Fig 3. Endocycle is required for ecdysone biosynthesis.
(A) Knockdown of CycE in the PG causes reduction in DNA content. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) of control (phm22 > dicer2) and CycE RNAi-1 (phm22 > dicer2, CycE RNAi-1) animals were labeled for Dib (green) and DNA (white) at 120 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (B and C) Knockdown of endocycle regulators in the PG causes reduction in DNA content. Each gene was knocked down using two independent RNAi lines. The C value (B) and number (C) of PG cells of control (black bars) and RNAi (phm22 > dicer2, RNAi; gray bars) larvae at 120 hAEL. Average C value in control is normalized to 64C. Error bars represent standard errors. Numbers of animals tested are in parentheses. Significance was calculated using Student’s t-test (* P < 0.001; n.s., Not significant). (D) Knockdown of endocycle regulators in the PG causes arrest at the 3rd instar larval stage. Developmental profiles of control and RNAi animals are shown. Numbers of animals tested are in parentheses. (E) Pupariated control animal (left) and CycE RNAi-1 larva arrested at the 3rd instar stage (right). (F) Knockdown of Fzr in the PG causes block of mitotic-to-endocycle transition. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) in control (phm22 > dicer2), Fzr RNAi (phm22 > dicer2, Fzr RNAi) and Fzr RNAi +InRCA (phm22 > dicer2, Fzr RNAi, InR.A1325D) animals were labeled for Dib (green) and DNA (white) at 120 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (G and H) The C value (G) and number (H) of PG cells of control, Fzr RNAi and Fzr RNAi +InRCA larvae at 120 hAEL. Average C value in control is normalized to 64C. Error bars represent standard errors. Numbers of animals tested are in parentheses. (I) Knockdown of Fzr causes reduction in expression of ecdysone biosynthetic genes. Expression of ecdysone biosynthetic genes in control, Fzr RNAi and Fzr RNAi +InRCA larvae at 120 hAEL was measured using qPCR. Average values of three independent data sets are shown with standard errors. (J) Knockdown of Fzr causes decrease in ecdysteroid level. Whole-body ecdysteroid levels in control, Fzr RNAi, and Fzr RNAi +InRCA larvae at 120 hAEL were measured using ELISA. Average values of five independent data sets are shown with standard errors. Statistical significance was calculated using Student’s t-test (* P < 0.05). (K) Knockdown of Fzr in the PG causes developmental arrest at the 3rd instar larval stage. Percentages of pupariated animals in control, Fzr RNAi and Fzr RNAi +InRCA animals are shown at indicated stages. Numbers of animals tested are in parentheses. (L) Pupariated control animal (left) and Fzr RNAi and Fzr RNAi +InRCA larvae arrested at the 3rd instar stage (middle and right, respectively). (M) 20E feeding rescues developmental arrest in Fzr RNAi. Percentages of pupariated Fzr RNAi animals reared on 20E-containing or control medium from 72 hAEL are shown at indicated stages. Numbers of animals tested are in parentheses.