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

Myofiber atrophy occurs with aging and in many diseases but the underlying mechanisms are incompletely understood. Here, we have used >1,100 muscle-targeted RNAi interventions to comprehensively assess the function of 447 transcription factors in the developmental growth of body wall skeletal muscles in Drosophila. This screen identifies new regulators of myofiber atrophy and hypertrophy, including the transcription factor Deaf1. Deaf1 RNAi increases myofiber size whereas Deaf1 overexpression induces atrophy. Consistent with its annotation as a Gsk3 phosphorylation substrate, Deaf1 and Gsk3 induce largely overlapping transcriptional changes that are opposed by Deaf1 RNAi. The top category of Deaf1-regulated genes consists of glycolytic enzymes, which are suppressed by Deaf1 and Gsk3 but are upregulated by Deaf1 RNAi. Similar to Deaf1 and Gsk3 overexpression, RNAi for glycolytic enzymes reduces myofiber growth. Altogether, this study defines the repertoire of transcription factors that regulate developmental myofiber growth and the role of Gsk3/Deaf1/glycolysis in this process.

Introduction

Skeletal muscle is a key tissue of the human body accounting for approximately 40–50% of the total body mass. A balance between muscle protein synthesis and breakdown is essential for maintaining the functionality and size of skeletal muscles [1]. When muscle protein synthesis exceeds protein degradation, this leads to skeletal muscle hypertrophy, which typically results from an increase in myofiber size. Conversely, myofiber atrophy occurs when protein breakdown is excessive or protein synthesis is insufficient [1,2]. This occurs following inactivity, fasting, as a side effect of many pharmacological treatments, and in the course of many degenerative diseases such as cancer cachexia, chronic heart disease, diabetes, sepsis, infections, chronic obstructive pulmonary disease, and renal failure [3]. Importantly, the loss of muscle mass is not just a side-effect of these conditions but a rather important contributor to morbidity and mortality. Strikingly, prevention of skeletal muscle mass loss in tumor-bearing mice results in increased survival even if cancer progression is not halted [4-6]. Despite great strides towards understanding the mechanisms responsible for muscle wasting, incomplete knowledge in this area has hampered the development of suitable therapies.

Gene expression changes are fundamental drivers of myofiber atrophy [7]. Many signaling pathways that induce atrophy impinge on key transcription factors to promote muscle protein degradation [1,8-10]. For example, forkhead box O (FoxO) transcription factors are activated in response to decreased insulin/IGF signaling and induce the expression of components of the autophagy-lysosome and ubiquitin-proteasome systems which in turn mediate protein degradation [11-17]. However, apart from a few transcription factors that have been extensively studied [1,8], much remains to be learnt on the role that the ~1,400 transcription factors encoded by the human genome [18] play in skeletal muscle mass homeostasis.

Drosophila body wall skeletal muscles have emerged as an important model system to determine the mechanisms of muscle growth and differentiation [19-34]. Previously, we have found that FoxO overexpression in larval body wall skeletal muscles leads to myofiber atrophy and reduces developmental muscle growth [35], suggesting that the fruit fly Drosophila melanogaster can be used to identify evolutionary-conserved regulators of myofiber size [10,36,37]. Here, by examining the impact of transgenic RNAi on developmental muscle growth, we have tested 1,114 RNAi lines targeting 447 of the 708 transcription factors encoded by the Drosophila genome [38]. Our study provides information on many novel transcription factors necessary for myofiber size determination. These include the transcription factor Deaf1 that is annotated as a phosphorylation target of the kinase GSK3-b [39], which is a known inducer of atrophy [40,41]. Similar to Gsk3, Deaf1 overexpression induces myofiber atrophy, whereas Deaf1 RNAi induces myofiber hypertrophy. Gene expression profiling further indicates that Gsk3/Deaf1-induced changes in myofiber size are associated with corresponding changes in the expression of glycolytic enzymes. Altogether, this study expands the repertoire of transcription factors that are implicated in myofiber size determination and indicates a possible role of Deaf1 in this process.

Results

A RNAi screen targeting transcription factors in Drosophila body wall skeletal muscles identifies regulators of muscle atrophy and hypertrophy

Previously, it was found that novel regulators of myofiber size can be uncovered by testing their function in Drosophila body wall larval skeletal muscles. For example, the vast increase in muscle size (~40-fold) that occurs over ~5 days of Drosophila larval development is modulated by the insulin/Akt/TOR signaling pathway [35], which is a well-known modulator of atrophy and hypertrophy in mammals [1]. Moreover, loss of UBR4, a ubiquitin ligase implicated in atrophy-associated muscle proteolysis [42,43], induces hypertrophy in Drosophila and in mammals [37]. Altogether, these studies indicate that homologous signaling pathways modulate myofiber size in mammals and in the developing Drosophila larvae. Moreover, Drosophila body wall muscles constitute an excellent setup for the identification of transcription factors that regulate myofiber size, as found for FoxO and Mnt [35].

On this basis, we took advantage of the simplicity of this system and the availability of transgenic RNAi resources for tissue-specific modulation of gene function to interrogate the role of evolutionary-conserved transcription factors in myofiber size regulation. For these studies with UAS/Gal4, the skeletal muscle-specific Mef2-Gal4 [44] was crossed with 1,114 transgenic RNAi lines (from the VDRC and Bloomington stock centers) to target 447 of the 708 transcription factors encoded by the Drosophila genome [38]. 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 a convenient readout to assess the outcome of muscle-specific RNAi interventions (Fig 1A).

