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An RNAi based screen in Drosophila larvae identifies fascin as a regulator of myoblast fusion and myotendinous junction structure

An RNAi based screen in Drosophila larvae identifies fascin as a regulator of myoblast fusion and... Background: A strength of Drosophila as a model system is its utility as a tool to screen for novel regulators of various functional and developmental processes. However, the utility of Drosophila as a screening tool is dependent on the speed and simplicity of the assay used. Methods: Here, we use larval locomotion as an assay to identify novel regulators of skeletal muscle function. We combined this assay with muscle-specific depletion of 82 genes to identify genes that impact muscle function by their expression in muscle cells. The data from the screen were supported with characterization of the muscle pattern in embryos and larvae that had disrupted expression of the strongest hit from the screen. Results: With this assay, we showed that 12/82 tested genes regulate muscle function. Intriguingly, the disruption of five genes caused an increase in muscle function, illustrating that mechanisms that reduce muscle function exist and that the larval locomotion assay is sufficiently quantitative to identify conditions that both increase and decrease muscle function. We extended the data from this screen and tested the mechanism by which the strongest hit, fascin, impacted muscle function. Compared to controls, animals in which fascin expression was disrupted with either a mutant allele or muscle-specific expression of RNAi had fewer muscles, smaller muscles, muscles with fewer nuclei, and muscles with disrupted myotendinous junctions. However, expression of RNAi against fascin only after the muscle had finished embryonic development did not recapitulate any of these phenotypes. Conclusions: These data suggest that muscle function is reduced due to impaired myoblast fusion, muscle growth, and muscle attachment. Together, these data demonstrate the utility of Drosophila larval locomotion as an assay for the identification of novel regulators of muscle development and implicate fascin as necessary for embryonic muscle development. Keywords: Nuclear movement, Drosophila, Myoblast fusion, Myotendinous junction, Fascin, Myogenesis * Correspondence: eric.folker@bc.edu Equal contributors Biology Department, Boston College, Chestnut Hill, MA 02467, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Camuglia et al. Skeletal Muscle (2018) 8:12 Page 2 of 13 Background for the identification of genes that are necessary for Skeletal muscle has a distinctive architecture that is gener- muscle development. Indeed, many screens for regulators ated by a unique set of developmental phases. Making the of muscle development have been completed. Researchers myofiber syncytium requires the fusion of mononucleated have used adult locomotion [17] and embryonic muscle myoblasts. In both Drosophila and mammalian systems, structure [18] as indicators of muscle development. Al- individual myoblasts invade growing myotubes and deposit though these strategies have proven effective, they each their nucleus into the common cytoplasm to drive myotube have drawbacks. Analysis of embryonic muscle structure growth [1, 2]. Myoblast fusion is an actin-dependent is labor intensive and requires significant expertise in process that is reminiscent of a cancer cell invading a tissue muscle biology. Analysis of adult locomotion is limited during metastasis [3]. The mononucleated myoblast ex- because the disruption of many genes is lethal during pu- tends a protrusive invadapodia-like structure that makes pation. Therefore, we have developed a simple assay for possible the penetration of the myotube and the mixing of muscle function based on the larval locomotion. We have cytoplasm. Many factors and signaling pathways that regu- used this assay to screen for novel regulators of muscle late myoblast fusion have been identified [2, 4, 5]. However, function and identified fascin as one such regulator. many of the genes necessary for invadapodia-like structures Subsequent cell biological analysis implicates fascin as a in other contexts have not yet been implicated in myoblast regulator of myoblast fusion and MTJ structure. fusion, suggesting that additional regulators remain to be identified. One glaring omission from the categories of pro- Methods teins that have been identified as regulators of myoblast Drosophila genetics fusion is proteins that stabilize filopodia. Invadapodia are All stocks were grown under standard conditions at 25 ° sn28 filopodia-like structures [6, 7], and they require several C. The Fascin allele was a generous gift from Tina factors that are known to stabilize filopodia. Furthermore, Tootle (University of Iowa). All UAS-RNAi Drosophila although loss-of-function data is lacking, dominant nega- lines were purchased from Bloomington Drosophila tive mutants of the formin Diaphanous inhibit myoblast Stock Center. UAS-RNAi constructs were driven specif- fusion and may suggest that filopodia are essential for ically in the mesoderm using twist-GAL4, which drives myoblast fusion [8, 9]. Thus, it is likely that additional expression in the mesoderm from stage 10 of embryonic factors that have been identified as capable of stabilizing development through stage 13 of embryonic develop- filopodia for the purpose of protrusion and invasion in ment, DMef2-GAL4 that drives expression in the other contexts contribute to myoblast fusion. muscles from stage 12 through adulthood, or MHC- Beyond myoblast fusion, there are several features of GAL4 which drives expression in muscle from stage 17 muscle development that either require, or have been hy- of embryonic development through adulthood. pothesized to require, precise regulation of the actin cyto- skeleton, including the positioning of nuclei and the Larval locomotion assay development of the myotendinous junction (MTJ). To We performed a modified version of the previously used date, evidence for actin-dependent nuclear movement in assay that has been used to measure larval locomotion in muscle is restricted to the squeezing of nuclei to the per- individual larvae [19]. Virgins expressing DMef2-GAL4 iphery of the muscle [10, 11], although it has been pro- were mixed with males that carried the UAS-RNAi for 1 h posed that actin may contribute to the movement of in a vial with standard Drosophila food. After 1 h, adults nuclei along the length of the muscle [12, 13]. The role of were moved to a new vial and the embryos laid during the actin in MTJ development is more established. MTJs are 1 h period were aged for 5 days until they were third- integrin-based adhesions that transmit force from the instar larvae (L3). Larvae were then floated from the food muscle to the skeleton [14]. The initial formation involves by the addition of 15% sucrose. Using a paintbrush, larvae extension of filopodia-like structures from the muscle cell were moved to a plate with wet yeast. After all of the ge- that interact with the tendon cell before forming a stable notypes had been collected, 10 larvae of each RNAi were and somewhat rigid attachment that enables effective moved to an arena that consisted of 3% agarose dyed black force transmission [15, 16]. All of these processes require with standard food color poured over the top of a 96-well linear actin-cables. The similarity in the actin-based struc- plate cover. Movement of larvae toward a stick dipped in tures suggests that the same molecular components may ethyl butyrate was captured using an iPhone mounted contribute to each of these aspects of muscle develop- above the arena. The speed of each larva was then ana- ment. Therefore, it is critical to determine how newly lyzed using ImageJ. identified genes and proteins contribute to each process. Because the developmental path and final architecture Viability assay of muscle cells is conserved from Drosophila to humans, Stage 16 embryos were picked and placed on an agarose flies provide a genetically tractable and inexpensive model plate. The selected embryos were incubated at 20 °C Camuglia et al. Skeletal Muscle (2018) 8:12 Page 3 of 13 overnight. After incubation, the number of unhatched βPS (1:50, Developmental Studies Hybridoma Bank CF. embryos were counted to determine embryonic lethality 6G11), mouse anti-Fascin (1:25, Developmental Studies and the L1 larvae from the hatched embryos were trans- Hybridoma Bank sn 7C, generous gift from Tina Tootle) ferred to a vial of standard fly food. Larvae were incu- , and mouse anti-αTubulin (1:200, Sigma-Aldrich bated at 25 °C until larvae began to pupate and eclose. T6199). Conjugated fluorescent secondary antibodies Adult flies were counted to determine pupal lethality used were Alexa Fluor 555 donkey-anti-rabbit (1:200), and removed from the vials of standard fly food upon Alexa Fluor 488 donkey-anti-rat (1:200), and Alexa Fluor eclosure. After 1 week of no eclosures, the number of 647 donkey-anti-mouse (1:200) (all Life Technologies) pupal cases were counted to determine larval lethality. and Alexa Fluor 488 donkey-anti-mouse (1:200, Life Technologies). Furthermore, Acti-stain 555 phalloidin Immunohistochemistry (1:400, Cytoskeleton PHDH1-A) and Hoechst 33342 Preparation of embryos (1 μg/ml) were used on larvae. Embryos and larvae were Embryos were collected at 25 °C and were dechorio- mounted in ProLong Gold (Life Technologies, P36930). nated by submersion in 50% bleach for 4 min. Embryos were then fixed in a solution of equal parts heptane and Microscopy 10% formalin (Sigma, Product # HT501128). Fixation All microscopy was performed on a Zeiss LSM700 with lasted for 20 min during which time the embryos were an oil-immersion × 40 APOCHROMAT, 1.4 NA object- placed on an orbital shaker that rotated at a rate of 250/ ive. All images of embryos, and the image of fascin min. Following fixation, the formalin and heptane were localization in Fig. 2, were acquired with a 1.0× optical removed and replaced with a solution of equal parts zoom. All other larval images were acquired with a 0.5× methanol and heptane. The embryos were vortexed for optical zoom. Image tiling was necessary to acquire im- 1 min to devitellinize the embryos. Embryos were stored ages of the full larval muscles and was completed using in methanol at − 20 °C until immunostaining. the tiling function in the ZEN software that controls the microscope. Preparation of larvae Dissection of larvae was carried out as previously de- Statistics scribed [18] with minor modifications. The primary dif- All statistics were performed using GraphPad Prism. All ference being that the buffer used was modified to data sets were compared to appropriate controls by a increase the preservation of muscle structure. The modi- Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < fied dissection buffer was 100 mM PIPES (Sigma-Al- 0.0001. drich, P6757), 115 mM D-sucrose (Fisher Scientific, BP220-1), 5 mM trehalose (Acros Organics, 182550250), Image analysis 10 mM sodium bicarbonate (Fisher Scientific, BP328- Analysis of nuclear position in larvae 500), 75 mM potassium chloride (Fisher Scientific, Although the field has traditionally measured the dis- P333-500), 4 mM magnesium chloride (Sigma-Aldrich, tance between nuclei [18, 20, 21], this measurement M1028), and 1 mM EGTA (Fisher Scientific, 28-071-G). does not account for changes in muscle size and nuclear Larvae were then fixed with 10% formalin (Sigma-Al- number. We have therefore modified this measurement drich, HT501128) for 20 min. Briefly, dissection involved to determine how evenly nuclei are spaced within a lateral cuts at the anterior and posterior end of the larva muscle [22]. First, the area and length of the muscle was that encompassed 70% of larval circumference. These measured. Next, the position and number of nuclei is were followed by a longitudinal cut through the dorsal calculated by using the multipoint tool in ImageJ to surface of the animal that connected the two lateral cuts. place a point in the center of each nucleus. The position The intestines, other internal tissues, and neurons were of each nucleus is used to calculate the actual inter- then removed, and the flaps of tissue composed of epi- nuclear distance. The maximal internuclear distance is dermis and muscle were pinned down and fixed. For