Fig. 3

Key methods that have been applied to P. tepidariorum. a Schematic representation of the typical procedure and timeline of pRNAi-mediated gene knockdown in P. tepidariorum. dsRNA for the target gene is repeatedly injected into the abdomen of a mated female. The RNAi effect typically appears in egg sacs that are produced more than 1 week after the first dsRNA injection. b The setup for microinjection and three examples of microinjection applications, depending on the materials injected. Bleached eggs are attached on a glass slide using double-sticky tape, covered with halocarbon oil, and then injected. 16- to 128-cell stages constitute the period when the injection can be performed relatively easily. For eRNAi, dsRNA for Pt-orthodenticle (Pt-otd) was co-injected with FITC-dextran, visualized in red in the fixed sample. The embryo was also stained for Pt-hh transcript, demonstrating that Pt-otd positively regulates Pt-hh expression in the anterior region of the nascent germ band [11]. The images showing histone-tdEosFP expression and the eRNAi are adapted and modified from Hemmi et al. [13] (CC BY 4.0) and Kanayama et al. [11] (CC BY-NC-SA 3.0), respectively. c Flat preparation of a germ band stained for Pt-hh (green) and Pt-noto1 (red) transcripts by multicolor FISH and counter-stained for DNA (blue). This image is adapted and modified from Hemmi et al. [13] (CC BY 4.0). d The infrared laser module used for laser ablation (left) and an example of a laser-irradiated embryo (right). The XYClone laser module is integrated with a 20× objective lens (Hamilton Thorne). In the embryo shown, which had been laser-irradiated 7 h before, dead cells derived from the irradiated region were apparent (asterisks). The left (L) and right (R) embryonic fields were developing into partially separate germ bands. The image of the embryo is adapted and modified from Oda et al. [22] (CC BY 4.0). Scale bars: 50 µm