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 size, indicating that these RNAi interventions do not impact developmental muscle growth. There were RNAi interventions that lead to larval lethality and various degrees of atrophy, indicating that transcription factors targeted by these RNAi are necessary for optimal skeletal muscle growth. Conversely, RNAi for another subset of transcriptional regulators lead to hypertrophy, indicating that the transcription factors targeted by these RNAi interventions normally limit muscle growth. Additionally, there were certain RNAi intervention that rather than affecting size primarily affected the shape of the larva, leading to thin or sickle-shaped larvae. (C) Although RNAi interventions that induce atrophy at the larval stage do not develop into adult flies, we have examined the adults that eclosed from all other RNAi interventions. The wings are kept at stereotypical positions in adult flies but developmental defects leading to muscle degeneration are known to lead to upheld and/or depressed wings, as found here for RNAi of several transcription factors. A full report of screen results is shown in S1 Table. (D-E) The area of larvae with muscle-specific expression of transgenic RNAi driven by Mef2-Gal4 (D) and by MhcK-Gal4 (E). Similar results are obtained with RNAi driven by both drivers, although some differences are found in the phenotypes induced by Mef2-Gal4 versus MhcK-Gal4, presumably because of differences in the potency and tissue-specificity of these Gal4 lines. Mean±SD and N = 6–31 (D) and N = 9–32 (E) are shown; **P<0.01, ***P<0.001, ****P<0.0001. The scheme in Fig 1 was drawn with BioRender.

Compared to control RNAi, ~88% RNAi interventions lead to 3rd instar larvae of normal size, indicating that these RNAi do not impact developmental muscle growth. However, there were RNAi interventions that lead to larval lethality (~1.6%) and various degrees of atrophy (~3.7%), indicating that transcription factors targeted by these RNAi are necessary for optimal skeletal muscle growth. Conversely, ~3% of RNAi lead to hypertrophy, indicating that the transcription factors targeted by these RNAi normally limit muscle growth. Additionally, there were ~3% of RNAi interventions that rather than affecting size primarily affected larval shape, leading to thin or sickle-shaped larvae (Fig 1B and S1 Table).

RNAi interventions that induce atrophy at the larval stage typically do not develop into adult flies [35]. Therefore, it is not surprising to find that ~16% of Mef2-Gal4-driven RNAi interventions did not yield any adults, as these include RNAi interventions that impact larval stages of muscle growth as well as pupal stages of muscle remodeling. However, there were some muscle-related phenotypes that were manifested in adult flies obtained from other RNAi crosses. These included early lethality of adult flies soon after eclosion, as well as defects in wing position. Normally, the wings are kept at stereotypical positions in adult flies but developmental defects that cause muscle degeneration lead to upheld and/or depressed wings, as found before for pink1/parkin loss [45] and here for ~1% of RNAi interventions that target transcription factors (Fig 1C and S1 Table). Altogether, by using muscle-restricted RNAi screening, we have here identified novel transcription factors that impact developmental skeletal muscle growth.

Among the many screen hits identified for their capacity to regulate myofiber size, there were 17 genes that scored consistently with 2 or more RNAi lines and that therefore are more likely to be robust regulators of muscle growth (S1 Table). To further test these genes, we re-screened them with Mef2-Gal4 and also with an additional muscle-specific driver, MhcK-Gal4, which drives strong transgene expression from the embryonic stage of muscle development [46]. However, different from Mef2-Gal4, InR overexpression with MhcK-Gal4 only marginally increased larval size (S1 Fig), suggesting that MhcK-Gal4 might not be suitable to uncover muscle hypertrophy phenotypes compared to Mef2-Gal4. On this basis, we have re-screened with MhcK-Gal4 only RNAi lines (from the Bloomington collection) with which we previously obtained a reduction in body size upon muscle-specific expression.

This follow-up analysis revealed that larval size is overall similarly affected by RNAi driven by Mef2-Gal4 (Fig 1D) and by MhcK-Gal4 (Fig 1E). However, some discrepancies in the phenotypes induced by Mef2-Gal4 versus MhcK-Gal4 were also observed, presumably because of differences in the potency and tissue-specificity of these Gal4 lines (Figs 1D–1E and S2). Specifically, RNAi for CG7839, taf1, mtTFB2, pdm3, Su(var)3-9, and myc (dm) consistently reduced larval body area with both Mef2-Gal4 and MhcK-Gal4, although pdm3 RNAi yielded stronger effects with MhcK-Gal4 versus Mef2-Gal4. However, RNAi for srp and PolrMT significantly reduced larval size with Mef2-Gal4 but not with MhcK-Gal4. Moreover, RNAi for CG6724 marginally reduced larval size with both Mef2-Gal4 and MhcK-Gal4, although this was statistically significant only with Mef2-Gal4. Conversely, RNAi for Not1 reduced larval size only when driven by MhcK-Gal4. Lastly, RNAi for Pc and trachealess (trh), which were previously classified as screen hits, did not significantly impact larval size when rescreened with Mef2-Gal4 (Fig 1D), suggesting that they are false positives. Altogether, despite some differences, RNAi driven by Mef2-Gal4 and MhcK-Gal4 similarly impacts myofiber size (Fig 1D and 1E), indicating that our screen strategy is appropriate for finding candidate regulators of muscle growth.