fix- determined by taking the square root of the muscle area ation, larvae were incubated in a solution of 10% divided by the nuclear number. This value represents formalin in PBS for 20 min. the distance between nuclei, if internuclear distance was fully maximized. The ratio between the actual inter- Immunostaining nuclear distance and the maximal internuclear distance Staining of embryos and larvae was identical. Antibodies ratio was then used to determine how evenly nuclei were were used at the following dilutions: rabbit anti-dsRed distributed. This method normalizes the internuclear (1:400, Clontech 632496), rat anti-tropomyosin (1:200, distance to both nuclear count and muscle area which Abcam ab50567), mouse anti-GFP (1:50, Developmental leads to a more representative means of comparison be- Studies Hybridoma Bank GFP-G1), mouse anti-integrin tween muscles, larvae, and genotypes. All viable (not Camuglia et al. Skeletal Muscle (2018) 8:12 Page 4 of 13 torn) ventral longitudinal (VL3) muscles were measured fluorescence intensity line scans were measured within a from each larva. At least four larvae from one experi- 10 ×10 μm box at the segment border using plot profile ment were measured for each genotype. Statistical ana- function in ImageJ. The width of the fluorescence peak lysis was performed with Prism 4.0 (GraphPad). composed of fluorescence intensities greater than 25% of Student’s t test was used to assess the statistical signifi- the maximum intensity (75% fluorescence intensity cance of differences in measurements between experi- peak) was measured. Each data points indicate the width mental genotypes and controls. of the 75% fluorescence intensity peak for a single myo- tendinous junction. A total of at least 50 MTJs from 20 Analysis of nuclear position in embryos different embryos were analyzed. The position of nuclei was measured in stage 16 em- bryos. This is the latest stage before cuticle development Analysis of muscle size in larvae blocks the ability to perform immunofluorescence mi- The area of the VL3 muscles were measured using the croscopy. Embryos were staged based primarily on gut polygon selection tool in ImageJ as previously described morphology as previously described [21]. At stage 16, [22]. Data points indicate the size of an individual the nuclei are reliably positioned adjacent to the muscle muscle. ends, and disruptions in this positioning can be easily determined as previously described [21, 23, 24]. Images, Analysis of general muscle architecture acquired as described above, were processed as max- Qualitative muscle phenotype analysis was completed on imum intensity projections of confocal z-stacks using embryos of each genotype. All analysis was based on the ImageJ. The position of the nuclei was determined by immunofluorescence staining pattern of Tropomyosin in using the line function in ImageJ to measure the dis- stage 16 embryos. The frequency of the following pheno- tance between either the dorsal end of the muscle and types were scored: the number of free myoblasts in an the nearest nucleus or the ventral end of the muscle and embryo that indicated a defect in myoblast fusion (small, the nearest nucleus. All four LT muscles were measured unfused circles stained by anti-tropomyosin) and the in four hemisegments from each embryo. At least 20 number of muscles in each hemisegment (> 4 defined as embryos from at least two independent experiments extra muscles, < 4 defined as missing muscles) indicating were measured for each genotype with each data point gross abnormalities in the specification of muscle tissue. representing the average for all muscles measured within For analysis of the unfused myoblasts, embryos were a single embryo. grouped into bins with a width of 5 and the first bin centered on zero. Analysis of nuclear count in embryos The number of nuclei was counted using apRed fluor- Results escence in the 4 lateral transverse (LT) muscles of Fascin is necessary for muscle function stage17embryos,oncenucleihad separatedfrom Animal movement provides a simple assay for muscle their clusters and into easily distinguished individual function. Although adult locomotion has been used to nuclei. Only hemisegments in which there were four perform a full-genome, RNAi-based screen for regula- lines of nuclei, corresponding to 4 LT muscles, were tors of muscle function in Drosophila adults [17], similar counted. Data points indicate the number of apRed screens have not been completed using Drosophila lar- positive nuclei within a single hemisegment. A total vae. The greatest advantage to evaluating muscle func- of at least 49 hemisegments from at least 19 individ- tion in larvae rather than adults is that pupation, and ual embryos were counted. the high probability of lethality during pupation, is bypassed. We have therefore modified published larval Analysis of nuclear count in larvae locomotion assays [18, 19] to identify regulators of The number of nuclei in the VL3 muscles were counted muscle function. To ensure that the identified genes had using Hoechst staining. Data points indicate the number a muscle-autonomous effect on muscle function, we of nuclei within an individual muscle. Careful analysis of used the GAL4/UAS system [25] to disrupt gene func- z-stacks was used to ensure that nuclei were in the tion in a muscle specific manner. Specifically, we used muscle that they were attributed to. DMef2-GAL4 to drive the expression of a small library of UAS-RNAi constructs. We measured movement of Analysis of muscle attachment in embryos larvae toward a chemo-attractant as previously described Integrin accumulation at the myotendinous junction of [19], with modifications to increase the throughput of stage 16 embryos were measured in dorsal muscle 2 the assay. First, we skipped the selection of stage 17 em- (DO2). Z-stack projection images that extended through bryos, which previously ensured that the ages of the the entire MTJ were used for these data. Integrin evaluated larvae were similar. We replaced this step, Camuglia et al. Skeletal Muscle (2018) 8:12 Page 5 of 13 which previously took ~ 60 min per genotype, per ex- For the proof-of-concept screen, we expressed RNAi periment, with a timed-lay. Briefly, virgins that against 82 genes (Additional file 1: Table S1). The se- expressed DMef2-GAL4 and males that carried the lected genes included those expected to impact muscle UAS-RNAi were mixed together in a vial for 1 h. The function (e.g., Dystrophin, Msp300) and many for which adults were then moved to another vial. The first vial, we did not have a prediction. The speed of locomotion which contained all of the embryos that were laid in larvae that expressed each RNAi was compared to during the 1-h period, was then used for the experi- control larvae in which DMef2-GAL4 drove the expres- ment to ensure that all larvae used in an experiment sion of mCherry RNAi. The disruption of 12/82 genes were of similar age. These vials were aged for 5 days significantly altered larval locomotion compared to con- until the animals were third-instar larvae (L3). Sec- trol larvae, indicating that these 12 genes regulate ond, we measured themovementofmanylarvaesim- muscle function. Of these 12 genes, disruption of 5 ultaneously rather than measuring locomotion for an caused larvae to move faster compared to controls, and individual larva. The movement of larvae was then the disruption of 7 caused the larvae to move more tracked using ImageJ (Fig. 1a). Measuring larval loco- slowly than controls (Fig. 1b). RNAi directed against the motion of many animals simultaneously provided two expression of singed (sn) which encodes for the actin- benefits. First, it increased the speed of the assay binding protein fascin [26, 27] caused the greatest from ~ 60 min/genotype to ~ 10 min/genotype. Coin- decrease in larval locomotion. Therefore, we investigated cident with the increased speed of the assay, there the impact that fascin depletion had on muscle structure was less variability in the age of larvae that were to identify the mechanism by which fascin regulates tracked in an experiment, thus increasing the preci- muscle function. sion of the data. Fascin is localized to the nucleus in Drosophila muscle As a first approach to determine how fascin regulates muscle function, we examined the localization of fascin in Drosophila larval muscles and found that fascin local- ized to the sarcomeres and to the nuclei. The nuclear localization is similar to the localization of fascin in nurse cells [28] (Fig. 2a). An emergent regulator of muscle function is the pos- ition of the many myonuclei within a single cytoplasm [12, 13, 29]. Based on the localization of fascin to the nucleus, we hypothesized that fascin may regulate nu- clear movement during muscle development. Consistent with this hypothesis, fascin interacts with the nuclear en- velope protein Nesprin-2 and regulates nuclear move- ment in migrating fibroblasts [30] and is necessary for the positioning of nuclei in developing Drosophila oocytes [28]. To determine whether nuclear position was affected in Drosophila muscle, we crossed apRed, a marker for the nuclei in the lateral transverse (LT) mus- cles [31] into the sn Drosophila and measured the position of nuclei as previously described [21]. In sn mutants, nuclei were closer to the ventral end of the muscle compared to controls (Fig. 2b–d). To determine whether the effect on nuclear position was muscle autonomous, we transiently expressed RNAi specifically Fig. 1 An RNAi screen for larval locomotion identifies fascin as a in the mesoderm during the early stages of muscle regulator of muscle function. a Cartoon illustrating the locomotion development using Twist-GAL4 or in a more sustained assay that was used to identify RNAi constructs that when expressed manner using DMef2-GAL4 and measured the position specifically in muscle, altered muscle function. b Graph indicating the speed of larval locomotion toward a chemoattractant when indicated of the nuclei. Muscle-specific depletion of fascin had no genes were depleted by expression of RNAi specifically in muscle. All impact on nuclear position (Fig. 2e–h). Together, these data were compared to their control by Student’s t test. *p <0.05; data indicate that although nuclei are closer to the **p <0.01; ****p < 0.0001 muscle end in fascin mutants, this is not regulated by Camuglia et al. Skeletal Muscle (2018) 8:12 Page 6 of 13 b c d Fig. 2 Fascin is localized to actin and the nucleus in muscle. a Images from different focal planes of dissected L3 larvae showing that fascin (sn) is colocalized with both Phalloidin (F-actin) (left two panels) and Hoechst (nuclei) (right two panels). b Immunofluorescence images from stage 16 embryos stained for Tropomyosin to identify the muscles (magenta) and the nuclei in the LT muscles (green) in control and sn mutant embryos. Scale bar, 10 μm. c Graph indicating the distance between the dorsal end of the muscle and the nearest nucleus in control and sn mutant embryos. d Graph indicating the distance between the ventral end of the muscle and the nearest nucleus in control and sn mutant embryos. e, f Immunofluorescence images from stage 16 embryos stained for Tropomyosin to indicate the muscles (magenta) and the nuclei within the LT muscles (green) in embryos where RNAi against either mCherry (control) or fascin (sn RNAi) was driven by Twist-GAL4 (e)or Dmef2- GAL4 (f). g, h Graphs indicating the average distance between the dorsal end of the muscle and the nearest nucleus (g) or the ventral end of the muscle and the nearest nucleus (h) in embryos of indicated genotypes. All data (c, d, g, h) were compared to their control by Student’s t test. **p < 0.01 fascin expressed in muscle during embryonic muscle de- mutant embryos. At embryonic stage 16, there are 30 velopment in Drosophila. well-characterized muscles per hemisegment in the Drosophila embryo [32]. We noted a number of dif- Fascin regulates myoblast fusion ferences between sn mutant embryos and controls Because the loss of fascin had a limited effect on nu- (Fig. 3a). First, there was a reduction in the number clear position in Drosophila embryonic muscles, we of muscles. Although, various muscles were missing looked at the general muscle pattern in the sn in individual hemisegments, we focused on the LT Camuglia et al. Skeletal Muscle (2018) 8:12 Page 7 of 13 de Fig. 3 Fascin