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Muscle length and myonuclear position are independently regulated by distinct Dynein pathways

Muscle length and myonuclear position are independently regulated by distinct Dynein pathways RESEARCH ARTICLE 3827 Development 139, 3827-3837 (2012) doi:10.1242/dev.079178 © 2012. Published by The Company of Biologists Ltd Muscle length and myonuclear position are independently regulated by distinct Dynein pathways 1 1,2 1,2, Eric S. Folker , Victoria K. Schulman and Mary K. Baylies * SUMMARY Various muscle diseases present with aberrant muscle cell morphologies characterized by smaller myofibers with mispositioned nuclei. The mechanisms that normally control these processes, whether they are linked, and their contribution to muscle weakness in disease, are not known. We examined the role of Dynein and Dynein-interacting proteins during Drosophila muscle development and found that several factors, including Dynein heavy chain, Dynein light chain and Partner of inscuteable, contribute to the regulation of both muscle length and myonuclear positioning. However, Lis1 contributes only to Dynein-dependent muscle length determination, whereas CLIP-190 and Glued contribute only to Dynein-dependent myonuclear positioning. Mechanistically, microtubule density at muscle poles is decreased in CLIP-190 mutants, suggesting that microtubule-cortex interactions facilitate myonuclear positioning. In Lis1 mutants, Dynein hyperaccumulates at the muscle poles with a sharper localization pattern, suggesting that retrograde trafficking contributes to muscle length. Both Lis1 and CLIP-190 act downstream of Dynein accumulation at the cortex, suggesting that they specify Dynein function within a single location. Finally, defects in muscle length or myonuclear positioning correlate with impaired muscle function in vivo, suggesting that both processes are essential for muscle function. KEY WORDS: Drosophila, Dynein, Nuclear positioning, Muscle size INTRODUCTION the number of fusion events (Bate, 1990), but it is unlikely that The size, shape and organization of tissues and individual cells fusion alone determines myofiber size. In Drosophila larvae and in directly impact their physiology. A pressing question in cell and mammals, a number of signaling pathways have been demonstrated developmental biology is how different morphogenetic processes to control muscle size through growth regulation (Demontis and that are necessary for the functional development of a common Perrimon, 2009; Rommel et al., 2001). Although several guidance tissue are coordinated. Skeletal muscle provides an ideal system cues and signaling mechanisms are known to regulate the direction with which to study structure-function relationships as it is highly of muscle growth (Schejter and Baylies, 2010; Schweitzer et al., organized with a physiologically testable output. 2010), the factors and mechanisms that directly provide the The cellular unit of all metazoan muscle is the myofiber, a necessary forces that promote oriented and regulated muscle syncytial cell that is formed from the iterative fusion of myoblasts. growth during early myogenesis are not known. The correlation between structure and function is highlighted by The importance of organelle positioning is highlighted by the the organization of myosin and actin within the sarcomere that localization of the myonuclei. In skeletal muscle, nuclei reside provides the contractile apparatus (Squire, 1997). However, other above the sarcomere at the periphery of the muscle fiber and are aspects of skeletal muscle structure, including myofiber size and positioned to maximize their internuclear distance. Moreover, organelle positioning, are also crucial for its function. mispositioned myonuclei, in conjunction with smaller myofibers, Myofiber size encompasses a variety of measurements, are a hallmark of many muscle disorders and have been used including length, width, depth and volume, and size is important diagnostically for decades (Pierson et al., 2007; Romero, 2010). for muscle output, as smaller myofibers provide less contractile The mechanisms by which nuclei are moved throughout the muscle force than larger muscles (Fitts et al., 1991). During muscle (Metzger et al., 2012) and anchored at the neuromuscular junction formation in Drosophila, muscle cell growth occurs in three (NMJ) (Grady et al., 2005; Lei et al., 2009) are beginning to distinct phases (Volk, 1999; Schnorrer and Dickson, 2004). As emerge; however, only a small number of factors necessary for fusion begins, the muscle cell breaks symmetry, adds mass, and these processes have been identified. Additionally, potential links elongates to form polarized structures with a long axis and a short between muscle size and myonuclear position have not been axis. Once the long axis is established, the muscle cell extends explored. In other systems, the microtubule cytoskeleton regulates filopodia at each end in search of an attachment with a tendon cell. both polarized growth and nuclear position. Cytoplasmic Dynein Finally, the filopodial protrusions stop, the muscle cell poles (Dynein) is a crucial factor in both processes. During nuclear become smooth, and stable muscle-tendon attachments are made. movement in the first cell division of C. elegans and in migrating In Drosophila embryos, muscle size has often been correlated with neurons, cortically anchored Dynein pulls microtubule minus ends and the attached nuclei towards itself (Gönczy, 2002; Tsai et al., 2007). During polarized cell growth, Dynein contributes to the establishment of the polarity axis (Etienne-Manneville and Hall, Program in Developmental Biology, Sloan Kettering Institute, New York, NY 10065, 2001; Palazzo et al., 2001) that is necessary to localize the factors USA. Cell and Developmental Biology, Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA. for cell protrusion (Prigozhina and Waterman-Storer, 2004; Schmoranzer et al., 2003). Given these functions, Dynein may *Author for correspondence ([email protected]) contribute to both myofiber size and myonuclear positioning and might integrate the two processes. Accepted 16 July 2012 DEVELOPMENT 3828 RESEARCH ARTICLE Development 139 (20) To investigate how myofiber size and myonuclear positioning (Sigma, HT501128-4L). Embryos and larvae were mounted in ProLong Gold (Invitrogen) for fluorescent stainings. Antibodies were preabsorbed are regulated, and whether the two processes are co-dependent, we where noted (PA) and used at the following final dilutions: rabbit anti- have studied the role of Dynein during muscle morphogenesis in dsRed (PA, 1:400, Clontech 632496), rat anti-Tropomyosin (PA, 1:500, Drosophila. We establish muscle length, which is an aspect of Abcam ab50567), mouse anti-GFP (PA, 1:200, Clontech 632381), mouse muscle size, and myonuclear positioning as independent aspects of anti-DHC (1:50, Developmental Studies Hybridoma Bank), mouse anti- muscle morphogenesis. We further show that the microtubule Tubulin (1:500, Sigma T9026), chicken anti--galactosidase (PA, 1:1000, cytoskeleton contributes to both processes. Specifically, Dynein Novus Biologicals NB100-2045), mouse anti--PS-Integrin (1:100, regulates muscle length and myonuclear positioning with Developmental Studies Hybridoma Bank) and mouse anti-Discs large overlapping, but distinct, sets of proteins. The Dynein heavy chain (1:200, Developmental Studies Hybridoma Bank). We used Alexa Fluor (Dhc64C), Dynein light chain (Dlc90F; Tctex1 in mammals) and 488-, Alexa Fluor 555- and Alexa Fluor 647-conjugated fluorescent Partner of inscuteable [Pins; Rapsynoid (Raps) – FlyBase] are secondary antibodies (1:200), Alexa Fluor 647-conjugated phalloidin necessary regulators of both muscle length and myonuclear (1:100) and Hoechst 33342 (1 g/ml) for fluorescent stains (all Invitrogen). Fluorescent images were acquired on a Leica SP5 laser-scanning confocal positioning. Subsequently, the two pathways become distinct: microscope equipped with a 63 1.4 NA HCX PL Apochromat oil muscle length is regulated by a Dynein-Lis1-dependent objective and LAS AF 2.2 software. Images were processed using Adobe mechanism, whereas myonuclear positioning is regulated by a Photoshop CS4. Maximum intensity projections of confocal z-stacks were Dynein–CLIP-190–Glued-dependent mechanism. rendered using Volocity Visualization software (Improvision). Mechanistically, in mutant embryos that lack Dynein 4-19 193 (Dhc64C ) or fail to localize Dynein to the muscle pole (raps ), Nuclear position and muscle length measurements both muscle length and myonuclear positioning are affected, Embryos were staged based on standard laboratory procedures: overall embryo shape, the intensity of the apRed and Tropomyosin signals, gut suggesting that Dynein activity at the muscle pole is required for morphology, and the morphology of the trachea (Beckett and Baylies, both processes. However, Dynein localizes to the muscle pole in 2007). Measurements were taken from confocal projections of embryos both Lis1 and CLIP-190 mutant embryos, indicating that Dynein- and acquired using the Line function of ImageJ software (NIH). For each dependent regulation of muscle length and Dynein-dependent genotype, all four lateral transverse (LT) muscles were measured in three regulation of myonuclear positioning are specified downstream of hemisegments from each of 20 embryos from four independent its arrival at the muscle pole. Lis1 mutant embryos exhibit experiments. Three measurements were taken within each LT muscle: (1) hyperaccumulation of Dynein at the muscle pole with a tighter myotube length and (2, 3) the shortest distance between the myotube poles focus of peak intensity, suggesting that Lis1-dependent activation and the outermost edge of the nearest nucleus (supplementary material Fig. of Dynein retrograde trafficking is essential for muscles to achieve S1). The edges of the nuclei and ends of the myotubes were defined by the their proper length. CLIP-190 mutant embryos have fewer clear change from signal to background within the relevant viewing microtubules near the myofiber poles despite normal Dynein channel. Similar measurements were performed using -PS-Integrin to identify the ends of the LT muscles. The statistical significance of localization, suggesting that CLIP-190 promotes microtubule-cell differences in measurements was assessed using a Student’s t-test to cortex interactions that are necessary for Dynein-dependent compare each experimental genotype with wild-type apRed controls. myonuclear positioning. Together, these data suggest that both Statistical analysis was performed with Prism 4.0 (GraphPad). CLIP-190 and Lis1 work downstream of Dynein localization to the Because the live stage 17 embryos offer few markers for accurate muscle pole and thus specify Dynein function within this single staging, staging was achieved by timed lays: after a pre-lay period, location. Differential regulation of Dynein activity within a single embryos were collected for 15 minutes and then aged for 16.5 hours. location is novel and likely to be important during morphogenetic Embryos were then dechorionated and mounted on coverslips in processes for which proper timing is essential. Finally, both muscle halocarbon oil and images were acquired on a Zeiss Axioplan 2 microscope length and myonuclear positioning are important for muscle with a 20 PlanNeoFluor 0.50 NA objective and an Axiocam MRm function, as defects in either process correlate with larvae that camera. Nuclear spread measurements were performed using the Line function in ImageJ to measure the distance between the dorsal edge of the crawl more slowly than controls. dorsal nucleus and the ventral edge of the ventral nucleus. Additionally, these same images of stage 17 embryos were used to count the number of MATERIALS AND METHODS nuclei per hemisegment, which was used as a measure of the number of Drosophila genetics myoblast fusion events. Stocks were grown under standard conditions. Stocks used were apME- 4-19 6-10 NLS::dsRed (Richardson et al., 2007), Dhc64C and Dhc64C (Gepner Microtubule organization measurements et al., 1996), UAS-Dhc64C-RNAi (Vienna Drosophila RNAi Center, 05089 K11702 Confocal z-stacks were sequentially