Abstract
Small RNAs specifically expressed in reproductive tissues are key regulators of germline development in eukaryotes. Rice microRNA2118 (miR2118), which is enriched during reproduction in grasses, is a trigger to produce phased small interfering RNAs (phasiRNAs). These phasiRNAs demonstrate the temporal regulation with premeiotic phasiRNAs and meiotic phasiRNAs in anther development. Furthermore, the site-specific regulation via miR2118 and phasiRNAs is of importance in soma and germ development in anthers. Accordingly, histological imaging methods are essential tools for understanding spatiotemporal regulation during reproduction and elucidating the reproductive roles of miRNAs and phasiRNAs. We successfully developed a method to visualize the three-dimensional (3D) structure of entire rice anthers, which can also be used for distinguishing the internal structure of the anthers in other plants. Here, we describe the detailed methods of in situ hybridization for miR2118 localization and the visualization of the 3D structure of entire anthers of rice.
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Acknowledgments
This work was supported by JST PRESTO Program (Grant number JPMJPR17Q3, Japan), JST FORESTO Program (Grant Number JPMJFR204U, Japan), KAKENHI Programs (Grant numbers JP17H05608 and JP15H01476), the Naito Foundation, and the Okinawa Institute of Science and Technology Graduate University, Japan, to R.K. We thank Ms. Saori Araki and Ms. Hinako Tamotsu for rice growth and sampling.
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The 3D imaging movie of the 1Â mm rice anther taken by a Lightsheet microscope. Cyan shows the fluorescence of the somatic anther walls stained with SR2200 dye. Magenta shows autofluorescence of the anther. Laser excitation/emission: 405Â nm/420-470Â nm (SR2200), 561Â nm/575Â nm LP (autofluorescence). (MOV 9549 kb)
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Koizumi, K., Komiya, R. (2022). 3D Imaging and In Situ Hybridization for Uncovering the Functions of MicroRNA in Rice Anther. In: Parrish, N.F., Iwasaki, Y.W. (eds) piRNA. Methods in Molecular Biology, vol 2509. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2380-0_6
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DOI: https://doi.org/10.1007/978-1-0716-2380-0_6
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