is necessary for myoblast fusion. a Immunofluorescence images of stage 16 embryos stained for Tropomyosin to indicate the muscles (magenta). Images are focused on the lateral transverse (LT) muscles in stage 16 embryos. Green arrowheads indicate unfused myoblasts. Scale bar, 10 μm. b Graph indicating the percentage of embryos that had at least one hemisegment with > 4 LT muscles (Extra LTs) or < 4 LT muscles (missing LTs). Values that exceed 100% indicate that some embryos had one hemisegment with > 4 LT muscles (extra muscles) and another hemisegment with < 4 LT muscles (missing muscles). c Graph indicating the number of apRed positive nuclei per hemisegment. Student’s t test was used for comparison to controls. ****p < 0.0001. d Graph indicating how frequently different numbers of unfused myoblasts 28 28 are seen in control (black) and sn mutant embryos (green). e Graph indicating the viability of the sn mutant animals (green) compared to controls (black) muscles because they are near the embryo surface increased to 7.5 in sn mutants (Fig. 3d). Additionally, and are the only muscles that are perfectly aligned on 70% of control embryos had two or fewer identifiable the dorsal-ventral axis of the embryo. These features unfused myoblasts whereas 75% sn mutant embryos make the LTs easy to count, image, and analyze. had at least three unfused myoblast and 50% of sn The typical hemisegment from a control embryo has mutant embryos eight or more unfused myoblasts. four LT muscles. Seventy percent of control embryos Based on the missing muscles and the abundance of had four LT muscles in every hemisegment, and 30% of unfused myoblasts in mutant embryos, we tested the controls had at least one hemisegment with greater than viability of the sn mutants and found that there was four LT muscles. In sn mutants, 70% of embryos had significant lethality during both the embryonic and four LT muscles in every hemisegment and 19% of larval stages (Fig. 3e). These data indicate that fascin embryos had at least one hemisegment with greater than regulates myoblast fusion. four LT muscles. Additionally, 19% of sn embryos had To determinewhether thesephenotypeswere at least one hemisegment with fewer than four LT muscle autonomous, we used the GAL4/UAS system muscles (Fig. 3b), and were therefore missing LT to deplete fascin specifically from the developing muscles. Eight percent of embryos had at least one mesoderm and muscle of the Drosophila embryo. The hemisegment with an extra muscle and one expression of a UAS-sn RNAi (fascin RNAi) was hemisegment with a missing muscle and were thus driven with each of three GAL4 drivers. Twist-GAL4 counted in both categories, pushing the total embryos was used to drive RNAi expression in the early meso- over 100%. Because the absence of muscles can indicate derm, DMef2-Gal4 was used to drive RNAi expression a defect in myoblast fusion, we counted the number of slightly later in muscle with sustained expression nuclei that were incorporated into the LT muscles per throughout development, and MHC-GAL4 was used hemisegment. This number was reduced from a mean of to drive RNAi expression beginning at the final stage 26 in the controls to a mean of 22 in the sn mutants of embryonic development and continuing throughout (Fig. 3c). Consistent with this, there was an increase in development. We then examined the general muscle unfused myoblasts. In controls, the median number of structure in stage 16 embryos as we had done for free myoblasts per embryo was 1, and that number sn mutant embryos. There were no defects in Camuglia et al. Skeletal Muscle (2018) 8:12 Page 8 of 13 muscle morphology when MHC-GAL4 was used to affected neither measure of fusion (Fig. 4d). However, drive RNAi expression suggesting that fascin must be DMef2-GAL4-mediated expression of RNAi had a depleted early during development to have significant greater effect on viability (Fig. 4e)suggestingthatthe impact (Additional file 2:Figure S1). absence of muscles was more detrimental to animal Early mesodermal expression of the RNAi under the viability. control of Twist-GAL4 and expression of RNAi in muscle under the control of DMef2-GAL4 both in- creased the percentage of embryos that were missing Fascin-dependent fusion effects are evident in larvae LT muscles, but DMef2-GAL4-mediated expression re- To determine the effects of fascin-depletion later in devel- sulted in a higher frequency of embryos with missing opment, larvae were dissected and stained with Phalloidin muscles (Fig. 4a, b). Conversely, only Twist-GAL4-me- to identify the muscles and Hoechst to identify the nuclei diated expression of RNAi against fascin caused a de- (Fig. 5). We examined the third ventral longitudinal muscle crease in the number of nuclei that were incorporated (VL3) because after dissection this muscle is on the surface into the LT muscles (Fig. 4c). This suggested that and therefore easily imaged. The distribution of myonuclei early expression of fascin RNAi was necessary to in- was similar in the controls and sn larvae (Fig. 5a, b). The hibit myoblast fusion. Consistent with this, Twist- size of the muscles (Fig. 5a, c), and the number of nuclei in GAL4-mediated RNAi expression increased both the each muscle (Fig. 5a, d) were both reduced in the sn percentage of embryos with unfused myoblasts and larvae compared to the controls, but the reductions were the number of unfused myoblasts per embryos. statistically insignificant. We hypothesized that the lack of DMef2-GAL4-mediated expression of fascin RNAi phenotype may be based on selection of the healthiest ab de Fig. 4 Fascin has muscle autonomous effects on myoblast fusion. a Stage 16 embryos stained for tropomyosin to identify the muscles (magenta). Embryos expressed RNAi against fascin under the control of Twist-GAL4 (top) or Dmef2-GAL4 (bottom). Green arrowheads indicate unfused myoblasts. Scale bar, 10 μm. b Graph indicating the percentage of embryos of indicated genotypes that have at least one hemisegment with either > 4 LT (extra LTs) muscles or < 4 LT muscles (missing LTs). c Graph indicating the number of apRed positive nuclei incorporated into LT muscles per hemisegment in indicated genotypes. Student’s t test was used for comparison to controls. *p <0.05. d Graph indicating how frequently different numbers of unfused myoblasts were seen in indicated genotypes. e Graph indicating the viability of animals with indicated genotypes Camuglia et al. Skeletal Muscle (2018) 8:12 Page 9 of 13 bc d Fig. 5 Genetic disruption of fascin did not affect larval muscle structure. a Immunofluorescence images of the third ventral longitudinal muscle (VL3) in L3 larvae of indicated genotypes. Sarcomeres (magenta) were stained with Phalloidin to identify the muscle and Hoechst (green) was used to identify the nuclei. Scale bar, 25 μm. b Graph indicating the actual internuclear distance divided by the maximal internuclear distance in indicated genotypes. c Graph indicating the area of the muscles as a proxy for muscle size in the indicated genotypes. d Graph indicating the number of nuclei in indicated genotypes. All data (b–c) were compared to their control by Student’s t test. All differences were statistically insignificant animals because they are the animals that survived until the MTJ of dorsal muscle 2 (DO2). Compared to controls, L3 stage. As such, we examined animals that expressed the signal was wider in sn mutants (Fig. 7a–c). RNAi specifically in the muscle, which are more viable Similarly, DMef2-GAL4-mediated expression of fascin (compare Fig. 4e to Fig. 3e). RNAi, but not Twist-GAL4-mediated expression of fas- We expressed RNAi against fascin under the control cin RNAi also increased the width of the βPS-integrin of Twist-GAL4 (early, transient expression), DMef2-Gal4 signal (Fig. 7d–h). These data suggest that sustained fas- (slightly later, sustained expression), or MHC-Gal4 (late, cin function is necessary for proper MTJ organization. sustained expression). The distribution of nuclei was the same in each genotype (Fig. 6a–d). Muscle size was de- Discussion creased when either Twist-GAL4 or DMef2-GAL4 was One of the many strengths of Drosophila as a model sys- used to express fascin RNAi (Fig. 6e) suggesting that tem is its utility as a tool to identify novel regulators of early fascin-dependent processes contribute to fascin- specific biological functions. This ability utilizes the im- dependent muscle growth. Finally, the number of nuclei mense genetic tools that are available and requires sim- in VL3 muscles was reduced by DMef2-GAL4-mediated ple and fast assays to screen many mutants and/or RNAi fascin depletion. Twist-GAL4-mediated depletion did re- lines. In this work, we adapted a published larval- duce the number of nuclei per muscle, but insignificantly tracking assay [19] to perform a proof-of-concept screen so. MHC-GAL4-mediated depletion had no impact on the for muscle function. We identified 12 genes that regulate number of nuclei per muscle (Fig. 6a–c, f). Thus, the de- muscle function, either positively or negatively. We con- fects in fusion are not transient, but are evident through- tinued these experiments by examining the mechanism out larval development. by which singed, Drosophila fascin, regulated muscle function because fascin-depletion had the strongest Fascin regulates muscle attachment effect on muscle function. DMef2-Gal4-mediated expression of sn RNAi did not re- We used a combination of mutant alleles and tissue- duce the number of nuclei incorporated into embryonic specific expression of RNAi against fascin to demonstrate LT muscles (Fig. 4c), but did reduce the total number of that fascin regulates both myoblast fusion and the struc- muscles in the embryo (Fig. 4b). This could be explained ture of the MTJ. Fascin is well-described as a protein that by an effect on the attachments between the muscle and can bundle F-actin filaments and increase their strength, the tendon cell at the myotendinous junction (MTJ). To and the strength of actin-based cellular protrusions [33]. determine whether fascin affected MTJ integrity, we im- Furthermore, by this mechanism, fascin contributes to cel- munostained embryos for tropomyosin to identify the lular invasions associated with cancer metastasis [34, 35]. muscles and βPS-integrin to identify the MTJ (Fig. 7). Myoblast fusion requires a similar organization of protru- We measured the width of the βPS-integrin signal at the sive F-actin structures that invade the growing myotube. Camuglia et al. Skeletal Muscle (2018) 8:12 Page 10 of 13 c f Fig. 6 Muscle-specific depletion of fascin results in smaller muscles with fewer nuclei. a–c Immunofluorescence images of the VL3 muscle in L3 larvae that expressed RNAi against either mCherry (control) or fascin (sn RNAi) under the control of Twist-GAL4 (a), DMef2-GAL4 (b), or MHC-GAL4 (c). Sarcomeres were identified by Phalloidin (magenta), and nuclei were identified by Hoechst (green). Scale bar, 25 μm. d Graph indicating the actual internuclear distance divided by the maximal internuclear distance in indicated genotypes. e Graph indicating the area of the muscles in larvae of indicated genotypes. f Graph indicating the number of nuclei per muscle in larvae of indicated genotypes. All data (d–e) were compared to their control by Student’s t test. *p <0.05; **p <0.01; ****p <0.0001 The most surprising aspect of the myoblast fusion Perhaps the most intriguing feature of these data is the data is the relatively minor effect that fascin has com- temporal separation of fascin-dependent myoblast fusion pared to other genes necessary for myoblast fusion and fascin-dependent MTJ stability. This conclusion is [31, 36–38]. The reason for this is not clear. One based on our finding that the time and duration of fascin possibility is that maternal loading provides sufficient depletion determines the phenotype that will emerge. fascin to facilitate the initial rounds of fusion. Alter- Transient depletion of fascin during early stages of natively, perhaps the final fusion events require muscle development disrupted myoblast fusion but not greater protrusive force and only then does the func- MTJ structure. Conversely, later, and sustained depletion tion of fascin become critical. of fascin affected MTJ structure, but not myoblast fu- The contribution of fascin to MTJ structure is consist- sion. These data are important because