The small set of high-confidence regulators of muscle growth includes genes with functional homologs in humans, i.e. CG7839/CEBPZ, taf1/TAF1, the mitochondrial transcription factor mtTFB2/TFB2M, pdm3/POU6F2, Su(var)3-9/SUV39H, and dm/MYC (Fig 1D and 1E and S1 Table). Although these transcription factors have not been previously implicated in muscle growth or wasting, apart dm/MYC [35], some were found to modulate muscle differentiation in mice. Specifically, TAF1 has been previously implicated in myogenesis via its capacity to bind Pax3 and modulate its ubiquitination and proteasomal degradation [47] whereas the histone methyltransferase SUV39H1 was found to repress MyoD-stimulated myogenic differentiation [48].

Altogether, these findings indicate that RNAi screening in Drosophila is a useful approach to identify novel candidate regulators of myofiber size determination.

Muscle-specific RNAi for screen hits identifies transcription factors that modulate developmental myofiber growth in Drosophila

We have conducted a large-scale RNAi screen for transcription factors that regulate developmental skeletal muscle growth in Drosophila. Because body wall skeletal muscles are located beneath the epidermis, genetic interventions that regulate muscle size correspondingly change the size of the larva [35,37]. Assessing larval size is an easily-scorable screen readout that has led to the identification of many regulators of developmental muscle growth (Fig 1).

To better test the impact of screen hits, we have determined their impact on myofiber size via larval dissections and analysis of body wall skeletal muscles. Specifically, the outcome of some muscle-specific interventions that affected larval size was validated via the analysis of a set of representative muscles, ventral longitudinal VL3 and VL4 muscles, which are each composed by a single myofiber with a stereotypical size [35,37].

For these studies, we selected a set of genes based on their extremely high evolutionary conservation (i.e., typically, a DIOPT score >7), consistent scoring with multiple RNAI lines, and/or novelty (Fig 2 and S1 Table). Compared to controls (whiteRNAi and mcherryRNAi), RNAi for screen hits driven in skeletal muscle by Mef2-Gal4 led to decreased (atrophy) and increased (hypertrophy) size of VL3 and VL4 skeletal muscles. Quantification 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. Conversely, muscle-specific RNAi for FoxO, Cnc, and Deaf1 increases the area of VL3+VL4 muscles (hypertrophy), indicating that these transcription factors normally limit developmental myofiber growth. Altogether, these histological analyses confirm that this muscle-targeted RNAi screen has identified novel transcription factors that regulate myofiber developmental growth.

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.

Deaf1 RNAi induces myofiber hypertrophy whereas Deaf1 overexpression causes myofiber atrophy

Among the many regulators of skeletal muscle homeostasis identified in this screen, RNAi interventions that induce myofiber hypertrophy are the most interesting as they highlight transcription factors that normally impede growth and that could be inhibited to contrast wasting. Among them, the transcription factor Deaf1 (deformed epidermal autoregulatory factor 1) has been previously implicated in early development and innate immunity in Drosophila [49-52] and in human neurodevelopmental disorders [53-55] but not in muscle growth. On the basis of this possible novel function of Deaf1 in muscle, we further examined its impact on myofiber size determination. Whereas Deaf1 RNAi induces hypertrophy compared to control RNAi (Fig 2D), muscle-restricted Deaf1 overexpression led to myofiber atrophy (Fig 3A). Specifically, the area of ventral longitudinal VL3 and VL4 muscles is lower upon Deaf1 overexpression (Fig 3B), whereas it increases upon insulin receptor (InR) overexpression, as expected [35].

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 Gsk3 transgenes with Mef2-Gal4 leads to a decrease (atrophy) in the size of myofibers, as indicated by the quantitation of the cumulative area of VL3 and VL4 muscles from multiple larvae (F); N = 9–19 and mean±SD is shown; ****P<0.0001. (G) Conversely, Gsk3 RNAi in skeletal muscle induces myofiber hypertrophy, as indicated by the the cumulative area of VL3 and VL4 muscles (H); N = 27–71 and mean±SD is shown; ****P<0.0001.

Gsk3 regulates myofiber size similar to Deaf1

Although Deaf1 has not been implicated in myofiber size determination, it was previously identified as a phosphorylation target of the glycogen synthase kinase GSK3 [39], which induces myofiber atrophy and muscle wasting in response to many catabolic stimuli via the phosphorylation of target proteins in mice [14,41,56]. Specifically, it was found that DEAF1 interacts with and is phosphorylated by GSK3A and GSK3B [39]. To determine whether shaggy (the Drosophila homolog of GSK3 and GSK3B) regulates myofiber size in Drosophila as found in mammals, we modulated its activity via overexpression of constitutive active versions. Specifically, we employed Mef2-Gal4 to drive expression of transgenes encoding for constitutive-active, wild-type, and kinase-dead Gsk3. As expected based on its function in higher organisms, we found that muscle-specific overexpression of constitutive active (CA) and wild-type (WT) Gsk3 led to reduced body size, which is indicative of muscle atrophy (Fig 3C and 3D). On the other hand, Gsk3 variants with reduced kinase activity minimally impacted larval size, and kinase-dead Gsk3 mutants had no effect (Fig 3C and S3 Table).

To corroborate these findings, larvae with skeletal muscle-specific Gsk3 overexpression were dissected and the area of ventral longitudinal VL3 and VL4 muscles analyzed. As expected based on the function of Gsk3 in mammalian systems [14,41,56] and our preliminary analyses (Fig 3C and 3D), Gsk3CA lead to significant decline in myofiber size (Fig 3E and 3F). To further assess the role that Gsk3 plays in Drosophila body wall muscle growth, we reduced its levels in skeletal muscle via RNAi. Conversely to Gsk3 activation (Fig 3C–3F), Gsk3 RNAi induced myofiber hypertrophy, compared to control RNAi lines against white and mcherry (Fig 3G and 3H).

