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Fig. S1. Insulin receptor signaling and dMyc function are required for muscle growth. (A-F′′) Body wall muscles from age-matched Drosophila larvae carrying hypomorphic alleles of (A-B′′) dmyc (diminutive) and (C-D′′) the Insulin receptor substrate chico, stained with phalloidin (green) and DAPI (blue). Body wall muscles VL3 and VL4 are encircled in red. Both (B-B′′) dMyc and (D-D′′) Insulin receptor signaling are required for muscle growth and variation of nuclear size, in comparison with (A-A′′,C-C′′) matched heterozygous controls and wild type (not shown). (F-F′′) Overexpression of Cyclin E (CycE) in muscles blocks muscle growth and variation of nuclear size, in comparison with control (E-E′′). This indicates that the endoreplicative cell cycle is involved in ploidy variation, which is necessary for muscle growth. Scale bars: 75 µm in A-F; 18.7 µm in A′-F′.
Fig. S2. RNAi treatment against dmyc, InR and Akt1 impairs muscle growth and endoreplication. Phalloidin (green) and DAPI (blue) staining of body wall muscles from age-matched larvae, in which hairpins against (B-B′′) dmyc, (C-C′′) InR or (D-D′′) Akt1 are driven in muscles. Dicer-2 (Dcr-2) was co-expressed to increase the potency of RNAi treatment (Dietzl et al., 2007). Knockdown of dMyc, InR and Akt1 in all cases results in impaired muscle growth and endoreplication compared with the control (A-A′′). Scale bars: 75 µm in A-D; 18.7 µm in A′-D′.
Fig. S3. Tissue specificity of transgene expression driven by Dmef2-Gal4 and Mhc-Gal4. (A,B) Micrographs of overview of larvae in which DsRed expression has been driven with the (A) Dmef2-Gal4 or (B) Mhc-Gal4 drivers. Auto-fluorescence (red channel) from wild-type larvae was analyzed as control (not shown). Immunofluorescence above background is detected in body wall muscles (somatic muscle lineage) but not in other endoreplicating tissues including fat body (FB) and salivary glands (SG), with both Dmef2-Gal4 and Mhc-Gal4. Dmef2-Gal4 also drives transgene expression in cardiac and visceral muscle lineages, in the muscle (adepithelial) layer of wing imaginal discs (WID), and in a few cells in the brain (not shown). Fluorescence in the gut (G) arises from the underlying muscle layer. Note that Mhc-Gal4 recapitulates the autonomous and non-autonomous phenotypes observed with Dmef2-Gal4, when sufficient transgene expression is driven. (C) InR overexpression in muscles (Dmef2-Gal4 UAS-InR) results in larger larvae and pupae, and increases the size of most endoreplicating organs and, to a lesser extent, of imaginal discs. Compare with control (UAS-Pten, Fig. 2), which does not significantly differ from Dmef2-Gal4 control flies (not shown).
Fig. S4. Overexpression of foxo and dmnt regulates myofiber growth and nuclear ploidy by dMyc. (A-F′′) Micrographs of some of the genotypes quantified in Fig. 5. (A-A′′) Staining of body wall muscles of Dmef2-Gal4 third instar larvae with phalloidin (green) and DAPI (blue). (B-B′′) The InR signaling repressor foxo was overexpressed in muscles using the Dmef2-Gal4 muscle driver. Repression of InR signaling results in a significant decrease in the area of myofibers VL3 and VL4 (encircled in red) with concomitant reduction of nuclear area. (C-C′′) Repression of dMyc signaling, following overexpression of dmnt, impairs muscle growth and endoreplication. (D-E′′) Co-overexpression of foxo and dmyc (dmyc tr2, where tr2 is transgene 2), impairs dMyc-dependent cell cycle progression, indicating that Foxo inhibits dMyc function (quantification in Fig. 5). (F-F′′) Co-expression of dsRed with dmyc does not impair dMyc function. Scale bars: 75 µm in A-F; 18.7 µm in A′-F′.