acquired to maximize the signal and v28053), dlc90F (Caggese et al., 2001), lis1 (Lei and Warrior, G10.14 resolution at the myofiber poles and to minimize interference from the 2000), lis1 (Liu et al., 1999), UAS-Lis1-RNAi (Vienna Drosophila KG06490 internal muscles and the epidermis. Images were acquired with a 63 1.4 RNAi Center, v6216), clip190 (Bloomington Drosophila Stock NA HCX PL Apochromat oil objective and a 10 optical zoom. z-stacks Center, 14493), UAS-Clip190-RNAi (Vienna Drosophila RNAi Center, 193 P49 through a depth of 2 m with a step size of 0.25 m were acquired for all v107176), raps (Parmentier et al., 2000), insc (Kraut and Campos- analyzed images. z-stacks were then assembled into confocal projections Ortega, 1996) and UAS-Glued-RNAi (Vienna Drosophila RNAi Center, using Volocity, which were then exported to ImageJ for analysis. Analysis v3785). Mutants were balanced and identified using CTG (CyO, twi-Gal4, was performed on 20 embryos from four independent experiments. To UAS-2xeGFP), TTG (TM3, twi-Gal4, UAS-2xeGFP), TM6B, CyO count the number of microtubules in contact with the muscle pole, a 22 P[w+wgen11lacZ] and TM3 Sb1 Dfd-lacZ. m box was drawn from the tip of the muscle toward the center and the Immunohistochemistry number of microtubules in this region were counted. Embryos were collected at 25°C and fixed with 4% EM-grade To determine the average intensity, the Measure Average Intensity paraformaldehyde (Polysciences, 00380) diluted in PBS (Roche, Function in ImageJ was performed on the distal 3 m of the myofiber. The intensity of Tubulin staining was standardized against the intensity of 11666789001) and an equal volume of heptane to allow permeabilization. In all cases, embryos were devitellinized by vortexing in a 1:1 Tropomyosin staining to control for sample variation. For each genotype, methanol:heptane solution. Larvae were dissected in ice-cold HL3.1 as all four LT muscles were measured in three hemisegments from each of 20 previously described (Brent et al., 2009) and fixed with 10% formalin embryos from four independent experiments. Student’s t-test was used to DEVELOPMENT Dynein-dependent muscle formation RESEARCH ARTICLE 3829 compare each experimental genotype with wild-type apRed controls. examined. Dhc64C mutant embryos had the same number of Statistical analysis was performed with Prism 4.0. apRed nuclei per hemisegment as controls, indicating that myoblast fusion was unaffected (Fig. 1A,B). Although the nuclei were Dynein localization measurements aligned in columns within each LT muscle in embryos of each Embryos were collected and fixed with 10% formalin diluted 1:1 in genotype, the distance between the most ventral and the most heptane for 20 minutes, then rinsed three times in PBS containing 0.6% dorsal nuclei was reduced in Dhc64C mutant embryos (Fig. 1A,C). Triton X-100, then fixed with 4% paraformaldehyde diluted 1:1 in heptane To determine whether the difference in the distribution of nuclei for 20 minutes. Antibody incubations were performed in PBS supplemented with 1% BSA and 0.6% Triton X-100. Image acquisition and was caused by defective nuclear positioning or effects on muscle processing were as for microtubule quantification. Dynein size, we examined late stage 16 embryos (16 hours AEL), which immunofluorescence intensity was assessed by linescan analysis using are amenable to whole-mount immunostaining. ImageJ. Positions of linescans were chosen randomly on the Tropomyosin The general muscle pattern as revealed by Tropomyosin staining image and the line then copied to the identical position on the Dynein (Fig. 1D) was normal in Dhc64C mutant embryos. Additionally, - image. The intensity of Dynein immunofluorescence at each point was PS-Integrin staining was normal. Thus, all muscles were present, measured relative to Tropomyosin intensity at that same point to control properly oriented and attached to tendon cells, indicating that for sample variation, and the ratios were multiplied by 100. Three specification, guidance and attachment mechanisms are functional. hemisegments from 20 embryos from four independent experiments were In late stage 16 (16 hours AEL) control embryos, the myonuclei analyzed. The greatest pixel intensity from each embryo was used to were in two clusters within each LT muscle: one cluster at the determine the peak intensity. To determine the width of peak intensity, the pixels that were ≥80% of peak intensity were determined and the distance dorsal pole and the other at the ventral pole (Fig. 1D; between the first pixel and the last pixel that fitted these criteria was supplementary material Fig. S1) (Metzger et al., 2012). In embryos measured. For each genotype, all four LT muscles were measured in three homozygous for either Dhc64C allele, the nuclei were also in two hemisegments from each of five embryos. Student’s t-test was used to clusters; however, the clusters were mispositioned 50% further compare each experimental genotype with wild-type apRed controls. from the muscle poles than in wild-type and heterozygous controls Statistical analysis was performed with Prism 4.0. The heat map (Fig. 1D,E; supplementary material Fig. S2). Additionally, the representation of Dynein immunofluorescence was generated using the length of the LT muscles was reduced by 15% in embryos Cool application of the standard Lookup tools in ImageJ. homozygous for either Dhc64C allele as compared with wild-type Larval behavior and heterozygous controls (Fig. 1D,F; supplementary material Fig. Larval speed was assessed as previously described (Louis et al., 2008; S2). To account for the difference in muscle length, the distance Metzger et al., 2012) with minor modifications. Stage 16 and 17 embryos from each cluster of nuclei to the nearest muscle pole was were selected for the absence of the fluorescent balancer and placed on calculated as a percentage of muscle length (Fig. 1G). This yeast-coated apple juice agar plates at 22°C overnight. L1 larvae were measurement was used with respect to all subsequent genotypes. selected the following day and placed into vials of standard fly food To validate the use of Tropomyosin as a marker for muscle ends, containing Bromophenol Blue (Bio-Rad, 161-0404). L3 larvae were picked we performed identical measurements using -PS-Integrin to mark from the vial 6 days later and individually tracked as they crawled towards the muscle ends. These analyses produced identical results (Fig. an odor source of ethyl butyrate (3.3%; Sigma, E15701) diluted in paraffin 1D; supplementary material Fig. S2C,D). oil (Fluka, 76235). Their movements were recorded with a CCD camera for either five total minutes or until they reached the odor source or the To confirm that the effects of Dynein were autonomous to walls of the apparatus. The tracks were processed using Ethovision muscle, we used the GAL4/UAS system to deplete Dynein software (Nodlus). At least 20 larvae were tracked for each genotype. specifically in the mesoderm using twist-GAL4 to express UAS- Tracked larvae were dissected, stained and analyzed as follows. Dhc64C-RNAi (Dietzl et al., 2007). Tissue-specific depletion of Internuclear distance was measured in ImageJ by drawing lines between Dhc64C recapitulated the effects of the Dhc64C alleles: the LT the center of each nucleus and its nearest neighbor using projection images. muscles were 12% shorter than controls and the nuclei were Muscle size was measured by determining the area of confocal projections positioned 40% further from the muscle poles than in controls (Fig. of each muscle. To calculate the internuclear distance as a function of 1D-G). These data indicate that Dynein is specifically required in muscle size, the average internuclear distance for a given muscle was the muscle to generate muscles of proper length and to properly divided by the area of that specific muscle. Nine muscles from three larvae position myonuclei. were used for quantification. Given that myonuclear position seems to be affected similarly in stage 16 and stage 17 embryos, we examined embryos at earlier RESULTS stages in their development to determine when the defects in Dynein regulates muscle length and myonuclear muscle length and myonuclear positioning become evident. Using position Tropomyosin to identify the muscles and a combination of trachea Each body wall muscle in the Drosophila embryo and larva morphology and gut morphology to assess embryo age, we consists of a single syncytial myofiber similar to the individual determined the length of muscles and the positioning of myonuclei myofibers in mammalian muscle. To assess the role of Dynein in late stage 14 (11 hours AEL), stage 15 (13 hours AEL) and stage during Drosophila muscle morphogenesis, the distribution of nuclei 16 (16 hours AEL) embryos (Fig. 2). At stage 14, the length of the 4-19 within the lateral transverse (LT) muscles was examined in wild- muscles in control and Dhc64C embryos was similar. However, 4-19 type embryos and in embryos in which Dynein was zygotically by stage 15 and through stage 16 the muscles in the Dhc64C 4-19 6-10 removed with either the Dhc64C (null) or the Dhc64C embryos were significantly shorter (17% and 14%, respectively) (hypomorphic) allele (Gepner et al., 1996). The myonuclei in the than those in the control embryos. LT muscles were visualized by expression of the apterous The effects on myonuclear positioning at the different stages mesodermal enhancer fused to a nuclear localization signal and the were more complex. The nuclei were 30% further from the muscle 4-19 fluorescent protein DsRed (apRed) (Richardson et al., 2007), which poles in late stage 14 Dhc64C embryos than in controls (Fig. specifically labels the nuclei of the LT muscles. Using this reporter, 2C); however, only the positioning relative to the dorsal muscle live stage 17 embryos [16.5-20 hours after egg lay (AEL)] were pole was statistically significant. At stage 15, myonuclei were DEVELOPMENT 3830 RESEARCH ARTICLE Development 139 (20) Fig. 1. Cytoplasmic Dynein regulates muscle length and myonuclear position. (A)Fluorescence images of apRed nuclei in the lateral transverse (LT) muscles of live stage 17 Drosophila embryos (16.5-20 hours AEL) of the indicated genotypes. (B)The number of nuclei per hemisegment in stage 17 embryos of the indicated genotypes. (C)The distance between the dorsalmost and ventralmost nuclei in stage 17 embryos of the indicated genotypes. (D)Immunofluorescence images of stage 16 (16 hours AEL) embryos of the genotypes noted to the left and antigen listed at the top of the first image. Green, Tropomyosin (muscle); red, DsRed (nuclei); blue, -PS-Integrin in merge. Arrows in the control panels denote points from which measurements were made for all genotypes. The distance between the points indicated by the yellow arrows in the Tropomyosin and -PS-Integrin panels was used to determine muscle length. The distance between the pair of gray arrows at the top of the merged image was used to determine the distance between the nuclei and the dorsal pole. The distance between the pair of white arrows at the bottom of the merged image was used to determine the distance between the nuclei and the ventral pole. (E)The shortest distance between the indicated pole of the LT muscles (gray, dorsal; white, ventral) and the nearest cluster of nuclei in stage 16 embryos. (F)LT muscle length in stage 16 embryos. (G)The shortest distance between the indicated pole of the LT muscles (gray, dorsal; white, ventral) and the nearest cluster of nuclei normalized for muscle length in stage 16 embryos of the indicated genotypes. Error bars indicate s.d.; **P<0.01, *P<0.05. Scale bars: 10m. 4-19 similarly positioned in control and Dhc64C embryos (Fig. 2D). and neurons (McKenney et al., 2010; Mesngon et al., 2006; In stage 16 embryos, the myonuclei were significantly Sheeman et al., 2003); and CLIP-190, which works with Lis1 to mispositioned (50% further from the poles) at both the dorsal and regulate Dynein activity (Tanenbaum et al., 2008). 