Drosophila genetics
All stocks were grown under standard conditions at 25 °C. The Fascinsn28 allele was a generous gift from Tina Tootle (University of Iowa). All UAS-RNAi Drosophila lines were purchased from Bloomington Drosophila Stock Center. UAS-RNAi constructs were driven specifically in the mesoderm using twist-GAL4, which drives expression in the mesoderm from stage 10 of embryonic development through stage 13 of embryonic development, DMef2-GAL4 that drives expression in the muscles from stage 12 through adulthood, or MHC-GAL4 which drives expression in muscle from stage 17 of embryonic development through adulthood.
Larval locomotion assay
We performed a modified version of the previously used assay that has been used to measure larval locomotion in individual larvae [19]. Virgins expressing DMef2-GAL4 were mixed with males that carried the UAS-RNAi for 1 h in a vial with standard Drosophila food. After 1 h, adults were moved to a new vial and the embryos laid during the 1 h period were aged for 5 days until they were third-instar larvae (L3). Larvae were then floated from the food by the addition of 15% sucrose. Using a paintbrush, larvae were moved to a plate with wet yeast. After all of the genotypes had been collected, 10 larvae of each RNAi were moved to an arena that consisted of 3% agarose dyed black with standard food color poured over the top of a 96-well plate cover. Movement of larvae toward a stick dipped in ethyl butyrate was captured using an iPhone mounted above the arena. The speed of each larva was then analyzed using ImageJ.
Viability assay
Stage 16 embryos were picked and placed on an agarose plate. The selected embryos were incubated at 20 °C overnight. After incubation, the number of unhatched embryos were counted to determine embryonic lethality and the L1 larvae from the hatched embryos were transferred to a vial of standard fly food. Larvae were incubated at 25 °C until larvae began to pupate and eclose. Adult flies were counted to determine pupal lethality and removed from the vials of standard fly food upon eclosure. After 1 week of no eclosures, the number of pupal cases were counted to determine larval lethality.
Immunohistochemistry
Preparation of embryos
Embryos were collected at 25 °C and were dechorionated by submersion in 50% bleach for 4 min. Embryos were then fixed in a solution of equal parts heptane and 10% formalin (Sigma, Product # HT501128). Fixation lasted for 20 min during which time the embryos were placed on an orbital shaker that rotated at a rate of 250/min. Following fixation, the formalin and heptane were removed and replaced with a solution of equal parts methanol and heptane. The embryos were vortexed for 1 min to devitellinize the embryos. Embryos were stored in methanol at − 20 °C until immunostaining.
Preparation of larvae
Dissection of larvae was carried out as previously described [18] with minor modifications. The primary difference being that the buffer used was modified to increase the preservation of muscle structure. The modified dissection buffer was 100 mM PIPES (Sigma-Aldrich, P6757), 115 mM d-sucrose (Fisher Scientific, BP220-1), 5 mM trehalose (Acros Organics, 182550250), 10 mM sodium bicarbonate (Fisher Scientific, BP328-500), 75 mM potassium chloride (Fisher Scientific, P333-500), 4 mM magnesium chloride (Sigma-Aldrich, M1028), and 1 mM EGTA (Fisher Scientific, 28-071-G). Larvae were then fixed with 10% formalin (Sigma-Aldrich, HT501128) for 20 min. Briefly, dissection involved lateral cuts at the anterior and posterior end of the larva that encompassed 70% of larval circumference. These were followed by a longitudinal cut through the dorsal surface of the animal that connected the two lateral cuts. The intestines, other internal tissues, and neurons were then removed, and the flaps of tissue composed of epidermis and muscle were pinned down and fixed. For fixation, larvae were incubated in a solution of 10% formalin in PBS for 20 min.
Immunostaining
Staining of embryos and larvae was identical. Antibodies were used at the following dilutions: rabbit anti-dsRed (1:400, Clontech 632496), rat anti-tropomyosin (1:200, Abcam ab50567), mouse anti-GFP (1:50, Developmental Studies Hybridoma Bank GFP-G1), mouse anti-integrin βPS (1:50, Developmental Studies Hybridoma Bank CF.6G11), mouse anti-Fascin (1:25, Developmental Studies Hybridoma Bank sn 7C, generous gift from Tina Tootle), and mouse anti-αTubulin (1:200, Sigma-Aldrich T6199). Conjugated fluorescent secondary antibodies used were Alexa Fluor 555 donkey-anti-rabbit (1:200), Alexa Fluor 488 donkey-anti-rat (1:200), and Alexa Fluor 647 donkey-anti-mouse (1:200) (all Life Technologies) and Alexa Fluor 488 donkey-anti-mouse (1:200, Life Technologies). Furthermore, Acti-stain 555 phalloidin (1:400, Cytoskeleton PHDH1-A) and Hoechst 33342 (1 μg/ml) were used on larvae. Embryos and larvae were mounted in ProLong Gold (Life Technologies, P36930).
