A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species

Abstract

In situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has proven to be essential to our understanding of biological processes, including developmental regulation. In situ protocols are today routine in numerous laboratories, and although details might change, they all include a hybridization step, where specific antisense RNA or DNA probes anneal to the target nucleic acids strand. This step, in general, is carried out at high temperatures and in a denaturing solution, the hybridization buffer, commonly containing 50% (v/v) formamide. An important drawback is that hot formamide poses a significant health risk and so must be handled with great care.We were prompted to test alternative hybridization solutions for in situ detection of gene expression in the medusa of the hydrozoan Clytia hemisphaerica, where traditional protocols caused extensive deterioration of the morphology and texture during hybridization, hindering observation and interpretation of results. Inspired by optimized protocols for Northern and Southern blot analysis, we substituted the 50% formamide with an equal volume of 8 M urea solution in the hybridization buffer. The new protocol yielded better morphologies and consistency of tissues, and also notably improved the resolution of the signal, allowing more precise localization of gene expression, as well as reduced staining at non-specific sites. Given the improved results using a less toxic hybridization solution, we tested the urea protocol on a number of other metazoans: two brachiopod species (Novocrania anomala and Terebratalia transversa) and the worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea in in situ hybridization offers safer alternative protocols, potentially useful in research, medical and teaching contexts. We encourage other workers to test this approach on their study organisms, and hope that they will also obtain better sample preservation, more precise expression patterns and fewer problems due to aspecific staining, as we report here for Clytia medusae and Novocrania and Terebratalia developing larvae.

Molecular characterisation of a cellular conveyor belt in Clytia medusae

“…The reciprocal procedure was used for rehydration before in situ hybridisation. Two different protocols were used for in situ hybridisation, using for the hybridisation step either formamide (as described in or urea (as described in Sinigaglia et al, 2018). Both protocols were used for all genes and the results were cross-checked for reproducibility.…”

A G protein–coupled receptor mediates neuropeptide-induced oocyte maturation in the jellyfish Clytia

“…No MIHR-expressing cells were detected in the manubrium, only nonspecific staining of the manubrium floor. Regulation of tentacle contractions by MIHR-expressing cells in the tentacles in response to MIH produced in the gastrovascular system, integrated in a wider neuronal system, for instance including RFamide expressing cells [32], could account for the sluggish movement and poor growth of MIHR mutant medusae. Specific assays should be devised in the future to examine this hypothesis in detail.…”

Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones

“…For in situ hybridisation, isolated gonads or whole jellyfish were processed as described previously (Fourrage et al, 2014) except that 4 M urea was used instead of 50% formamide in the hybridisation buffer (Sinigaglia et al, 2017). For double fluorescent in situ hybridisation, female Clytia gonads were fixed overnight at 18°C in HEM buffer containing 3.7% formaldehyde, washed five times in PBS-T, then dehydrated on ice using 50% methanol/PBS-T then 100% methanol.…”

“…For double fluorescent in situ hybridisation, female Clytia gonads were fixed overnight at 18°C in HEM buffer containing 3.7% formaldehyde, washed five times in PBS-T, then dehydrated on ice using 50% methanol/PBS-T then 100% methanol. In situ hybridisation (Lapébie et al, 2014;Sinigaglia et al, 2017) was performed using a DIG-labelled probe for Che-pp1 and a fluorescein-labelled probe for Che-pp4. A 3 h incubation with a peroxidase-labelled anti-DIG antibody was followed by washes in MABT (100 mM maleic acid pH 7.5, 150 mM NaCl, 0.1% Triton X-100).…”