they demonstrate ent with previously published data. Fascin contributes to that although both fusion and MTJ structure require fas- filopodia formation [35] and MTJ development is cin function, they are not codependent features of dependent on filopodia-like extensions. Furthermore, al- muscle development. though the MTJ forms as a smooth attachment during pu- It is not clear whether either function is more critical pation [16], the MTJ in the embryo is dynamic [39]. Thus, than the other. Certainly, sustained depletion of fascin, perhaps fascin is continually necessary for the turnover which disrupts MTJ integrity has a greater effect on and the integrity of the MTJ. animal survival than does the transient depletion that Camuglia et al. Skeletal Muscle (2018) 8:12 Page 11 of 13 a b c d e f h Fig. 7 Fascin is necessary for proper myotendinous junction organization a Immunofluorescence images of the MTJ of muscle DO2 stained for tropomyosin and βPS-integrin in control (top) and sn mutant embryos (bottom). Scale bar, 10 μm. b Representative intensity profile of the βPS-integrin signal in control (black) and sn mutant embryos (green). c Graph indicating the width of the βPS-integrin signal defined by the points at which the signal is 25% of the maximum. d, e Immunofluorescence images of the MTJ of muscle DO2 stained for tropomyosin and βPS-integrin in animals in which twist-GAL4 was used (d) or DMef2-GAL4 was used (e) to express RNAi against either mCherry (control) or fascin (sn RNAi). f, g Representative intensity profiles of the βPS-integrin signal in indicated genotypes. h Graph indicating the width of the βPS-integrin signal in indicated genotypes as defined by the points at which the signal is 25% of the maximum. All data (c, h) were compared to their control by Student’s t test. **p < 0.01; ****p < 0.0001 disrupts fusion. However, this conclusion is limited be- larval muscles in Drosophila, we suspect that this re- cause the impact that a small reduction in nuclear number duction is based in muscle damage that may be has on muscle organization is not clear. Reduced nuclear linked to improper attachments and poor mechanical numbers do correlate with reduced muscle size [40, 41] stability. However, further work is necessary to under- and therefore likely cause reduced muscle function. Data stand the mechanism by which nuclei are lost so that in embryos indicated that DMef2-GAL4-mediated the impact of individual fascin-dependent functions expression of fascin RNAi only affected MTJs and would can be determined. therefore allow us to isolate the impact of the MTJ versus the impact of myoblast fusion. However, we see that in lar- Conclusions vae, there is a reduction in the number of nuclei per In total, we have used a larval locomotion assay to iden- muscle. Because there is no repair of embryonic and tify novel regulators of muscle function. Furthermore, Camuglia et al. Skeletal Muscle (2018) 8:12 Page 12 of 13 we have described that the strongest hit from the screen, Received: 16 October 2017 Accepted: 22 March 2018 fascin, contributes to muscle function by regulating myoblast fusion and MTJ structure. Both of these func- References tions are consistent with known biochemical abilities for 1. Abmayr SM, Pavlath GK. Myoblast fusion: lessons from flies and mice. fascin and suggest that fascin has multiple essential Development. 2012;139:641–56. https://doi.org/10.1242/dev.068353. functions for muscle development that are separated in 2. Kim JH, Jin P, Duan R, Chen EH. ScienceDirect mechanisms of myoblast fusion during muscle development. Curr Opin Genet Dev. 2015;32:162–70. time. More generally, this work outlines a new strategy https://doi.org/10.1016/j.gde.2015.03.006. for the identification of genes and pathways that can be 3. Sens KL, Zhang S, Jin P, Duan R, Zhang G, Luo F, et al. An invasive manipulated to either increase or decrease muscle podosome-like structure promotes fusion pore formation during myoblast fusion. J Cell Biol. 2010;191:1013–27. https://doi.org/10.1083/jcb.201006006. function. 4. Bothe I, Deng S, Baylies M. PI(4,5)P2 regulates myoblast fusion through Arp2/3 regulator localization at the fusion site. Development. 2014;141: 2289–301. https://doi.org/10.1242/dev.100743. Additional files 5. Kim JH, Ren Y, Ng WP, Li S, Son S, Kee Y-S, et al. Mechanical tension drives cell membrane fusion. Dev Cell. 2015;32:561–73. https://doi.org/10.1016/j. Additional file 1: Table S1. All of the data acquired during the limited devcel.2015.01.005. RNAi-based screen for regulators of muscle function. (PDF 139 kb) 6. Gimona M, Buccione R, Courtneidge SA, Linder S. Assembly and biological Additional file 2: Figure S1. Expression of RNAi against fascin late in role of podosomes and invadopodia. Curr Opin Cell Biol. 2008;20:235–41. embryonic development does not affect muscle development a https://doi.org/10.1016/j.ceb.2008.01.005. Immunofluorescence images showing the muscle pattern in animals 7. McNiven MA. Breaking away: matrix remodeling from the leading edge. expressing mCherry RNAi (control) and animals expressing fascin RNAi (sn Trends Cell Biol. 2013;23:16–21. https://doi.org/10.1016/j.tcb.2012.08.009. RNAi) under the control of the MHC-GAL4 driver. b Graph comparing the 8. Deng S, Bothe I, Baylies MK. The formin diaphanous regulates myoblast frequency of embryos with extra muscles in each genotype. No embryos fusion through actin polymerization and Arp2/3 regulation. PLoS Genet. with missing muscles were observed in either genotype. c Graph 2015;11:e1005381–29. https://doi.org/10.1371/journal.pgen.1005381. comparing the frequency at which embryos were found to have unfused 9. Deng S, Bothe I, Baylies M. Diaphanous regulates SCAR complex localization myoblasts in each genotype. (PDF 360 kb) during Drosophila myoblast fusion. Fly. 2016;10:178–86. https://doi.org/10. 1080/19336934.2016.1195938. 10. D’Alessandro M, Hnia K, Gache V, Koch C, Gavriilidis C, Rodriguez D, et al. Acknowledgements Amphiphysin 2 orchestrates nucleus positioning and shape by linking the The authors would like to thank Tina Tootle (University of Iowa) for providing nuclear envelope to the actin and microtubule cytoskeleton. Dev Cell. 2015; sn28 the fascin allele and her advice. Drosophila stocks obtained from the 35:186–98. https://doi.org/10.1016/j.devcel.2015.09.018. Bloomington Drosophila Stock Center (NIHP400D018537) were used in this 11. Roman W, Martins JP, Carvalho FA, Voituriez R, Abella JVG, Santos NC, et al. study. The integrin betaPS antibody developed by D. Brower at Harvard Myofibril contraction and crosslinking drive nuclear movement to the Medical School was obtained from the Developmental Studies Hybridoma periphery of skeletal muscle. Nat Cell Biol. 2017;19:1189–201. https://doi.org/ Bank, created by the NICHD of the NIH and maintained at the University of 10.1038/ncb3605. Iowa, Department of Biology, Iowa City, IA 52242, USA. 12. Folker ES, Baylies MK. Nuclear positioning in muscle development and disease. Front Physiol. 2013;4:363. https://doi.org/10.3389/fphys.2013.00363. Funding 13. Cadot B, Gache V, Gomes ER. Moving and positioning the nucleus in This work was funded by grants from the American Heart Association to skeletal muscle—one step at a time. Nucleus. 2015;6:373–81. https://doi.org/ E.S.F. and by startup funds provided by Boston College to E.S.F. 10.1080/19491034.2015.1090073. 14. Brown NH, Gregory SL, Martin-Bermudo MD. Integrins as mediators of morphogenesis in Drosophila. Dev Biol. 2000;223:1–16. https://doi.org/10. Availability of data and materials 1006/dbio.2000.9711. All data are presented in the manuscript. Raw data, if requested, will be provided 15. Schnorrer F, Kalchhauser I, Dickson BJ. The transmembrane protein Kon-tiki by the corresponding author. Similarly, all reagents that are not commercially couples to Dgrip to mediate myotube targeting in Drosophila. Dev Cell. available will be made available upon request to the corresponding author. 2007;12:751–66. https://doi.org/10.1016/j.devcel.2007.02.017. 16. Weitkunat M, Kaya-Çopur A, Grill SW, Schnorrer F. Tension and force- Authors’ contributions resistant attachment are essential for myofibrillogenesis in Drosophila flight JMC and TRM designed, completed, and analyzed the experiments and muscle. Curr Biol. 2014;24:705–16. contributed to the writing of the manuscript. RM designed, completed, and 17. Schnorrer F, Schönbauer C, Langer CCH, Dietzl G, Novatchkova M, analyzed the experiments related to larval locomotion. JM completed and Schernhuber K, et al. Systematic genetic analysis of muscle morphogenesis analyzed the experiments. CHH and DS contributed to the development of and function in Drosophila. Nature. 2010;464:287–91. https://doi.org/10. the tracking assay. ESF designed the experiments, interpreted the results, 1038/nature08799. and wrote the manuscript. All authors read and approved the final 18. Metzger T, Gache V, Xu M, Cadot B, Folker ES, Richardson BE, et al. MAP and manuscript. kinesin-dependent nuclear positioning is required for skeletal muscle function. Nature. 2012;484:120–4. https://doi.org/10.1038/nature10914. Ethics approval and consent to participate 19. Louis M, Piccinotti S, Vosshall LB. High-resolution measurement of odor- Not applicable driven behavior in Drosophila larvae. J Vis Exp. 2008;11:638. https://doi.org/ 10.3791/638. Consent for publication 20. Elhanany-Tamir H, Yu YV, Shnayder M, Jain A, Welte M, Volk T. Organelle Not applicable positioning in muscles requires cooperation between two KASH proteins and microtubules. J Cell Biol. 2012;198:833–46. https://doi.org/10.1083/jcb. Competing interests 21. Folker ES, Schulman VK, Baylies MK. Muscle length and myonuclear position The authors declare that they have no competing interests. are independently regulated by distinct Dynein pathways. Development. 2012;139:3827–37. https://doi.org/10.1242/dev.079178. Publisher’sNote 22. Collins MA, Mandigo TR, Camuglia JM, Vazquez GA, Anderson AJ, Hudson Springer Nature remains neutral with regard to jurisdictional claims in CH, et al. Emery-Dreifuss muscular dystrophy-linked genes and published maps and institutional affiliations. centronuclear myopathy-linked genes regulate myonuclear movement by Camuglia et al. Skeletal Muscle (2018) 8:12 Page 13 of 13 distinct mechanisms. Mol Biol Cell. 2017; https://doi.org/10.1091/mbc.E16- 10-0721. 23. Folker ES, Schulman VK, Baylies MK. Translocating myonuclei have distinct leading and lagging edges that require kinesin and dynein. Development. 2014;141:355–66. https://doi.org/10.1242/dev.095612. 24. Schulman VK, Folker ES, Rosen JN, Baylies MK. Syd/JIP3 and JNK signaling are required for myonuclear positioning and muscle function. PLoS Genet. 2014;10:e1004880–15. https://doi.org/10.1371/journal.pgen.1004880. 25. Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993;118:401–15. 26. Paterson J, O'Hare K. Structure and transcription of the singed locus of Drosophila melanogaster. Genetics. 1991;129:1073–84. 27. Bryan J, Edwards R, Matsudaira P, Otto J, Wulfkuhle J. Fascin, an echinoid actin- bundling protein, is a homolog of the Drosophila singed gene product. 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Int J Dev Biol. 1998;42:283–90. 33. Jayo A, Parsons M. Fascin: a key regulator of cytoskeletal dynamics. Int J Biochem Cell Biol. 2010;42:1614–7. https://doi.org/10.1016/j.biocel.2010.06.019. 34. Hashimoto Y, Kim DJ, Adams JC. The roles of fascins in health and disease. J Pathol. 2011;224:289–300. https://doi.org/10.1002/path.2894. 35. Zanet J, Jayo A, Plaza S, Millard T, Parsons M, Stramer B. Fascin promotes filopodia formation independent of its role in actin bundling. J Cell Biol. 2012;197:477–86. https://doi.org/10.1083/jcb.201110135. 36. Erickson MR, Galletta BJ, Abmayr SM. Drosophila myoblast city encodes a conserved protein that is essential for myoblast fusion, dorsal closure, and cytoskeletal organization. J Cell Biol. 1997;138:589–603. 37. Chen EH, Olson EN. Antisocial, an intracellular adaptor protein, is required for myoblast fusion in Drosophila. Dev Cell. 2001;1:705–15. 38. Chen EH, Pryce BA, Tzeng JA, Gonzalez GA, Olson EN. 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An RNAi based screen in Drosophila larvae identifies fascin as a regulator of myoblast fusion and myotendinous junction structure

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Copyright © 2018 by The Author(s).