Altogether, these findings indicate that Gsk3, similar to Deaf1, regulates muscle mass in Drosophila (Fig 3), as observed in mammals [14,41,56]. Because Deaf1 was found to be a phosphorylation target of Gsk3 [39], these findings suggest that Deaf1 may negatively regulate myofiber size in Drosophila skeletal muscle by acting downstream of Gsk3 signaling.

Gsk3 and Deaf1 induce similar gene expression changes in Drosophila body wall skeletal muscles

Because the transcription factor DEAF1 has been previously reported to interact with and to be phosphorylated by GSK3A/B [39], and it similarly regulates myofiber size (Fig 3), we next examined whether Gsk3 and Deaf1 induce similar gene expression changes in Drosophila body wall skeletal muscle. For these studies, we used Mef2-Gal4 to modulate the levels of Deaf1 and Gsk3 in muscle. As expected, Deaf1 mRNA levels were significantly lower upon Deaf1 RNAi and higher upon Deaf1 overexpression, respectively. Similarly, higher Gsk3 levels were found upon its overexpression (Fig 4A).

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 results of RNA sequencing.

RNA sequencing from filleted larvae (which consist primarily of body wall skeletal muscles and the associated epidermis) identified many transcriptional changes that occur upon Deaf1 RNAi in comparison to control white RNAi. Cross-comparison with gene expression changes induced by Deaf1 overexpression revealed that significantly regulated genes (p<0.05) are largely regulated in opposite fashions by Deaf1 RNAi and Deaf1 overexpression (R2 = 0.46), each normalized to its respective control (Fig 4B). Moreover, comparison of the muscle transcriptomes revealed that gene expression changes induced by Deaf1 overexpression are highly overlapping (R2 = 0.49) with those induced by constitutive active Gsk3 (Fig 4C).

GO term analysis of categories enriched among genes upregulated by Deaf1 RNAi indicates that Deaf1 RNAi may induce myofiber hypertrophy by promoting glycolysis, sarcomere organization, and by modulating the function of histone deacetylases (Fig 4D). In particular, glycolysis represents the top category of genes upregulated by Deaf1 RNAi and, consistent with transcriptome cross-comparisons (Fig 4B and 4C), Deaf1 and Gsk3 overexpression induce converse changes, i.e. significantly reduce expression of most glycolytic enzymes (Fig 4E). Altogether, these findings suggest that the transcription factor Deaf1 may regulate myofiber size via the transcriptional modulation of several target genes, including glycolytic enzymes.

Expression of glycolytic enzymes sustains growth of larval body wall skeletal muscles

We have found that Deaf1 RNAi, which induces myofiber hypertrophy, promotes the expression of glycolytic enzymes whereas Deaf1 overexpression, similar to Gsk3, reduces their expression (Fig 4). On this basis, we next tested the impact of glycolysis on skeletal muscle growth. To this purpose, we screened 44 RNAi lines targeting glycolytic enzymes with Mef2-Gal4 and found that many of them led to small larval size (Fig 5A and S3 Table), indicative of muscle atrophy [35]. We further analyzed some of the RNAi lines that target glycolytic enzymes. As expected, small body size due to expression of transgenic RNAi for Eno and Pglym78 in skeletal muscle (Fig 5A) was associated with reduced size of VL3 and VL4 muscles (Fig 5B and 5C). These findings indicate that glycolysis is necessary for myofiber growth, in line with previous studies [57,58], and that it may indeed be a primary means by which Gsk3 and Deaf1 modulate myofiber growth in Drosophila body wall skeletal muscles.

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.

Discussion

In this study, we took advantage of a simple, genetically tractable, model organism, Drosophila melanogaster [59], to expand the knowledge about transcription factors that regulate myofiber size. Specifically, we have used transgenic RNAi to knock down the levels of evolutionary-conserved transcription factors in Drosophila larval body wall skeletal muscles which, by growing ~40-fold during few days of larval development, offer an ideal setup for identifying modulators of myofiber growth. Because these muscles are located right beneath and surround the larval epidermis, changes in muscle mass result in changes in the overall larval body size, making this system amenable for visual phenotypic screens [35,37]. Moreover, the reduced genetic redundancy of Drosophila melanogaster compared to mice and humans constitutes another advantage for uncovering regulators of myofiber size [22], as demonstrated by the lower number of transcription factors present in Drosophila compared to humans (708 versus ~1,400).

The RNAi screen here done provides insight into transcription factors that regulate myofiber growth. Because this screen included both DNA-binding transcription factors and transcriptional regulators that are part of larger nuclear complexes, we expect that the screen hits here identified may modulate myofiber size via a plethora of mechanisms. For example, the nucleosome remodeling factor Nurf-38 catalyzes ATP-dependent nucleosome sliding and facilitates transcription of chromatin [60] and this may constitute a mechanism by which it is necessary for myofiber growth. Another example is e(y)1/Taf9, i.e. TBP-associated factor 9, which encodes for a component of the transcription factor IID complex, which regulates transcription from core promoters and enhancer-promoter interactions [61] but also regulates lipid metabolism [62], which has been implicated in muscle wasting [63]. Overall, despite phenotypic similarity, the transcription factors and transcriptional regulators here identified may regulate myofiber size via distinct target genes and transcriptional/chromatin remodeling mechanisms.

