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. 2017 Jan 25;13(1):e1006583. doi: 10.1371/journal.pgen.1006583

Fig 4. TOR is required for endocycle progression to activate ecdysone biosynthesis.

Fig 4

(A) Expression of TORDN in the PG causes arrest in DNA content increase at the 3rd instar larval stage. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) of control (phm22 > +) and TORDN (phm22 > TOR.TED) animals were labeled for Dib (green) and DNA (white) at 78 and 114 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (B and C) The C value (B) and number (C) of PG cells of control and TORDN larvae at indicated stages. The CW checkpoint in control (81 hAEL, see S4O Fig) is indicated by a dashed line in B. 11–19 PGs were analyzed for each group. Average C value in control at 114 hAEL is normalized to 64C. Error bars represent standard errors. (D) Expression of TORDN in the PG causes reduction in CycE expression and EdU incorporation at the 3rd instar larval stage. The PGs of control +GFP (phm22 > mCD8::GFP) and TORDN +GFP (phm22 > mCD8::GFP, TOR.TED) larvae were labeled for GFP (green) and CycE or EdU (magenta) at indicated stages. The PGs are outlined by dashed lines. Scale bars, 50 μm. (E and F) Percentages of CycE-positive (E) and EdU-positive (F) PG cells in control +GFP and TORDN +GFP larvae at indicated stages. All data are shown as box plot (see Fig 2E and 2F). The CW checkpoint in control is indicated by dashed lines. 17–26 PGs were analyzed for each group. (G) Expression of CycE in the PG of TORDN restores CycE protein expression. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) in TORDN +GFP, TORDN +CycE-1 +GFP (phm22 > mCD8::GFP, TOR.TED, CycE-1) and TORDN +S6KTE +GFP (phm22 > mCD8::GFP, TOR.TED, S6K.TE) larvae were labeled for GFP (green) and CycE (magenta) at 84 hAEL. Scale bars, 50 μm. (H) Percentages of CycE-positive PG cells in TORDN +GFP, TORDN +CycE-1 +GFP and TORDN +S6KTE +GFP larvae at 84 hAEL. All data are shown as box plot. Numbers of animals tested are in parentheses. (I) Expression of CycE in the PG of TORDN rescues reduction in DNA content. The PGs (upper panels, outlined) and nuclei of PG cells (lower panels, outlined) in TORDN, TORDN +CycE-1 (phm22 > TOR.TED, CycE-1) and TORDN +S6KTE (phm22 > TOR.TED, S6K.TE) animals were labeled for Dib (green) and DNA (white) at 120 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (J) The C value of PG cells of TORDN, TORDN +CycE-1 and TORDN +S6KTE larvae at 120 hAEL. Average C value in TORDN is normalized to 11C, according to data in B. Error bars represent standard errors. Numbers of animals tested are in parentheses. (K) Expression of CycE in the PG of TORDN restores expression of ecdysone biosynthetic genes. Expression of ecdysone biosynthetic genes in TORDN, TORDN +CycE-1 and TORDN +S6KTE larvae at 120 hAEL was measured using qPCR. Average values of three independent data sets are shown with standard errors. (L) Expression of CycE in the PG of TORDN rescues decrease in ecdysteroid level. Whole-body ecdysteroid levels in TORDN, TORDN +CycE-1 and TORDN +S6KTE 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 ANOVA with Tukey’s post hoc test (* P < 0.05). (M) Expression of CycE in the PG of TORDN rescues developmental arrest. Percentages of pupariated TORDN, TORDN +CycE-1 and TORDN +S6KTE animals are shown at indicated stages. Numbers of animals tested are in parentheses. (N) TORDN animal arrested at 3rd instar larval stage (left) and pupariated TORDN +CycE-1 animal (right).