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First published online September 1, 2004
doi: 10.1242/10.1242/dev.01309

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kette and blown fuse interact genetically during the second fusion step of myogenesis in Drosophila

Roxane H. Schröter^1,*, Simone Lier^1,*,, Anne Holz^1,, Sven Bogdan³, Christian Klämbt³, Lothar Beck² and Renate Renkawitz-Pohl^1,

¹ Philipps-Universität Marburg, Fachbereich Biologie, Entwicklungsbiologie, Karl-von-Frisch Strasse 8, 35043 Marburg, Germany
² Philipps-Universität Marburg, Fachbereich Biologie, Spezielle Zoologie, Karl-von-Frisch Strasse 8, 35043 Marburg, Germany
³ Institut für Neuround Verhaltensbiologie, Westfälische Wilhelms-Universität Münster, Badestrasse 9, 48149 Münster, Germany

View larger version (121K): [in a new window]   Fig. 1. Kette has a mesoderm intrinsic essential function for myoblast fusion. kette mutants show a strong defect in muscle fusion that is due to an intrinsic mesodermal function of Kette. (A-F) Anti-ß3-Tubulin fluorescent staining of stage 16 embryos shows the myogenic defects of kette mutants. (A) Wild-type muscle pattern; (B) ketteJ4-48 (null allele), which shows many unfused myoblasts, even at stage 16. (C) Hypomorphic allele ketteG1-37. (D-F) Detailed magnifications of A-C. The dorsal vessel (dv) is formed correctly in all kette mutants. (E) ketteJ4-48 null mutant: mini-muscles are indicated by arrows. (F) ketteG1-37 stage 16 embryo: the unfused myoblasts have vanished. Large gaps in the muscle pattern and attachment defects (arrow) are visible. (G,H) Anti-Kette antibody staining on wild-type embryos shows the mesodermal expression of Kette. (G) In stage 16 embryos, the protein concentrates towards the muscle tips. (H) In stage 14 embryos, when muscle fusion takes place, Kette can be found in the whole somatic mesoderm. (I,J) Overexpression of Kette in the mesoderm in a kette mutant background with the help of a twi-GAL4 driver line rescues the kette phenotype. (I) Ventrolateral view of a rescued stage 16 embryo. (J) Higher magnification and lateral view of a rescued stage 16 embryo; only a few unfused myoblasts can be detected (arrow). (K) Anti-Kette (green) anti-ß-galactosidase (red) double labelling of rP298-expressing stage 13 wild-type embryo. (L,M) Anti-ß3-Tubulin and anti-Alien double labelling, monitoring the muscle attachment to the epidermis. (L) Wild-type stage 16 embryo with properly attached muscles. (M) Stage 16 ketteG1-37 mutant showing partly attached (arrowhead) and partly unattached (arrows) muscles. Unless otherwise stated, embryos in all figures are orientated with anterior towards the left.

View larger version (122K): [in a new window]   Fig. 2. Founders and fusion-competent myoblasts are determined in kette-null mutants. kette mutants were analysed for expression of the enhancer trap rP298-lacZ (A-D) and localisation of the sns-transcript (E,F) to examine the determination of founders and fusion-competent myoblasts. (A-D) Expression pattern of the rP298-lacZ enhancer trap (green), which resembles the founder cell marker duf/kirre expression pattern, shown by anti ß-galactosidase fluorescent staining. ß3-Tubulin fluorescent staining is red. (A) Stage 13 heterozygous wild-type embryo with TDLZ blue-balancer. (B) Stage 13 ketteJ4-48-null mutant. (C) Late stage 13 wild-type embryos. After fusion, all nuclei of the syncytia express rP298. The forming dorsal muscles contain more than four nuclei and muscle structures is visible. (D) In stage 13 ketteJ4-48-null mutants, founder cells are determined but no muscle structure are visible. Compared with wild type, less rP298-positive cells are detected that are surrounded by many fusion-competent ß3-Tubulin-positive myoblasts (arrow). (E,F) Fluorescence in situ hybridisation with sns-antisense probe shows the correct determination of fusion-competent myoblasts in the somatic (sm) and visceral mesoderm (vm) of kette mutants. (E) Stage 11 wild-type embryo; (F) stage 11 ketteJ4-48-null mutant embryo.

