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First published online March 21, 2008
doi: 10.1242/10.1242/dev.012849

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RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance

¹ Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
² Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
³ Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA 01605, USA.

View larger version (61K): [in this window] [in a new window]   Fig. 1. Myogenesis in primary cultures derived from Drosophila embryos. (A-F) Fluorescence micrographs of freshly dissociated cells obtained from Drosophila gastrulating embryos carrying rp298-lacZ immediately following plating. Cells are stained using DAPI for nuclei (A, and blue in C), and antibodies targeting Dmef2 (B, and green in C,E), β-galactosidase (D, and red in E,F) and Lmd (green in F). (G-I) Primary cells derived from Dmef2-Gal4 embryos were mixed with those from UAS-2EGFP and allowed to develop for 48 hours at 18°C in culture. The GFP-positive myotube (G, and green in I) resulted from fusion of cells supplied by two genetically different embryos, and the GFP-negative one is most likely derived from the fusion of cells from two genetically identical embryos. Both myotubes expressed Mhc (H, and red in I). (J-L) Multinucleated myotubes are identified by staining for Mhc (J, and green in L) and for Dmef2 (K, and red in L). Note that not all Dmef2-positive nuclei are found in myotubes. The percentage of myotube nuclei among the total number of Dmef2-positive nuclei was used as an indication of the amount of fusion. (M) Time-course of myoblast fusion at 18°C and 25°C. Primary cell cultures were fixed and stained for Dmef2, Mhc or Actin at the times indicated. The number of Dmef2-positive nuclei was counted using Autoscope and Metamorph software. The number of nuclei in the myotubes was determined manually. The percentage of myotube nuclei was estimated by the number of myotube nuclei among the total Dmef2-positive nuclei, and used as an indication of the extent of myoblast fusion. Each point represents the average results of two or three trials. Arrows point to the time when fusion is nearly complete (pink for 25°C and blue for 18°C). (N) Fluorescence micrograph of a primary myotube from a 2-day culture at 18°C stained for Mhc. The white arrowhead points to the immature myofibril that formed along the side of the myotube. (O-R) Primary myotubes from 11-day cultures at 18°C, stained for Mhc (O), Actin (as detected using phalloidin) (P), Actn (Q) and Tropomyosin (R). The short arrow in O indicates the bundled myofibrils. Scale bars: 20 µm, in A for A-L and in N for N-Q.

View larger version (60K): [in this window] [in a new window]   Fig. 2. Primary cultures derived from Drosophila embryonic cells contain a mixture of different cell populations that include muscles and neurons. (A-D) Primary myotubes strongly stained by phalloidin (A, red in the merged image in D) are all stained for Mhc (B, green in the merged image in D). Note that other cell types whose nuclei are revealed by DAPI (C) are faintly visible by phalloidin staining. As myotubes mature they become more contractile, some detach from the tissue culture surface, and are seen as round muscles (arrows in A,B,D). (E-H) Primary cells were isolated from Dmef2-Gal4, D42-Gal4, UAS-mito-GFP embryos in which Dmef2-Gal4 and D42-Gal4 drive expression of mito-GFP, a mitochondrial marker transgene that fuses the mitochondrial targeting signal to the N-terminus of EGFP, in muscles and motoneurons, respectively. Muscle structure is visualized by phalloidin staining of Actin (E, red in the merged image in H), and neurons can be seen in F (green in the merged image in H) as they stain strongly with mito-GFP but not phalloidin (triangles in F,H). In addition to neurons and muscles, other cells are present in the culture, as revealed by the staining with DAPI (G, blue in the merged image in H). In H, muscles are shown in red and yellow, neurons and their extensions in green only. Scale bar: 50 µm.

View larger version (59K): [in this window] [in a new window]   Fig. 3. Gene-specific RNAi effects in primary cells. Primary cells were isolated from G053 Drosophila embryos expressing the SLS-GFP fusion protein and were treated with dsRNAs targeting lacZ (A,D), sls (B,E) and Mhc (C,F). SLS-GFP expression was detected by GFP (A-C, green in D-F). Muscle structure was revealed by SLS-GFP in green, phalloidin staining of Actin in red, and DAPI staining of nuclei in blue (D,E,F). Scale bar: 20 µm.

View larger version (72K): [in this window] [in a new window]   Fig. 4. Phenotypic classes identified from the RNAi screen. (A) Protocol for RNAi screening in primary cultures. (B-F) Four distinct classes of muscle phenotype were distinguished based on the staining of Actin using phalloidin (left panels, and red in right panels), Mhc (middle panels) and of nuclei with DAPI (blue in right panels). (B) Wild-type control myotubes treated with dsRNAs targeting lacZ. (C) Class I (treated with mew dsRNAs). (D) Class II (treated with sls dsRNAs). (E) Class III (treated with Mlc2 dsRNAs). (F) Class IV (treated with Actin dsRNAs simultaneously targeting all Actin isoforms, including Act42A, Act57B, Act5C, Act79B, Act87E and Act88F). Note that the four phenotypic classes did not result from fewer fusions, as muscles contained the same number of nuclei as controls. Arrowheads point to the nuclei. Scale bar: 15 µm.

