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First published online 23 January 2008
doi: 10.1242/dev.015529

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Mind bomb 2, a founder myoblast-specific protein, regulates myoblast fusion and muscle stability

Centro de Biología Molecular Severo Ochoa, CSIC and UAM, Universidad Autónoma de Madrid, 28049, Madrid, Spain.

View larger version (84K): [in this window] [in a new window]   Fig. 1. Embryonic expression of mib2 in Drosophila. (A-D) mib2 RNA in situ hybridisations to wild-type embryos. (A,B) In stage 11 embryos, mib2 is expressed in founders of the somatic (arrowheads) and circular visceral (arrow) muscles. (C) Detail of a late stage 12 embryo (inset). mib2 expression occurs in founders of the longitudinal visceral muscles (arrow). (D) At stage 14, mib2 expression is maintained in muscle precursors and is also detected in cardioblasts (asterisk). (E-G) Mib2 (green) accumulates in founders of circular visceral muscles (E, arrow) and in somatic founders, revealed by anti-β-gal accumulating in the nuclei (red) in rP298-lacZ embryos (F, enlarged in G). (H-I'') Mib2 (green) accumulates in the cytoplasm but not in Connectin-labelled membranes (red, arrows in H-H''). Mib2 is not enriched at muscle attachments (anti-Kettin, red, arrows in I-I''). See Fig. 3 for a diagram of the muscle pattern. In all embryos, anterior is to the left and dorsal to the top, except for the ventral view in E.

View larger version (31K): [in this window] [in a new window]   Fig. 2. Organisation of the mib2 locus and the Mib2 protein. (A) The Drosophila mib2 locus, as summarised by the BDGP and our sequence analysis of mib2 alleles and cDNA. The horizontal line represents genomic DNA; the white boxes represent genes mapping in the 37B region; the green boxes represent exons of mib2. Positions of P-element insertion in mib2KG10508 (red arrowhead), nonsense codon in mib21 (red arrow) and mib2r14 deletion (bracket) are indicated. Below the cDNA, coloured lines indicate the extent of Mib2 domains. Modular organisation of Mib2 protein (1049 amino acids) and comparison with Mib1 and SKE. The red line represents the region of mib2 sequence used to make the RNAi construct. (B) Alignment of ankyrin (framed in orange) and RING (framed in blue) domains of Mib2, SKE and Mib1 proteins. Percentage identity between Mib2 and SKE or MIb1 is shown in the column on the right. Consensus sequence for the RING-finger motif is indicated below the blue box.

View larger version (114K): [in this window] [in a new window]   Fig. 3. Embryonic loss-of-function phenotypes of mib2 alleles. (A-C) Lateral views of the ventral region of stage 16 wild-type (A), mib21 (B) and mib21 (C) germline clone Drosophila embryos stained with anti-myosin (A,B) or anti-Tropomyosin (C). Positions of VA2, VT1 and VO muscles are indicated. Note the presence of unfused myoblasts (black arrowheads in B,C), the absence of muscles in the VL region (asterisks in B,C, and muscle scheme in Fig. 4G), and presence of myospheres (arrow, B). Whole-embryo pictures (below) are focused on the gut. White arrowheads point to the position of the first midgut constriction (A) and to the unfolded midgut (B,C). (D,E) Dorsal views focused on the DA1 muscle of mib21/CyO and mib21 stage 15 embryos, respectively. Arrowheads point to Eve-labelled nuclei (green). Asterisks mark nuclei of pericardial cells. (F) Number of eve-expressing nuclei in DA1 muscles of embryos of the indicated genotype (n, number of hemisegments quantified). (G-J) Progressive degeneration of muscles in mib21 embryos. Muscles were revealed by Mhc--GFP (G,I,J) and by anti-Tm staining (H). At stage 15 (H), all muscles are present and attached to the apodemes (arrowhead, unfused myoblast). At stage 16 (I), some muscle attachments are becoming thinner (arrowhead), while others, many in the VL region, have detached (arrows). In L1 larvae, most muscles are missing (J). (K) Scheme indicating percentages of specific muscles that remain attached to apodemes in mib21 first instar larvae: red (80-100%), blue (50-80%), yellow (10-25%) and white (<10%) (n=50 abdominal hemisegments; 2 to 5 examined).