4-19 ventral poles of the muscle in Dhc64C mutant embryos relative We tested two Dynein light chains, Cdlc2 and Dlc90F, which are to controls (Fig. 2E). the Drosophila orthologs of Dynein light chain 1/2 and Tctex1, These data indicate that the defects in muscle length arise prior respectively (Caggese et al., 2001; Goldstein and Gunawardena, to muscle-tendon attachment and, together with the muscle-specific 2000), by RNAi-mediated depletion (Dietzl et al., 2007). Depletion depletion of Dhc64C, suggest that the effects are muscle of Cdlc2 specifically in mesoderm and muscle did not affect autonomous and not due to defects in tendon specification. muscle length or myonuclear positioning, whereas depletion of Dlc90F resulted in shorter muscles with mispositioned nuclei. Dynein-interacting proteins contribute to Dynein- Additionally, embryos that were homozygous for dlc90F dependent regulation of muscle length and (Caggese et al., 2001) or the pins allele raps (Parmentier et al., myonuclear positioning 2000), produced muscles that were 12% shorter with nuclei that To determine whether the roles of Dynein in specifying muscle were 40-50% further from the muscle poles than in wild-type and length and myonuclear positioning are functionally coupled, we heterozygous control embryos (Fig. 3A-C; supplementary material tested known Dynein-interacting proteins for their contributions to Fig. S2). These data indicate that Dlc90F and Pins contribute to each process. We focused on factors that are general regulators of both muscle length and myonuclear positioning. K11702 Dynein activity or have been found to specifically contribute to Embryos homozygous for either of two Lis1 alleles, lis1 G10.14 Dynein-mediated nuclear movements in other systems. We (Lei and Warrior, 2000) and lis1 (Liu et al., 1999), produced examined Dynein light chains, which can regulate Dynein activity muscles that were 15% shorter than those of wild-type and both positively (Sönnichsen et al., 2005) and negatively (O’Rourke heterozygous controls. However, myonuclear positioning was et al., 2007); Glued, which increases Dynein motor processivity normal in Lis1 homozygous embryos. Conversely, embryos that and mediates Dynein-cargo interactions (Gill et al., 1991; Vaughan were depleted of Glued (Dietzl et al., 2007) in muscle and embryos KG06490 et al., 2002; Waterman-Storer et al., 1997); Pins, which anchors that were homozygous for clip190 (Bellen et al., 2004) Dynein at the cortex to move nuclei in other systems (Gotta et al., produced muscles of similar length to controls. However, in these 2003; Hughes et al., 2004); Lis1, which maintains Dynein in an embryos the nuclei were positioned 50% further from the muscle active state and has been implicated in nuclear movements in yeast poles than in wild-type and heterozygous controls (Fig. 3A-C; DEVELOPMENT Dynein-dependent muscle formation RESEARCH ARTICLE 3831 Df(2L)BSC294, Df(2L)Exel7068 and Df(2L)Exel8036 (supplementary material Fig. S2). Additionally, we measured the ventral longitudinal muscle VL3 and found that its length in the embryo was similar in each genetic background (supplementary material Fig. S2). These data suggest that a unique feature of the LT muscles makes them more susceptible to Dynein levels and/or activity with respect to muscle growth in the embryo. The number and position of nuclei in stage 17 embryos were also examined. Embryos from each genotype had the same number of apRed nuclei per hemisegment, indicating that the defects in muscle length did not result from impaired fusion (Fig. 1B). The clusters of nuclei in each genotype did resolve into columns during stage 17, but the distance between the ventral and dorsal nuclei was reduced by 15% compared with controls. Moreover, the distance between the most distal nuclei was similar in stage 16 and stage 17 embryos for each genotype (supplementary material Fig. S3). To determine whether the Dynein-interacting proteins work with, or independently of, Dynein, we examined embryos that were doubly heterozygous for Dhc64C mutations and mutations in each of the associated genes. In these, and all subsequent genetic interactions, maternal effects were controlled for by providing each allele from 4-19 the mother or the father in reciprocal crosses. Both Dhc64C and 6-10 Fig. 2. Stage-dependent effects of Dynein on muscle length and Dhc64C were used in combination with alleles for each putative myonuclear positioning. (A)Immunofluorescence images of the 05089 Dynein-interacting gene. Double heterozygotes of dlc90F and muscles (green, Tropomyosin) and nuclei (red, DsRed) at stage 14, 15 4-19 05089 6-10 Dhc64C or of dlc90F and Dhc64C produced muscles that 4-19 and 16 in control and Dhc64C Drosophila embryos. Scale bar: were 15% shorter than controls and with their nuclei 50% further 4-19 10m. (B)The length of the muscles in control (gray) and Dhc64C from the muscle poles (Fig. 4A-C). These data indicate that Dlc90F (black) embryos at stage 14, 15 and 16. (C-E)The distance between the (Tctex1) regulates Dynein activity for both muscle length and nearest nucleus and either the dorsal (gray) or ventral (white) pole of – – myonuclear positioning. All Lis1 /+; Dhc64C /+ doubly the muscle in stage 14 (C), 15 (D) and 16 (E) embryos. Error bars indicate s.d.; *P<0.05, **P<0.01. heterozygous embryos produced muscles that were 15% shorter than those in control embryos, but with properly positioned myonuclei at – – the muscle poles (Fig. 4A-C). Conversely, CLIP-190 /+; Dhc64C /+ supplementary material Fig. S2). The effects seen on myonuclear doubly heterozygous embryos produced muscles that were the same KG06490 positioning in the clip190 homozygote were phenocopied in length as those of control embryos but with nuclei 40% further from KG06490 embryos that were transheterozygous for clip190 and each the muscle poles (Fig. 4A-C; supplementary material Fig. S3). – 193 of three different deficiencies that removed CLIP-190: Dhc64C , +/+, raps doubly heterozygous embryos and embryos Fig. 3. Dynein-interacting proteins regulate myotube length and/or myonuclear position. (A)Immunofluorescence images of stage 16 Drosophila embryos of the indicated genotypes. Green, Tropomyosin; red, DsRed; blue, -PS- Integrin in merge. The antigen for grayscale images is listed at the top of the first image. Scale bar: 10m. (B)The length of the LT muscles in stage 16 embryos. (C)The shortest distance between the indicated pole of the LT muscles (gray, dorsal; white, ventral) and the nearest cluster of nuclei normalized for muscle length. Error bars indicate s.d.; *P<0.05, **P<0.01. DEVELOPMENT 3832 RESEARCH ARTICLE Development 139 (20) Fig. 4. Interactions between Dynein and associated genes during muscle development. (A)Immunofluorescence images of stage 16 4-19 Drosophila embryos that are doubly heterozygous for Dhc64C and the indicated allele. Green, Tropomyosin; red, DsRed. Scale bar: 10m. (B)LT muscle length in stage 16 embryos that are doubly heterozygous for the Dhc64C allele shown beneath the histogram and the listed alleles. (C)The shortest distance between the indicated pole of the LT muscles (gray, dorsal; white, ventral) and the nearest cluster of nuclei normalized for muscle length in stage 16 embryos that are doubly heterozygous for the Dhc64C allele indicated beneath the histogram and the listed alleles. Error bars indicate s.d.; **P<0.01. – 05089 depleted of Glued in the Dhc64C /+ background died prior to stage interacted with dlc90F , and these interactions recapitulated K11702 05089 16 and could not be evaluated. These data indicate that Lis1 and their interactions with Dhc64C: lis1 /+; dlc90F /+ and G10.14 05089 CLIP-190 regulate muscle length and myonuclear position, lis1 /+; dlc90F /+ double heterozygotes had muscles that respectively, via interactions with Dynein, and not by Dynein- were 20% shorter than those of control embryos, whereas KG06490 05089 independent mechanisms. clip190 /+; dlc90F /+ double heterozygotes had muscles in which the nuclei were mispositioned, 30% further from the The two Dynein-dependent pathways are muscle poles compared with controls. Similarly, pins functionally K11702 193 separable interacted with both Lis1 and CLIP-190: lis1 /+; raps /+ To confirm that Dynein-dependent myonuclear positioning and double heterozygotes had muscles that were 20% shorter than those KG06490 193 Dynein-dependent determination of muscle length are regulated by of control embryos and clip190 /+; raps /+ double separate mechanisms, we examined the interactions between heterozygotes had normal sized muscles with mispositioned Dynein regulatory proteins. Both Lis1 and CLIP-190 functionally myonuclei that were 60% further from the muscle poles compared Fig. 5. The pathways controlling muscle length and myonuclear position do not genetically interact. (A)Immunofluorescence images of stage 16 Drosophila embryos that are doubly heterozygous for the indicated alleles. Green, Tropomyosin; red, DsRed. Scale bar: 10m. (B)LT muscle length in stage 16 embryos that are doubly heterozygous for the indicated alleles. (C)The shortest distance between the indicated pole of the LT muscles (gray, dorsal; white, ventral) and the nearest cluster of nuclei in stage 16 embryos normalized for muscle length. Error bars indicate s.d.; *P<0.05, **P<0.01. DEVELOPMENT Dynein-dependent muscle formation RESEARCH ARTICLE 3833 Fig. 6. Dynein hyperaccumulates in Lis1 mutant embryos. (A)Confocal projections of a single hemisegment from stage 16 Drosophila embryos immunostained for Tropomyosin (green) and Dynein heavy chain (red). The boxed regions are shown at higher magnification to the right, with Tropomyosin shown in grayscale and Dynein shown as a heat map (the scale indicates relative intensity). The regions outlined by the green dotted lines were used for linescan analysis. Scale bars: left-hand panel, 5m; right-hand panel, 10m. (B)Representative linescan analysis indicating the intensity of Dynein heavy chain immunofluorescence as a function of position across the muscle pole. (C)The peak intensity signal for Dynein heavy chain immunofluorescence in the indicated genotypes. (D)The width of the peak intensity of Dynein heavy chain immunofluorescence. Error bars indicate s.d.; *P<0.05, **P<0.01, compared with control. with controls (Fig. 5A-C; supplementary material Fig. S3). The muscle poles. In Lis1 mutant embryos, the peak intensity of Dynein interaction of dlc90F with pins was also examined but resulted immunofluorescence was increased compared with controls and the in embryonic death prior to stage 16 and could not be evaluated. width of peak intensity was slightly decreased (Fig. 6A-D). The Lis1 contributes only to muscle length, whereas CLIP-190 higher peak intensity combined with the sharper localization contributes solely to myonuclear positioning. We tested whether suggested that Lis1 does not contribute to the initial localization of Lis1 functionally interacts with CLIP-190. Lis1 , +/+, Dynein to the muscle pole, but rather to the retrograde trafficking KG06490 clip190 double heterozygotes were similar to control of Dynein away from the muscle pole. Together, these data indicate embryos in both muscle length and myonuclear positioning. This that proper muscle length requires Dynein localization to the indicates that Dynein-dependent muscle length and Dynein- muscle pole, and that raps results in shorter muscles due to poor dependent myonuclear positioning are indeed mechanistically accumulation of Dynein at the myofiber pole. Additionally, tightly distinct processes (Fig. 5A-C; supplementary material Fig. S3). focused hyperaccumulation of Dynein, as seen in Lis1 mutant embryos, also affects muscle length, suggesting that trafficking of Lis1 affects Dynein localization and CLIP-190 Dynein away from the myofiber pole is mediated by Lis1 and is affects microtubule-cortex interactions necessary to generate muscles of the proper length. We next examined the effects of Dhc64C, Lis1, CLIP-190 and raps Similarly, confocal projections of embryos immunostained for mutants on Dynein localization (Fig. 6A) and microtubule Tubulin and Tropomyosin were used to assess the presence of organization (Fig. 7A) to understand how Dynein differentially microtubules near myofiber poles (Fig. 7A). Two different regulates muscle length and myonuclear positioning. Using measures – the number of microtubules in contact with the confocal projections, linescans of Dynein immunofluorescence myofiber pole (Fig. 7B) and the average intensity