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First published online 11 February 2009
doi: 10.1242/dev.027466

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Integration of Insulin receptor/Foxo signaling and dMyc activity during muscle growth regulates body size in Drosophila

¹ Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
² Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

View larger version (39K): [in this window] [in a new window]   Fig. 1. Body wall muscles of Drosophila larvae consist of single, syncytial myofibers containing several polyploid nuclei. (A) Phalloidin staining of body wall muscles from a wild-type third instar (L3) larva. Several muscle cells (myofibers) of different sizes can be seen. The red box delineates VL3 and VL4 muscles of an abdominal segment. (B,C) The outline (B) and staining for F-actin (phalloidin, green) and nuclei (DAPI, blue) (C) of body wall muscles VL3 and VL4. Each muscle is composed of a single syncytial cell (myofiber), which differs in size and number of nuclei. (D-H) Quantification of (D) the number of nuclei, (E) myofiber area, (F) ratio of myofiber area/nucleus, (G) nuclear area, and (H) intensity of DAPI staining in VL3 and VL4 muscles. There is no significant difference in the nuclear area between body wall muscles VL3 and VL4, suggesting that ploidy is not a significant cause of differential growth. n(muscles)=10, n(nuclei muscle VL3)=100, n(nuclei muscle VL4)=50; ***P<0.001. (I-K) Variation of (I) myofiber area and (J) nuclear area during larval growth, and (K) quantification of these results. Note the correlation between the extent of muscle growth, the increase in nuclear size, and the intensity of DAPI staining. For statistical analysis in K, n(muscles)=9, n(nuclei)=100 for nuclear size, and n(nuclei)=10 for intensity of DAPI staining. Error bars indicate s.d. Scale bars: 300 µm in A; 47.5 µm in C; 75 µm in I; 22 µm in J.

View larger version (58K): [in this window] [in a new window]   Fig. 2. Inhibition of muscle growth affects the size of the entire body and of other tissues. (A-C'') Staining of body wall muscles of L3 Drosophila larvae with phalloidin (green) and DAPI (blue). (A-A'') Matched controls (Dmef2-Gal4). (B-C'') Activators [B, Insulin-like receptor (InR)] and repressors [C, Insulin-like receptor dominant-negative (InR DN)] were overexpressed in muscles using the Dmef2-Gal4 muscle driver. (B-B'') Activation of InR signaling results in a significant increase in the area of myofibers VL3 and VL4 (encircled in red) with a concomitant increase in nuclear area. (C-C'') Inhibition of InR signaling exerts converse effects. Scale bars: 75 µm in A-C; 18.7 µm in A'-C'. (D) Quantification of average larval weight (n>20), larval length (n>15), diameter (n>15) and volume (n>15) of larvae in which InR, InR DN, Pten, foxo or Tsc1 and Tsc2 have been overexpressed using Dmef2-Gal4. A decrease in muscle growth (see Fig. 5) always correlates with a reduction in larval body size. Consistent with these results, overexpression of InR, which promotes an increase in muscle growth (B), also increases larval body size. (E) Quantification of length (n>10), diameter (n>10) and volume (n>15) of pupae arising from larvae in which InR signaling has been modulated in muscles (see Fig. 5). Error bars indicate s.d.; *P<0.05, **P<0.01, ***P<0.001. (F) Overexpression of Pten in muscles using Dmef2-Gal4 (Dmef2-Gal4 UAS-Pten) or Mhc-Gal4 (Mhc-Gal4 UAS-Pten) results in smaller larvae and pupae when compared with the control (UAS-Pten). (G)The size of internal tissues and organs, visualized with the lipophilic dye FM4-64, is also affected. Note, especially, the reduction in size of endoreplicating tissues. Magnification: 10x, except for gut (3x). For a full description of genotypes, see Table S1 in the supplementary material.

