A Whole Mount In Situ Hybridization Method for the Gastropod Mollusc Lymnaea stagnalis

摘要

Whole mount in situ hybridization (WMISH) is a technique that allows for the spatial resolution of nucleic acid molecules (often mRNAs) within a 'whole mount' tissue preparation, or developmental stage (such as an embryo or larva) of interest. WMISH is extremely powerful because it can significantly contribute to the functional characterization of complex metazoan genomes, a challenge that is becoming more of a bottleneck with the deluge of next generation sequence data. Despite the conceptual simplicity of the technique much time is often needed to optimize the various parameters inherent to WMISH experiments for novel model systems; subtle differences in the cellular and biochemical properties between tissue types and developmental stages mean that a single WMISH method may not be appropriate for all situations. We have developed a set of WMISH methods for the re-emerging gastropod model Lymnaea stagnalis that generate consistent and clear WMISH signals for a range of genes, and across all developmental stages. These methods include the assignment of larvae of unknown chronological age to an ontogenetic window, the efficient removal of embryos and larvae from their egg capsules, the application of an appropriate Proteinase-K treatment for each ontogenetic window, and hybridization, post-hybridization and immunodetection steps. These methods provide a foundation from which the resulting signal for a given RNA transcript can be further refined with probe specific adjustments (primarily probe concentration and hybridization temperature).