Multi-probe in situ hybridization to whole mount Arabidopsis seedlings

Abstract

In situ RNA-RNA hybridization (ISH) is a molecular method for localization of gene transcripts at the cellular level and is widely used to provide spatial and temporal information regarding gene expression. However, standard protocols are complex and laborious to implement, restricting analysis to one or a few genes at any one time, each one observed on separate ISH preparations. Multi-probe whole-mount in situ hybridization is a powerful technique to compare the expression patterns of two or more genes simultaneously in the same tissue or organ. We describe for the first time in plants, the detection of three different mRNAs in a single fixed whole mount Arabidopsis seedling. A combination of bright fluorescent secondary antibodies was used for the detection of riboprobes differentially labeled by digoxigenin, biotin and fluorescein. The 3-D detection of each of the multiple fluorescent hybridization signals or in combination was obtained through confocal laser-scanning microscopy. The reliability of the method was tested in the root, using the PINFORMED (PIN) genes with non-overlapping temporal and spatial expression patterns. In the shoot, a class-I KNOTTED -like homeobox gene from Arabidopsis (KNAT1) with expression restricted to the shoot apical meristem was used in combination with ELONGATOR3 (ELO3) gene. In addition, the expression patterns of ELONGATOR complex gene (ELO2, ELO3) and HISTONE MONOUBIQUITINATION1 (HUB1) genes were analyzed in both shoot and root and a partial overlapping was observed. The whole procedure takes only 6 days.

Analysis of AtGUS1 and AtGUS2 in Arabidopsis root apex by a highly sensitive TSA-MISH method

“…4) fully matches that previously observed (Nelissen et al, 2010;Bruno et al, 2011). Indeed in both the present and the previous experiments (Nelissen et al, 2010;Bruno et al, 2011) ELO3 transcripts are present in the shoot meristematic dome, in the emerging leaf primordia, in the blastozone of developing organs and provascular strands (Fig.…”

“…In conclusion, the TSA-MISH technique expands upon the previously one established in Bruno L et al, (2011) and provides the following advantages: a) brilliant hybridization signal with reduced background; b) amplification of signal with low-abundance genes expression which could be underestimated or missed by using MISH; c) reliable concomitant detection of genes with very different levels of expression; namely, in the last case, it could be hard to establish hybridization conditions adapted to avoid either over-or under-estimation of "highy expressed" and "lowly expressed" genes, respectively. Similar difficulty could occur facing single gene expressed at different level in the different organs and tissues.…”

“…Previously, tissue-specific expression pattern of ELO3 was investigated through ISH and MISH techniques in both A. thaliana seedlings as well as during embryo development (Nelissen et al, 2010;Bruno et al, 2011;Himanen et al, 2012) According to the role of ELONGATOR complex in cell proliferation, ELO3 expression in seedling was confined to meristematic regions and proliferating cells.…”

Transcriptional Regulation of Arabidopsis Polycomb Repressive Complex 2 Coordinates Cell-Type Proliferation and Differentiation

“…Fluorescein-labeled sense and antisense probes were performed according to the manufacturer's indications (Fluorescein RNA labeling mix; Roche). Tissue fixation, permeabilization, probe hybridization, and detection were adapted from Bruno et al (2011). Probe detection was performed using horseradish peroxidase-conjugated anti-FITC antibody (1:100 dilution) (AB6656; Abcam), followed by tyramide signal amplification (TSA reagent, Alexa Fluor 488 Tyramide; Molecular Probes).…”

Visualization of Protein Coding, Long Noncoding, and Nuclear RNAs by Fluorescence in Situ Hybridization in Sections of Shoot Apical Meristems and Developing Flowers

“…In plants, FISH has been used to visualize entire chromosomes or individual genetic loci (Lamb et al, 2007;Tirichine et al, 2009;Jiang, 2019) as well as pathogen DNA (Shargil et al, 2015). However, although there are notable examples using whole-mounted tissues (Bruno et al, 2011(Bruno et al, , 2015Himanen et al, 2012;Rozier et al, 2014;Bleckmann and Dresselhaus, 2016;Woloszynska et al, 2019), the use of FISH to investigate plant RNA localization at high resolution and deep in tissues remains to be fully exploited. We have established RNA FISH in the Arabidopsis shoot apex based on the tyramide signal amplification (TSA) system applied to sectioned tissue (Yang et al, 2017).…”