View larger version (88K): [in this window] [in a new window]   Fig. 3. Inhibition of InR signaling in muscles induces catabolic programs in endoreplicating tissues and modulates larval feeding behavior. (A-B') Transmitted light microscopy images of fat body and salivary gland cells from (A,B) control Drosophila larvae (Dmef2-Gal4) and (A',B') Dmef2-Gal4 UAS-Pten larvae. Lipid remobilization and catabolic events, possibly related to autophagy, are detected respectively in (A') fat body and (B') salivary gland cells of larvae in which Pten is overexpressed in muscles, in comparison with matched controls (A,B). Note the reduction in cell size (encircled in green) and nuclear size (indicative of nuclear ploidy, encircled in red) in salivary gland. Magnification: 40x (fat body) and 63x (salivary gland). (C) Modulation of InR signaling in muscles regulates larval feeding behavior. The number of mouth hook contractions every 30 seconds is significantly reduced in larvae that overexpress Pten, Tsc1 and Tsc2, or foxo in muscles, and is increased upon InR overexpression. n>50; error bars indicate s.d.; ***P<0.001. A similar regulation of feeding behavior was observed in Mhc-Gal4 UAS-Pten larvae (not shown).

View larger version (50K): [in this window] [in a new window]   Fig. 4. Modulation of InR signaling during muscle growth affects the final size of adult flies. (A) Adult flies in which muscle growth has been altered by either activating (InR) or inhibiting (InR DN) InR signaling. (B) Significant changes are correspondingly observed in fly body weight, eye size, abdomen length and wing area. Changes in wing area result from an increase (InR) or decrease (InR DN) in cell size and possibly also cell number. Note that because the growth of distinct body parts is differentially affected, body proportions are also altered. Error bars indicate s.d.; **P<0.01, ***P<0.001; n(weight)>20, n(eye)>12, n(abdomen)>22, n(wing)=10.

View larger version (62K): [in this window] [in a new window]   Fig. 5. InR/Tor signaling regulates muscle growth and nuclear ploidy by inhibiting dMyc function. (A,A') Expression of dmyc is promoted by InR and antagonized by Foxo. qRT-PCR analysis of dmyc transcript levels in Drosophila L3 larvae in which either InR or foxo is overexpressed in body wall muscles. (A) A 2-fold increase in dmyc levels is observed upon InR overexpression in muscles, whereas (A') foxo activation results in a significant 2.5-fold reduction of dmyc transcripts. Error bars indicate s.d. (n=4); **P<0.01. (B-G'') Staining of body wall muscles of L3 larvae with phalloidin (green) and DAPI (blue). (B-B'') Dmef2-Gal4. Overexpression of the InR signaling negative regulators (C) Pten and (D) Tsc1 and Tsc2 in muscles using Dmef2-Gal4. (B-D'') Repression of InR signaling results in all cases in a significant decrease in the area of muscles VL3 and VL4 (encircled in red) with a concomitant reduction in nuclear area. (E) Overexpression of dmyc results in a significant increase in nuclear area without a proportional increase in myofiber area. (F) Co-expression of dmyc with Pten, or (G) with Tsc1 and Tsc2, is sufficient to suppress dMyc-driven polyploidization, indicating that Insulin signaling antagonizes dMyc. For additional examples of muscle phenotypes, generated by overexpressing dmnt and foxo,see Fig. S4 in the supplementary material. Scale bars: 75 µm in B-G; 18.7 µm in B'-G'. (H-K) Quantification of (H) the number of nuclei, (I) myofiber area, (J) nuclear area and (K) intensity of DAPI staining in muscles VL3 (blue) and VL4 (red). Modulation of InR/Tor signaling in muscles is sufficient to promote significant changes in muscle size, which parallel variation in nuclear area and intensity of DAPI staining, but not in the number of nuclei. For statistical analysis, n(myofibers)=10, n(nuclei muscle VL3)=100, n(nuclei muscle VL4)=50; n(nuclei)=10 for intensity of DAPI staining; error bars indicate s.d.