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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10.1186/s13395-018-0159-9
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29625624
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Abstract

Background: A strength of Drosophila as a model system is its utility as a tool to screen for novel regulators of various functional and developmental processes. However, the utility of Drosophila as a screening tool is dependent on the speed and simplicity of the assay used. Methods: Here, we use larval locomotion as an assay to identify novel regulators of skeletal muscle function. We combined this assay with muscle-specific depletion of 82 genes to identify genes that impact muscle function by their expression in muscle cells. The data from the screen were supported with characterization of the muscle pattern in embryos and larvae that had disrupted expression of the strongest hit from the screen. Results: With this assay, we showed that 12/82 tested genes regulate muscle function. Intriguingly, the disruption of five genes caused an increase in muscle function, illustrating that mechanisms that reduce muscle function exist and that the larval locomotion assay is sufficiently quantitative to identify conditions that both increase and decrease muscle function. We extended the data from this screen and tested the mechanism by which the strongest hit, fascin, impacted muscle function. Compared to controls, animals in which fascin expression was disrupted with either a mutant allele or muscle-specific expression of RNAi had fewer muscles, smaller muscles, muscles with fewer nuclei, and muscles with disrupted myotendinous junctions. However, expression of RNAi against fascin only after the muscle had finished embryonic development did not recapitulate any of these phenotypes. Conclusions: These data suggest that muscle function is reduced due to impaired myoblast fusion, muscle growth, and muscle attachment. Together, these data demonstrate the utility of Drosophila larval locomotion as an assay for the identification of novel regulators of muscle development and implicate fascin as necessary for embryonic muscle development. Keywords: Nuclear movement, Drosophila, Myoblast fusion, Myotendinous junction, Fascin, Myogenesis * Correspondence: eric.folker@bc.edu Equal contributors Biology Department, Boston College, Chestnut Hill, MA 02467, USA © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Camuglia et al. Skeletal Muscle (2018) 8:12 Page 2 of 13 Background for the identification of genes that are necessary for Skeletal muscle has a distinctive architecture that is gener- muscle development. Indeed, many screens for regulators ated by a unique set of developmental phases. Making the of muscle development have been completed. Researchers myofiber syncytium requires the fusion of mononucleated have used adult locomotion [17] and embryonic muscle myoblasts. In both Drosophila and mammalian systems, structure [18] as indicators of muscle development. Al- individual myoblasts invade growing myotubes and deposit though these strategies have proven effective, they each their nucleus into the common cytoplasm to drive myotube have drawbacks. Analysis of embryonic muscle structure growth [1, 2]. Myoblast fusion is an actin-dependent is labor intensive and requires significant expertise in process that is reminiscent of a cancer cell invading a tissue muscle biology. Analysis of adult locomotion is limited during metastasis [3]. The mononucleated myoblast ex- because the disruption of many genes is lethal during pu- tends a protrusive invadapodia-like structure that makes pation. Therefore, we have developed a simple assay for possible the penetration of the myotube and the mixing of muscle function based on the larval locomotion. We have cytoplasm. Many factors and signaling pathways that regu- used this assay to screen for novel regulators of muscle late myoblast fusion have been identified [2, 4, 5]. However, function and identified fascin as one such regulator. many of the genes necessary for invadapodia-like structures Subsequent cell biological analysis implicates fascin as a in other contexts have not yet been implicated in myoblast regulator of myoblast fusion and MTJ structure. fusion, suggesting that additional regulators remain to be identified. One glaring omission from the categories of pro- Methods teins that have been identified as regulators of myoblast Drosophila genetics fusion is proteins that stabilize filopodia. Invadapodia are All stocks were grown under standard conditions at 25 ° sn28 filopodia-like structures [6, 7], and they require several C. The Fascin allele was a generous gift from Tina factors that are known to stabilize filopodia. Furthermore, Tootle (University of Iowa). All UAS-RNAi Drosophila although loss-of-function data is lacking, dominant nega- lines were purchased from Bloomington Drosophila tive mutants of the formin Diaphanous inhibit myoblast Stock Center. UAS-RNAi constructs were driven specif- fusion and may suggest that filopodia are essential for ically in the mesoderm using twist-GAL4, which drives myoblast fusion [8, 9]. Thus, it is likely that additional expression in the mesoderm from stage 10 of embryonic factors that have been identified as capable of stabilizing development through stage 13 of embryonic develop- filopodia for the purpose of protrusion and invasion in ment, DMef2-GAL4 that drives expression in the other contexts contribute to myoblast fusion. muscles from stage 12 through adulthood, or MHC- Beyond myoblast fusion, there are several features of GAL4 which drives expression in muscle from stage 17 muscle development that either require, or have been hy- of embryonic development through adulthood. pothesized to require, precise regulation of the actin cyto- skeleton, including the positioning of nuclei and the Larval locomotion assay development of the myotendinous junction (MTJ). To We performed a modified version of the previously used date, evidence for actin-dependent nuclear movement in assay that has been used to measure larval locomotion in muscle is restricted to the squeezing of nuclei to the per- individual larvae [19]. Virgins expressing DMef2-GAL4 iphery of the muscle [10, 11], although it has been pro- were mixed with males that carried the UAS-RNAi for 1 h posed that actin may contribute to the movement of in a vial with standard Drosophila food. After 1 h, adults nuclei along the length of the muscle [12, 13]. The role of were moved to a new vial and the embryos laid during the actin in MTJ development is more established. MTJs are 1 h period were aged for 5 days until they were third- integrin-based adhesions that transmit force from the instar larvae (L3). Larvae were then floated from the food muscle to the skeleton [14]. The initial formation involves by the addition of 15% sucrose. Using a paintbrush, larvae extension of filopodia-like structures from the muscle cell were moved to a plate with wet yeast. After all of the ge- that interact with the tendon cell before forming a stable notypes had been collected, 10 larvae of each RNAi were and somewhat rigid attachment that enables effective moved to an arena that consisted of 3% agarose dyed black force transmission [15, 16]. All of these processes require with standard food color poured over the top of a 96-well linear actin-cables. The similarity in the actin-based struc- plate cover. Movement of larvae toward a stick dipped in tures suggests that the same molecular components may ethyl butyrate was captured using an iPhone mounted contribute to each of these aspects of muscle develop- above the arena. The speed of each larva was then ana- ment. Therefore, it is critical to determine how newly lyzed using ImageJ. identified genes and proteins contribute to each process. Because the developmental path and final architecture Viability assay of muscle cells is conserved from Drosophila to humans, Stage 16 embryos were picked and placed on an agarose flies provide a genetically tractable and inexpensive model plate. The selected embryos were incubated at 20 °C Camuglia et al. Skeletal Muscle (2018) 8:12 Page 3 of 13 overnight. After incubation, the number of unhatched βPS (1:50, Developmental Studies Hybridoma Bank CF. embryos were counted to determine embryonic lethality 6G11), mouse anti-Fascin (1:25, Developmental Studies and the L1 larvae from the hatched embryos were trans- Hybridoma Bank sn 7C, generous gift from Tina Tootle) ferred to a vial of standard fly food. Larvae were incu- , and mouse anti-αTubulin (1:200, Sigma-Aldrich bated at 25 °C until larvae began to pupate and eclose. T6199). Conjugated fluorescent secondary antibodies Adult flies were counted to determine pupal lethality used were Alexa Fluor 555 donkey-anti-rabbit (1:200), and removed from the vials of standard fly food upon Alexa Fluor 488 donkey-anti-rat (1:200), and Alexa Fluor eclosure. After 1 week of no eclosures, the number of 647 donkey-anti-mouse (1:200) (all Life Technologies) pupal cases were counted to determine larval lethality. and Alexa Fluor 488 donkey-anti-mouse (1:200, Life Technologies). Furthermore, Acti-stain 555 phalloidin Immunohistochemistry (1:400, Cytoskeleton PHDH1-A) and Hoechst 33342 Preparation of embryos (1 μg/ml) were used on larvae. Embryos and larvae were Embryos were collected at 25 °C and were dechorio- mounted in ProLong Gold (Life Technologies, P36930). nated by submersion in 50% bleach for 4 min. Embryos were then fixed in a solution of equal parts heptane and Microscopy 10% formalin (Sigma, Product # HT501128). Fixation All microscopy was performed on a Zeiss LSM700 with lasted for 20 min during which time the embryos were an oil-immersion × 40 APOCHROMAT, 1.4 NA object- placed on an orbital shaker that rotated at a rate of 250/ ive. All images of embryos, and the image of fascin min. Following fixation, the formalin and heptane were localization in Fig. 2, were acquired with a 1.0× optical removed and replaced with a solution of equal parts zoom. All other larval images were acquired with a 0.5× methanol and heptane. The embryos were vortexed for optical zoom. Image tiling was necessary to acquire im- 1 min to devitellinize the embryos. Embryos were stored ages of the full larval muscles and was completed using in methanol at − 20 °C until immunostaining. the tiling function in the ZEN software that controls the microscope. Preparation of larvae Dissection of larvae was carried out as previously de- Statistics scribed [18] with minor modifications. The primary dif- All statistics were performed using GraphPad Prism. All ference being that the buffer used was modified to data sets were compared to appropriate controls by a increase the preservation of muscle structure. The modi- Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < fied dissection buffer was 100 mM PIPES (Sigma-Al- 0.0001. drich, P6757), 115 mM D-sucrose (Fisher Scientific, BP220-1), 5 mM trehalose (Acros Organics, 182550250), Image analysis 10 mM sodium bicarbonate (Fisher Scientific, BP328- Analysis of nuclear position in larvae 500), 75 mM potassium chloride (Fisher