The RNAi screen we have conducted has identified several candidate regulators of muscle growth. However, as found in other screens [64], some of these hits could be false positives. For example, trachealess (trh), a transcription factor necessary for the development of the Drosophila airway system [65], was initially identified as a screen hit but subsequent re-testing with Mef2-Gal4 uncovered only minor phenotypes induced by trh RNAi (Fig 1D), suggesting that this is a false positive. While transcription factors that scored consistently with multiple RNAi lines (such as Deaf1 RNAi) are less likely to be false positives, screen hits identified with a single RNAi line most likely also consist in large part of bona fide muscle growth regulators (indeed in some cases only a single RNAi line was available to test the function of a given transcription factor).

Conversely, other transcription factors that are important regulators of muscle growth may not have scored because of technical limitations, i.e. they could be false negatives. Although the potency of transgenic RNAi depends on the Gal4 line used and specific RNAi collection, a previous study in the Drosophila embryo has found that phenotypes were observed only among the RNAi interventions that yielded a target gene knockdown greater than 50% [66]. Specifically, this study has found that, out of ~450 RNAi lines targeting kinases, ~29% were not functional (~12% did not display any knockdown, and an additional ~17% displayed a knockdown of less than 50% that did not yield a phenotype; [66]). Therefore, it is possible that around 1/3 of the lines tested in our screen is not functional and that therefore the muscle growth regulators targeted by these RNAi lines are not uncovered by our screen. On this basis, rather than being an exhaustive and definitive determination of the transcription factors that regulate muscle growth, our study provides a list of candidate regulators of myofiber size which should be further tested in Drosophila and other model organisms.

Another limitation of the screen consists in the tissue specificity of the Mef2-Gal4 line that we have used. As originally described in ref. [35], this Mef2-Gal4 line drives transgene expression in body wall skeletal muscles but also in visceral muscles and in some cells in the brain [35]. Closer analysis of such brain cells suggests that they consist of mushroom body neurons (S2 Fig), which have been found to express endogenous Mef2 [67]. However, Mef2-Gal4 fluorescence is most strongly observed in skeletal muscle compared to non-muscle cells and therefore it is unclear if sufficient target gene knockdown is achieved in non-muscle cells to generate a phenotype. Moreover, the overall similarity of phenotypes induced by transgenic RNAi driven by complementary muscle drivers (i.e. Mef2-Gal4 and MhcK-Gal4) suggests that the observed muscle atrophy phenotypes are indeed due to RNAi expression in skeletal muscle (Fig 1D and 1E). Nonetheless, the changes in larval body size and myofiber growth observed in our study may in some cases depend on the modulation of the target gene outside of skeletal muscle. As in the case of false and negative screen hits, this constitutes a technical limitation of the study that will be resolved by complementary approaches for testing the function of the candidate regulators of myofiber size here identified.

Among the many screen hits identified, we have examined in more detail the function of Deaf1, an evolutionary conserved transcription factor that had not been previously implicated in skeletal muscle growth. Specifically, we have found that Deaf1 RNAi induces myofiber hypertrophy whereas Deaf1 overexpression causes atrophy. Mechanistically, Deaf1 RNAi promotes the expression of glycolytic enzymes whereas Deaf1 overexpression reduces it, suggesting that glycolysis is necessary for optimal skeletal muscle growth, as previously found in Drosophila [57] and in other contexts [68].

We also find that similar transcriptional responses, including expression of glycolytic enzymes, are induced by Deaf1 and Gsk3, a known inducer of myofiber atrophy [40,69-71] that interacts with and phosphorylates Deaf1 [39]. Many phosphorylation targets of GSK3 have been identified in mammals, including several transcription factors, such as MITF, NF-κB, and CREB [72-77]. However, much remains to understand about the GSK3 targets that are most necessary for GSK3 output in distinct tissues and disease conditions. In particular, although it is well established that GSK3 promotes muscle wasting [40,69-71], it is incompletely understood how GSK3 promotes transcriptional changes that drive muscle protein catabolism during atrophy. Our findings now suggest that Deaf1 may contribute at least in part to the transcriptional changes induced by Gsk3 activity in muscle, and that these may include the modulation of glycolysis.

Altogether, this study has expanded the repertoire of transcription factors that regulate myofiber growth and highlights a possible role for Gsk3, Deaf1, and glycolysis in this process.

Materials and methods

RNAi screening

The list of fly stocks used for RNAi screening is reported in S1 Table and refers to RNAi for Drosophila transcription factors that are evolutionarily conserved in humans, as defined based on a homology DIOPT [78] score of ≥2. For each screen cross, 10 Mef2-Gal4 virgin females were crossed with 5 males for each RNAi line tested. Progenies were reared at 25°C and transferred to new food every 4 days. Subsequently, the size of 3rd instar wandering larvae was scored in comparison with negative controls (whiteRNAi) and positive controls, which consisted in FoxO overexpression (which induces atrophy) and overexpression of insulin receptor (InR, which induces hypertrophy), as previously shown [35,37]. Specifically, larval size phenotypes due to Mef2-Gal4-drived RNAi in muscle were scored as follows: A- no larvae/no pupae; B- very small larvae/no pupae (i.e. smaller than FoxO overexpression); C- small larvae/small pupae (i.e. similar to FoxO overexpression); D- normal (no visible phenotype); E- increased larval/pupal size (i.e. similar to InR overexpression); and F- thin or sickle-shaped larvae with locomotor defects.

