An optimised whole mount in situ hybridisation protocol for the mollusc Lymnaea stagnalis. 2015

Jennifer Hohagen, and Ines Herlitze, and Daniel John Jackson
Department of Geobiology, Geosciences Centre, Georg-August University of Göttingen, Goldschmidtstrasse 3, 37077, Göttingen, Germany. jhohage@uni-goettingen.de.

BACKGROUND The ability to visualise the expression of individual genes in situ is an invaluable tool for developmental and evolutionary biologists; it allows for the characterisation of gene function, gene regulation and through inter-specific comparisons, the evolutionary history of unique morphological features. For well-established model organisms (e.g., flies, worms, sea urchins) this technique has been optimised to an extent where it can be automated for high-throughput analyses. While the overall concept of in situ hybridisation is simple (hybridise a single-stranded, labelled nucleic acid probe complementary to a target of interest, and then detect the label immunologically using colorimetric or fluorescent methods), there are many parameters in the technique that can significantly affect the final result. Furthermore, due to variation in the biochemical and biophysical properties of different cells and tissues, an in situ technique optimised for one species is often not suitable for another, and often varies depending on the ontogenetic stage within a species. RESULTS Using a variety of pre-hybridisation treatments we have identified a set of treatments that greatly increases both whole mount in situ hybridisation (WMISH) signal intensity and consistency while maintaining morphological integrity for early larval stages of Lymnaea stagnalis. These treatments function well for a set of genes with presumably significantly different levels of expression (beta tubulin, engrailed and COE) and for colorimetric as well as fluorescent WMISH. We also identify a tissue-specific background stain in the larval shell field of L. stagnalis and a treatment, which eliminates this signal. CONCLUSIONS This method that we present here will be of value to investigators employing L. stagnalis as a model for a variety of research themes (e.g. evolutionary biology, developmental biology, neurobiology, ecotoxicology), and brings a valuable tool to a species in a much understudied clade of animals collectively known as the Spiralia.

UI MeSH Term Description Entries
D008195 Lymnaea A genus of dextrally coiled freshwater snails that includes some species of importance as intermediate hosts of parasitic flukes. Lymnea,Lymnaeas,Lymneas
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D017403 In Situ Hybridization A technique that localizes specific nucleic acid sequences within intact chromosomes, eukaryotic cells, or bacterial cells through the use of specific nucleic acid-labeled probes. Hybridization in Situ,Hybridization, In Situ,Hybridizations, In Situ,In Situ Hybridizations
D018507 Gene Expression Regulation, Developmental Any of the processes by which nuclear, cytoplasmic, or intercellular factors influence the differential control of gene action during the developmental stages of an organism. Developmental Gene Expression Regulation,Embryologic Gene Expression Regulation,Gene Expression Regulation, Embryologic,Regulation of Gene Expression, Developmental,Regulation of Gene Expression, Embryologic,Regulation, Gene Expression, Developmental,Regulation, Gene Expression, Embryologic

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