View larger version (66K): [in this window] [in a new window]   Fig. 6. dMyc is autonomously antagonized by InR/Tor signaling. (A-F') Mosaic analysis of Drosophila body wall muscles. (A) PG157-Gal4 drives the expression of transgenes in only a subset of body wall muscles (green for concurrent GFP expression; yellow in merge). Body wall muscles with no GFP expression (red) serve as control. DAPI (blue) and phalloidin (red) staining are used to outline nuclei and muscles, respectively. In A'-F', only DAPI staining is shown (white) and the red line demarcates transgene-expressing (above) from non-expressing (below) muscles. (A,A') Expression of GFP alone does not alter muscle and nuclear area. (B,B') Expression of GFP concomitantly with Pten, or (C,C') Tsc1 and Tsc2, results in a marked decrease in muscle growth (see Fig. S6 in the supplementary material) and nuclear size (green, above in micrographs), in comparison with neighboring VL3 and VL4 muscles in which no transgene expression occurs (red, below in micrographs). (D) dmyc expression results in a marked increase in nuclear size, in comparison with neighboring control muscles. (E,E') Concomitant expression of dmyc with Pten, or (F,F') with Tsc1 and Tsc2, results in decreased muscle and nuclear size, indicating that dMyc function is autonomously controlled by Pten and Tsc. Representative nuclei are shown in the insets. Scale bar: 37.5 µm. (G) Foxo inhibits dMyc transcriptional activity. Luciferase assays performed using three different dMyc transcriptional reporters (CG4364, CG5033 and CG5033 E-box) and overexpression of dmyc and foxo. Transfection of S2R+ cells, with or without subsequent serum starvation, was performed with dmyc (pMT-dmyc), foxo (pMT-foxo), or both in combination. Activation of endogenous Foxo by serum starvation, or following foxo overexpression, suppresses transcription of the dMyc Luciferase reporters. (H) Luciferase assays using dMyc reporters in dmyc and foxo RNAi-treated cells. dmyc RNAi suppresses, whereas foxo RNAi promotes, Luciferase expression. However, in combination with dmyc RNAi, foxo RNAi does not bring about a similar transcriptional regulation. Relative Luciferase activity corresponds to the firefly:Renilla luminescence ratio. The s.e.m. is indicated (n=4).

View larger version (52K): [in this window] [in a new window]   Fig. 7. dMyc primes muscle cells for growth. dMyc promotes biogenesis of nucleoli and expression of genes required for protein synthesis. (A) Fibrillarin immunoreactivity (red) stains the nucleoli of transgene-expressing muscles (green for concurrent GFP expression, above in micrographs; nuclei identified by DAPI staining, blue) and of neighboring control myofibers [outlined in white, based on phalloidin staining (not shown), below in micrographs]. (B) dmyc overexpression promotes nucleolus biogenesis that is, however, insufficient to drive muscle growth. (A',B') Fibrillarin immunoreactivity, together with representative nuclei (blue) and nucleoli (red; insets). Scale bar: 37.5 µm. (C)qRT-PCR analysis of dMyc target genes involved in growth. Significant induction of gene expression is observed upon dmyc overexpression and, to a lesser extent, upon overexpression of InR. Error bars indicate s.d. (n=4).

View larger version (43K): [in this window] [in a new window]   Fig. 8. Interplay of growth signals during muscle growth regulates Drosophila body size. Integration of growth signals controls endoreplication, muscle growth and body size. Inhibition of InR/Tor signaling is accompanied by activation of Foxo, which inhibits transcription of dmyc. In turn, dMyc protein requires input from InR/Tor signaling for its maximal function. dMyc promotes endoreplication, biogenesis of nucleoli and expression of genes required for protein synthesis. However, dMyc is necessary, but not sufficient, to sustain extensive muscle growth, which also requires concurrent activation of InR/Tor signaling to drive protein synthesis and other anabolic processes. A decrease or increase in muscle mass in turn perturbs the growth of other tissues and, indeed, the whole body, at least in part by regulating the feeding behavior of the larva.

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