of tubulin intensity (Fig. 6B) were employed to measure Dynein localization. immunofluorescence in the distal 3 m of the myofiber (Fig. 7C) KG06490 To control for sample variation, the intensity of Dynein – indicated that only clip190 homozygotes had significantly immunofluorescence was measured relative to the intensity of fewer microtubules near the muscle pole than all other genotypes. Tropomyosin immunofluorescence at the same point. Both the peak These data suggest that CLIP-190 is necessary to mediate of Dynein immunofluorescence (Fig. 6C) and width of peak interactions between microtubules and the myofiber cortex that are immunofluorescence intensity (Fig. 6D) were quantified. then utilized by Dynein to move nuclei towards the muscle pole. KG06490 clip190 did not significantly affect Dynein localization. Lis1 and raps mutants did affect Dynein localization, although Defects in muscle size and myonuclear position differently. In raps mutant embryos, the peak intensity of Dynein impair muscle function immunofluorescence was decreased compared with controls, Larval crawling towards an odorant stimulus was used to assess the whereas the width of peak intensity was increased (Fig. 6A-D), importance of Dynein-dependent muscle size regulation and suggesting that Pins is required for Dynein localization to the Dynein-dependent myonuclear positioning to muscle physiology. DEVELOPMENT 3834 RESEARCH ARTICLE Development 139 (20) Fig. 7. Microtubule organization is disrupted in CLIP-190 mutant embryos. (A)Confocal projections of a single hemisegment from stage 16 Drosophila embryos immunostained for Tropomyosin (green) and Tubulin (grayscale). The boxed regions are shown at higher magnification to the right. Yellow arrows indicate individual and/or bundles of microtubules that reach the muscle pole. Scale bars: 10m. (B)The number of microtubules within 3m of the myofiber pole in the indicated genotypes. (C)The average intensity of Tubulin immunofluorescence in the 3m near the myotube pole in the indicated genotypes. Error bars represent s.d.; *P<0.05, compared with control. Each genotype tested crawled towards the stimulus, indicating that size and myonuclear position, suggesting that the defects seen in all genotypes perceived and responded to the stimulus. However, the embryo are bona fide effects and not merely developmental KG06940 G10.14 L3 larvae that were homozygous for clip190 or lis1 delays. crawled towards the stimulus 33% more slowly than controls (Fig. 4-19 6-10 K11702 8A). Dhc64C , Dhc64C and lis1 were early larval lethal, DISCUSSION thus their locomotive ability could not be tested. However, when We have used the Drosophila musculature to investigate the Dhc64C was depleted during embryonic and larval development mechanisms that control muscle size and intracellular organization. by driving the UAS-Dhc64C-RNAi construct with the muscle- We find that muscle length is regulated independently of the specific driver DMef2-Gal4, these larvae also crawled towards the number of fusion events and demonstrate that perturbations that stimulus 40% more slowly than controls (Fig. 8A,B). Additionally, affect embryonic muscle length correlate with decreased larval muscle-specific depletion of CLIP-190 and Lis1 inhibited larval muscle size and poor muscle function. Additionally, we find that locomotion by 20% (Fig. 8A,B). That the depletion was muscle intracellular organization, specifically myonuclear positioning, is specific confirms that the effects on crawling are muscle essential for muscle function, consistent with our recent autonomous. Furthermore, the NMJ, as measured by bouton observations (Metzger et al., 2012). Moreover, we have shown that number and distribution, was similar to that of the control in each the length and intracellular organization of the myofiber are of the genotypes tested (supplementary material Fig. S4). Finally, mechanistically independent. Although a number of factors link G10.14 KG06490 lis1 , +/+, clip190 doubly heterozygous larvae crawled both processes, we have identified factors that contribute solely to towards the stimulus at the same speed as control larvae (Fig. muscle length or myonuclear position and demonstrate that we can 8A,B), further indicating the independence of these two genetic independently manipulate each feature. pathways. Dynein regulates both muscle length and myonuclear The larvae were then dissected and their musculature was positioning. Some Dynein-interacting proteins, such as Dlc90F and examined. The internuclear distance was reduced by 50-60% in Pins, are necessary for both processes. That these factors regulate G10.14 KG06490 lis1 and clip190 larvae and in muscle depleted of both muscle length and myonuclear positioning suggests that Dhc64C, Lis1 or CLIP-190 (Fig. 8C,D). However, the muscles in specific aspects of muscle growth and the positioning of myonuclei G10.14 lis1 , Lis1-depleted and Dhc64C-depleted larvae were 35% can indeed be coordinated. However, Lis1 affects muscle length KG06490 smaller than in control and clip190 larvae (Fig. 8E). Because specifically, whereas CLIP-190 and Glued specifically affect the muscle size was variable between genotypes, internuclear myonuclear position. Within the contexts of muscle length and distance was normalized as a function of muscle size. Using this myonuclear position CLIP-190 and Lis1 do not genetically interact, measure, both muscle size and internuclear distance were illustrating that, although linked, the two processes are diminished in Dhc64C-depleted larvae compared with controls. mechanistically distinct. However, internuclear distance was specifically reduced in Our identification of CLIP-190 and Lis1 as regulators of Dynein KG06490 clip190 and CLIP-190-depleted larvae, whereas muscle size is not novel. CLIP-190 and Lis1 are known to interact with Dynein G10.14 was specifically reduced in lis1 (Fig. 8D-F). These data both physically and functionally, and they usually cooperate toward demonstrate a correlation between muscle output and both muscle a single goal (Coquelle et al., 2002). Here, we provide the first DEVELOPMENT Dynein-dependent muscle formation RESEARCH ARTICLE 3835 retrograde trafficking of Dynein away from the myofiber pole. We hypothesize that, in the absence of Dynein retrograde trafficking, cellular components accumulate at the extending muscle end, thus inhibiting the trafficking of factors necessary for further directed growth. Thus, when Dynein is incapable of moving cargo away from the myofiber pole, muscles are shorter than in control embryos. Indeed, a similar correlation between retrograde trafficking and directed growth was recently reported during mechanosensory bristle growth and axonal transport (Otani et al., 2011; Yi et al., 2011). Interestingly, it was recently reported that decreased retrograde transport of Dynein results in longer processes in both fibroblasts and neurons in culture (Rishal et al., 2012). This contradictory finding raises several interesting questions. Is there an opposing pathway in muscle or is another aspect of muscle size increased? For example, does the length of the muscle impact its volume? Complications due to muscle contraction in the live embryo make such analysis difficult, however. Likewise, working in vivo on a dynamically changing tissue located 30-150 m below the surface of the developing embryo presents challenges for imaging the rapid cellular processes that underlie motor activity, microtubule organization, organelle positioning and cell size. Nevertheless, the link between different aspects of cell size and their relationships to each other and to motor activity are being explored. It is not clear why LT muscle growth/length is more sensitive to Dynein activity than the VL muscles in the embryo. A simple explanation is that the maternally loaded Dynein persists longer in the VL muscles than in the LT muscles. Additionally there might be a physical explanation. Although a cluster of potential tendon cells for the VL muscles exist, they are clustered at the segment border. Therefore, the size of the hemisegments, and thus the distance from segment border to segment border, determines muscle length. Conversely, the cluster of potential tendon cells for the LT muscles might be more broadly dispersed and the location of the muscle pole at the time of tendon specification determines the length of the muscle. Under this hypothesis, inefficient extension of the LT muscles would result in the muscles being shorter during tendon specification/maturation and therefore shorter Fig. 8. Larval muscle organization and physiology are affected by throughout embryonic development. Alternatively, differences in Dynein and associated proteins. (A,B)The average (A) and guidance/signaling systems could explain why the LT muscles, but maximum (B) speed of Drosophila larvae as they crawl towards a stimulus. (C)Fluorescence images of VL3 muscles from L3 larvae that not the VL muscles, are shorter when Dynein activity is were used in locomotion assays just prior to dissection. White, compromised. Different signaling mechanisms are employed by phalloidin; green, Hoechst. Scale bar: 20m. (D)The average distance these different muscle types (Schnorrer and Dickson, 2004; Volk, between nuclei in larval muscles from the indicated genotypes. (E)The 1999; Schweitzer et al., 2010; Schejter and Baylies, 2010). For surface area of the muscles in larvae of the indicated genotypes. (F)The example, Derailed (Drl) plays a crucial role in the ability of LT distance between nuclei in the larval muscles normalized for muscle muscles to recognize their target (Callahan et al., 1996). Perhaps, size. Error bars indicate s.d. *P<0.05, **P<0.01. altered trafficking of Drl or another factor in the signaling pathway causes slight, but significant, changes in LT muscle length. It is interesting that the LT muscles are smaller in Dhc64C, example of CLIP-190 and Lis1 serving completely independent, Dlc90F, pins and Lis1 mutant embryos, but that other muscles are Dynein-dependent functions. unaffected, whereas in larvae all muscles appear to be smaller. Mechanistically, our data suggest that Dynein function, with During the larval stages, muscles remain stably attached to tendon respect to the regulation of muscle length and myonuclear cells and grow through insulin signaling/Foxo- and dMyc- positioning, is specified by Lis1 and CLIP-190 downstream of its dependent pathways (Demontis and Perrimon, 2009). We interpret localization. That Dynein localization to the myofiber pole is our observations to mean that Dynein and its regulatory proteins important is illustrated by pins (raps ) mutants in which Dynein Dlc90F, Pins and Lis1 contribute to insulin receptor-mediated does not accumulate at the myofiber pole, and we observe defects muscle growth as previously described (Huang et al., 2001; Varadi in both muscle length and myonuclear positioning. This suggests et al., 2003). Indeed, it would not be surprising to find that Dynein- that Pins recruits and stabilizes Dynein at the cortex as it does dependent cellular trafficking is essential to that signaling pathway. during mitosis (Gotta et al., 2003; Hughes et al., 2004). Lis1 also CLIP-190 does not dramatically affect Dynein localization, but affects Dynein localization, but in Lis1 mutants Dynein is localized it does affect microtubule organization, which, in turn, affects to the muscle pole and is tightly restricted to the pole compared nuclear positioning. 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Determinants of S. cerevisiae dynein localization and and Vallee, R. B. (2011). High-resolution imaging reveals indirect coordination activation: implications for the mechanism of spindle positioning. Curr. Biol. 13, of opposite motors and a role for LIS1 in high-load axonal transport. J. Cell Biol. 364-372. 195, 193-201. DEVELOPMENT http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Development The Company of Biologists

Muscle length and myonuclear position are independently regulated by distinct Dynein pathways

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10.1242/dev.079178
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Abstract

