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Analysis of 3D gene expression patterns in plants using whole-mount RNA in situ hybridization

Nature Protocols volume 9, pages 2464–2475 (2014)Cite this article

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Abstract

In situ mRNA hybridization is one of the most powerful techniques for analyzing patterns of gene expression. However, its usefulness is limited in complex plant tissues by the need to fix, embed and section samples before localizing the desired mRNA. Here we present a robust whole-mount in situ hybridization method that allows easy access to patterns of gene expression in intact, complex tissues, such as the inflorescence apex of Arabidopsis thaliana. The tissue is first fixed and then permeabilized by treatment with a cocktail of cell wall–digesting enzymes that has been optimized to preserve the integrity of tissue structures, while also permitting the detection of expression patterns in deep tissues. In addition to colorimetric staining, fluorimetric staining that can be imaged by confocal microscopy can also be used with this protocol, thus providing full 3D resolution. The entire procedure can take <3 d from tissue preparation to image acquisition.

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Figure 1: Flowchart of the whole-mount in situ protocol.
Figure 2: General methodology for the detection of labeled mRNA using either NBT/BCIP colorimetric or Alexa Fluor tyramide (TSA) fluorimetric methods.
Figure 3: Schematic for mounting samples.
Figure 4: Examples of whole-mount in situ mRNA localizations in Arabidopsis inflorescences.

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Acknowledgements

We thank A. Lacroix and P. Villand for help with plant growth, C. Lionnet (PLATIM imaging platform) for help with imaging and J.H. Doonan for his insights. P.D. was supported by an INRA Chaire d'excellence grant, and T.V. was supported by the ANR-12-BSV6-0005 (AuxiFlo) grant.

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Authors and Affiliations

  1. Laboratoire Reproduction et Développement des Plantes, Ecole Normale Supérieure Lyon, Lyon, France

    Frédérique Rozier, Vincent Mirabet, Teva Vernoux & Pradeep Das

  2. Laboratoire Joliot Curie, Ecole Normale Supérieure Lyon, Lyon, France

    Vincent Mirabet & Pradeep Das

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  1. Frédérique Rozier
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  2. Vincent Mirabet
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Contributions

T.V. and P.D. designed and supervised experiments; F.R. performed the experiments with the help of V.M. and P.D.; F.R., V.M., T.V. and P.D. analyzed the data; and F.R., V.M., T.V. and P.D. wrote the paper.

Corresponding authors

Correspondence to Teva Vernoux or Pradeep Das.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Expected results for variations in the tissue permeabilization and probe hydrolysis steps.

(a–b) Examples of whole-mount in situ experiments to localize WUSCHEL mRNA in tissue that has been subjected to enzymatic permeabilization of either 0 min (a) or 6 min (b). Compare the absent or reduced signal in the thicker parts of the tissue, such as the center of the SAM (red arrowheads) or in older flowers, either at stage 3 (blue arrows) or stage 2 (red arrows). (c) Lane 1 shows the products of a transcription reaction (for 1 μl of the 20 μl reaction volume) from a PCR-amplified full-length (291 bp) CLAVATA3 template. Note the presence of an additional larger band (solid triangle), which is visible in most transcription reactions. Lane 2 shows the results of a successful hydrolysis reaction (for 5 μl of the 80-100 μl reaction volume), where a smear centered at roughly 0.1 kb is typically observed (open triangle). In the case of an unsuccessful hydrolysis, lane 2 would look identical to lane 1.

Supplementary information

Supplementary Figure 1

Expected results for variations in the tissue permeabilization and probe hydrolysis steps. (PDF 502 kb)

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Rozier, F., Mirabet, V., Vernoux, T. et al. Analysis of 3D gene expression patterns in plants using whole-mount RNA in situ hybridization. Nat Protoc 9, 2464–2475 (2014). https://doi.org/10.1038/nprot.2014.162

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