View larger version (76K): [in a new window]   Fig. 3. kette and blow mutants form precursor cells with three or four nuclei. kette and blow mutants were analysed for founder/precursor cell status (using Eve and Kr) at (A-C) stage 15-16 and (D-G) stage 13. As a control for the Kr pattern of expression, we used mbc mutant embryos, which do not undergo any fusion step and arrest at the founder cell stage. It can be clearly seen that both kette and blow mutants build precursors that contain three or four nuclei, corresponding to what is seen in the wild type at stage 13. (A-C) Anti-Eve fluorescent staining in red (pc, pericardial cells). (A) Stage 16 wild-type embryo containing up to 14 Eve- and rP298-positive nuclei in DA1 muscle after fusion is completed. (B) Stage 15 ketteJ4-48 null mutant embryo containing three or four Eve-positive nuclei in muscle DA1, corresponding to precursor cells. (C) Stage 15 blow2 mutant with two or three nuclei in DA1 muscle, which also indicates the presence of precursor cells. (D-G) Anti-Kr fluorescent staining. (D) Stage 13 wild-type embryo displays precursor cells of muscles DA1 and DO1 with three or four nuclei after the first fusion step occurs. (E) Stage 13 mbc mutant embryo does not undergo the first fusion step, indicated by the single kr-expressing cells that correspond to the founder cells of the muscles. (F) Stage 13 ketteJ4-48-null mutant embryo with three or four nuclei in precursors of muscles DA1 and DO1. (G) Stage 13 blow2 mutant embryo with two or three nuclei in precursors of DA1 and DO1. Arrows in D-G indicate precursors of lateral muscles that are developing.

View larger version (173K): [in a new window]   Fig. 4. kette mutants stop fusion during formation of electron-dense plaques. (A-C) Transmission electron microscope analyses of ketteJ4-48, blow2 and mbc mutants confirm that ketteJ4-48 and blow2 mutants do form muscle precursor cells, while mbc mutants do not. Scale bars: 2 µm. (A) Stage 14 ketteJ4-48 mutant embryo; asterisks indicate precursors with two or three nuclei. (B) Stage 12-13 blow2 mutant embryo; developing precursors with two nuclei are clearly visible. (C) Stage 13-14 mbc mutant embryo. (D-F) Stage 13 wild-type embryo. (D) A muscle precursor has established contact with fusion-competent myoblasts, while groups of electron dense vesicles start to build the prefusion complex of paired vesicles (arrowheads). Nearby, a prefusion complex has already started to dissolve and will form a electron-dense plaque (arrow). Scale bar: 1.5 µm. (E,F) Detailed view of a group of electron dense vesicles in D. (F) Dissolving prefusion complex and developing electron dense-plaque (arrow) forming within a cloud of vesicles. Scale bar: 500 nm. (G) Developing electron-dense plaque in a stage 15 ketteJ4-48 embryo. Remains of the dissolving prefusion complex are still visible (arrow); the length of the plaque is nearly twice that of the wild type plaque described by Doberstein et al. (Doberstein et al., 1997). Scale bar: 150 nm.

View larger version (68K): [in a new window]   Fig. 5. Kette and Blown fuse interact genetically. (A-D) Anti-ß3-Tubulin fluorescent staining of stage 15 embryos. (A) Homozygous ketteG1-37 hypomorphic mutant. (B) Homozygous blow2 null mutant. (C) Homozygous ketteG1-37 mutant with only one intact copy of blow (an enhancement of fusion defect takes place). (D) Double homozygous blow2 and ketteG1-37 mutant (further enhancement of fusion defects leads to a phenotype that resembles the original blow2 phenotype more than the ketteG1-37 phenotype). (E,F) Anti-ß3-Tubulin fluorescent staining (red); anti-ß-galactosidase staining (green) of rP298-lacZ enhancer trap. (E) Stage 16 blow2 mutant embryo (only minimuscles, presumably representing precursors, are formed, lareg gaps in the somatic mesoderm allow a direct view of the gut); ß3-Tubulin-positive cardioblasts are properly arranged. At this stage, unfused myoblasts have been engulfed by macrophages. (F) Mesodermal overexpression of UAS-hem with a twist-GAL4 driver line in blow2 mutant background rescues the blow phenotype, at least partially, at stage 16. The embryo forms clearly fused and attached muscles, although defects in muscle number, size and attachment occur.

View larger version (31K): [in a new window]   Fig. 6. Model of the two-step myoblast fusion process. The cell-cell recognition is mediated by Duf in the founder/precursor cells (yellow) and Sns in the fusion-competent myoblasts (orange). The first fusion step crucially depends on Mbc and Loner (Chen and Olson, 2003; Erickson et al., 1997). The second fusion step is characterised by formation of prefusion complexes, electron-dense plaques and membrane breakdown (Doberstein et al., 1997). This requires proper function of Rols, Blow and Kette. In this process, blow, kette and sns15 mutants are involved at different times. Kette acts during the formation of the electron-dense plaques shortly after Blow.

View larger version (31K): [in a new window]   Fig. 7. Hypothesis for the function of Kette during the second myoblast fusion step compared with the known function of Kette during axonal outgrowth. We propose that the function of Dock/NCK, which mediates Kette function during neurogenesis (Bogdan and Klämbt, 2003), is taken over by Crk, which has also been shown to interact with Blow and Mbc, and to be involved in Rac1 activation and the formation of lamellipodia (see text for details).