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

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.

Formin Is Associated with Left-Right Asymmetry in the Pond Snail and the Frog

“…Expression of candidate loci was examined in early L. stagnalis embryos using whole-mount in situ hybridization (WMISH; [ 23 ]). The first two embryonic cleavages in L. stagnalis are equal, so that the four macromeres that are formed from the first two cleavages are indistinguishable [ 3 , 4 , 5 ], until contact between the third quartet of micromeres induces one of the macromeres to become the future D blastomere (but see 24 ]).…”

An Antarctic molluscan biomineralisation tool-kit

“…Digoxigenin(DIG)-labelled riboprobes were synthesised as previously described in ref. 21. Adult mantle tissues (n = 3) from L. elliptica were fixed for 12 h in freshly prepared Davidson fixative (22% formalin, 33% ethyl alcohol, 12% glacial acetic acid and 33% sterile sea water) and transferred to 70% (RT) ethanol for storage.…”

A Whole Mount <em>In Situ</em> Hybridization Method for the Gastropod Mollusc <em>Lymnaea stagnalis</em>

“…Despite their morphological diversity 1 , species richness (second only to the Arthropods in terms of species number 2 ) and relevance to a wide range of commercial 3 , medical 4 and scientific issues [5][6][7][8] , there are relatively few molluscan species that can claim to be both well-equipped scientific models and easy to maintain in a laboratory environment. One mollusc that is much used by disciplines such as neurobiology 9 , ecotoxicology 10 and more recently evolutionary biology 11,12 , is Lymnaea stagnalis, primarily because of its widespread distribution and extreme ease of maintenance. Despite its popularity as a 'model' organism and its long history of use by developmental biologists [13][14][15][16][17][18][19] , the range and power of molecular tools available to the L. stagnalis scientific community lies far behind that of more traditional animal models (Drosophila, mouse, sea urchin, nematodes).…”

“…Lymnaea occupies a position within the Metazoa that is extremely under-represented in terms of model organisms. As a representative Spiralian, Lymnaea can bring insight into the evolution of distinct morphological features such as shell formation 12 and body handedness [33][34][35] and is also a valuable neuroethology 36 and neurophysiology model 9,37 . Powerful techniques such as the ability to efficiently visualize gene expression patterns in situ increases the functionality of Lymnaea as a model organism, and broadens the variety of questions that it can be used to address.…”