Wholemount In Situ Hybridization to Amphioxus Embryos

  • Peter W. H. Holland
Part of the METHODS IN MOLECULAR BIOLOGY™ book series (MIMB, volume 461)

1. Introduction

Once a gene has been cloned the spatial distribution of its transcripts may be determined by in situ hybridization. This involves applying a labeled antisense RNA probe, with complimentary nucleotide sequence to the mRNA, either to tissue sections or to whole fixed specimens. The cephalochordate amphioxus has small embryos (100–300 μm in length) that are Particularly well suited to whole mount in situ hybridization. The method has been successfully used to examine expression of many developmental expressed genes in amphioxus embryos, giving insight into the evolutionary conservation and divergence of gene expression between amphioxus and vertebrates.

2. Materials

  1. 1.

    MOPS buffer: 0.5 M NaCl, 1 mM MgSO4, 2 mM EGTA, 0.1 M morpholinopro-panesulfonic acid buffer, pH 7.5. Sterile.

  2. 2.

    Paraformaldehyde: electron microscopy-grade (TAAB, Aldermastan, UK).

  3. 3.

    Nunclon four-well multiwell dishes (Available from Sigma-Aldrich, catalogue number D6789. Nunclon is a trademark of...


Acetic Anhydride Hybridization Buffer Nitro Blue Tetrazolium Wash Solution Change Solution 
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  1. 1.
    Holland, P. W. H., Holland, L. Z., Williams, N. A., and Holland, N. D. (1992) An amphioxus homeobox gene: sequence conservation, spatial expression during development and insights into vertebrate evolution. Development 116, 653–661.PubMedGoogle Scholar
  2. 2.
    Yasuo, H. and Satoh, N. (1994) An ascidian homolog of the mouse Brachyury (T) gene is expressed exclusively in notochord cells at the fate restricted stage. Dev. Growth Differ. 36, 9–18.CrossRefGoogle Scholar

Copyright information

© Humana Press, a Part of Springer Science + Business Media, LLC 2008

Authors and Affiliations

  • Peter W. H. Holland
    • 1
  1. 1.DePartment of ZoologyUniversity of OxfordOxfordUK

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