3D Imaging and In Situ Hybridization for Uncovering the Functions of MicroRNA in Rice Anther.

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.