A large-scale transgenic RNAi screen identifies transcription factors that modulate myofiber size in Drosophila

Figures

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  Fig 1. Muscle-specific RNAi screening identifies transcription factors that modulate developmental growth of Drosophila body wall skeletal muscles. (A) Scheme for the identification of transcription factors that modulate developmental skeletal muscle growth in Drosophila. The skeletal muscle-specific Mef2-Gal4 was crossed with a collection of 1114 transgenic RNAi lines that target 447 transcription factors and transcriptional regulators to test their function in developmental muscle growth. Mef2-Gal4 drives transgene expression in the body wall musculature located beneath the epidermis, and composed of muscles with stereotypical sizes, each consisting of a single myofiber. Because skeletal muscle-specific interventions that regulate the size of body wall muscles correspondingly change the size of the larva [35,37], we have scored the size of larvae as convenient readout to assess the outcome of muscle-specific RNAi interventions. Moreover, in cases where adult flies eclosed, also wing positioning was scored as upheld or depressed wings can indicate muscle developmental defects and degeneration. (B) Compared to control RNAi, most RNAi interventions lead to 3rd instar larvae of normal…

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  Fig 2. Muscle-specific RNAi for screen hits identifies transcription factors that modulate developmental myofiber growth in Drosophila. (A) Validation of RNAi screen hits via dissection of 3rd instar larvae and confocal imaging of ventral longitudinal VL3 and VL4 body wall skeletal muscles, which have stereotypical sizes. (B) Compared to controls (whiteRNAi and mcherryRNAi), RNAi for screen hits driven in skeletal muscle by Mef2-Gal4 leads to a decrease (atrophy) and an increase (hypertrophy) in the size of VL3 and VL4 skeletal muscles, each consisting of a single myofiber. (C) Quantitation of the cumulative area of VL3 and VL4 muscles from multiple larvae indicates that RNAi for Nurf-38, e(y)1, alien, CG7839, Taf1, MBD-R2, mtTFB2, pdm3, and Dati reduces VL3+VL4 muscle area (atrophy). This indicates that these transcription factors are necessary for optimal myofiber growth during larval development. (D) Conversely, muscle-specific RNAi for Foxo, Cnc, and Deaf1 increases the area of VL3+VL4 muscles (hypertrophy), indicating that these transcription factors limit developmental myofiber growth. N = 12–70 and mean±SD is shown; **P<0.01, ***P<0.001.

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  Fig 3. Muscle-restricted activity of Deaf1 and Gsk3 impedes developmental muscle growth. (A) Transgenic overexpression of the transcription factor Deaf1, driven specifically in skeletal muscle with Mef2-Gal4, leads to a reduction in the area of larval muscles, each consisting of a single myofiber, compared to controls with no transgene expression (Mef2>+). (B) The area of ventral longitudinal VL3 and VL4 muscles is lower upon Deaf1 overexpression, whereas it increases upon insulin receptor (InR) overexpression. N = 9–19 and mean±SD is shown; ****P<0.001. (C) Gsk3 transgenic overexpression driven in skeletal muscle with Mef2-Gal4 leads to small size of 3rd instar larvae in a manner proportional to Gsk3 kinase activity: Gsk3 CA (constitutive active) and WT (wild-type) induce atrophy, hypomorphic (HYP) mutants with only limited kinase activity have little impact, whereas KD (kinase-dead) Gsk3 mutants do not affect developmental muscle growth. (D) Gsk3 activity in muscle reduces body size, as indicated by larvae where body wall skeletal muscle are shown by expression of Mhc-GFP. (E) Compared to controls (no transgene and mcherry overexpression), overexpression of 2 different Gsk3CA…

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  Fig 4. GSK3 and Deaf1 induce similar gene expression changes in larval skeletal muscles. (A) Validation of genetic interventions. Muscle-restricted Deaf1 overexpression leads to an increase in Deaf1 mRNA levels, opposite to Deaf1 RNAi. As expected, GSK3 overexpression also increases Gsk3 mRNA levels. (B) Coincident with their opposite roles in regulating myofiber size, converse gene expression changes (R2 = 0.46; genes regulated with p<0.05) are induced in larval body wall muscles by Deaf1 overexpression (OE) and Deaf RNAi, each normalized to their respective controls, i.e. no transgene overexpression and control white RNAi. (C) Largely similar gene expression changes are induced by overexpression of Deaf1 and of constitutive active (CA) Gsk3 (R2 = 0.49; p<0.05). (D) DAVID GO term analysis reveals gene categories that are enriched among Deaf1-regulated genes, which include glycolysis. (E) Analysis of glycolytic genes reveals that most of them are significantly (p<0.05) and concordantly regulated by Deaf1 and Gsk3. Specifically, glycolytic enzymes are upregulated by Deaf1 RNAi compared to control whereas their expression is suppressed by Deaf1 OE and Gsk3CA OE. S2 Table reports the…

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  Fig 5. RNAi for glycolytic enzymes impedes developmental growth of larval body wall skeletal muscles. (A) RNAi for glycolytic enzymes driven in skeletal muscle with Mef2-Gal4 leads to small size of 3rd instar larvae, indicating that glycolysis is needed to sustain developmental muscle growth. A full list of phenotypes obtained with 44 lines targeting glycolysis is reported in S3 Table. (B) Transgenic RNAi for the glycolytic enzymes Eno and Pglym78 reduces myofiber size, compared to control whiteRNAi and cherryRNAi. (C) The area of ventral longitudinal VL3 and VL4 muscles, each consisting of a single myofiber, is reduced upon EnoRNAi and Pglym78RNAi. N = 12–29 and mean±SD is shown; **P<0.01 and ***P<0.001.