Spatial Activation of TORC1 Is Regulated by Hedgehog and E2F1 Signaling in the Drosophila Eye  Wonho Kim, Yoon-Gu Jang, Jinsung Yang, Jongkyeong Chung  Developmental Cell  Volume 42, Issue 4, Pages 363-375.e4 (August 2017) DOI: 10.1016/j.devcel.2017.07.020 Copyright © 2017 Elsevier Inc. Terms and Conditions

Developmental Cell 2017 42, 363-375. e4DOI: (10. 1016/j. devcel. 2017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 TORC1 Is Selectively Activated in the S-Phase Cells of the SMW (A) S2 cells were subjected to amino acid and serum starvation for an hour, and then they were replenished with amino acid for 20 min. Lysate samples were prepared, separated by SDS-PAGE, and immunoblotted with the anti-pS6, -pS6K, -S6K, and -β-tubulin antibodies. (B) Specific dsRNAs were used to knock down S6K, TOR, Raptor, Rheb, or tsc2 in S2 cells as indicated. dsRNA for Luciferase was used as a control. After 4 days, the S2 cells were serum-starved for 4 hr. Lysate samples were separated by SDS-PAGE and subsequently immunoblotted by the anti-pS6, -pS6K, -S6K, and -β-tubulin antibodies. (C) The eye discs of wandering w1118 larvae were immunostained with Hoechst (DNA, blue) and the anti-pS6 (red) antibody. Arrowhead indicates the MF. (D) Boxed area of (C) was magnified. (E) The eye disc expressing a GFP-fused PCNA via endogenous PCNA promoter was immunostained with the antibody against pS6 (red). (F) The boxed area of (E) was magnified. (G and H) S6K (G) and TOR (H) mutant clones were generated and immunostained with the anti-pS6 antibody (red). The absence of GFP indicates the mutant clones. (I) GFP-positive Raptor RNAi-expressing clones were stained with the anti-pS6 antibody (red). (J) Rheb mutant clones marked by the absence of GFP were examined by immunostaining with anti-pS6 antibody (red). (K) GFP-positive Rheb-expressing clones were immunostained with the anti-pS6 antibody (red). (L) GFP-negative clones in which tsc2 was mutated were immunostained with the anti-pS6 antibody (red). (K and L) Due to the strong anti-pS6 immunostaining signals from clones, the pS6 signals in the SMW looked very faint. The boxed areas in (G)–(L) are magnified in the rightmost panels, where the boundary of the clones is indicated by white dotted lines. Scale bars, 30 μm. See also Figures S1–S3. Developmental Cell 2017 42, 363-375.e4DOI: (10.1016/j.devcel.2017.07.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Insulin and Amino Acid Signalings Are Required for TORC1 Activity in the SMW (A) Wild-type eye disc was immunostained with the anti-pAkt antibody (red). (B–D) InRDN- (B), PI3KDN- (C), and Akt RNAi (D)-expressing clones were marked by GFP and immunostained with the anti-pS6 antibody (red). (E) A dominant negative form of RagA was clonally expressed in the eye disc, which is indicated by GFP. RpS6 phosphorylation (red) was examined. (F) Phosphorylated RpS6 (red) was immunostained in the eye disc in which RagC mutant clones (GFP-negative) were generated. (G–I) RNAi targeting HBXIP (G), p18 (H), or mio (I) was expressed using DE-GAL4, and RpS6 phosphorylation (red) was examined. Dicer2 was expressed together for the higher knockdown efficiency. (J) The eye disc of seh1 mutant larvae was immunostained with the anti-pS6 antibody (red). (A and G–J) DNA was marked by Hoechst (blue). (B–F) The boxed areas are magnified in the rightmost panels, where the boundary of the clones is indicated by white dotted lines. (G–I) The dorsal regions in which transgenes were expressed by DE-GAL4 are above the dotted lines. Scale bars, 30 μm. See also Figure S4. Developmental Cell 2017 42, 363-375.e4DOI: (10.1016/j.devcel.2017.07.