View larger version (97K): [in this window] [in a new window]   Fig. 5. In vivo validation of Fit1 and Fit2 using dsRNA injection. (A-E) Micrographs of whole-mount in situ hybridizations of Drosophila embryos with Dig-labeled antisense probes specifically targeting Fit1 (A-C) and Fit2 (D,E), oriented anterior to the left. (A,D) Lateral view of stage 14 embryos. (B) Dorsal view of a stage 16 embryo focusing on visceral muscle and somatic body wall muscles. (C) High-magnification image showing Fit1 somatic body wall muscle expression. Arrowhead, somatic body wall muscles; arrow, visceral gut muscles. (E) Lateral view of a stage 16 embryo. (F-J) Fluorescence micrographs of stage 17 embryos carrying MHC-GFP. dsRNAs targeting (F) lacZ (2 µg/µl), (G) mys (2 µg/µl), (H) Fit1 (2 µg/µl), (I) Fit2 (2 µg/µl) and (J) Fit1 (1 µg/µl) + Fit2 (1 µg/µl) were injected into MHC-GFP embryos. MHC-GFP allows visualization of all somatic muscles, as shown in F, where the embryos were injected with a negative control dsRNA targeting lacZ (n=67, none showed muscle phenotypes). Note that severely rounded muscles are present in the embryos injected with dsRNAs targeting mys (G) (100% penetrance, n=87, where n is the number of embryos injected) and Fit1 + Fit2 (J) (96% penetrance, n=150), whereas dsRNAs targeting either Fit1 (H) or Fit2 (I) alone only caused some muscles to round up (short arrows in H,I). Long arrows in H-J point to the ventral acute muscles that are still present as fibers in H and I, but round up in J. Scale bars: 50 µm in A for A-E, in F for F-J.

View larger version (72K): [in this window] [in a new window]   Fig. 6. In vivo validation of CG2165 using transgenic RNAi. (A-E) Wild-type (A,B) and CG2165 RNAi (C-E) primary muscle phenotypes at 18°C cultured for 4 days (A,C), 8 days (E) and 11 days (B,D), revealed by phalloidin staining for Actin. (F) Western blots probed with rabbit anti-Drosophila PMCA (top) and mouse anti-tubulin (bottom, as loading controls). Note that the expression of PMCA was significantly reduced in muscle-specific CG2165 RNAi larvae (lane 2) compared with wild type (lane 1). (G) Comparison of the body size in wild-type (top) and muscle-specific CG2165 RNAi (bottom) first instar larvae of the same age (30 hours AEL at 25°C). Note the short size and hypercontracted appearance of muscle-specific CG2165 RNAi larvae. (H,I) Confocal fluorescent micrographs showing the ventral internal muscles of first instar larvae of UAS-Dcr-2; Dmef2-Gal4 (H) and muscle-specific CG2165 RNAi (I) stained for Mhc and Actn. Arrows in I point to the rounded-up muscles. Note that although both control and muscle-specific CG2165 RNAi VL4 muscles (brackets) contained a comparable number of sarcomeres longitudinally, the length of CG2165 RNAi VL4 muscles is only half that of the wild type (lines labeled L), and thus the sarcomere size was only 50% of that in wild type. Also note that the transverse distance (T) between two VL4 muscles in the same segment in muscle-specific CG2165 RNAi larvae is much greater than that in the wild type (lines labeled T). (J) Fluorescent micrograph showing a larva with almost complete rounded-up muscles as revealed by staining for Mhc. (K) Fura PE 3 ratiometric calcium imaging micrographs of primary muscles derived from embryos of wild type (top) and UAS-Dcr-2/+; Dmef2-Gal4/UAS-CG2165 hp (bottom) and cultured at 25°C for 3 days. The color indicates the ratio between the emission intensities excited at 340 nm and 380 nm, and reflects a measurement of calcium concentration. (L) Bar chart showing [Ca2+]i as average±s.e.m. for wild-type control cells (0.344±0.0162 µM; n=35 muscle cells in two representative experiments, white bar), and for muscle-specific CG2165 RNAi (0.0105±0.0012 µM; n=71 in three representative experiments, gray bar). Scale bars: 50 µm in A for A-E; 20 µm in H for H,I; 75 µm in J.