Abstract
Whole mount in situ hybridization (WMISH) is a method for detecting specific messenger ribonucleic acids (mRNAs) at their site of expression within an embryo or intact tissue fragment. Like conventional in situ hybridization, WMISH depends upon the availability of a complementary nucleic acid probe which can be labeled, annealed to the mRNA fixed within the tissue, and subsequently detected. Whereas the conventional approach uses radioactive probes hybridized to tissue sections, WMISH refers to hybridization to intact tissue or embryos using nonradioactive probes that are detected using standard immunohistochemical methods. For developmental biologists, WMISH offers the advantage of visualizing the domain of expression of a gene within the context of the entire embryo. In addition, WMISH can be used to study more than one gene in the same embryo, allowing spatial and temporal overlaps in expression to be clearly discerned (Fig 1). Similar to conventional in situ hybridization, one can determine the precise cellular distribution of expression by embedding and sectioning embryos following WMISH. For these reasons WMISH has become an essential and standard tool for studying gene expression during embryonic development (e.g., see 1).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lowe, L. A., Supp, D. M., Sampath, K., Yokoyama, T., Wright, C. V., Potter, S. S., Overbeek, P., and Kuehn, M. R. (1996) Conserved left-right asymmetry of nodal expression and alterations in murine situs inversus. Nature 381, 158–161.
Hemmati-Brivanlou, A., Frank, D., Bolce, M. E., Brown, B. D., Sive, H. L., and Harland, R. M. (1990) Localization of specific mRNAs in Xenopus embryos by whole-mount in situ hybridization. Development. 110, 325–330.
Conlon, R. A. and Herrmann, B. G. (1993) Detection of messenger RNA by in situ hybridization to postimplantation embryo whole mounts, in Methods in Enzymology, vol. 225, Guide to Techniques in Mouse Development (Wassarman, P. M. and DePamphilis, M. L., eds.), Academic, San Diego,CA, pp. 373–383.
Hogan, B., Beddington, R., Costantini, F., and Lacy, E. (1994) Manipulating the Mouse Embryo. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Plainview,NY.
Jowett, T. and Lettice, L. (1994) Whole-mount in situ hybridizations on zebrafish embryos using a mixture of digoxigenin-and fluorescein-labelled probes. Trends Genet. 10, 73–74.
Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J., and Ish-Horowicz, D. (1995) Expression of a Delta homologue in prospective neurons in the chick. Nature 375, 787–790.
Nieto, M. A., Patel, K., and Wilkinson, D. G. (1996) In situ hybridization analysis of chick embryos in whole mount and tissue sections. Methods Cell Biol. 51, 219–235.
Conlon, R. A. (1997) Whole mount in situ hybridization to embryos and embryonic tissues, in Methods in Molecular Biology, vol. 63, Recombinant Protein Protocols: Detection and Isolation(Tuan, R. S., ed.), Humana Press, Totowa, NJ, pp. 257–262.
Hsu, C. W. and Tuan, R. S. (1997) Whole-mount in situ hybridization for developing chick embryos using digoxygenin-labeled RNA probes, in Methods in Molecular Biology, vol. 63, Recombinant Protein Protocols: Detection and Isolation (Tuan, R. S., ed.), Humana Press, Totowa, NJ, pp. 263–270.
Stern, C. D. (1998) Detection of multiple gene products simultaneously by in situ hybridizationand immunohistochemistry in whole mounts of avian embryos. Curr. Top. Dev. Biol. 36, 223–243.
Stachel, S. E., Grunwald, D. J., and Myers, P. Z. (1993) Lithium perturbation and goosecoid expression identify a dorsal specification pathway in the pregastrula zebrafish. Development 117, 1261–1274.
Cockroft, D. L. (1990) Dissection and culture of postimplantation embryos, in PostimplantationMammalian Embryos-A Practical Approach, (Copp, A. J. and Cockroft, D. L., eds.), IRL, Oxford, UK, pp. 15–39.
Sturm, K. and Tam, P. P. L. (1993) Isolation and culture of whole postimplantation embryos and germ layer derivatives, in Methods in Enzymology, vol. 225, Guide to Techniques in Mouse Development (Wassarman, P. M. and DePamphilis, M. L., eds.), Academic, San Diego, CA, pp. 164–190.
Martins-Green, M. M. (1990) Transmission electron microscopy and immunolabelling for light and electron microscopy, in Postimplantation Mammalian Embryos-A Practical Approach, (Copp, A. J. and Cockroft, D. L.,eds.), IRL, Oxford, UK, pp. 127–154.
Burgess, R., Cserjesi, P., Ligon, K. L., and Olson, E. N. (1995) Paraxis: a basic helixloop-helix protein expressed in paraxial mesoderm and developing somites. Dev. Biol. 168, 296–306.
Herrmann, B. G. and Kispert, A. (1994) The T genes in embryogenesis. Trends Genet. 10, 280–286.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Lowe, L.A., Kuehn, M.R. (2000). Whole Mount In Situ Hybridization to Study Gene Expression During Mouse Development. In: Tuan, R.S., Lo, C.W. (eds) Developmental Biology Protocols. Methods in Molecular Biology™, vol 137. Humana Press. https://doi.org/10.1385/1-59259-066-7:125
Download citation
DOI: https://doi.org/10.1385/1-59259-066-7:125
Publisher Name: Humana Press
Print ISBN: 978-0-89603-854-7
Online ISBN: 978-1-59259-066-7
eBook Packages: Springer Protocols