Whole-Mount In Situ Hybridization (WISH) Optimized for Gene Expression Analysis in Mouse Embryos and Embryoid Bodies

  • Eleni DakouEmail author
  • Nele Vanbekbergen
  • Sara Corradi
  • Caroline R. Kemp
  • Erik Willems
  • Luc LeynsEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1211)


Whole-mount in situ hybridization (WISH) is a technique widely used in developmental biology to study the localization of RNA sequences in intact tissues or whole organisms. In this chapter we present a detailed protocol that was optimized for gene expression analysis in early stage mouse embryos (5.5–10.5 days post-coitum) and embryoid bodies formed by differentiating embryonic stem cells and can be used for the detection of up to two distinct RNA sequences simultaneously. The initial steps of the procedure are the generation of the labeled riboprobe(s) and the embryo or embryoid body preparation, which can be completed in less than 2 days. The actual WISH procedure, comprised of the hybridization, the post-hybridization washes, and the immunological staining, can be completed in 3 days.

Key words

WISH In situ hybridization mRNA Mouse embryo Embryoid bodies Gene expression pattern 



Eleni Dakou is funded by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen). This research is supported by an Interuniversity Attraction Pole grant (IAP-P7-07).


  1. 1.
    Tautz D, Pfeifle C (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98(2):81–85PubMedCrossRefGoogle Scholar
  2. 2.
    Kemp C, Willems E, Abdo S et al (2005) Expression of all Wnt genes and their secreted antagonists during mouse blastocyst and postimplantation development. Dev Dyn 233(3):1064–1075PubMedCrossRefGoogle Scholar
  3. 3.
    Kemp CR, Willems E, Wawrzak D et al (2007) Expression of Frizzled5, Frizzled7, and Frizzled10 during early mouse development and interactions with canonical Wnt signaling. Dev Dyn 236(7):2011–2019PubMedCrossRefGoogle Scholar
  4. 4.
    Willems E, Leyns L (2008) Patterning of mouse embryonic stem cell-derived pan-mesoderm by Activin A/Nodal and Bmp4 signaling requires Fibroblast Growth Factor activity. Differentiation 76(7):745–759PubMedCrossRefGoogle Scholar
  5. 5.
    Hargrave M, Koopman P (2000) In situ hybridization of whole-mount embryos. In: Darby IA (ed) Methods in molecular biology, vol 123, In situ hybridization protocols. Humana Press, Totowa, NJ, pp 279–289Google Scholar
  6. 6.
    Lowe L, Kuehn M (2000) Whole mount in situ hybridization to study gene expression during mouse development. In: Tuan RS, Lo CW (eds) Methods in molecular biology, vol 137, Developmental biology protocols, vol III. Humana Press, Totowa, NJ, pp 125–137Google Scholar
  7. 7.
    Pollet N, Niehrs C (2001) Expression profiling by systematic high-throughput in situ hybridization to whole-mount embryos. In: Starkey MP, Elaswarapu R (eds) Methods in molecular biology, vol 175, Genomics protocols. Humana Press, Totowa, NJ, pp 309–321Google Scholar
  8. 8.
    Acloque H, Wilkinson DG, Nieto MA (2008) In situ hybridization analysis of chick embryos in whole-mount and tissue sections. In: Bronner-Fraser M (ed) Methods in cell biology, vol 87. Academic, New York, pp 169–185Google Scholar
  9. 9.
    Piette D, Hendrickx M, Willems E et al (2008) An optimized procedure for whole-mount in situ hybridization on mouse embryos and embryoid bodies. Nat Protoc 3(7):1194–1201PubMedCrossRefGoogle Scholar
  10. 10.
    Weiszmann R, Hammonds AS, Celniker SE (2009) Determination of gene expression patterns using high-throughput RNA in situ hybridization to whole-mount Drosophila embryos. Nat Protoc 4(5):605–618PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Lagendijk AK, Moulton JD, Bakkers J (2012) Revealing details: whole mount microRNA in situ hybridization protocol for zebrafish embryos and adult tissues. Biol Open 1(6):566–569PubMedCrossRefPubMedCentralGoogle Scholar
  12. 12.
    Coucouvanis E, Martin GR (1995) Signals for death and survival: a two-step mechanism for cavitation in the vertebrate embryo. Cell 83(2):279–287PubMedCrossRefGoogle Scholar
  13. 13.
    Coucouvanis E, Martin GR (1999) BMP signaling plays a role in visceral endoderm differentiation and cavitation in the early mouse embryo. Development 126(3):535–546PubMedGoogle Scholar
  14. 14.
    Leahy A, Xiong JW, Kuhnert F et al (1999) Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation. J Exp Zool 284(1):67–81PubMedCrossRefGoogle Scholar
  15. 15.
    Desbaillets I, Ziegler U, Groscurth P et al (2000) Embryoid bodies: an in vitro model of mouse embryogenesis. Exp Physiol 85(6):645–651PubMedCrossRefGoogle Scholar
  16. 16.
    ten Berge D, Koole W, Fuerer C et al (2008) Wnt signaling mediates self-organization and axis formation in embryoid bodies. Cell Stem Cell 3(5):508–518PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Höpfl G, Gassmann M, Desbaillets I (2004) Differentiating embryonic stem cells into embryoid bodies. In: Schatten H (ed) Methods in molecular biology, vol 254, Germ cell protocols. Humana Press, Totowa, NJ, pp 79–98Google Scholar
  18. 18.
    Kurosawa H (2007) Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng 103(5):389–398PubMedCrossRefGoogle Scholar
  19. 19.
    Nagy A, Gertsenstein M, Vintersten K et al (2003) Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Biology, Lab of Cell GeneticsVrije Universiteit BrusselBrusselsBelgium
  2. 2.Muscle Development and Regeneration ProgramSanford-Burnham Medical Research InstituteLa JollaUSA

Personalised recommendations