Adult flies obtained from RNAi crosses were scored based on the following categories: H- normal; I- no adults eclosed, i.e. developmental lethal; J- upheld or depressed wings in the majority of flies in the tube (i.e. similar to pink1 RNAi or parkin RNAi); and K- early lethality of eclosed flies. Normal muscle development results in stereotypical wing positioning, which is present in whiteRNAi control flies, whereas upheld/depressed wings are an indication of improper muscle development and/or muscle degeneration [45]. RNAi interventions that lead to small larvae and pupae (A-C) and larvae with aberrant shape (F) typically do not give rise to adult flies, as previously shown [35].

Drosophila stocks

In addition to Mef2-Gal4 [44], the following fly stocks were used: UAS-Deaf1 [50], UAS-foxo and UAS-InR [35,79], and stocks for overexpression of wild-type, constitutive-active, and kinase-dead Gsk3 transgenes [80], which are reported in S3 Table. MhcK-Gal4 (Mhc-GAL4.K, BL#55133; [46]) was used for studies in Fig 1E. The list of fly stocks used for RNAi screening is reported in S1 Table whereas the list of RNAi lines that target glycolytic enzymes is reported in S3 Table.

The fly stocks utilized for VL3+VL4 muscle analyses (Fig 2) are the following: UAS-whiteRNAi (BL#33623), UAS-cherryRNAi (BL#35785), UAS-Nurf-38RNAi (BL#31341), UAS-e(y)1RNAi (BL#32345), UAS-alienRNAi (BL#28908), UAS-SsrpRNAi (BL#26222), UAS-CG7839RNAi (BL#25992), UAS-Taf1RNAi (BL#32421), UAS-MBD-R2RNAi (BL#27029), UAS-mtTFB2RNAi (BL#27055), UAS-pdm3RNAi (BL#35726), UAS-datiRNAi (BL#26711), UAS-foxoRNAi (BL#27656 and BL#32993), UAS-CncRNAi (BL#32863), and UAS-Deaf1RNAi (BL#32512).

The fly stocks utilized for RNA-seq (Fig 4) are the following: UAS-Deaf1RNAi (BL#32512), UAS-whiteRNAi (BL#33623), UAS-Deaf1 [50], UAS-Gsk3CA (BL#5255), and w1118 (+).

Staining of body wall skeletal muscles, confocal microscopy, and image analysis

Male larvae were dissected in ice-cold Ca2+-free, MgCl2-free PBS (Gibco) and filleted larval samples were fixed for 20 minutes in PBS with 4% paraformaldehyde, as previously done [35]. After washing with PBS, body wall skeletal muscles were stained overnight with DAPI (4’,6-diamidino-2phenylindole, 1μg/mL) to visualize nuclei, and imaged to detect the endogenous fluorescence of a Mhc-GFP fusion protein. Body wall muscles were mounted on microscope slides and the VL3/4 muscles were imaged using a Zeiss LSM880 confocal laser-scanning microscope. Confocal images were analyzed using the measure tools of the ImageJ software to quantitate the area of VL3+VL4 muscles for each sample. Larval body size was quantified with ImageJ.

RNA sequencing

RNA-seq was done following similar procedures as before [81,82]. Specifically, total RNA was extracted from filleted Drosophila larvae, which consist primarily of body wall skeletal muscles. Three biological replicates were prepared for RNA-seq with the TruSeq stranded mRNA library preparation kit (Illumina) and sequenced on the Illumina HiSeq 4000 platform, with six samples in each lane. Multiplexing was done on a per flowcell basis. Approximately 100 million reads were obtained for each sample. FASTQ sequences derived from mRNA paired-end 100-bp sequences were mapped to the Drosophila melanogaster genome (BDGP5) with the STAR aligner (version 2.5.3a) [83]. Transcript level data were counted using HTSeq (version 0.6.1p1) [84] based on the BDGP5 GTF release 75. The TMM method [85] was used to calculate the normalization factors. Then, linear modeling was carried out on the log2(CPM) (count per million) values where the mean-variance relationship is accommodated using precision weights calculated by the voom function [86] of the limma package in R 3.2.3 (R Core Team 2013; [87]). A q-value (FDR) was calculated for multiple comparison adjustments of RNA-seq data. The lmFit, eBayes, and contrasts.fit functions from the limma package were used for the linear modeling. Statistical analyses were performed using log2(FPKM) values in Partek Genomic Suite 6.6 (www.partek.com/partek-genomics-suite/). The gene sets were analyzed by DAVID (Database for Annotation Visualization and Integrated Discovery) to identify enriched functional classes of genes [88].

The RNA-seq comparisons refer to Mef2>Deaf1RNAi(BL#32512) versus Mef2>whiteRNAi(BL#33623), Mef2>Deaf1 versus Mef2>+, and Mef2>Gsk3CA(BL#5255) versus Mef2>+; (n = 3/genotype). The RNA-seq data is reported in S2 Table and has been deposited to the Gene Expression Omnibus with accession number GSE174637.

Statistical analysis

All data points refer to biological replicates and the number is indicated in the figure legends. Each biological replicate refers to data obtained from a different larva; typically, larvae from 2 or more crosses were analyzed for each genotype. The larvae analyzed in Figs 2–5 were obtained from crosses different from those used for the screen. The unpaired two-tailed Student’s t-test was used to compare the means of two independent groups to each other. One-way ANOVA with Tukey’s post hoc test was used for multiple comparisons of more than two groups of normally distributed data. Bar graphs present the mean ± SEM or ± SD, as indicated in the figure legends. Throughout the figures, asterisks indicate the significance of the p value: *p<0.05; **p<0.01; ***p<0.001. A significant result was defined as p<0.05. Statistical analyses were done with Excel and GraphPad Prism.

