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

Abstract

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).

An Antarctic molluscan biomineralisation tool-kit

“…Dried tissue sections were rehydrated through a graded ethanol series before being transferred to an Invatis in situ -Pro robot for all subsequent treatments as described in ref. 22. In brief, tissue sections were treated with proteinase K (50 μg mL −1 , 10 min, RT), stopped with 0.2% glycine (5 min, RT), washed with phosphate buffered saline with 0.1% Tween20 (PBTw, 5 min, RT) re-fixed with 4% paraformaldehyde (20 min, RT), incubated with hybridisation buffer (2 h, 55 °C), incubated with specific riboprobe in hybridisation buffer (500 ng μL −1 , 26 h, 55 °C) and washed with a series of saline-sodium citrate (SSC) buffers (4x, 2x, 1x, 1x with 0.0 1% Tween20, 15 min each, 55 °C).…”

A technical review and guide to RNA fluorescence in situ hybridization

“…Zebrafish embryos are treated with 10 µg/mL proteinase K in PBST for 2-20 min depending on the age (Oxtoby & Jowett, 1993;Marra et al, 2017). The same treatment is also recommended for snail embryos as well as whole-mount planarian worms and is sometimes applied to fruit fly embryos, although several other permeabilization strategies including acetone are also frequently used for Drosophila (Paré et al, 2009;Pearson et al, 2009;Jackson, Herlitze & Hohagen, 2016;Hauptmann et al, 2016;Trcek et al, 2017). Some protocols call for brain sections to be treated with proteinase K, however, many protocols omit this step as permeability is less of an issue with sectioned material (Kasai et al, 2016;Hua et al, 2018).…”

“…A treatment of 1 M HCl at 37 C for 30-50 min is effective to improve permeability of mycolic-acid-containing bacterial cells whereas other bacteria (including Escherichia coli) can be permeabilized in only 10 min (Macnaughton, O'Donnell & Embley, 1994). The addition of Triton X-100 or other detergent directly to the fixative in the initial fixation protocol has also been used to improve the permeability of bacterial cells through its interaction with cell envelope lipid molecules (Jackson, Herlitze & Hohagen, 2016;Rocha, Almeida & Azevedo, 2018). Protease-free detergent-based methods have also been successful for permeabilization of Drosophila embryos (Boettiger & Levine, 2013).…”

“…The stringency of the hybridization is affected by the concentration of salt in the hybridization solution (lower concentrations are more stringent) as well as the hybridization temperature (higher temperatures are more stringent). It is most common to keep the salt concentration constant (750 mM NaCl, 87.5 mM sodium citrate), with pH roughly between 7.0 and 8.5, and simply adjust the hybridization temperature to achieve the ideal stringency (Pearson et al, 2009;Zhang et al, 2012;Jackson, Herlitze & Hohagen, 2016). An initial denaturation step of 75 C for 10 min can be used to denature all target and probe RNA to facilitate hybridization, the sample is then immediately adjusted to the designated hybridization temperature (Jékely & Arendt, 2007;Jackson, Herlitze & Hohagen, 2016).…”

“…The optimal hybridization temperature is dependent on the length and composition of the probe. Although the hybridization temperature should be empirically optimized for every probe individually, short oligonucleotide probes (20-50 nucleotides) typically require lower hybridization temperatures of 37 C whereas longer riboprobes of 1,000+ nucleotides may hybridize at temperatures >55 C (Pearson et al, 2009;Jackson, Herlitze & Hohagen, 2016;Fontenete et al, 2016). Generally, the hybridization step cannot be over-incubated.…”