Abstract
Tissue clearing methods combined with confocal microscopy have been widely used for studying developmental biology. In plants, ClearSee is a reliable clearing method that is applicable to a wide range of tissues and is suitable for gene expression analysis using fluorescent reporters, but its application to the Arabidopsis thaliana embryo, a model system to study morphogenesis and pattern formation, has not been described in the original literature. Here, we describe a ClearSee-based clearing protocol which is suitable for obtaining 3D images of Arabidopsis thaliana embryos. The method consists of embryo dissection, fixation, washing, clearing, and cell wall staining and enables high-quality 3D imaging of embryo morphology and expression of fluorescent reporters with the cellular resolution. Our protocol provides a reliable method that is applicable to the analysis of morphogenesis and gene expression patterns in Arabidopsis thaliana embryos.
Highlights
The embryogenesis of Arabidopsis thaliana, in which a relatively small number of tissues and organs are arranged in a simple pattern, provides an excellent system to study morphogenesis and pattern formation, and many regulatory factors that affect these processes have been identified and studied extensively [2,3]
Because patterns of cell division and elongation are significantly regular during Arabidopsis embryogenesis [4,5,6], their possible roles in development and the underlying mechanisms for oriented cell division and elongation have been an important subject [7,8,9,10]
We first applied the original ClearSee protocol [11] to whole seeds with the expectation of visualizing embryos without dissection. Seeds processed with this protocol exhibited brown color in the endothelium (Figure 1A), preventing us from imaging internal embryos
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. In plant development, oriented cell division and expansion play essential roles in morphogenesis and pattern formation [1]. TOMEI-II [13] and ClearSee [11] have an advantage in visualizing gene expression patterns, as these methods well preserve the fluorescence of various fluorescent proteins. Both methods can be applicable to a wide range of tissue types and to various plant species, whether they can work with embryos has not been reported. A recent application of ClearSee to Arabidopsis thaliana ovules [15] confirms that the method has potential to visualize plant internal structures with high quality. We established a protocol to apply the ClearSee method to the embryo of Arabidopsis thaliana and to demonstrate that the protocol can visualize cellular arrangement and the signal of various fluorescent reporters in 3D
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