Scientific, Although the field has traditionally measured the dis- P333-500), 4 mM magnesium chloride (Sigma-Aldrich, tance between nuclei [18, 20, 21], this measurement M1028), and 1 mM EGTA (Fisher Scientific, 28-071-G). does not account for changes in muscle size and nuclear Larvae were then fixed with 10% formalin (Sigma-Al- number. We have therefore modified this measurement drich, HT501128) for 20 min. Briefly, dissection involved to determine how evenly nuclei are spaced within a lateral cuts at the anterior and posterior end of the larva muscle [22]. First, the area and length of the muscle was that encompassed 70% of larval circumference. These measured. Next, the position and number of nuclei is were followed by a longitudinal cut through the dorsal calculated by using the multipoint tool in ImageJ to surface of the animal that connected the two lateral cuts. place a point in the center of each nucleus. The position The intestines, other internal tissues, and neurons were of each nucleus is used to calculate the actual inter- then removed, and the flaps of tissue composed of epi- nuclear distance. The maximal internuclear distance is dermis and muscle were pinned down and fixed. For fix- determined by taking the square root of the muscle area ation, larvae were incubated in a solution of 10% divided by the nuclear number. This value represents formalin in PBS for 20 min. the distance between nuclei, if internuclear distance was fully maximized. The ratio between the actual inter- Immunostaining nuclear distance and the maximal internuclear distance Staining of embryos and larvae was identical. Antibodies ratio was then used to determine how evenly nuclei were were used at the following dilutions: rabbit anti-dsRed distributed. This method normalizes the internuclear (1:400, Clontech 632496), rat anti-tropomyosin (1:200, distance to both nuclear count and muscle area which Abcam ab50567), mouse anti-GFP (1:50, Developmental leads to a more representative means of comparison be- Studies Hybridoma Bank GFP-G1), mouse anti-integrin tween muscles, larvae, and genotypes. All viable (not Camuglia et al. Skeletal Muscle (2018) 8:12 Page 4 of 13 torn) ventral longitudinal (VL3) muscles were measured fluorescence intensity line scans were measured within a from each larva. At least four larvae from one experi- 10 ×10 μm box at the segment border using plot profile ment were measured for each genotype. Statistical ana- function in ImageJ. The width of the fluorescence peak lysis was performed with Prism 4.0 (GraphPad). composed of fluorescence intensities greater than 25% of Student’s t test was used to assess the statistical signifi- the maximum intensity (75% fluorescence intensity cance of differences in measurements between experi- peak) was measured. Each data points indicate the width mental genotypes and controls. of the 75% fluorescence intensity peak for a single myo- tendinous junction. A total of at least 50 MTJs from 20 Analysis of nuclear position in embryos different embryos were analyzed. The position of nuclei was measured in stage 16 em- bryos. This is the latest stage before cuticle development Analysis of muscle size in larvae blocks the ability to perform immunofluorescence mi- The area of the VL3 muscles were measured using the croscopy. Embryos were staged based primarily on gut polygon selection tool in ImageJ as previously described morphology as previously described [21]. At stage 16, [22]. Data points indicate the size of an individual the nuclei are reliably positioned adjacent to the muscle muscle. ends, and disruptions in this positioning can be easily determined as previously described [21, 23, 24]. Images, Analysis of general muscle architecture acquired as described above, were processed as max- Qualitative muscle phenotype analysis was completed on imum intensity projections of confocal z-stacks using embryos of each genotype. All analysis was based on the ImageJ. The position of the nuclei was determined by immunofluorescence staining pattern of Tropomyosin in using the line function in ImageJ to measure the dis- stage 16 embryos. The frequency of the following pheno- tance between either the dorsal end of the muscle and types were scored: the number of free myoblasts in an the nearest nucleus or the ventral end of the muscle and embryo that indicated a defect in myoblast fusion (small, the nearest nucleus. All four LT muscles were measured unfused circles stained by anti-tropomyosin) and the in four hemisegments from each embryo. At least 20 number of muscles in each hemisegment (> 4 defined as embryos from at least two independent experiments extra muscles, < 4 defined as missing muscles) indicating were measured for each genotype with each data point gross abnormalities in the specification of muscle tissue. representing the average for all muscles measured within For analysis of the unfused myoblasts, embryos were a single embryo. grouped into bins with a width of 5 and the first bin centered on zero. Analysis of nuclear count in embryos The number of nuclei was counted using apRed fluor- Results escence in the 4 lateral transverse (LT) muscles of Fascin is necessary for muscle function stage17embryos,oncenucleihad separatedfrom Animal movement provides a simple assay for muscle their clusters and into easily distinguished individual function. Although adult locomotion has been used to nuclei. Only hemisegments in which there were four perform a full-genome, RNAi-based screen for regula- lines of nuclei, corresponding to 4 LT muscles, were tors of muscle function in Drosophila adults [17], similar counted. Data points indicate the number of apRed screens have not been completed using Drosophila lar- positive nuclei within a single hemisegment. A total vae. The greatest advantage to evaluating muscle func- of at least 49 hemisegments from at least 19 individ- tion in larvae rather than adults is that pupation, and ual embryos were counted. the high probability of lethality during pupation, is bypassed. We have therefore modified published larval Analysis of nuclear count in larvae locomotion assays [18, 19] to identify regulators of The number of nuclei in the VL3 muscles were counted muscle function. To ensure that the identified genes had using Hoechst staining. Data points indicate the number a muscle-autonomous effect on muscle function, we of nuclei within an individual muscle. Careful analysis of used the GAL4/UAS system [25] to disrupt gene func- z-stacks was used to ensure that nuclei were in the tion in a muscle specific manner. Specifically, we used muscle that they were attributed to. DMef2-GAL4 to drive the expression of a small library of UAS-RNAi constructs. We measured movement of Analysis of muscle attachment in embryos larvae toward a chemo-attractant as previously described Integrin accumulation at the myotendinous junction of [19], with modifications to increase the throughput of stage 16 embryos were measured in dorsal muscle 2 the assay. First, we skipped the selection of stage 17 em- (DO2). Z-stack projection images that extended through bryos, which previously ensured that the ages of the the entire MTJ were used for these data. Integrin evaluated larvae were similar. We replaced this step, Camuglia et al. Skeletal Muscle (2018) 8:12 Page 5 of 13 which previously took ~ 60 min per genotype, per ex- For the proof-of-concept screen, we expressed RNAi periment, with a timed-lay. Briefly, virgins that against 82 genes (Additional file 1: Table S1). The se- expressed DMef2-GAL4 and males that carried the lected genes included those expected to impact muscle UAS-RNAi were mixed together in a vial for 1 h. The function (e.g., Dystrophin, Msp300) and many for which adults were then moved to another vial. The first vial, we did not have a prediction. The speed of locomotion which contained all of the embryos that were laid in larvae that expressed each RNAi was compared to during the 1-h period, was then used for the experi- control larvae in which DMef2-GAL4 drove the expres- ment to ensure that all larvae used in an experiment sion of mCherry RNAi. The disruption of 12/82 genes were of similar age. These vials were aged for 5 days significantly altered larval locomotion compared to con- until the animals were third-instar larvae (L3). Sec- trol larvae, indicating that these 12 genes regulate ond, we measured themovementofmanylarvaesim- muscle function. Of these 12 genes, disruption of 5 ultaneously rather than measuring locomotion for an caused larvae to move faster compared to controls, and individual larva. The movement of larvae was then the disruption of 7 caused the larvae to move more tracked using ImageJ (Fig. 1a). Measuring larval loco- slowly than controls (Fig. 1b). RNAi directed against the motion of many animals simultaneously provided two expression of singed (sn) which encodes for the actin- benefits. First, it increased the speed of the assay binding protein fascin [26, 27] caused the greatest from ~ 60 min/genotype to ~ 10 min/genotype. Coin- decrease in larval locomotion. Therefore, we investigated cident with the increased speed of the assay, there the impact that fascin depletion had on muscle structure was less variability in the age of larvae that were to identify the mechanism by which fascin regulates tracked in an experiment, thus increasing the preci- muscle function. sion of the data. Fascin is localized to the nucleus in Drosophila muscle As a first approach to determine how fascin regulates muscle function, we examined the localization of fascin in Drosophila larval muscles and found that fascin local- ized to the sarcomeres and to the nuclei. The nuclear localization is similar to the localization of fascin in nurse cells [28] (Fig. 2a). An emergent regulator of muscle function is the pos- ition of the many myonuclei within a single cytoplasm [12, 13, 29]. Based on the localization of fascin to the nucleus, we hypothesized that fascin may regulate nu- clear movement during muscle development. Consistent with this hypothesis, fascin interacts with the nuclear en- velope protein Nesprin-2 and regulates nuclear move- ment in migrating fibroblasts [30] and is necessary for the positioning of nuclei in developing Drosophila oocytes [28]. To determine whether nuclear position was affected in Drosophila muscle, we crossed apRed, a marker for the nuclei in the lateral transverse (LT) mus- cles [31] into the sn Drosophila and measured the position of nuclei as previously described [21]. In sn mutants, nuclei were closer to the ventral end of the muscle compared to controls (Fig. 2b–d). To determine whether the effect on nuclear position was muscle autonomous, we transiently expressed RNAi specifically Fig. 1 An RNAi screen for larval locomotion identifies fascin as a in the mesoderm during the early stages of muscle regulator of muscle function. a Cartoon illustrating the locomotion development using Twist-GAL4 or in a more sustained assay that was used to identify RNAi constructs that when expressed manner using DMef2-GAL4 and measured the position specifically in muscle, altered muscle function. b Graph indicating the speed of larval locomotion toward