Comparison of Insulin Receptor overexpression with Mef2-Gal4 and MhcK-Gal4.

Consistent with previous studies in Drosophila and mammals, overexpression of insulin/IGF receptor (InR) in skeletal muscle via Mef2-Gal4 induces skeletal muscle hypertrophy, as indicated by the increase in body size. However, a relatively minor increase is found with InR overexpression via MhcK-Gal4 (Mhc-Gal4.K, BL#55133) suggesting that this Gal4 line is not ideal for uncovering muscle hypertrophy phenotypes.

(TIF)

Click here for additional data file.

Characterization of the tissue-specificity of transgenic expression with Mef2-Gal4.

Red fluorescence due to transgenic DsRed expression is detected primarily in body wall skeletal muscles but also in visceral muscles and few cells in the brain. We find no evidence for Mef2-Gal4-driven DsRed expression in insulin producing cells (ipc) with this line. However, DsRed expression driven by Mef2-Gal4 is detected in brain cells of the mushroom body (mb), consistent with our original characterization of this driver (Demontis and Perrimon, 2009, Development; PMID:19211682) and a more recent study that has found endogenous Mef2 expression in a subset of Kenyon cells of the mushroom body (Crittenden et al. 2018, Biology Open; PMID:30115617).

(TIF)

Click here for additional data file.

RNAi screen data.

(XLSX)

Click here for additional data file.

RNA-seq data.

(XLSX)

Click here for additional data file.

Phenotypes of RNAi lines for glycolytic enzymes.

(TIF)

Click here for additional data file.

Additional primary data, related to Figs 2 and 3.

(XLSX)

Click here for additional data file.

16 Jun 2021

Dear Dr Demontis,

Thank you very much for submitting your Research Article entitled 'A global transgenic RNAi screen identifies transcription factors that modulate myofiber size in Drosophila.' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The manuscript by Graca et al., titled A global transgenic RNAi screen identifies transcription factors that modulate myofiber size in Drosophila does most of what is suggested by the title. The authors do a fairly large and systematic RNAi screen with the goal of identifying novel transcription factors that regulate muscle growth to better understand the mechanisms that underly muscle atrophy and hypertrophy. In the opinion of this reviewer, the manuscript is exceptionally well written, provides clear and easy to interpret figures, and puts forth a mechanisms by which Glycolysis is regulated for the purpose of muscle growth. Although there are some revisions that are necessary to make this manuscript suitable for publication they are relatively minor in scope.

Questions and Comments

1. I personally would not use the word “global” in the title. As stated, 447 out of 708 transcription factors were investigated. Global to me suggests that all transcription factors were examined. I’d recommend “large-scale” or “systematic”

2. Figure 1 and the associated methods suggest that larvae were just categorized as large, small, mishapen, or normal. Additional details indicating the threshold for variation from control necessary to categorize a larva would be helpful, particularly for readers who may try to follow up on this work.

3. The methods associated with Figure 1 list the control as “such as white RNAi”. If multiple different controls were used they should all be listed along with the rationale for using each.

4. Figure 2 represents my biggest and most important questions regarding this manuscript.

4A. It seems that only a subset of the hits from the screen were examined in figure 2. I certainly recognize that completing this analysis of a large number of different conditions might be prohibitive. However, I think it is essential that the authors provide a rationale for choosing the hits they follow up on and ignoring the hits that they did not. If in fact all of the hits were examined, it should be stated explicitly and data for all should be provided in the supplement at the very least.

4B. The hemisegment chosen for muscle size analysis will impact the data. Therefore it is critical to know which hemisegments were used. Additionally, and perhaps more importantly, the hemisegments near the anterior and posterior end of the animal are significantly smaller and more variable than the central hemisegments. Based on figure 2A it seems that a subset of muscles from these hemisegments may have been used. The authors should remove these data points and rely on the data from central hemisegments.

5. I hesitate to suggest this because it has the feel of making the paper what I would like it to be rather than what it is. Nevertheless, figure 5 just seems an anticlimactic way to end a very nice manuscript. It is suggestive, along with the transcriptomic data in figure 4, that Deaf1 works through regulation of glycolysis to regulate muscle growth. But, we already knew that disruption of glycolysis would impact muscle size. If the glycolysis disruption experiments could be done in the background of altered Deaf1 and/or GSK3 levels, it would be a more fitting conclusion to this manuscript. Even selecting just two glycolytic enzymes to do in this background would be a much more convincing way to show that glycolysis works downstream of Deaf1 and GSK3 to regulate muscle growth.

Minor Comments

1. Citations vary between author, year and numbers. I presume this will be fixed and is not a concern, rather something I noticed.

2. At the bottom of page 6, the authors cite two papers as evidence that fusion is not responsible for the variations in muscle size. I do believe that this is not a fusion effect, but I do not believe that these specific disruptions were looked at in those papers. If I am correct, the authors should be clear and precise in their language.

3. Regarding the statistical analysis, the authors state that all data points indicate a biological replicate. I think that this is vague and means different things to different researchers. For example, in figure 2 one might assume that each point refers to a different mating of males and virgins whereas another might think it means a different animal from a single mating. A more precise description will be helpful in understanding the variability of the data.

4. The last sentence of the first paragraph on page 4 suggests that the sickle shape is indicative of defective contractile properties. Without something to reference, or data to support this conclusion, the authors should just report their finding.

