Abstract
BackgroundThe 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.ResultsUsing 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0068-7) contains supplementary material, which is available to authorized users.
Highlights
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
Using a variety of pre-hybridisation treatments we have identified a set of conditions that allow for high whole mount in situ hybridisation (WMISH) signal intensity and consistency in colorimetric as well as fluorescent WMISH
These include a treatment with 0.5% sodium dodecyl sulfate (SDS), treatment of one to two dpfc larvae with 15 μg/ml Proteinase K (Pro-K), two to three dpfc larvae with 15–20 μg/ml Pro-K and three to five dpfc larvae with 20–30 μg/ml, followed by treatment with triethanolamine + acetic anhydride (TEAAA)
Summary
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. The most commonly employed technique for the study of spatial gene expression in a given tissue or developmental stage is in situ hybridisation (ISH), often applied to whole embryos or larvae as whole mount in situ hybridisation (WMISH). L. stagnalis embryos develop individually within egg capsules filled with a fluid that serves a nutritive function and is uptaken by pinocytosis during development [10,11,12] This viscous intra-capsular fluid, which consists of a complex mixture of ions, polysaccharides, proteoglycans and other polymers [13], can be seen to stick to the embryo following decapsulation, and likely interferes with any WMISH procedure. From 52 hours post first cleavage onwards the first insoluble material associated with shell formation is secreted [8] This material nonspecifically binds some nucleic acid probes and generates a characteristic background signal. Described WMISH protocols for larvae of L. stagnalis produced WMISH signals with low signal to noise ratios, making some previously reported gene expression patterns difficult to interpret [14,15,16]
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