RESEARCH ARTICLE 3827 Development 139, 3827-3837 (2012) doi:10.1242/dev.079178 © 2012. Published by The Company of Biologists Ltd Muscle length and myonuclear position are independently regulated by distinct Dynein pathways 1 1,2 1,2, Eric S. Folker , Victoria K. Schulman and Mary K. Baylies * SUMMARY Various muscle diseases present with aberrant muscle cell morphologies characterized by smaller myofibers with mispositioned nuclei. The mechanisms that normally control these processes, whether they are linked, and their contribution to muscle weakness in disease, are not known. We examined the role of Dynein and Dynein-interacting proteins during Drosophila muscle development and found that several factors, including Dynein heavy chain, Dynein light chain and Partner of inscuteable, contribute to the regulation of both muscle length and myonuclear positioning. However, Lis1 contributes only to Dynein-dependent muscle length determination, whereas CLIP-190 and Glued contribute only to Dynein-dependent myonuclear positioning. Mechanistically, microtubule density at muscle poles is decreased in CLIP-190 mutants, suggesting that microtubule-cortex interactions facilitate myonuclear positioning. In Lis1 mutants, Dynein hyperaccumulates at the muscle poles with a sharper localization pattern, suggesting that retrograde trafficking contributes to muscle length. Both Lis1 and CLIP-190 act downstream of Dynein accumulation at the cortex, suggesting that they specify Dynein function within a single location. Finally, defects in muscle length or myonuclear positioning correlate with impaired muscle function in vivo, suggesting that both processes are essential for muscle function. KEY WORDS: Drosophila, Dynein, Nuclear positioning, Muscle size INTRODUCTION the number of fusion events (Bate, 1990), but it is unlikely that The size, shape and organization of tissues and individual cells fusion alone determines myofiber size. In Drosophila larvae and in directly impact their physiology. A pressing question in cell and mammals, a number of signaling pathways have been demonstrated developmental biology is how different morphogenetic processes to control muscle size through growth regulation (Demontis and that are necessary for the functional development of a common Perrimon, 2009; Rommel et al., 2001). Although several guidance tissue are coordinated. Skeletal muscle provides an ideal system cues and signaling mechanisms are known to regulate the direction with which to study structure-function relationships as it is highly of muscle growth (Schejter and Baylies, 2010; Schweitzer et al., organized with a physiologically testable output. 2010), the factors and mechanisms that directly provide the The cellular unit of all metazoan muscle is the myofiber, a necessary forces that promote oriented and regulated muscle syncytial cell that is formed from the iterative fusion of myoblasts. growth during early myogenesis are not known. The correlation between structure and function is highlighted by The importance of organelle positioning is highlighted by the the organization of myosin and actin within the sarcomere that localization of the myonuclei. In skeletal muscle, nuclei reside provides the contractile apparatus (Squire, 1997). However, other above the sarcomere at the periphery of the muscle fiber and are aspects of skeletal muscle structure, including myofiber size and positioned to maximize their internuclear distance. Moreover, organelle positioning, are also crucial for its function. mispositioned myonuclei, in conjunction with smaller myofibers, Myofiber size encompasses a variety of measurements, are a hallmark of many muscle disorders and have been used including length, width, depth and volume, and size is important diagnostically for decades (Pierson et al., 2007; Romero, 2010). for muscle output, as smaller myofibers provide less contractile The mechanisms by which nuclei are moved throughout the muscle force than larger muscles (Fitts et al., 1991). During muscle (Metzger et al., 2012) and anchored at the neuromuscular junction formation in Drosophila, muscle cell growth occurs in three (NMJ) (Grady et al., 2005; Lei et al., 2009) are beginning to distinct phases (Volk, 1999; Schnorrer and Dickson, 2004). As emerge; however, only a small number of factors necessary for fusion begins, the muscle cell breaks symmetry, adds mass, and these processes have been identified. Additionally, potential links elongates to form polarized structures with a long axis and a short between muscle size and myonuclear position have not been axis. Once the long axis is established, the muscle cell extends explored. In other systems, the microtubule cytoskeleton regulates filopodia at each end in search of an attachment with a tendon cell. both polarized growth and nuclear position. Cytoplasmic Dynein Finally, the filopodial protrusions stop, the muscle cell poles (Dynein) is a crucial factor in both processes. During nuclear become smooth, and stable muscle-tendon attachments are made. movement in the first cell division of C. elegans and in migrating In Drosophila embryos, muscle size has often been correlated with neurons, cortically anchored Dynein pulls microtubule minus ends and the attached nuclei towards itself (Gönczy, 2002; Tsai et al., 2007). During polarized cell growth, Dynein contributes to the establishment of the polarity axis (Etienne-Manneville and Hall, Program in Developmental Biology, Sloan Kettering Institute, New York, NY 10065, 2001; Palazzo et al., 2001) that is necessary to localize the factors USA. Cell and Developmental Biology, Weill Cornell Graduate School of Medical Sciences, Cornell University, New York, NY 10065, USA. for cell protrusion (Prigozhina and Waterman-Storer, 2004; Schmoranzer et al., 2003). Given these functions, Dynein may *Author for correspondence ([email protected]) contribute to both myofiber size and myonuclear positioning and might integrate the two processes. Accepted 16 July 2012 DEVELOPMENT 3828 RESEARCH ARTICLE Development 139 (20) To investigate how myofiber size and myonuclear positioning (Sigma, HT501128-4L). Embryos and larvae were mounted in ProLong Gold (Invitrogen) for fluorescent stainings. Antibodies were preabsorbed are regulated, and whether the two processes are co-dependent, we where noted (PA) and used at the following final dilutions: rabbit anti- have studied the role of Dynein during muscle morphogenesis in dsRed (PA, 1:400, Clontech 632496), rat anti-Tropomyosin (PA, 1:500, Drosophila. We establish muscle length, which is an aspect of Abcam ab50567), mouse anti-GFP (PA, 1:200, Clontech 632381), mouse muscle size, and myonuclear positioning as independent aspects of anti-DHC (1:50, Developmental Studies Hybridoma Bank), mouse anti- muscle morphogenesis. We further show that the microtubule Tubulin (1:500, Sigma T9026), chicken anti--galactosidase (PA, 1:1000, cytoskeleton contributes to both processes. Specifically, Dynein Novus Biologicals NB100-2045), mouse anti--PS-Integrin (1:100, regulates muscle length and myonuclear positioning with Developmental Studies Hybridoma Bank) and mouse anti-Discs large overlapping, but distinct, sets of proteins. The Dynein heavy chain (1:200, Developmental Studies Hybridoma Bank). We used Alexa Fluor (Dhc64C), Dynein light chain (Dlc90F; Tctex1 in mammals) and 488-, Alexa Fluor 555- and Alexa Fluor 647-conjugated fluorescent Partner of inscuteable [Pins; Rapsynoid (Raps) – FlyBase] are secondary antibodies (1:200), Alexa Fluor 647-conjugated phalloidin necessary regulators of both muscle length and myonuclear (1:100) and Hoechst 33342 (1 g/ml) for fluorescent stains (all Invitrogen). Fluorescent images were acquired on a Leica SP5 laser-scanning confocal positioning. Subsequently, the two pathways become distinct: microscope equipped with a 63 1.4 NA HCX PL Apochromat oil muscle length is regulated by a Dynein-Lis1-dependent objective and LAS AF 2.2 software. Images were processed using Adobe mechanism, whereas myonuclear positioning is regulated by a Photoshop CS4. Maximum intensity projections of confocal z-stacks were Dynein–CLIP-190–Glued-dependent mechanism. rendered using Volocity Visualization software (Improvision). Mechanistically, in mutant embryos that lack Dynein 4-19 193 (Dhc64C ) or fail to localize Dynein to the muscle pole (raps ), Nuclear position and muscle length measurements both muscle length and myonuclear positioning are affected, Embryos were staged based on standard laboratory procedures: overall embryo shape, the intensity of the apRed and Tropomyosin signals, gut suggesting that Dynein activity at the muscle pole is required for morphology, and the morphology of the trachea (Beckett and Baylies, both processes. However, Dynein localizes to the muscle pole in 2007). Measurements were taken from confocal projections of embryos both Lis1 and CLIP-190 mutant embryos, indicating that Dynein- and acquired using the Line function of ImageJ software (NIH). For each dependent regulation of muscle length and Dynein-dependent genotype, all four lateral transverse (LT) muscles were measured in three regulation of myonuclear positioning are specified downstream of hemisegments from each of 20 embryos from four independent its arrival at the muscle pole. Lis1 mutant embryos exhibit experiments. Three measurements were taken within each LT muscle: (1) hyperaccumulation of Dynein at the muscle pole with a tighter myotube length and (2, 3) the shortest distance between the myotube poles focus of peak intensity, suggesting that Lis1-dependent activation and the outermost edge of the nearest nucleus (supplementary material Fig. of Dynein retrograde trafficking is essential for muscles to achieve S1). The edges of the nuclei and ends of the myotubes were defined by the their proper length. CLIP-190 mutant embryos have fewer clear change from signal to background within the relevant viewing microtubules near the myofiber poles despite normal Dynein channel. Similar measurements were performed using -PS-Integrin to identify the ends of the LT muscles. The statistical significance of localization, suggesting that CLIP-190 promotes microtubule-cell differences in measurements was assessed using a Student’s t-test to cortex interactions that are necessary for Dynein-dependent compare each experimental genotype with wild-type apRed controls. myonuclear positioning. Together, these data suggest that both Statistical analysis was performed with Prism 4.0 (GraphPad). CLIP-190 and Lis1 work downstream of Dynein localization to the Because the live stage 17 embryos offer few markers for accurate muscle pole and thus specify Dynein function within this single staging, staging was achieved by timed lays: after a pre-lay period, location. Differential regulation of Dynein activity within a single embryos were collected for 15 minutes and then aged for 16.5 hours. location is novel and likely to be important during morphogenetic Embryos were then dechorionated and mounted on coverslips in processes for which proper timing is essential. Finally, both muscle halocarbon oil and images were acquired on a Zeiss Axioplan 2 microscope length and myonuclear positioning are important for muscle with a 20 PlanNeoFluor 0.50 NA objective and an Axiocam MRm function, as defects in either process correlate with larvae that camera. Nuclear spread measurements were performed using the Line function in ImageJ to measure the distance between the dorsal edge of the crawl more slowly than controls. dorsal nucleus and the ventral edge of the ventral nucleus. Additionally, these same images of stage 17 embryos were used to count the number of MATERIALS AND METHODS nuclei per hemisegment, which was used as a measure of the number of Drosophila genetics myoblast fusion events. Stocks were grown under standard conditions. Stocks used were apME- 4-19 6-10 NLS::dsRed (Richardson et al., 2007), Dhc64C and Dhc64C (Gepner Microtubule organization measurements et al., 1996), UAS-Dhc64C-RNAi (Vienna Drosophila RNAi Center, 05089 K11702 Confocal z-stacks were sequentially acquired to maximize the signal and v28053), dlc90F (Caggese et al., 2001), lis1 (Lei and Warrior, G10.14 resolution at the myofiber poles and to minimize interference from the 2000), lis1 (Liu et al., 1999), UAS-Lis1-RNAi (Vienna Drosophila KG06490 internal muscles and the