View larger version (97K): [in this window] [in a new window]   Fig. 4. Embryonic rescue of mib2 phenotypes and ultrastructure organisation of mib2 larval muscles. (A-C) Dorsal region of stage 17 kettin-GFP (A), mib21; kettin-GFP (B) and mib21; kettin-GFP rescued with UAS-mib2-C935S-C1020S (C) embryos. Note the alignment of Z bands in the DO1/2 muscles in A and C (arrowheads), and the distorted (arrow), irregular (asterisk) or absent (arrowhead) bands in B. Images in A and C also show the Z bands of the immediately overlying DA1/2 muscles (see F for dorsal muscle scheme). (D-E') Mesodermal phenotype of stage 16 mib21 embryos (anti-myosin stained), expressing UAS-mib2-FL (D,D') and UAS-mib2-C935S-C1020S (E,E') (24BGAL4 driver). Gut constrictions (D,E) and external (D',E', left) and internal (D',E', right) views of the ventral muscles schematised in G are shown. Mib2-FL completely rescues the mutant phenotype (D,D', compare with Fig. 3A,B). Mib2-C935S-C1020S rescues all embryonic phenotypes except the fusion defect, indicated by the presence of unfused myoblasts (arrowhead in E') and number of DA1 nuclei (Fig. 3F). Some embryos also failed to complete the first gut constriction (arrowhead, E), a phenotype common to most fusion mutants. (F) Scheme indicating dorsal muscles. (G) Scheme indicating ventral muscles (brown) missing in Fig. 3B,C.

View larger version (92K): [in this window] [in a new window]   Fig. 5. Effects of Mib2 mis-expression in FCMs and its requirement in FMs to eliminate Lmd from myotubes. In all embryos, the lateral-ventral region is shown and anterior is to the left. (A) Mef2GAL4; 2xUAS-mib2, (B) snsGAL4; 2xUAS-mib2 and (C) Mef2GAL4; 2xUAS-mib2-C935S-C1020S Drosophila embryos. (A',B',C') Higher magnification views of corresponding embryos. Note the presence in A' and B' (but not in C') of thin (mono/bi-nucleated) myotubes (anti-myosin staining). (D-F) Mef2GAL4; 2xUAS-mib2 embryos. (D) Duf (green) and Rols (red) co-localise to the membrane of mini-myotubes (arrowheads) induced by overexpression of Mib2-FL. (E,F) Overexpression promotes death (activated-Caspase 3 expression, red) of FCMs (Kettin labelling, green) starting at stage 12 (E) and most prevalent at stage 14 (F). Mini-myotubes did not express activated-Caspase 3 (arrowhead in F). (G,H) Late stage 13 yw and mib21 embryos, respectively, stained with anti-Connectin (red) and anti-Lmd (green). (I) Schematic representation of late stage 13 Connectin-expressing precursors. (J,K) Stage 16 mib21/CyO and mib21 embryos stained with anti-Tm (red) and anti-Lmd (green). Lmd is not detected in early precursors (G) or late myotubes (J) of wild-type embryos, but it is not eliminated in mib21 embryos (H,K).

View larger version (39K): [in this window] [in a new window]   Fig. 6. Mib2 localisation and functional analysis in Drosophila adult muscle. (A-A''') Mib2 (green) accumulates at the Z band (Kettin staining, blue) of myofibrils and to lesser an extent in the M band (absence of actin). (B-D') Electron micrographs of longitudinal sections of IFM from 3-day-old wild-type (B,B'), 1-day-old (C,C') and 3-day-old (D,D') 1151-GAL4/UAS-RNAi-mib2 adults. IFMs of 1151-GAL4/UAS-RNAi-mib2 young adults show poorly defined M bands and irregular Z disks (C'), compared with the wild type (B'). Three-day-old 1151-GAL4/UAS-RNAi-mib2 IFMs lack sarcomeres and display remains of dense material that probably corresponds to distorted and split Z bands (D', arrow and arrowhead, respectively). (E-G) Co-immunoprecipitation experiments showing a physical interaction between Mib2 and nonmuscle myosin. Protein extracts from sqh-GFP thoraces were immunoprecipitated with anti-Mib2 (E), anti-GFP (F) or anti-nonmuscle Mhc (G) antibodies, followed by immunoblotting with anti-GFP (E) or anti-Mib2 (F,G). Note the presence of bands of 47 kDa corresponding to Sqh-GFP (arrowhead in E) and 115 kDa corresponding to Mib2 (arrowheads in F,G) in the experimental and their absence in the control P lanes. In E, the weak bands detected in P lanes correspond to immunoglobulins. Input, immunoprecipitation reaction samples; P, immunoprecipitated pellets; S, supernatants. Scale bars: 0.5 µm in B; 0.1 µm in B'.