An optimised whole mount in situ hybridization protocol for the mollusc Lymnaea stagnalis

Abstract

Background: The ability to visualise the expression of individual genes in situ is an invaluable tool for developmenta and evolutionary biologists; it allows for the characterisation of gene function, gene regulation and throug inter-specific comparisons, the evolutionary history of unique morphological features. For well-established mode organisms (e.g., flies, worms, sea urchins) this technique has been optimised to an extent where it can be automate 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 usin colorimetric or fluorescent methods), there are many parameters in the technique that can significantly affect the fina result. Furthermore, due to variation in the biochemical and biophysical properties of different cells and tissues, an i situ technique optimised for one species is often not suitable for another, and often varies depending on the ontogeneti stage within a species Results: Using a variety of pre-hybridisation treatments we have identified a set of treatments that greatly increases bot whole mount in situ hybridisation (WMISH) signal intensity and consistency while maintaining morphological integrit for early larval stages of Lymnaea stagnalis. These treatments function well for a set of genes with presumabl significantly different levels of expression (beta tubulin, engrailed and COE) and for colorimetric as well as fluorescen WMISH. We also identify a tissue-specific background stain in the larval shell field of L. stagnalis and a treatment, whic eliminates this signal Conclusions: This method that we present here will be of value to investigators employing L. stagnalis as a mode for a variety of research themes (e.g. evolutionary biology, developmental biology, neurobiology, ecotoxicology), an brings a valuable tool to a species in a much understudied clade of animals collectively known as the Spiralia.