epidermis. Images were acquired with a 63 1.4 RNAi Center, v6216), clip190 (Bloomington Drosophila Stock NA HCX PL Apochromat oil objective and a 10 optical zoom. z-stacks Center, 14493), UAS-Clip190-RNAi (Vienna Drosophila RNAi Center, 193 P49 through a depth of 2 m with a step size of 0.25 m were acquired for all v107176), raps (Parmentier et al., 2000), insc (Kraut and Campos- analyzed images. z-stacks were then assembled into confocal projections Ortega, 1996) and UAS-Glued-RNAi (Vienna Drosophila RNAi Center, using Volocity, which were then exported to ImageJ for analysis. Analysis v3785). Mutants were balanced and identified using CTG (CyO, twi-Gal4, was performed on 20 embryos from four independent experiments. To UAS-2xeGFP), TTG (TM3, twi-Gal4, UAS-2xeGFP), TM6B, CyO count the number of microtubules in contact with the muscle pole, a 22 P[w+wgen11lacZ] and TM3 Sb1 Dfd-lacZ. m box was drawn from the tip of the muscle toward the center and the Immunohistochemistry number of microtubules in this region were counted. Embryos were collected at 25°C and fixed with 4% EM-grade To determine the average intensity, the Measure Average Intensity paraformaldehyde (Polysciences, 00380) diluted in PBS (Roche, Function in ImageJ was performed on the distal 3 m of the myofiber. The intensity of Tubulin staining was standardized against the intensity of 11666789001) and an equal volume of heptane to allow permeabilization. In all cases, embryos were devitellinized by vortexing in a 1:1 Tropomyosin staining to control for sample variation. For each genotype, methanol:heptane solution. Larvae were dissected in ice-cold HL3.1 as all four LT muscles were measured in three hemisegments from each of 20 previously described (Brent et al., 2009) and fixed with 10% formalin embryos from four independent experiments. Student’s t-test was used to DEVELOPMENT Dynein-dependent muscle formation RESEARCH ARTICLE 3829 compare each experimental genotype with wild-type apRed controls. examined. Dhc64C mutant embryos had the same number of Statistical analysis was performed with Prism 4.0. apRed nuclei per hemisegment as controls, indicating that myoblast fusion was unaffected (Fig. 1A,B). Although the nuclei were Dynein localization measurements aligned in columns within each LT muscle in embryos of each Embryos were collected and fixed with 10% formalin diluted 1:1 in genotype, the distance between the most ventral and the most heptane for 20 minutes, then rinsed three times in PBS containing 0.6% dorsal nuclei was reduced in Dhc64C mutant embryos (Fig. 1A,C). Triton X-100, then fixed with 4% paraformaldehyde diluted 1:1 in heptane To determine whether the difference in the distribution of nuclei for 20 minutes. Antibody incubations were performed in PBS supplemented with 1% BSA and 0.6% Triton X-100. Image acquisition and was caused by defective nuclear positioning or effects on muscle processing were as for microtubule quantification. Dynein size, we examined late stage 16 embryos (16 hours AEL), which immunofluorescence intensity was assessed by linescan analysis using are amenable to whole-mount immunostaining. ImageJ. Positions of linescans were chosen randomly on the Tropomyosin The general muscle pattern as revealed by Tropomyosin staining image and the line then copied to the identical position on the Dynein (Fig. 1D) was normal in Dhc64C mutant embryos. Additionally, - image. The intensity of Dynein immunofluorescence at each point was PS-Integrin staining was normal. Thus, all muscles were present, measured relative to Tropomyosin intensity at that same point to control properly oriented and attached to tendon cells, indicating that for sample variation, and the ratios were multiplied by 100. Three specification, guidance and attachment mechanisms are functional. hemisegments from 20 embryos from four independent experiments were In late stage 16 (16 hours AEL) control embryos, the myonuclei analyzed. The greatest pixel intensity from each embryo was used to were in two clusters within each LT muscle: one cluster at the determine the peak intensity. To determine the width of peak intensity, the pixels that were ≥80% of peak intensity were determined and the distance dorsal pole and the other at the ventral pole (Fig. 1D; between the first pixel and the last pixel that fitted these criteria was supplementary material Fig. S1) (Metzger et al., 2012). In embryos measured. For each genotype, all four LT muscles were measured in three homozygous for either Dhc64C allele, the nuclei were also in two hemisegments from each of five embryos. Student’s t-test was used to clusters; however, the clusters were mispositioned 50% further compare each experimental genotype with wild-type apRed controls. from the muscle poles than in wild-type and heterozygous controls Statistical analysis was performed with Prism 4.0. The heat map (Fig. 1D,E; supplementary material Fig. S2). Additionally, the representation of Dynein immunofluorescence was generated using the length of the LT muscles was reduced by 15% in embryos Cool application of the standard Lookup tools in ImageJ. homozygous for either Dhc64C allele as compared with wild-type Larval behavior and heterozygous controls (Fig. 1D,F; supplementary material Fig. Larval speed was assessed as previously described (Louis et al., 2008; S2). To account for the difference in muscle length, the distance Metzger et al., 2012) with minor modifications. Stage 16 and 17 embryos from each cluster of nuclei to the nearest muscle pole was were selected for the absence of the fluorescent balancer and placed on calculated as a percentage of muscle length (Fig. 1G). This yeast-coated apple juice agar plates at 22°C overnight. L1 larvae were measurement was used with respect to all subsequent genotypes. selected the following day and placed into vials of standard fly food To validate the use of Tropomyosin as a marker for muscle ends, containing Bromophenol Blue (Bio-Rad, 161-0404). L3 larvae were picked we performed identical measurements using -PS-Integrin to mark from the vial 6 days later and individually tracked as they crawled towards the muscle ends. These analyses produced identical results (Fig. an odor source of ethyl butyrate (3.3%; Sigma, E15701) diluted in paraffin 1D; supplementary material Fig. S2C,D). oil (Fluka, 76235). Their movements were recorded with a CCD camera for either five total minutes or until they reached the odor source or the To confirm that the effects of Dynein were autonomous to walls of the apparatus. The tracks were processed using Ethovision muscle, we used the GAL4/UAS system to deplete Dynein software (Nodlus). At least 20 larvae were tracked for each genotype. specifically in the mesoderm using twist-GAL4 to express UAS- Tracked larvae were dissected, stained and analyzed as follows. Dhc64C-RNAi (Dietzl et al., 2007). Tissue-specific depletion of Internuclear distance was measured in ImageJ by drawing lines between Dhc64C recapitulated the effects of the Dhc64C alleles: the LT the center of each nucleus and its nearest neighbor using projection images. muscles were 12% shorter than controls and the nuclei were Muscle size was measured by determining the area of confocal projections positioned 40% further from the muscle poles than in controls (Fig. of each muscle. To calculate the internuclear distance as a function of 1D-G). These data indicate that Dynein is specifically required in muscle size, the average internuclear distance for a given muscle was the muscle to generate muscles of proper length and to properly divided by the area of that specific muscle. Nine muscles from three larvae position myonuclei. were used for quantification. Given that myonuclear position seems to be affected similarly in stage 16 and stage 17 embryos, we examined embryos at earlier RESULTS stages in their development to determine when the defects in Dynein regulates muscle length and myonuclear muscle length and myonuclear positioning become evident. Using position Tropomyosin to identify the muscles and a combination of trachea Each body wall muscle in the Drosophila embryo and larva morphology and gut morphology to assess embryo age, we consists of a single syncytial myofiber similar to the individual determined the length of muscles and the positioning of myonuclei myofibers in mammalian muscle. To assess the role of Dynein in late stage 14 (11 hours AEL), stage 15 (13 hours AEL) and stage during Drosophila muscle morphogenesis, the distribution of nuclei 16 (16 hours AEL) embryos (Fig. 2). At stage 14, the length of the 4-19 within the lateral transverse (LT) muscles was examined in wild- muscles in control and Dhc64C embryos was similar. However, 4-19 type embryos and in embryos in which Dynein was zygotically by stage 15 and through stage 16 the muscles in the Dhc64C 4-19 6-10 removed with either the Dhc64C (null) or the Dhc64C embryos were significantly shorter (17% and 14%, respectively) (hypomorphic) allele (Gepner et al., 1996). The myonuclei in the than those in the control embryos. LT muscles were visualized by expression of the apterous The effects on myonuclear positioning at the different stages mesodermal enhancer fused to a nuclear localization signal and the were more complex. The nuclei were 30% further from the muscle 4-19 fluorescent protein DsRed (apRed) (Richardson et al., 2007), which poles in late stage 14 Dhc64C embryos than in controls (Fig. specifically labels the nuclei of the LT muscles. Using this reporter, 2C); however, only the positioning relative to the dorsal muscle live stage 17 embryos [16.5-20 hours after egg lay (AEL)] were pole was statistically significant. At stage 15, myonuclei were DEVELOPMENT 3830 RESEARCH ARTICLE Development 139 (20) Fig. 1. Cytoplasmic Dynein regulates muscle length and myonuclear position. (A)Fluorescence images of apRed nuclei in the lateral transverse (LT) muscles of live stage 17 Drosophila embryos (16.5-20 hours AEL) of the indicated genotypes. (B)The number of nuclei per hemisegment in stage 17 embryos of the indicated genotypes. (C)The distance between the dorsalmost and ventralmost nuclei in stage 17 embryos of the indicated genotypes. (D)Immunofluorescence images of stage 16 (16 hours AEL) embryos of the genotypes noted to the left and antigen listed at the top of the first image. Green, Tropomyosin (muscle); red, DsRed (nuclei); blue, -PS-Integrin in merge. Arrows in the control panels denote points from which measurements were made for all genotypes. The distance between the points indicated by the yellow arrows in the Tropomyosin and -PS-Integrin panels was used to determine muscle length. The distance between the pair of gray arrows at the top of the merged image was used to determine the distance between the nuclei and the dorsal pole. The distance between the pair of white arrows at the bottom of the merged image was used to determine the distance between the nuclei and the ventral pole. (E)The shortest distance between the indicated pole of the LT muscles (gray, dorsal; white, ventral) and the nearest cluster of nuclei in stage 16 embryos. (F)LT muscle length in stage 16 embryos. (G)The shortest distance between the indicated pole of the LT muscles (gray, dorsal; white, ventral) and the nearest cluster of nuclei normalized for muscle length in stage 16 embryos of the indicated genotypes. Error bars indicate s.d.; **P<0.01, *P<0.05. Scale bars: 10m. 4-19 similarly positioned in control and Dhc64C embryos (Fig. 2D). and neurons (McKenney et al., 2010; Mesngon et al., 2006; In stage 16 embryos, the myonuclei were significantly Sheeman et al., 2003); and CLIP-190, which works with Lis1 to mispositioned (50% further from the poles) at both the dorsal and regulate Dynein activity (Tanenbaum et al., 2008). 