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Hh Signaling Is Required for TORC1 Activity through Inhibiting the Formation of CiR (A) RpS6 phosphorylation (red) was examined in Smo mutant clones (GFP-negative). (B) Smo mutant clones expressing Ci RNAi (GFP-positive) were generated in the eye disc, which was stained with the anti-pS6 (red) and -Ci (yellow) antibodies. (C) CiR was clonally expressed with GFP in the eye disc, and phosphorylated RpS6 (red) was immunostained. (D) The eye disc in which Ci RNAi and Dicer2 were expressed using DE-GAL4 was immunostained with the anti-pS6 (red) and -Ci (green) antibodies. The dorsal region in which transgenes were expressed by DE-GAL4 is above the dashed line. (A–C) The boxed areas are magnified in the rightmost panels, where the boundary of the clones is indicated by white dotted lines. Scale bars, 30 μm. See also Figure S5. Developmental Cell 2017 42, 363-375.e4DOI: (10.1016/j.devcel.2017.07.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 E2F1 Transcription Factor Positively Regulates TORC1 Downstream of Smoothened (A) E2F1 RNAi was expressed in the eye disc using DE-GAL4, and RpS6 phosphorylation (red) was examined by immunostaining. (B) Rbf1 RNAi was expressed with Dicer2 in the eye disc using DE-GAL4, and phosphorylated RpS6 (red) was examined. (C) Smo mutant clones (absence of RFP) were generated in the eye disc in which a GFP-fused PCNA was expressed via endogenous PCNA promoter. (D) DE-GAL4 was used to express Rbf1 RNAi in the eye disc in which Smo mutant clones (absence of GFP) were generated. The eye disc was immunostained with the anti-pS6 antibody (red). (E and F) RNAi for cyclin D (E) or Cdk4 (F) was expressed with RFP (not shown in the figure) by DE-GAL4, and RpS6 phosphorylation (red) was examined. (G) Cyclin D and Cdk4 were expressed with RFP (not shown in the figure) by DE-GAL4, and the eye disc was immunostained with the anti-pS6 antibody (red). (H) E2F1 RNAi was expressed with cyclin D and Cdk4 by DE-GAL4, and RpS6 phosphorylation (red) was examined. EGFP was expressed together as a marker (not shown in the figure). (A, B, and D–H) The dorsal regions in which transgenes were expressed by DE-GAL4 are above the dotted lines. (A, B, and E–H) DNA was marked by Hoechst (blue). (C and D) The boxed areas are magnified in the rightmost panels, where the boundary of the clones is indicated by white dotted lines. Scale bars, 30 μm. Developmental Cell 2017 42, 363-375.e4DOI: (10.1016/j.devcel.2017.07.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Genetic Epistasis of Hh-E2F1 Signaling to the TSC Complex and Insulin/Amino Acid Signaling (A) Smo mutant clones in which Rheb was overexpressed with GFP were generated in the eye disc, and RpS6 phosphorylation (red) was examined. (B) Smo mutant clones (GFP-negative) were generated in the eye disc in which tsc2 RNAi and Dicer2 were expressed using DE-GAL4, and the eye disc was stained with the anti-pS6 antibody (red). (C) tsc2 RNAi was expressed with Dicer2 using DE-GAL4, and RpS6 phosphorylation (green) was examined. (D) E2F1 RNAi and tsc2 RNAi were simultaneously expressed with Dicer2 using DE-GAL4, and phosphorylated RpS6 (green) was immunostained. (E) The eye disc in which Smo mutant clones (GFP-negative) was generated and immunostained with the anti-pAkt antibody (red). (F and G) E2F1 RNAi (F) or Rbf1 RNAi (G) was expressed with Dicer2 by DE-GAL4, and the eye disc was immunostained with the anti-pAkt antibody (green). (H and I) PTEN mutant clones (absence of GFP) (H) or Smo and PTEN double-mutant clones (absence of GFP) (I) were generated and immunostained with