The previously described whole mount in situ hybridisation techniques for plants (de Almeida Engler et al., 1998; Bennett et al., 1996; Ludevid et al., 1992) did not yield entirely satisfactory results for us, especially for less abundant mRNAs and mRNAs with spatially less restricted localisation patterns. In order to improve the signal/background ratio, which was a limiting factor for these methods, we developed a new procedure based on these protocols as well as on protocols used for the mRNA localisation in Drosophila embryos (Tautz and Pfeifle, 1989) and hydras (Gajewski et al., 1996). The typical in situ hybridisation experiment consists of several steps: (i) tissue fixation and permeation, (ii) hybridisation and (iii) signal detection. Most in situ hybridisation protocols start with a fixation step using cross-linking by aldehyde. This is followed by proteinase K treatment for degrading the protein bound to the RNA. This standard step can be fully replaced by a brief heat denaturation as has been described for animal species (Plickert et al., 1997). However, because of the specific properties of plant material, namely the presence of cell walls and a waxy layer at the surface of most of the plant body, additional permeation steps have to be included. These include heptane treatment during material fixation, alcohol treatment, which also removes chlorophyll and other pigments, and finally xylene treatment. In case of persisting penetration problems, the duration of these treatments was simply prolonged as required by the particular plant material.
For hybridisation, different types of probes such as radioactive or digoxigenin-labelled antisense RNAs can be used. We used the latter, and obtained the best results with short probes (150–220 base pairs) produced by alkaline hydrolysis of longer transcripts. For most of the probes, the optimal hybridisation temperature was 50°C, which ensured sufficient hybridisation specificity. However, with less abundant transcripts or less specific probes, the temperature was decreased to 45°C or increased to 55°C, respectively. Detection of the bound probe was performed by using commercially available anti-DIG antibody linked to alkaline phosphatase.
The crucial question of how to ensure that observed signals specifically reflect the localisation pattern of RNA of interest in every in situ hybridisation protocol remains. This is particularly important for whole mount techniques because, with most of the probes, after a sufficiently long staining time, signals can almost always be obtained, especially in the meristem regions. This is also often true for typical sense probe controls. Therefore, the best way to validate results obtained by in situ detection is with a parallel experiment using the corresponding knock-out mutant. If this is not available, simultaneous use of a probe with a known staining pattern as a positive control and a non-plant RNA probe as a negative control is helpful in ensuring that the observed experimental staining is not artifactual. In that case, an additional independent confirmation of staining pattern, e.g. by promotor::GUS analysis, is highly recommended. We used both manual and automated protocols for the detection of various mRNA transcripts in Arabidopsis seedlings with fully satisfactory results. For example, the localisation of root-specific putative auxin efflux regulator AtPIN2 mRNA showed the strongest signals in cortex cells, accompanied by epidermal and lateral root cap staining (Figure 1b). This localisation pattern correlated well with the pattern of AtPIN2 promotor activity monitored in AtPIN2::GUS transgenic plants (Figure 1a) and was similar to previously reported results (Chen et al., 1998; Müller et al., 1998). However, we achieved better signal resolution with our protocol. Additionally, we examined the specific expression pattern of another auxin-related gene, PINOID (PID), encoding the Ser/Thr kinase (Christensen et al., 2000) in root tissue. The PID mRNA was found in the differentiating vasculature of the root (Figure 2a) in accordance with the previous reports (Benjamins et al., 2001). The specificity of this signal was further indicated by the lack of staining in a parallel experiment with pid knock-out mutant (Figure 2b). The method presented here can be applied not only to the root tips, but also to all other organs of the Arabidopsis seedlings. To demonstrate this, we investigated the expression of another auxin efflux regulator, AtPIN1, in aerial seedling tissue. In addition to confirming the reported expression in root (Friml et al., 2002a) and stem (Gälweiler et al., 1998), our results show that AtPIN1 is also expressed in the cotyledon vasculature (Figure 2c). As another example, we analysed the aerial tissue expression of the small ribosomal protein RPS5A (Weijers et al., 2001). We detected RPS5A mRNA localisation in shoot apical meristem and developing leaf primordia (Figure 2d). Both aerial expression patterns corresponded with promotor activity studies (data not shown; Weijers et al., 2001). The protocol described here has also been successfully used by several other laboratories to localise various transcripts in Arabidopsis (Benjamins et al., 2001; Friml et al., 2002a,b; Weijers et al., 2001; Bennett, Hejátko, Scheres, personal communication), thus demonstrating reliability of this method.
Figure 1. AtPIN2 expression and localisation pattern in root.
(a) Staining for GUS activity in AtPIN2::GUS transgenic plants with subsequent tissue clearing. Signals (depicted in blue colour) were detected in outer layers of the root, especially in the epidermis and the cortex.
(b) AtPIN2-specific whole mount in situ hybridisation. The AtPIN2 mRNA (depicted in brown) was detected in lateral root cap, epidermis and cortex cells.
(c) AtPIN2 whole mount immunolocalisation. The AtPIN2 protein (depicted in green) was detected at the upper (basipetal) side of the lateral root cap and epidermis cells and more homogenously at the boundaries of cortex cells.
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Figure 2. Examples of whole mount in situ hybridisation experiments.
(a) PINOID-specific whole mount in situ hybridisation. The PINOID mRNA was detected in the differentiated vascular tissue proximal to the root meristem.
(b) PINOID mRNA could not be detected in the pinoid mutant root.
(c) AtPIN1-specific whole mount in situ hybridisation. The AtPIN1 mRNA was detected associated with cotyledon vasculature.
(d) AtRPS5A whole mount in situ hybridisation. The AtRPS5A mRNA was detected in shoot apical meristem and leaf primordia.
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