4-19 ventral poles of the muscle in Dhc64C mutant embryos relative We tested two Dynein light chains, Cdlc2 and Dlc90F, which are to controls (Fig. 2E). the Drosophila orthologs of Dynein light chain 1/2 and Tctex1, These data indicate that the defects in muscle length arise prior respectively (Caggese et al., 2001; Goldstein and Gunawardena, to muscle-tendon attachment and, together with the muscle-specific 2000), by RNAi-mediated depletion (Dietzl et al., 2007). Depletion depletion of Dhc64C, suggest that the effects are muscle of Cdlc2 specifically in mesoderm and muscle did not affect autonomous and not due to defects in tendon specification. muscle length or myonuclear positioning, whereas depletion of Dlc90F resulted in shorter muscles with mispositioned nuclei. Dynein-interacting proteins contribute to Dynein- Additionally, embryos that were homozygous for dlc90F dependent regulation of muscle length and (Caggese et al., 2001) or the pins allele raps (Parmentier et al., myonuclear positioning 2000), produced muscles that were 12% shorter with nuclei that To determine whether the roles of Dynein in specifying muscle were 40-50% further from the muscle poles than in wild-type and length and myonuclear positioning are functionally coupled, we heterozygous control embryos (Fig. 3A-C; supplementary material tested known Dynein-interacting proteins for their contributions to Fig. S2). These data indicate that Dlc90F and Pins contribute to each process. We focused on factors that are general regulators of both muscle length and myonuclear positioning. K11702 Dynein activity or have been found to specifically contribute to Embryos homozygous for either of two Lis1 alleles, lis1 G10.14 Dynein-mediated nuclear movements in other systems. We (Lei and Warrior, 2000) and lis1 (Liu et al., 1999), produced examined Dynein light chains, which can regulate Dynein activity muscles that were 15% shorter than those of wild-type and both positively (Sönnichsen et al., 2005) and negatively (O’Rourke heterozygous controls. However, myonuclear positioning was et al., 2007); Glued, which increases Dynein motor processivity normal in Lis1 homozygous embryos. Conversely, embryos that and mediates Dynein-cargo interactions (Gill et al., 1991; Vaughan were depleted of Glued (Dietzl et al., 2007) in muscle and embryos KG06490 et al., 2002; Waterman-Storer et al., 1997); Pins, which anchors that were homozygous for clip190 (Bellen et al., 2004) Dynein at the cortex to move nuclei in other systems (Gotta et al., produced muscles of similar length to controls. However, in these 2003; Hughes et al., 2004); Lis1, which maintains Dynein in an embryos the nuclei were positioned 50% further from the muscle active state and has been implicated in nuclear movements in yeast poles than in wild-type and heterozygous controls (Fig. 3A-C; DEVELOPMENT Dynein-dependent muscle formation RESEARCH ARTICLE 3831 Df(2L)BSC294, Df(2L)Exel7068 and Df(2L)Exel8036 (supplementary material Fig. S2). Additionally, we measured the ventral longitudinal muscle VL3 and found that its length in the embryo was similar in each genetic background (supplementary material Fig. S2). These data suggest that a unique feature of the LT muscles makes them more susceptible to Dynein levels and/or activity with respect to muscle growth in the embryo. The number and position of nuclei in stage 17 embryos were also examined. Embryos from each genotype had the same number of apRed nuclei per hemisegment, indicating that the defects in muscle length did not result from impaired fusion (Fig. 1B). The clusters of nuclei in each genotype did resolve into columns during stage 17, but the distance between the ventral and dorsal nuclei was reduced by 15% compared with controls. Moreover, the distance between the most distal nuclei was similar in stage 16 and stage 17 embryos for each genotype (supplementary material Fig. S3). To determine whether the Dynein-interacting proteins work with, or independently of, Dynein, we examined embryos that were doubly heterozygous for Dhc64C mutations and mutations in each of the associated genes. In these, and all subsequent genetic interactions, maternal effects were controlled for by providing each allele from 4-19 the mother or the father in reciprocal crosses. Both Dhc64C and 6-10 Fig. 2. Stage-dependent effects of Dynein on muscle length and Dhc64C were used in combination with alleles for each putative myonuclear positioning. (A)Immunofluorescence images of the 05089 Dynein-interacting gene. Double heterozygotes of dlc90F and muscles (green, Tropomyosin) and nuclei (red, DsRed) at stage 14, 15 4-19 05089 6-10 Dhc64C or of dlc90F and Dhc64C produced muscles that 4-19 and 16 in control and Dhc64C Drosophila embryos. Scale bar: were 15% shorter than controls and with their nuclei 50% further 4-19 10m. (B)The length of the muscles in control (gray) and Dhc64C from the muscle poles (Fig. 4A-C). These data indicate that Dlc90F (black) embryos at stage 14, 15 and 16. (C-E)The distance between the (Tctex1) regulates Dynein activity for both muscle length and nearest nucleus and either the dorsal (gray) or ventral (white) pole of – – myonuclear positioning. All Lis1 /+; Dhc64C /+ doubly the muscle in stage 14 (C), 15 (D) and 16 (E) embryos. Error bars indicate s.d.; *P<0.05, **P<0.01. heterozygous embryos produced muscles that were 15% shorter than those in control embryos, but with properly positioned myonuclei at – – the muscle poles (Fig. 4A-C). Conversely, CLIP-190 /+; Dhc64C /+ supplementary material Fig. S2). The effects seen on myonuclear doubly heterozygous embryos produced muscles that were the same KG06490 positioning in the clip190 homozygote were phenocopied in length as those of control embryos but with nuclei 40% further from KG06490 embryos that were transheterozygous for clip190 and each the muscle poles (Fig. 4A-C; supplementary material Fig. S3). – 193 of three different deficiencies that removed CLIP-190: Dhc64C , +/+, raps doubly heterozygous embryos and embryos Fig. 3. Dynein-interacting proteins regulate myotube length and/or myonuclear position. (A)Immunofluorescence images of stage 16 Drosophila embryos of the indicated genotypes. Green, Tropomyosin; red, DsRed; blue, -PS- Integrin in merge. The antigen for grayscale images is listed at the top of the first image. Scale bar: 10m. (B)The length of the LT muscles in stage 16 embryos. (C)The shortest distance between the indicated pole of the LT muscles (gray, dorsal; white, ventral) and the nearest cluster of nuclei normalized for muscle length. Error bars indicate s.d.; *P<0.05, **P<0.01. DEVELOPMENT 3832 RESEARCH ARTICLE Development 139 (20) Fig. 4. Interactions between Dynein and associated genes during muscle development. (A)Immunofluorescence images of stage 16 4-19 Drosophila embryos that are doubly heterozygous for Dhc64C and the indicated allele. Green, Tropomyosin; red, DsRed. Scale bar: 10m. (B)LT muscle length in stage 16 embryos that are doubly heterozygous for the Dhc64C allele shown beneath the histogram and the listed alleles. (C)The shortest distance between the indicated pole of the LT muscles (gray, dorsal; white, ventral) and the nearest cluster of nuclei normalized for muscle length in stage 16 embryos that are doubly heterozygous for the Dhc64C allele indicated beneath the histogram and the listed alleles. Error bars indicate s.d.; **P<0.01. – 05089 depleted of Glued in the Dhc64C /+ background died prior to stage interacted with dlc90F , and these interactions recapitulated K11702 05089 16 and could not be evaluated. These data indicate that Lis1 and their interactions with Dhc64C: lis1 /+; dlc90F /+ and G10.14 05089 CLIP-190 regulate muscle length and myonuclear position, lis1 /+; dlc90F /+ double heterozygotes had muscles that respectively, via interactions with Dynein, and not by Dynein- were 20% shorter than those of control embryos, whereas KG06490 05089 independent mechanisms. clip190 /+; dlc90F /+ double heterozygotes had muscles in which the nuclei were mispositioned, 30% further from the The two Dynein-dependent pathways are muscle poles compared with controls. Similarly, pins functionally K11702 193 separable interacted with both Lis1 and CLIP-190: lis1 /+; raps /+ To confirm that Dynein-dependent myonuclear positioning and double heterozygotes had muscles that were 20% shorter than those KG06490 193 Dynein-dependent determination of muscle length are regulated by of control embryos and clip190 /+; raps /+ double separate mechanisms, we examined the interactions between heterozygotes had normal sized muscles with mispositioned Dynein regulatory proteins. Both Lis1 and CLIP-190 functionally myonuclei that were 60% further from the muscle poles compared Fig. 5. The pathways controlling muscle length and myonuclear position do not genetically interact. (A)Immunofluorescence images of stage 16 Drosophila embryos that are doubly heterozygous for the indicated alleles. Green, Tropomyosin; red, DsRed. Scale bar: 10m. (B)LT muscle length in stage 16 embryos that are doubly heterozygous for the indicated alleles. (C)The shortest distance between the indicated pole of the LT muscles (gray, dorsal; white, ventral) and the nearest cluster of nuclei in stage 16 embryos normalized for muscle length. Error bars indicate s.d.; *P<0.05, **P<0.01. DEVELOPMENT Dynein-dependent muscle formation RESEARCH ARTICLE 3833 Fig. 6. Dynein hyperaccumulates in Lis1 mutant embryos. (A)Confocal projections of a single hemisegment from stage 16 Drosophila embryos immunostained for Tropomyosin (green) and Dynein heavy chain (red). The boxed regions are shown at higher magnification to the right, with Tropomyosin shown in grayscale and Dynein shown as a heat map (the scale indicates relative intensity). The regions outlined by the green dotted lines were used for linescan analysis. Scale bars: left-hand panel, 5m; right-hand panel, 10m. (B)Representative linescan analysis indicating the intensity of Dynein heavy chain immunofluorescence as a function of position across the muscle pole. (C)The peak intensity signal for Dynein heavy chain immunofluorescence in the indicated genotypes. (D)The width of the peak intensity of Dynein heavy chain immunofluorescence. Error bars indicate s.d.; *P<0.05, **P<0.01, compared with control. with controls (Fig. 5A-C; supplementary material Fig. S3). The muscle poles. In Lis1 mutant embryos, the peak intensity of Dynein interaction of dlc90F with pins was also examined but resulted immunofluorescence was increased compared with controls and the in embryonic death prior to stage 16 and could not be evaluated. width of peak intensity was slightly decreased (Fig. 6A-D). The Lis1 contributes only to muscle length, whereas CLIP-190 higher peak intensity combined with the sharper localization contributes solely to myonuclear positioning. We tested whether suggested that Lis1 does not contribute to the initial localization of Lis1 functionally interacts with CLIP-190. Lis1 , +/+, Dynein to the muscle pole, but rather to the retrograde trafficking KG06490 clip190 double heterozygotes were similar to control of Dynein away from the muscle pole. Together, these data indicate embryos in both muscle length and myonuclear positioning. This that proper muscle length requires Dynein localization to the indicates that Dynein-dependent muscle length and Dynein- muscle pole, and that raps results in shorter muscles due to poor dependent myonuclear positioning are indeed mechanistically accumulation of Dynein at the myofiber pole. Additionally, tightly distinct processes (Fig. 5A-C; supplementary material Fig. S3). focused hyperaccumulation of Dynein, as seen in Lis1 mutant embryos, also affects muscle length, suggesting that trafficking of Lis1 affects Dynein localization and CLIP-190 Dynein away from the myofiber pole is mediated by Lis1 and is affects microtubule-cortex interactions necessary to generate muscles of the proper length. We next examined the effects of Dhc64C, Lis1, CLIP-190 and raps Similarly, confocal projections of embryos immunostained for mutants on Dynein localization (Fig. 6A) and microtubule Tubulin and Tropomyosin were used to assess the presence of organization (Fig. 7A) to understand how Dynein differentially microtubules near myofiber poles (Fig. 7A). Two different regulates muscle length and myonuclear positioning. Using measures – the number of microtubules in contact with the confocal projections, linescans of Dynein immunofluorescence myofiber pole (Fig. 7B) and the average intensity of tubulin intensity (Fig. 6B) were employed to measure Dynein localization. immunofluorescence in the distal 3 m of the myofiber (Fig. 7C) KG06490 To control for sample variation, the intensity of Dynein – indicated that only clip190 homozygotes had significantly immunofluorescence