Az100 microscope

GUS staining was performed according to a standard protocol [68 (link)]. Transgenic rice tissues were incubated overnight at 37 °C in GUS staining buffer (0.1 M K₂HPO₄ (pH 7.0), 0.1 mM KH₂PO₄ (pH 7.0), 5 mM K₃Fe (CN)₆, 5 mM K₄Fe(CN)₆·3H₂O, 0.1% Triton X-100, 20% methanol, 1 mg mL⁻¹ X-Gluc). After staining, the tissues were soaked in 70% ethanol to remove chlorophyll and surface dyes. Images were captured under a stereomicroscope (Nikon AZ100 microscope, Nikon, Kyoto, Japan).

The morphology of the zebrafish larvae (otolith and body curvature) was imaged at 4 dpf using a Nikon AZ100 microscope (Nikon, Tokyo, Japan). The larvae were raised in Petri dishes and transferred to a new dish at 5 dpf for behavioral analysis. After allowing adaptation to the new environment for 5 min, locomotion was video-recorded for 3 min using a Nikon DS-Fil1 digital camera and processed with NIS-Elements F3.0 (Nikon).

Whole-mount in situ hybridization (WISH) was performed using probes for cdkn1a, mdm2, aaas, eftud2, gss, huwe1, mettl16, ice1, prpf3, prpf4, snrnp200, prpf6, snapc4, prpf8, snrpe, prpf31, sf3b4, eif4a3, tp53, pcna, her4.1 (Supplementary Table 3)^{60 (link)}. Staged embryos were fixed overnight in 4% PFA, and then dehydrated in a methanol gradient. Embryos were then rehydrated in phosphate-buffered saline containing 0.1% Tween-20 (PBST). Embryos were permeabilized by proteinase K digestion and then hybridized with digoxin-labeled probes overnight at 70 °C. The next day, embryos were washed in a preheated mixture of 50% saline sodium citrate containing 0.1% Tween-20 and 50% hybridization solution at 70 °C. Embryos were washed again at room temperature and incubated in staining solution in the dark until sufficient staining appeared. Embryos were mounted in glycerol and were visualized using a Nikon AZ100 microscope (Nikon, Tokyo, Japan). Images were captured using a Nikon DIGITAL SIGHT DS-Fil1 digital camera (Nikon) and processed with NIS-Elements F 3.0 (Nikon).

Specific primers were designed using Primer 5.0 (Table 1), and linear DNA with T7 and SP6 promoters at both ends was amplified as templates. In vitro transcription to synthesize DIG-labeled sense and antisense probes, respectively, was carried out a DIG RNA labeling kit (SP6/T7; Roche, Germany) and the probes were purified.
Muscle tissues were fixed in 4% paraformaldehyde, gradient dehydrated, embedded, sectioned, and then hybridized using a DIG Nucleic Acid Detection Kit (SP6/T7; Roche, according to the manufacturer’s instructions. After DAB color development, the tissues were photographed and observed using a Nikon AZ100 microscope (Nikon, Japan).

Animals expressing cameleon YC2.60 were imaged with a ×2 AZ-Plan Fluor objective (Nikon) on a Nikon AZ100 microscope fitted with ORCA-Flash4.0 digital cameras (Hamamatsu). Excitation light was provided from an Intensilight C-HGFI (Nikon), through a 438/24 nm filter and an FF458DiO2 dichroic (Semrock). Emission light was split using a TwinCam dual camera adapter (Cairn Research) and passed through CFP (483/32 nm) and YFP (542/27) filters, and a DC/T510LPXRXTUf2 dichroic. We acquired movies using NIS-Elements (Nikon), with 100 ms exposure time.
To image neural activity in freely moving animals (Supplementary Fig. 4g), single young adults were transferred to peptone-free agar plates spotted with 4 µl of concentrated OP50 food in M9 buffer, and imaged at 2× zoom. For all other figures, 4–8 young adults were transferred to peptone-free agar plates, immobilized on a 2 µl patch of concentrated OP50 in M9 buffer using Dermabond adhesive, leaving the nose exposed, and imaged at 4× zoom.

Stomatal anatomy and density from both abaxial and adaxial surface of the leaf were assessed by taking imprints from the first primary leaflet from the second uppermost fully unfolded leaf on both sides of the leaves in three biological replicates per treatment using the silicon rubber impression technique [59 (link)]. Imprints were made using impression material (elite HD+, Zhermack, Badia Polesine, Italy), and imprints were transferred to microscopic slides using nail varnish on the original imprints. Images were acquired at six different locations for each imprint using a Nikon AZ100 microscope (Nikon Corp., Tokyo, Japan) equipped with a Nikon DS-Fi1 camera. Images were analyzed using ImageJ software (version 1.51). For each picture, the stomata number was counted to determine the stomatal density. For measurements of stomatal anatomy, stomatal length, stomatal width, pore length and pore width were determined for all stomata in a randomly selected quarter of three images per biological replicate. The pore area was estimated assuming the shape of an ellipse, and stomata size was estimated by the area of the rectangle encasing the stomata.