Reviewer #2: The manuscript by Graca et al. reports the results of a straightforward RNAi screen in Drosophila designed to uncover roles for conserved transcription factors in muscle size control. The authors employed 1114 RNAi lines that targeted 447 transcription factors that showed some similarity to human transcription factors, for knockdown in developing muscles and then noted size defects at larval and adult stages as well as some wing positioning defects that have been noted to result from muscle abnormalities. In all about 12% of the targeted genes generated a phenotype consistent with and effect on muscle growth. The authors then performed followed up proof of principle studies on DEAf1 a conserved factor that was not known to affect muscle growth. Loss of Deaf1 was found to increase muscle size while overexpression led to smaller muscles. These phenotypes where opposite to those exhibited by manipulation of GSK3, a kinase that has been shown in mammal system to associate with and phosphorylate DEAf1. Interestingly transcriptome analysis of Deaf1 verses GSK3 knockdown and overexpression n lead to a strikingly similar regulation profile for the core genes involved in glycolysis consistent with pervious observations that inhibition of normal glycolytic flux leads to smaller muscle size.

Overall, this is a short but informative communication that will provide a useful resource to those that study muscle size control in both Drosophila and mammals. In general, the experimental design and outcomes are well described and the results easy to follow. That said I think there are a few points that the authors should address before publication.

1) There should be some discussion of the limitations of the screen. Obviously, there will be some false positives as well as false negatives. It is a bit hard to know what these limits are but one criterion for a true hit might be that more than one RNAi targeting a given gene gives a similar phenotype. By my count there are ~34 genes in supplement table 1 that satisfy this criterion. I think it would be useful to list these genes in a separate table within the main results and provide some limited discussion about these hits such has home many of these have been previously linked to muscle development. Likewise, it is worth pointing out that those genes with only one identified RNAI hit might simply be off target effects. Using this same data set the authors should also take the opportunity to provide an example of another type of possible false positive. I note that trachealess might be one such gene. It satisfies the two-hit criterion, but it is a big surprise, since as far as I am aware, there is no known role for trachealess in muscle development. Rather it seems to be specific for regulating genes involved in the formation of the insect oxygen delivery system. Obviously, if the mef-2 Gal4 driver has some off target expression at some stage in trachea, then it could explain the small larval size phenotype. This brings up my main concern with this study which is that only one “muscle-specific” driver has been used (understandable to keep the work effort down). However, one imposed limitation of this methodology is that the results are only interpretable if the driver expression is tight with respect to the targeted tissue. Mef-2 is pretty good, but there is a big problem with some versions of this driver which is that it is also strongly expressed in the ipc neurons (at least in late third instar larvae) that produce and release several key insulin-like peptides. It is incumbent on the authors to explicitly state which mef2 driver line they are using and confirm by crossing to a UAS reporter that it is indeed muscle specific and does not show expression in other tissues especially the IPCs. Without such confirmation, using mef-2 as the sole driver in the screen could produce false positives by alterations in insulin signaling which is a central regulator of body size through several mechanisms. Obviously, rescreening all hits with an ipc “specific driver” to rule out possible effects on insulin signaling is a large amount of work. However, if the authors Mef2 Gal4 line is expressed in IPCs, then perhaps they could just rescreen the 34 genes that satisfy the more than one RNAi hit criteria described above to determine if any of the phenotypes in this class might be caused by the off target Mef-2 expression.

2) A second issue is that the Deaf and GSK3 work leads to suggesting that they work in the same pathway but in opposite ways to modulate glycolysis. One simple test to determine their order of action is genetic epitasis. If GSK3’s effect on muscle size and glycolytic enzyme expression is primarily through altering Deaf 1 activity, then muscle directed overexpression of GSK3CA together with Deaf1 RNAi should produce a Deaf1 phenotype (higher levels of glycolytic enzyme expression and larger muscle size. Have the authors tried such an experiment?

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

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19 Oct 2021

Submitted filename: REBUTTAL LETTER_PLoS Genetics.pdf

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29 Oct 2021

Dear Dr Demontis,

Thank you very much for submitting your Research Article entitled 'A large-scale transgenic RNAi screen identifies transcription factors that modulate myofiber size in Drosophila.' to PLOS Genetics.

The revised manuscript was fully evaluated at the editorial level and by independent peer reviewers. Both reviewers are generally satisfied with the revision, but reviewer 1 has raised additional minor comments on the manuscript.

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The revised manuscript by Graca et al. addressed my questions and is suitable for publication with the potential of minor modifications to address one question I have.

In the last full paragraph of page 4, the authors compare the data from the Mef2 based RNAi screen to the MhcK based RNAi screen. Specifically, the similarities and the differences are highlighted. However, I question whether strict reliance on the statistical measures is appropriate here. For example, CG6724 is said to reduce larval size in Mef2 but not MhcK. But, the MhcK looks very similar to the Mef2. I do understand that the MhcK data can't be compared to the Mef2 control, but I think that this could be added to the new discussion section on the limitations of the screen. Similarly, Pc and trachealess are listed as "marginally impacted". If the authors are going to stick strictly to statistical measures, they should do so in this case also and list those RNAi lines as having no effect. Finally, the variation in the pdm3 phenotype could also be a point of discussion.

Reviewer #2: The authors have done a very through job of addressing my concerns. I think it is a nice study that will be of interest to many in the muscle biology field.

**********

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Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

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29 Oct 2021

Submitted filename: RESPONSE TO THE REVIEWERS.pdf

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4 Nov 2021

Dear Dr Demontis,

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9 Nov 2021

PGENETICS-D-21-00604R2

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

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We are pleased to inform you that your manuscript entitled "A large-scale transgenic RNAi screen identifies transcription factors that modulate myofiber size in Drosophila." has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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