was measured relative to the intensity of fewer microtubules near the muscle pole than all other genotypes. Tropomyosin immunofluorescence at the same point. Both the peak These data suggest that CLIP-190 is necessary to mediate of Dynein immunofluorescence (Fig. 6C) and width of peak interactions between microtubules and the myofiber cortex that are immunofluorescence intensity (Fig. 6D) were quantified. then utilized by Dynein to move nuclei towards the muscle pole. KG06490 clip190 did not significantly affect Dynein localization. Lis1 and raps mutants did affect Dynein localization, although Defects in muscle size and myonuclear position differently. In raps mutant embryos, the peak intensity of Dynein impair muscle function immunofluorescence was decreased compared with controls, Larval crawling towards an odorant stimulus was used to assess the whereas the width of peak intensity was increased (Fig. 6A-D), importance of Dynein-dependent muscle size regulation and suggesting that Pins is required for Dynein localization to the Dynein-dependent myonuclear positioning to muscle physiology. DEVELOPMENT 3834 RESEARCH ARTICLE Development 139 (20) Fig. 7. Microtubule organization is disrupted in CLIP-190 mutant embryos. (A)Confocal projections of a single hemisegment from stage 16 Drosophila embryos immunostained for Tropomyosin (green) and Tubulin (grayscale). The boxed regions are shown at higher magnification to the right. Yellow arrows indicate individual and/or bundles of microtubules that reach the muscle pole. Scale bars: 10m. (B)The number of microtubules within 3m of the myofiber pole in the indicated genotypes. (C)The average intensity of Tubulin immunofluorescence in the 3m near the myotube pole in the indicated genotypes. Error bars represent s.d.; *P<0.05, compared with control. Each genotype tested crawled towards the stimulus, indicating that size and myonuclear position, suggesting that the defects seen in all genotypes perceived and responded to the stimulus. However, the embryo are bona fide effects and not merely developmental KG06940 G10.14 L3 larvae that were homozygous for clip190 or lis1 delays. crawled towards the stimulus 33% more slowly than controls (Fig. 4-19 6-10 K11702 8A). Dhc64C , Dhc64C and lis1 were early larval lethal, DISCUSSION thus their locomotive ability could not be tested. However, when We have used the Drosophila musculature to investigate the Dhc64C was depleted during embryonic and larval development mechanisms that control muscle size and intracellular organization. by driving the UAS-Dhc64C-RNAi construct with the muscle- We find that muscle length is regulated independently of the specific driver DMef2-Gal4, these larvae also crawled towards the number of fusion events and demonstrate that perturbations that stimulus 40% more slowly than controls (Fig. 8A,B). Additionally, affect embryonic muscle length correlate with decreased larval muscle-specific depletion of CLIP-190 and Lis1 inhibited larval muscle size and poor muscle function. Additionally, we find that locomotion by 20% (Fig. 8A,B). That the depletion was muscle intracellular organization, specifically myonuclear positioning, is specific confirms that the effects on crawling are muscle essential for muscle function, consistent with our recent autonomous. Furthermore, the NMJ, as measured by bouton observations (Metzger et al., 2012). Moreover, we have shown that number and distribution, was similar to that of the control in each the length and intracellular organization of the myofiber are of the genotypes tested (supplementary material Fig. S4). Finally, mechanistically independent. Although a number of factors link G10.14 KG06490 lis1 , +/+, clip190 doubly heterozygous larvae crawled both processes, we have identified factors that contribute solely to towards the stimulus at the same speed as control larvae (Fig. muscle length or myonuclear position and demonstrate that we can 8A,B), further indicating the independence of these two genetic independently manipulate each feature. pathways. Dynein regulates both muscle length and myonuclear The larvae were then dissected and their musculature was positioning. Some Dynein-interacting proteins, such as Dlc90F and examined. The internuclear distance was reduced by 50-60% in Pins, are necessary for both processes. That these factors regulate G10.14 KG06490 lis1 and clip190 larvae and in muscle depleted of both muscle length and myonuclear positioning suggests that Dhc64C, Lis1 or CLIP-190 (Fig. 8C,D). However, the muscles in specific aspects of muscle growth and the positioning of myonuclei G10.14 lis1 , Lis1-depleted and Dhc64C-depleted larvae were 35% can indeed be coordinated. However, Lis1 affects muscle length KG06490 smaller than in control and clip190 larvae (Fig. 8E). Because specifically, whereas CLIP-190 and Glued specifically affect the muscle size was variable between genotypes, internuclear myonuclear position. Within the contexts of muscle length and distance was normalized as a function of muscle size. Using this myonuclear position CLIP-190 and Lis1 do not genetically interact, measure, both muscle size and internuclear distance were illustrating that, although linked, the two processes are diminished in Dhc64C-depleted larvae compared with controls. mechanistically distinct. However, internuclear distance was specifically reduced in Our identification of CLIP-190 and Lis1 as regulators of Dynein KG06490 clip190 and CLIP-190-depleted larvae, whereas muscle size is not novel. CLIP-190 and Lis1 are known to interact with Dynein G10.14 was specifically reduced in lis1 (Fig. 8D-F). These data both physically and functionally, and they usually cooperate toward demonstrate a correlation between muscle output and both muscle a single goal (Coquelle et al., 2002). Here, we provide the first DEVELOPMENT Dynein-dependent muscle formation RESEARCH ARTICLE 3835 retrograde trafficking of Dynein away from the myofiber pole. We hypothesize that, in the absence of Dynein retrograde trafficking, cellular components accumulate at the extending muscle end, thus inhibiting the trafficking of factors necessary for further directed growth. Thus, when Dynein is incapable of moving cargo away from the myofiber pole, muscles are shorter than in control embryos. Indeed, a similar correlation between retrograde trafficking and directed growth was recently reported during mechanosensory bristle growth and axonal transport (Otani et al., 2011; Yi et al., 2011). Interestingly, it was recently reported that decreased retrograde transport of Dynein results in longer processes in both fibroblasts and neurons in culture (Rishal et al., 2012). This contradictory finding raises several interesting questions. Is there an opposing pathway in muscle or is another aspect of muscle size increased? For example, does the length of the muscle impact its volume? Complications due to muscle contraction in the live embryo make such analysis difficult, however. Likewise, working in vivo on a dynamically changing tissue located 30-150 m below the surface of the developing embryo presents challenges for imaging the rapid cellular processes that underlie motor activity, microtubule organization, organelle positioning and cell size. Nevertheless, the link between different aspects of cell size and their relationships to each other and to motor activity are being explored. It is not clear why LT muscle growth/length is more sensitive to Dynein activity than the VL muscles in the embryo. A simple explanation is that the maternally loaded Dynein persists longer in the VL muscles than in the LT muscles. Additionally there might be a physical explanation. Although a cluster of potential tendon cells for the VL muscles exist, they are clustered at the segment border. Therefore, the size of the hemisegments, and thus the distance from segment border to segment border, determines muscle length. Conversely, the cluster of potential tendon cells for the LT muscles might be more broadly dispersed and the location of the muscle pole at the time of tendon specification determines the length of the muscle. Under this hypothesis, inefficient extension of the LT muscles would result in the muscles being shorter during tendon specification/maturation and therefore shorter Fig. 8. Larval muscle organization and physiology are affected by throughout embryonic development. Alternatively, differences in Dynein and associated proteins. (A,B)The average (A) and guidance/signaling systems could explain why the LT muscles, but maximum (B) speed of Drosophila larvae as they crawl towards a stimulus. (C)Fluorescence images of VL3 muscles from L3 larvae that not the VL muscles, are shorter when Dynein activity is were used in locomotion assays just prior to dissection. White, compromised. Different signaling mechanisms are employed by phalloidin; green, Hoechst. Scale bar: 20m. (D)The average distance these different muscle types (Schnorrer and Dickson, 2004; Volk, between nuclei in larval muscles from the indicated genotypes. (E)The 1999; Schweitzer et al., 2010; Schejter and Baylies, 2010). For surface area of the muscles in larvae of the indicated genotypes. (F)The example, Derailed (Drl) plays a crucial role in the ability of LT distance between nuclei in the larval muscles normalized for muscle muscles to recognize their target (Callahan et al., 1996). Perhaps, size. Error bars indicate s.d. *P<0.05, **P<0.01. altered trafficking of Drl or another factor in the signaling pathway causes slight, but significant, changes in LT muscle length. It is interesting that the LT muscles are smaller in Dhc64C, example of CLIP-190 and Lis1 serving completely independent, Dlc90F, pins and Lis1 mutant embryos, but that other muscles are Dynein-dependent functions. unaffected, whereas in larvae all muscles appear to be smaller. Mechanistically, our data suggest that Dynein function, with During the larval stages, muscles remain stably attached to tendon respect to the regulation of muscle length and myonuclear cells and grow through insulin signaling/Foxo- and dMyc- positioning, is specified by Lis1 and CLIP-190 downstream of its dependent pathways (Demontis and Perrimon, 2009). We interpret localization. That Dynein localization to the myofiber pole is our observations to mean that Dynein and its regulatory proteins important is illustrated by pins (raps ) mutants in which Dynein Dlc90F, Pins and Lis1 contribute to insulin receptor-mediated does not accumulate at the myofiber pole, and we observe defects muscle growth as previously described (Huang et al., 2001; Varadi in both muscle length and myonuclear positioning. This suggests et al., 2003). Indeed, it would not be surprising to find that Dynein- that Pins recruits and stabilizes Dynein at the cortex as it does dependent cellular trafficking is essential to that signaling pathway. during mitosis (Gotta et al., 2003; Hughes et al., 2004). Lis1 also CLIP-190 does not dramatically affect Dynein localization, but affects Dynein localization, but in Lis1 mutants Dynein is localized it does affect microtubule organization, which, in turn, affects to the muscle pole and is tightly restricted to the pole compared nuclear positioning. CLIP-190 mutant embryos have fewer with controls. We interpret this to mean that Lis1 is necessary for microtubules at the myofiber pole, suggesting that, similar to its DEVELOPMENT 3836 RESEARCH ARTICLE Development 139 (20) functions in other systems, the role of CLIP-190 is to stabilize Callahan, C. A., Bonkovsky, J. L., Scully, A. L. and Thomas, J. B. (1996). derailed is required for muscle attachment site selection in Drosophila. microtubule-cortex interactions (Neukirchen and Bradke, 2011; Development 122, 2761-2777. Watanabe et al., 2004), which Dynein then uses to move nuclei Coquelle, F. M., Caspi, M., Cordelières, F. P., Dompierre, J. P., Dujardin, D. L., towards the myofiber pole. The specification of Dynein function, Koifman, C., Martin, P., Hoogenraad, C. C., Akhmanova, A., Galjart, N. et al. (2002). LIS1, CLIP-170’s key to the dynein/dynactin pathway. Mol. Cell. Biol. downstream of its localization, is novel. 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Published: Oct 15, 2012

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