Fig. S5. Expression pattern of the PG157-Gal4 driver. (A) View of dissected PG157-Gal4 UAS-GFP L3 wandering larvae, with internal organs exposed. (A′) GFP fluorescence outlines the tissues where PG157-Gal4 drives transgene expression. GFP fluorescence is detected in salivary glands (sg, A,A′), cellular clusters in the brain (br, B,B′), trachea (tr, C,C′), and body wall muscles VI1 of the first abdominal segment and of thoracic segments (M, C,C′). See Materials and methods for details.
Fig. S6. Overview of genetic mosaics in muscles and quantification of the intensity of DAPI staining. Overexpression of (A) GFP alone, or GFP together with (B) Pten, (C) Tsc1 and Tsc2, (D) dmyc, (E) dmyc and Pten, or (F) dmyc and Tsc1 and Tsc2 with PG157-Gal4. Transgene expression is driven in muscle VI1 (up, green due to co-expression of GFP), but not in neighboring muscles VL3 and VL4 (down, not green), where no transgene expression is driven. Muscle growth and endoreplication are modulated upon expression of Pten, Tsc1 and Tsc2, or dmyc. Pten, and Tsc1 and Tsc2 antagonize dMyc activity. See Fig. 6 for a detailed analysis. (G) Quantification of relative intensity of DAPI staining in genetic mosaics. Transgene expression in muscle VI1 results in changes in the intensity of DAPI staining, which are normalized in each micrograph by comparison with muscles VL3 and VL4, where no transgene expression is driven. Values refer to the ratio of average intensity of DAPI staining per nucleus in muscle VI1 versus muscles VL3 and VL4, with n>9. Note that variation in the intensity of DAPI staining parallels variation in nuclear size. dMyc promotes an increase in the intensity of DAPI staining, which is indicative of nuclear DNA content, that is antagonized by Pten, and Tsc1 and Tsc2.
Fig. S7. Characterization of the Foxo-dMyc interaction. (A) Densitometric analysis of dMyc protein levels from western blot experiments upon modulation of InR signaling and dmyc overexpression in muscles with Dmef2-Gal4. dMyc protein levels are increased upon dmyc overexpression. (B) Foxo and dMyc do not apparently co-immunoprecipitate. Immunoprecipitation of dMyc and Foxo proteins from S2R+ cells upon no transfection (mock), or transfected with pMT-foxo. Cell extracts were immunoprecipitated with mouse anti-dMyc antibodies. Input, mock and immunoprecipitated materials were western blotted with rabbit anti-dMyc or anti-Foxo antibodies, as indicated. No significant co-immunoprecipitation of Foxo and dMyc is observed, suggesting that they might not physically interact. (C,D) Analysis by qRT-PCR of dmyc and foxo mRNA levels, upon (C) overexpression and (D) RNAi treatment. dmyc and foxo overexpression were induced for 2 days, whereas RNAi treatment was performed for 3 days. Changes in dmyc and foxo mRNA levels are observed, consistent with the treatment that is performed. Levels of α-Tubulin 84B are detected as a normalization control.
Fig. S8. Overexpression of Cyclin E in muscles non-autonomously regulates the growth of other internal organs. (A) Overview of L3 larvae and pupae. Overexpression of Cyclin E (CycE) in muscles (Dmef2-Gal4 UAS-CycE) or RNAi-mediated knockdown of Akt1 levels (Dmef2-Gal4 UAS-Akt1 hp) results in decreased body size, in comparison with matched controls (Dmef2-Gal4). (B) Internal organs of L3 larvae were stained with the lipophilic dye FM4-64 to outline their size. Overexpression of Cyclin E or Akt1 knockdown in muscles results in decreased size of most internal organs, which are affected to different extents. Magnification is 3× (gut) or 10×. (C) The size of the eyes from pupae with decreased Akt1 levels is also affected, in comparison with controls. (D) Impairment of larval feeding behavior in flies overexpressing Cyclin E or with decreased levels of Akt1 in muscles, in comparison with age-matched L3 control larvae (n=25, P<0.001). Transgene expression was driven at 25°C.
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