Microscopy
All microscopy was performed on a Zeiss LSM700 with an oil-immersion × 40 APOCHROMAT, 1.4 NA objective. All images of embryos, and the image of fascin localization in Fig. 2, were acquired with a 1.0× optical zoom. All other larval images were acquired with a 0.5× optical zoom. Image tiling was necessary to acquire images of the full larval muscles and was completed using the tiling function in the ZEN software that controls the microscope.
Statistics
All statistics were performed using GraphPad Prism. All data sets were compared to appropriate controls by a Student’s t test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.
Image analysis
Analysis of nuclear position in larvae
Although the field has traditionally measured the distance between nuclei [18, 20, 21], this measurement does not account for changes in muscle size and nuclear number. We have therefore modified this measurement to determine how evenly nuclei are spaced within a muscle [22]. First, the area and length of the muscle was measured. Next, the position and number of nuclei is calculated by using the multipoint tool in ImageJ to place a point in the center of each nucleus. The position of each nucleus is used to calculate the actual internuclear distance. The maximal internuclear distance is determined by taking the square root of the muscle area divided by the nuclear number. This value represents the distance between nuclei, if internuclear distance was fully maximized. The ratio between the actual internuclear distance and the maximal internuclear distance ratio was then used to determine how evenly nuclei were distributed. This method normalizes the internuclear distance to both nuclear count and muscle area which leads to a more representative means of comparison between muscles, larvae, and genotypes. All viable (not torn) ventral longitudinal (VL3) muscles were measured from each larva. At least four larvae from one experiment were measured for each genotype. Statistical analysis was performed with Prism 4.0 (GraphPad). Student’s t test was used to assess the statistical significance of differences in measurements between experimental genotypes and controls.
Analysis of nuclear position in embryos
The position of nuclei was measured in stage 16 embryos. This is the latest stage before cuticle development blocks the ability to perform immunofluorescence microscopy. Embryos were staged based primarily on gut morphology as previously described [21]. At stage 16, the nuclei are reliably positioned adjacent to the muscle ends, and disruptions in this positioning can be easily determined as previously described [21, 23, 24]. Images, acquired as described above, were processed as maximum intensity projections of confocal z-stacks using ImageJ. The position of the nuclei was determined by using the line function in ImageJ to measure the distance between either the dorsal end of the muscle and the nearest nucleus or the ventral end of the muscle and the nearest nucleus. All four LT muscles were measured in four hemisegments from each embryo. At least 20 embryos from at least two independent experiments were measured for each genotype with each data point representing the average for all muscles measured within a single embryo.
Analysis of nuclear count in embryos
The number of nuclei was counted using apRed fluorescence in the 4 lateral transverse (LT) muscles of stage 17 embryos, once nuclei had separated from their clusters and into easily distinguished individual nuclei. Only hemisegments in which there were four lines of nuclei, corresponding to 4 LT muscles, were counted. Data points indicate the number of apRed positive nuclei within a single hemisegment. A total of at least 49 hemisegments from at least 19 individual embryos were counted.
Analysis of nuclear count in larvae
The number of nuclei in the VL3 muscles were counted using Hoechst staining. Data points indicate the number of nuclei within an individual muscle. Careful analysis of z-stacks was used to ensure that nuclei were in the muscle that they were attributed to.
Analysis of muscle attachment in embryos
Integrin accumulation at the myotendinous junction of stage 16 embryos were measured in dorsal muscle 2 (DO2). Z-stack projection images that extended through the entire MTJ were used for these data. Integrin fluorescence intensity line scans were measured within a 10 ×10 μm box at the segment border using plot profile function in ImageJ. The width of the fluorescence peak composed of fluorescence intensities greater than 25% of the maximum intensity (75% fluorescence intensity peak) was measured. Each data points indicate the width of the 75% fluorescence intensity peak for a single myotendinous junction. A total of at least 50 MTJs from 20 different embryos were analyzed.
Analysis of muscle size in larvae
The area of the VL3 muscles were measured using the polygon selection tool in ImageJ as previously described [22]. Data points indicate the size of an individual muscle.
Analysis of general muscle architecture
Qualitative muscle phenotype analysis was completed on embryos of each genotype. All analysis was based on the immunofluorescence staining pattern of Tropomyosin in stage 16 embryos. The frequency of the following phenotypes were scored: the number of free myoblasts in an embryo that indicated a defect in myoblast fusion (small, unfused circles stained by anti-tropomyosin) and the number of muscles in each hemisegment (> 4 defined as extra muscles, < 4 defined as missing muscles) indicating gross abnormalities in the specification of muscle tissue. For analysis of the unfused myoblasts, embryos were grouped into bins with a width of 5 and the first bin centered on zero.