a chemoattractant when indicated of the nuclei. Muscle-specific depletion of fascin had no genes were depleted by expression of RNAi specifically in muscle. All impact on nuclear position (Fig. 2e–h). Together, these data were compared to their control by Student’s t test. *p <0.05; data indicate that although nuclei are closer to the **p <0.01; ****p < 0.0001 muscle end in fascin mutants, this is not regulated by Camuglia et al. Skeletal Muscle (2018) 8:12 Page 6 of 13 b c d Fig. 2 Fascin is localized to actin and the nucleus in muscle. a Images from different focal planes of dissected L3 larvae showing that fascin (sn) is colocalized with both Phalloidin (F-actin) (left two panels) and Hoechst (nuclei) (right two panels). b Immunofluorescence images from stage 16 embryos stained for Tropomyosin to identify the muscles (magenta) and the nuclei in the LT muscles (green) in control and sn mutant embryos. Scale bar, 10 μm. c Graph indicating the distance between the dorsal end of the muscle and the nearest nucleus in control and sn mutant embryos. d Graph indicating the distance between the ventral end of the muscle and the nearest nucleus in control and sn mutant embryos. e, f Immunofluorescence images from stage 16 embryos stained for Tropomyosin to indicate the muscles (magenta) and the nuclei within the LT muscles (green) in embryos where RNAi against either mCherry (control) or fascin (sn RNAi) was driven by Twist-GAL4 (e)or Dmef2- GAL4 (f). g, h Graphs indicating the average distance between the dorsal end of the muscle and the nearest nucleus (g) or the ventral end of the muscle and the nearest nucleus (h) in embryos of indicated genotypes. All data (c, d, g, h) were compared to their control by Student’s t test. **p < 0.01 fascin expressed in muscle during embryonic muscle de- mutant embryos. At embryonic stage 16, there are 30 velopment in Drosophila. well-characterized muscles per hemisegment in the Drosophila embryo [32]. We noted a number of dif- Fascin regulates myoblast fusion ferences between sn mutant embryos and controls Because the loss of fascin had a limited effect on nu- (Fig. 3a). First, there was a reduction in the number clear position in Drosophila embryonic muscles, we of muscles. Although, various muscles were missing looked at the general muscle pattern in the sn in individual hemisegments, we focused on the LT Camuglia et al. Skeletal Muscle (2018) 8:12 Page 7 of 13 de Fig. 3 Fascin is necessary for myoblast fusion. a Immunofluorescence images of stage 16 embryos stained for Tropomyosin to indicate the muscles (magenta). Images are focused on the lateral transverse (LT) muscles in stage 16 embryos. Green arrowheads indicate unfused myoblasts. Scale bar, 10 μm. b Graph indicating the percentage of embryos that had at least one hemisegment with > 4 LT muscles (Extra LTs) or < 4 LT muscles (missing LTs). Values that exceed 100% indicate that some embryos had one hemisegment with > 4 LT muscles (extra muscles) and another hemisegment with < 4 LT muscles (missing muscles). c Graph indicating the number of apRed positive nuclei per hemisegment. Student’s t test was used for comparison to controls. ****p < 0.0001. d Graph indicating how frequently different numbers of unfused myoblasts 28 28 are seen in control (black) and sn mutant embryos (green). e Graph indicating the viability of the sn mutant animals (green) compared to controls (black) muscles because they are near the embryo surface increased to 7.5 in sn mutants (Fig. 3d). Additionally, and are the only muscles that are perfectly aligned on 70% of control embryos had two or fewer identifiable the dorsal-ventral axis of the embryo. These features unfused myoblasts whereas 75% sn mutant embryos make the LTs easy to count, image, and analyze. had at least three unfused myoblast and 50% of sn The typical hemisegment from a control embryo has mutant embryos eight or more unfused myoblasts. four LT muscles. Seventy percent of control embryos Based on the missing muscles and the abundance of had four LT muscles in every hemisegment, and 30% of unfused myoblasts in mutant embryos, we tested the controls had at least one hemisegment with greater than viability of the sn mutants and found that there was four LT muscles. In sn mutants, 70% of embryos had significant lethality during both the embryonic and four LT muscles in every hemisegment and 19% of larval stages (Fig. 3e). These data indicate that fascin embryos had at least one hemisegment with greater than regulates myoblast fusion. four LT muscles. Additionally, 19% of sn embryos had To determinewhether thesephenotypeswere at least one hemisegment with fewer than four LT muscle autonomous, we used the GAL4/UAS system muscles (Fig. 3b), and were therefore missing LT to deplete fascin specifically from the developing muscles. Eight percent of embryos had at least one mesoderm and muscle of the Drosophila embryo. The hemisegment with an extra muscle and one expression of a UAS-sn RNAi (fascin RNAi) was hemisegment with a missing muscle and were thus driven with each of three GAL4 drivers. Twist-GAL4 counted in both categories, pushing the total embryos was used to drive RNAi expression in the early meso- over 100%. Because the absence of muscles can indicate derm, DMef2-Gal4 was used to drive RNAi expression a defect in myoblast fusion, we counted the number of slightly later in muscle with sustained expression nuclei that were incorporated into the LT muscles per throughout development, and MHC-GAL4 was used hemisegment. This number was reduced from a mean of to drive RNAi expression beginning at the final stage 26 in the controls to a mean of 22 in the sn mutants of embryonic development and continuing throughout (Fig. 3c). Consistent with this, there was an increase in development. We then examined the general muscle unfused myoblasts. In controls, the median number of structure in stage 16 embryos as we had done for free myoblasts per embryo was 1, and that number sn mutant embryos. There were no defects in Camuglia et al. Skeletal Muscle (2018) 8:12 Page 8 of 13 muscle morphology when MHC-GAL4 was used to affected neither measure of fusion (Fig. 4d). However, drive RNAi expression suggesting that fascin must be DMef2-GAL4-mediated expression of RNAi had a depleted early during development to have significant greater effect on viability (Fig. 4e)suggestingthatthe impact (Additional file 2:Figure S1). absence of muscles was more detrimental to animal Early mesodermal expression of the RNAi under the viability. control of Twist-GAL4 and expression of RNAi in muscle under the control of DMef2-GAL4 both in- creased the percentage of embryos that were missing Fascin-dependent fusion effects are evident in larvae LT muscles, but DMef2-GAL4-mediated expression re- To determine the effects of fascin-depletion later in devel- sulted in a higher frequency of embryos with missing opment, larvae were dissected and stained with Phalloidin muscles (Fig. 4a, b). Conversely, only Twist-GAL4-me- to identify the muscles and Hoechst to identify the nuclei diated expression of RNAi against fascin caused a de- (Fig. 5). We examined the third ventral longitudinal muscle crease in the number of nuclei that were incorporated (VL3) because after dissection this muscle is on the surface into the LT muscles (Fig. 4c). This suggested that and therefore easily imaged. The distribution of myonuclei early expression of fascin RNAi was necessary to in- was similar in the controls and sn larvae (Fig. 5a, b). The hibit myoblast fusion. Consistent with this, Twist- size of the muscles (Fig. 5a, c), and the number of nuclei in GAL4-mediated RNAi expression increased both the each muscle (Fig. 5a, d) were both reduced in the sn percentage of embryos with unfused myoblasts and larvae compared to the controls, but the reductions were the number of unfused myoblasts per embryos. statistically insignificant. We hypothesized that the lack of DMef2-GAL4-mediated expression of fascin RNAi phenotype may be based on selection of the healthiest ab de Fig. 4 Fascin has muscle autonomous effects on myoblast fusion. a Stage 16 embryos stained for tropomyosin to identify the muscles (magenta). Embryos expressed RNAi against fascin under the control of Twist-GAL4 (top) or Dmef2-GAL4 (bottom). Green arrowheads indicate unfused myoblasts. Scale bar, 10 μm. b Graph indicating the percentage of embryos of indicated genotypes that have at least one hemisegment with either > 4 LT (extra LTs) muscles or < 4 LT muscles (missing LTs). c Graph indicating the number of apRed positive nuclei incorporated into LT muscles per hemisegment in indicated genotypes. Student’s t test was used for comparison to controls. *p <0.05. d Graph indicating how frequently different numbers of unfused myoblasts were seen in indicated genotypes. e Graph indicating the viability of animals with indicated genotypes Camuglia et al. Skeletal Muscle (2018) 8:12 Page 9 of 13 bc d Fig. 5 Genetic disruption of fascin did not affect larval muscle structure. a Immunofluorescence images of the third ventral longitudinal muscle (VL3) in L3 larvae of indicated genotypes. Sarcomeres (magenta) were stained with Phalloidin to identify the muscle and Hoechst (green) was used to identify the nuclei. Scale bar, 25 μm. b Graph indicating the actual internuclear distance divided by the maximal internuclear distance in indicated genotypes. c Graph indicating the area of the muscles as a proxy for muscle size in the indicated genotypes. d Graph indicating the number of nuclei in indicated genotypes. All data (b–c) were compared to their control by Student’s t test. All differences were statistically insignificant animals because they are the animals that survived until the MTJ of dorsal muscle 2 (DO2). Compared to controls, L3 stage. As such, we examined animals that expressed the signal was wider in sn mutants (Fig. 7a–c). RNAi specifically in the muscle, which are more viable Similarly, DMef2-GAL4-mediated expression of fascin (compare Fig. 4e to Fig. 3e). RNAi, but not Twist-GAL4-mediated expression of fas- We expressed RNAi against fascin under the control cin RNAi also increased the width of the βPS-integrin of Twist-GAL4 (early, transient expression), DMef2-Gal4 signal (Fig. 7d–h). These data suggest that sustained fas- (slightly later, sustained expression), or MHC-Gal4 (late, cin function is necessary for proper MTJ organization. sustained expression). The distribution of nuclei was the same in each genotype (Fig. 6a–d). Muscle size was de- Discussion creased when either Twist-GAL4 or DMef2-GAL4 was One of the many strengths of Drosophila as a model sys- used to express fascin RNAi (Fig. 6e) suggesting that tem is its utility as a tool to identify novel regulators of early fascin-dependent processes contribute to fascin- specific biological functions. This ability utilizes the im- dependent muscle growth. Finally, the number of nuclei mense genetic tools that are available and requires sim- in VL3 muscles was reduced by DMef2-GAL4-mediated ple and fast assays to screen many mutants and/or RNAi fascin depletion. Twist-GAL4-mediated depletion did re- lines. In this work, we adapted a published