anti-pS6 antibody (red). (J) RagACA and E2F1 RNAi were expressed with Dicer2 by DE-GAL4 and phosphorylated RpS6 (red) was immunostained. (B–D, F, G, and J) The dorsal regions in which transgenes were expressed by DE-GAL4 are above the dotted lines. (C, D, F, G, and J) DNA was marked by Hoechst (blue). (A, B, E, H, and I) The boxed areas are magnified in the rightmost panels, where the boundary of the clones is indicated by white dotted lines. Scale bars, 30 μm. See also Figure S2. Developmental Cell 2017 42, 363-375.e4DOI: (10.1016/j.devcel.2017.07.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 TORC1 Promotes the G1/S Transition of the Cells in the SMW Downstream of Hh Signaling (A–C) BrdU was incorporated in the eye disc in which mutant clones of TOR (A), S6K (B), or both TOR and 4E-BP (C) (GFP-negative) were generated, and the eye disc was immunostained with the anti-BrdU antibody (red). (D) 4E-BP-expressing S6K mutant clones, which were marked by GFP, were generated and BrdU incorporation (red) was examined. (E and F) The eye disc in which TOR mutant clones (GFP-negative) were generated was immunostained with the anti-cyclin E (E) or -cyclin A (F) antibodies (red). (G) Smo mutant clones (GFP-negative) were generated in the eye disc and the larvae were fed with BrdU for 2 hr. The eye disc was fixed and immunostained with the anti-BrdU (magenta) and -pS6 (red) antibodies. Arrows indicate Smo mutant cells at the clone boundary which entered S phase. Note that the TORC1 activity is still inhibited in these cells (no red signal in GFP-negative clones regardless of BrdU incorporation), as it is in other Smo mutant cells. (H) tsc2 RNAi was expressed with Dicer2 using DE-GAL4, and Smo mutant clones (absence of GFP) were generated in the eye disc. BrdU was incorporated in the eye disc, and the eye disc was immunostained with the anti-BrdU antibody (red). The dorsal region in which transgenes was expressed by DE-GAL4 is above the dotted line. (A–H) The boxed areas are magnified in the rightmost panels, where the boundary of the clones is indicated by white dotted lines. Scale bars, 10 μm (A–C, E, and F) or 30 μm (D, G, and H). See also Figure S6. Developmental Cell 2017 42, 363-375.e4DOI: (10.1016/j.devcel.2017.07.020) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Hh-E2F1-cyclin D/Cdk4-mTORC1 Signaling Axis Is Conserved in Mammalian Cells (A) HeLa cells were treated with recombinant sonic Hedgehog (Shh, 4 μg/mL) or starved with amino-acid-free DMEM (stv) for 2 hr and the lysate samples were immunoblotted with anti-pS6, -S6, and -β-tubulin antibodies. (B) HeLa cells were treated with purmorphamine (Pur, 20 μM) and/or starved with amino-acid-free DMEM. The lysate samples were immunoblotted with the same antibodies as in (A). (C–E) E2F1/2/3 (C), CDK4/6 (D), or cyclin D1/2/3 (E) knockdown cells were treated with Pur and/or starved with amino-acid-free DMEM. The lysate samples were immunoblotted with the same antibodies as in (A). (F) Comparison of cyclin D1 mRNA expression levels in E2F1/2/3 knockdown HeLa cells with or without Pur treatment (n = 3). Significance was determined by Student's two-tailed t test (∗∗∗p < 0.001; ns, not significant). Error bars indicate SD. (G) Cyclin D1-overexpressed and/or E2F1/2/3 knockdown cells were treated with Pur and/or starved with amino-acid-free DMEM. The lysate samples were immunoblotted with anti-pS6, -S6, -HA, and -β-tubulin antibodies. (B–G) The indicated cells were treated with 20 μM Pur for 72 hr. See also Figure S7. Developmental Cell 2017 42, 363-375.e4DOI: (10.1016/j.devcel.2017.07.020) Copyright © 2017 Elsevier Inc. Terms and Conditions