Clay, H. and Ramakrishnan, L. (2005) Multiplex fluorescent in situ hybridization in zebrafish embryos using tyramide signal amplification. Zebrafish 2, 105–111.
Welten, M. C., de Haan, S. B., van den Boogert, N., Noordermeer, J. N., Lamers, G. E., Spaink, H. P., Meijer, A. H. and Verbeek, F. J. (2006) ZebraFISH: fluorescent in situ hybridization protocol and three-dimensional imaging of gene expression patterns. Zebrafish 3, 465–476.
Thisse, C, Thisse, B. (2008) High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat. Protoc. 3, 59–69.
Broadbent, J. and Read, E. M. (1999) Wholemount in situ hybridization of Xenopus and zebrafish embryos. Methods Mol. Biol. 27, 57–67.
Schulte-Merker, S., Ho, R. K., Herrmann, B. G. and Nüsslein-Volhard, C. (1992) The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo. Development 116, 1021–1032.
Thisse, C., Thisse, B., Schilling, T. F. and Postlethwait, J. H. (1993) Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos. Development 119, 1203–15.
Sprague, J., Bayraktaroglu, L., Clements, D., Conlin, T., Fashena, D., Frazer, K., Haendel, M., Howe, D. G., Mani, P., Ramachandran, S., Schaper, K., Segerdell, E., Song, P., Sprunger, B., Taylor, S., Van Slyke, C. E. and Westerfield, M. (2006) The Zebrafish Information Network: the zebrafish model organism database. Nucleic Acids Res. 34, D581–585.
Paragas, V. B., Zhang, Y. Z., Haugland, R. P. and Singer, V. L. (1997) The ELF-97 alkaline phosphatase substrate provides a bright, photostable, fluorescent signal amplification method for FISH. J. Histochem. Cytochem. 45, 345–357.
Jékely, G. and Arendt, D. (2007) Cellular resolution expression profiling using confocal detection of NBT/BCIP precipitate by reflection microscopy. Biotechniques 42, 751–755.
Trinh, le A., McCutchen, M. D., Bonner-Fraser, M., Fraser, S. E., Bumm, L. A. and McCauley, D. W. (2007) Fluorescent in situ hybridization employing the conventional NBT/BCIP chromogenic stain. Biotechniques 42, 756–759.
Brend, T. and Holley, S.A. (2009) Zebrafish whole mount high-resolution double fluorescent in situ. J. Vis. Exp. 25, pii: 1229. doi: 10.3791/1229.
Tessmar-Raible, K., Steinmetz, P. R., Snyman, H., Hassel, M. and Arendt, D. (2005) Fluorescent two-color whole mount in situ hybridization in Platynereis dumerilii (Polychaeta, Annelida), an emerging marine molecular model for evolution and development. Biotechniques 39, 460–462.
Hargrave, M., Bowles, J. and Koopman P. (2006) In situ hybridization of whole-mount embryos. Methods Mol. Biol. 326, 103–113.
Coverdale, L. E., Burton, L. E. and Martin, C. C. (2008) High-throughput whole mount in situ hybridization of zebrafish embryos for analysis of tissue-specific gene expression changes after environmental perturbation. Methods Mol. Biol. 410, 3–14.
Acknowledgments
We are grateful to Nataliya Borodovsky and Amos Gutnick for stimulating discussion and comments on this manuscript. Nataliya Borodovsky also provided the ISH images. The research in the Levkowitz lab is supported by the German-Israeli Foundation (grant number 183/2007); Israel Science Foundation (grant number 928/08) and the Harriet & Marcel Dekker Foundation. G.L. is an incumbent of the Tauro Career Development Chair in Biomedical Research.
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Machluf, Y., Levkowitz, G. (2011). Visualization of mRNA Expression in the Zebrafish Embryo. In: Gerst, J. (eds) RNA Detection and Visualization. Methods in Molecular Biology, vol 714. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-005-8_6
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DOI: https://doi.org/10.1007/978-1-61779-005-8_6
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