larval- duce the number of nuclei per muscle, but insignificantly tracking assay [19] to perform a proof-of-concept screen so. MHC-GAL4-mediated depletion had no impact on the for muscle function. We identified 12 genes that regulate number of nuclei per muscle (Fig. 6a–c, f). Thus, the de- muscle function, either positively or negatively. We con- fects in fusion are not transient, but are evident through- tinued these experiments by examining the mechanism out larval development. by which singed, Drosophila fascin, regulated muscle function because fascin-depletion had the strongest Fascin regulates muscle attachment effect on muscle function. DMef2-Gal4-mediated expression of sn RNAi did not re- We used a combination of mutant alleles and tissue- duce the number of nuclei incorporated into embryonic specific expression of RNAi against fascin to demonstrate LT muscles (Fig. 4c), but did reduce the total number of that fascin regulates both myoblast fusion and the struc- muscles in the embryo (Fig. 4b). This could be explained ture of the MTJ. Fascin is well-described as a protein that by an effect on the attachments between the muscle and can bundle F-actin filaments and increase their strength, the tendon cell at the myotendinous junction (MTJ). To and the strength of actin-based cellular protrusions [33]. determine whether fascin affected MTJ integrity, we im- Furthermore, by this mechanism, fascin contributes to cel- munostained embryos for tropomyosin to identify the lular invasions associated with cancer metastasis [34, 35]. muscles and βPS-integrin to identify the MTJ (Fig. 7). Myoblast fusion requires a similar organization of protru- We measured the width of the βPS-integrin signal at the sive F-actin structures that invade the growing myotube. Camuglia et al. Skeletal Muscle (2018) 8:12 Page 10 of 13 c f Fig. 6 Muscle-specific depletion of fascin results in smaller muscles with fewer nuclei. a–c Immunofluorescence images of the VL3 muscle in L3 larvae that expressed RNAi against either mCherry (control) or fascin (sn RNAi) under the control of Twist-GAL4 (a), DMef2-GAL4 (b), or MHC-GAL4 (c). Sarcomeres were identified by Phalloidin (magenta), and nuclei were identified by Hoechst (green). Scale bar, 25 μm. d Graph indicating the actual internuclear distance divided by the maximal internuclear distance in indicated genotypes. e Graph indicating the area of the muscles in larvae of indicated genotypes. f Graph indicating the number of nuclei per muscle in larvae of indicated genotypes. All data (d–e) were compared to their control by Student’s t test. *p <0.05; **p <0.01; ****p <0.0001 The most surprising aspect of the myoblast fusion Perhaps the most intriguing feature of these data is the data is the relatively minor effect that fascin has com- temporal separation of fascin-dependent myoblast fusion pared to other genes necessary for myoblast fusion and fascin-dependent MTJ stability. This conclusion is [31, 36–38]. The reason for this is not clear. One based on our finding that the time and duration of fascin possibility is that maternal loading provides sufficient depletion determines the phenotype that will emerge. fascin to facilitate the initial rounds of fusion. Alter- Transient depletion of fascin during early stages of natively, perhaps the final fusion events require muscle development disrupted myoblast fusion but not greater protrusive force and only then does the func- MTJ structure. Conversely, later, and sustained depletion tion of fascin become critical. of fascin affected MTJ structure, but not myoblast fu- The contribution of fascin to MTJ structure is consist- sion. These data are important because they demonstrate ent with previously published data. Fascin contributes to that although both fusion and MTJ structure require fas- filopodia formation [35] and MTJ development is cin function, they are not codependent features of dependent on filopodia-like extensions. Furthermore, al- muscle development. though the MTJ forms as a smooth attachment during pu- It is not clear whether either function is more critical pation [16], the MTJ in the embryo is dynamic [39]. Thus, than the other. Certainly, sustained depletion of fascin, perhaps fascin is continually necessary for the turnover which disrupts MTJ integrity has a greater effect on and the integrity of the MTJ. animal survival than does the transient depletion that Camuglia et al. Skeletal Muscle (2018) 8:12 Page 11 of 13 a b c d e f h Fig. 7 Fascin is necessary for proper myotendinous junction organization a Immunofluorescence images of the MTJ of muscle DO2 stained for tropomyosin and βPS-integrin in control (top) and sn mutant embryos (bottom). Scale bar, 10 μm. b Representative intensity profile of the βPS-integrin signal in control (black) and sn mutant embryos (green). c Graph indicating the width of the βPS-integrin signal defined by the points at which the signal is 25% of the maximum. d, e Immunofluorescence images of the MTJ of muscle DO2 stained for tropomyosin and βPS-integrin in animals in which twist-GAL4 was used (d) or DMef2-GAL4 was used (e) to express RNAi against either mCherry (control) or fascin (sn RNAi). f, g Representative intensity profiles of the βPS-integrin signal in indicated genotypes. h Graph indicating the width of the βPS-integrin signal in indicated genotypes as defined by the points at which the signal is 25% of the maximum. All data (c, h) were compared to their control by Student’s t test. **p < 0.01; ****p < 0.0001 disrupts fusion. However, this conclusion is limited be- larval muscles in Drosophila, we suspect that this re- cause the impact that a small reduction in nuclear number duction is based in muscle damage that may be has on muscle organization is not clear. Reduced nuclear linked to improper attachments and poor mechanical numbers do correlate with reduced muscle size [40, 41] stability. However, further work is necessary to under- and therefore likely cause reduced muscle function. Data stand the mechanism by which nuclei are lost so that in embryos indicated that DMef2-GAL4-mediated the impact of individual fascin-dependent functions expression of fascin RNAi only affected MTJs and would can be determined. therefore allow us to isolate the impact of the MTJ versus the impact of myoblast fusion. However, we see that in lar- Conclusions vae, there is a reduction in the number of nuclei per In total, we have used a larval locomotion assay to iden- muscle. Because there is no repair of embryonic and tify novel regulators of muscle function. Furthermore, Camuglia et al. Skeletal Muscle (2018) 8:12 Page 12 of 13 we have described that the strongest hit from the screen, Received: 16 October 2017 Accepted: 22 March 2018 fascin, contributes to muscle function by regulating myoblast fusion and MTJ structure. Both of these func- References tions are consistent with known biochemical abilities for 1. Abmayr SM, Pavlath GK. Myoblast fusion: lessons from flies and mice. fascin and suggest that fascin has multiple essential Development. 2012;139:641–56. https://doi.org/10.1242/dev.068353. functions for muscle development that are separated in 2. Kim JH, Jin P, Duan R, Chen EH. ScienceDirect mechanisms of myoblast fusion during muscle development. Curr Opin Genet Dev. 2015;32:162–70. time. More generally, this work outlines a new strategy https://doi.org/10.1016/j.gde.2015.03.006. for the identification of genes and pathways that can be 3. Sens KL, Zhang S, Jin P, Duan R, Zhang G, Luo F, et al. An invasive manipulated to either increase or decrease muscle podosome-like structure promotes fusion pore formation during myoblast fusion. J Cell Biol. 2010;191:1013–27. https://doi.org/10.1083/jcb.201006006. function. 4. Bothe I, Deng S, Baylies M. PI(4,5)P2 regulates myoblast fusion through Arp2/3 regulator localization at the fusion site. Development. 2014;141: 2289–301. https://doi.org/10.1242/dev.100743. Additional files 5. Kim JH, Ren Y, Ng WP, Li S, Son S, Kee Y-S, et al. Mechanical tension drives cell membrane fusion. Dev Cell. 2015;32:561–73. https://doi.org/10.1016/j. Additional file 1: Table S1. All of the data acquired during the limited devcel.2015.01.005. RNAi-based screen for regulators of muscle function. (PDF 139 kb) 6. Gimona M, Buccione R, Courtneidge SA, Linder S. Assembly and biological Additional file 2: Figure S1. Expression of RNAi against fascin late in role of podosomes and invadopodia. Curr Opin Cell Biol. 2008;20:235–41. embryonic development does not affect muscle development a https://doi.org/10.1016/j.ceb.2008.01.005. Immunofluorescence images showing the muscle pattern in animals 7. McNiven MA. Breaking away: matrix remodeling from the leading edge. expressing mCherry RNAi (control) and animals expressing fascin RNAi (sn Trends Cell Biol. 2013;23:16–21. https://doi.org/10.1016/j.tcb.2012.08.009. RNAi) under the control of the MHC-GAL4 driver. b Graph comparing the 8. Deng S, Bothe I, Baylies MK. The formin diaphanous regulates myoblast frequency of embryos with extra muscles in each genotype. No embryos fusion through actin polymerization and Arp2/3 regulation. PLoS Genet. with missing muscles were observed in either genotype. c Graph 2015;11:e1005381–29. https://doi.org/10.1371/journal.pgen.1005381. comparing the frequency at which embryos were found to have unfused 9. Deng S, Bothe I, Baylies M. Diaphanous regulates SCAR complex localization myoblasts in each genotype. (PDF 360 kb) during Drosophila myoblast fusion. Fly. 2016;10:178–86. https://doi.org/10. 1080/19336934.2016.1195938. 10. D’Alessandro M, Hnia K, Gache V, Koch C, Gavriilidis C, Rodriguez D, et al. Acknowledgements Amphiphysin 2 orchestrates nucleus positioning and shape by linking the The authors would like to thank Tina Tootle (University of Iowa) for providing nuclear envelope to the actin and microtubule cytoskeleton. Dev Cell. 2015; sn28 the fascin allele and her advice. Drosophila stocks obtained from the 35:186–98. https://doi.org/10.1016/j.devcel.2015.09.018. Bloomington Drosophila Stock Center (NIHP400D018537) were used in this 11. Roman W, Martins JP, Carvalho FA, Voituriez R, Abella JVG, Santos NC, et al. study. The integrin betaPS antibody developed by D. Brower at Harvard Myofibril contraction and crosslinking drive nuclear movement to the Medical School was obtained from the Developmental Studies Hybridoma periphery of skeletal muscle. Nat Cell Biol. 2017;19:1189–201. https://doi.org/ Bank, created by the NICHD of the NIH and maintained at the University of 10.1038/ncb3605. Iowa, Department of Biology, Iowa City, IA 52242, USA. 12. Folker ES, Baylies MK. Nuclear positioning in muscle development and disease. Front Physiol. 2013;4:363. https://doi.org/10.3389/fphys.2013.00363. Funding 13. Cadot B, Gache V, Gomes ER. Moving and positioning the nucleus in This work was funded by grants from the American Heart Association to skeletal muscle—one step at a time. Nucleus. 2015;6:373–81. https://doi.org/ E.S.F. and by startup funds provided by Boston College to E.S.F. 10.1080/19491034.2015.1090073. 14. 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Skeletal MuscleSpringer Journals

Published: Apr 6, 2018

References