Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos

Abstract

BackgroundWhole-mount in situ hybridization (WISH) is a fundamental tool for studying the spatio-temporal expression pattern of RNA molecules in intact embryos and tissues. The available methodologies for detecting mRNAs in embryos rely on enzymatic activities and chemical reactions that generate diffusible products, which are not fixed to the detected RNA, thereby reducing the spatial resolution of the technique. In addition, current WISH techniques are time-consuming and are usually not combined with methods reporting the expression of protein molecules.ResultsThe protocol we have developed and present here is based on the RNAscope technology that is currently employed on formalin-fixed, paraffin-embedded and frozen tissue sections for research and clinical applications. By using zebrafish embryos as an example, we provide a robust and rapid method that allows the simultaneous visualization of multiple transcripts, demonstrated here for three different RNA molecules. The optimized procedure allows the preservation of embryo integrity, while exhibiting excellent signal-to-noise ratios. Employing this method thus allows the determination of the spatial expression pattern and subcellular localization of multiple RNA molecules relative to each other at high resolution, in the three-dimensional context of the developing embryo or tissue under investigation. Lastly, we show that this method preserves the function of fluorescent proteins that are expressed in specific cells or cellular organelles and conserves antigenicity, allowing protein detection using antibodies.ConclusionsBy fine-tuning the RNAscope technology, we have successfully redesigned the protocol to be compatible with whole-mount embryo samples. Using this robust method for zebrafish and extending it to other organisms would have a strong impact on research in developmental, molecular and cell biology. Of similar significance would be the adaptation of the method to whole-mount clinical samples. Such a protocol would contribute to biomedical research and clinical diagnostics by providing information regarding the three-dimensional expression pattern of clinical markers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-014-0055-7) contains supplementary material, which is available to authorized users.

Temporally coordinated signals progressively pattern the anteroposterior and dorsoventral body axes

“…In situ transcript detection methods are qualitative and describe a transcript domain spatially, but do not provide quantitative data of transcript levels. However, recent advances in quantitative, single-molecule RNA transcript detection offer new possibilities [240–242]. For example, there are reports that bmp ligand expression during gastrulation is graded across the ventral half embryo, with highest expression ventrally and lowest expression laterally [144,178], while others do not report a difference in bmp expression [127,128,130,137,243].…”

Resolving in vivo gene expression during collective cell migration using an integrated RNAscope, immunohistochemistry and tissue clearing method

“…In addition to fixation, Gross-Thebing and colleagues (Gross-Thebing et al, 2014) reported that air drying zebrafish embryos for 30 minutes was crucial for maintaining embryo integrity. However, our tests showed that air drying was not necessary to maintain chick embryo integrity (HHSt11-18) and yield strong RNAscope signal.…”

“…Our RNAscope protocol for whole chick embryos was based upon published work that was the first to report using the novel RNAscope multiplex fluorescence in situ hybridization technology in a zebrafish embryo model system (Gross-Thebing et al, 2014). Optimization for the chick embryo required us to make several adaptations that are detailed in Suppl.…”

“…Novel fluorescence in situ hybridization methods (FISH) have emerged, including hybridized chain reaction (HCR; Choi et al, 2010, Choi et al, 2014, Choi et al, 2016) and RNAscope (Gross-Thebing et al, 2014). Both of these methods have strategies to amplify target-specific signals and reduce background noise from non-specific hybridization.…”

“…In addition, recent efforts have been made to either integrate mRNA expression detection with immunohistochemistry (IHC) in thick tissue sections (Grabinski et al, 2015) or optimize tissue clearing methods to visualize cells deep within thick adult tissue sections or intact embryos using light microscopy (Richardson and Lichtman, 2015). By multiplexing fluorescent probes, both RNAscope and HCR allow mRNA expression detection of up to 3 to 5 gene targets in the same tissue or embryo (Choi et al, 2014; Gross-Thebing et al, 2014). …”