Myotube migration to cover and shape the testis of Drosophila depends on Heartless, Cadherin/Catenin, and myosin II.

During Drosophila metamorphosis, nascent testis myotubes migrate from the prospective seminal vesicle of the genital disc onto pupal testes and then further to cover the testes with multinucleated smooth-like muscles. Here we show that DWnt2 is likely required for determination of testis-relevant myoblasts on the genital disc. Knock down of fibroblast growth factor receptor (FGFR) heartless by RNAi and a dominant-negative version revealed multiple functions of Heartless, namely regulation of the amount of myoblasts on the genital disc, connection of seminal vesicles and testes, and migration of muscles along the testes. Live imaging indicated that the downstream effector Stumps is required for migration of testis myotubes on the testis towards the apical tip. After myoblast fusion, myosin II is needed for migration of nascent testis myotubes, in which Thisbe-dependent fibroblast growth factor (FGF) signaling is activated. Cadherin-N is essential for connecting these single myofibers and for creating a firm testis muscle sheath that shapes and stabilizes the testis tubule. Based on these results, we propose a model for the migration of testis myotubes in which nascent testis myotubes migrate as a collective onto and along the testis, dependent on FGF-regulated expression of myosin II.

A new level of plasticity: Drosophila smooth-like testes muscles compensate failure of myoblast fusion.

The testis of Drosophila resembles an individual testis tubule of mammals. Both are surrounded by a sheath of smooth muscles, which in Drosophila are multinuclear and originate from a pool of myoblasts that are set aside in the embryo and accumulate on the genital disc later in development. These muscle stem cells start to differentiate early during metamorphosis and give rise to all muscles of the inner male reproductive system. Shortly before the genital disc and the developing testes connect, multinuclear nascent myotubes appear on the anterior tips of the seminal vesicles. Here, we show that adhesion molecules are distinctly localized on the seminal vesicles; founder cell (FC)-like myoblasts express Dumbfounded (Duf) and Roughest (Rst), and fusion-competent myoblast (FCM)-like cells mainly express Sticks and stones (Sns). The smooth but multinuclear myotubes of the testes arose by myoblast fusion. RNAi-mediated attenuation of Sns or both Duf and Rst severely reduced the number of nuclei in the testes muscles. Duf and Rst probably act independently in this context. Despite reduced fusion in all of these RNAi-treated animals, myotubes migrated onto the testes, testes were shaped and coiled, muscle filaments were arranged as in the wild type and spermatogenesis proceeded normally. Hence, the testes muscles compensate for fusion defects so that the myofibres encircling the adult testes are indistinguishable from those of the wild type and male fertility is guaranteed.

Distinct genetic programs guide Drosophila circular and longitudinal visceral myoblast fusion.

BACKGROUND: The visceral musculature of Drosophila larvae comprises circular visceral muscles tightly interwoven with longitudinal visceral muscles. During myogenesis, the circular muscles arise by one-to-one fusion of a circular visceral founder cell (FC) with a visceral fusion-competent myoblast (FCM) from the trunk visceral mesoderm, and longitudinal muscles arise from FCs of the caudal visceral mesoderm. Longitudinal FCs migrate anteriorly under guidance of fibroblast growth factors during embryogenesis; it is proposed that they fuse with FCMs from the trunk visceral mesoderm to give rise to syncytia containing up to six nuclei. RESULTS: Using fluorescence in situ hybridization and immunochemical analyses, we investigated whether these fusion events during migration use the same molecular repertoire and cellular components as fusion-restricted myogenic adhesive structure (FuRMAS), the adhesive signaling center that mediates myoblast fusion in the somatic mesoderm. Longitudinal muscles were formed by the fusion of one FC with Sns-positive FCMs, and defects in FCM specification led to defects in longitudinal muscle formation. At the fusion sites, Duf/Kirre and the adaptor protein Rols7 accumulated in longitudinal FCs, and Blow and F-actin accumulated in FCMs. The accumulation of these four proteins at the fusion sites argues for FuRMAS-like adhesion and signaling centers. Longitudinal fusion was disturbed in rols and blow single, and scar wip double mutants. Mutants of wasp or its interaction partner wip had no defects in longitudinal fusion. CONCLUSIONS: Our results indicated that all embryonic fusion events depend on the same cell-adhesion molecules, but that the need for Rols7 and regulators of F-actin distinctly differs. Rols7 was required for longitudinal visceral and somatic myoblast fusion but not for circular visceral fusion. Importantly, longitudinal fusion depended on Kette and SCAR/Wave but was independent of WASp-dependent Arp2/3 activation. Thus, the complexity of the players involved in muscle formation increases from binucleated circular muscles to longitudinal visceral muscles to somatic muscles.

Myosin heavy chain-like localizes at cell contact sites during Drosophila myoblast fusion and interacts in vitro with Rolling pebbles 7.

Besides representing the sarcomeric thick filaments, myosins are involved in many cellular transport and motility processes. Myosin heavy chains are grouped into 18 classes. Here we show that in Drosophila, the unconventional group XVIII myosin heavy chain-like (Mhcl) is transcribed in the mesoderm of embryos, most prominently in founder cells (FCs). An ectopically expressed GFP-tagged Mhcl localizes in the growing muscle at cell-cell contacts towards the attached fusion competent myoblast (FCM). We further show that Mhcl interacts in vitro with the essential fusion protein Rolling pebbles 7 (Rols7), which is part of a protein complex established at cell contact sites (Fusion-restricted Myogenic-Adhesive Structure or FuRMAS). Here, branched F-actin is likely needed to widen the fusion pore and to integrate the myoblast into the growing muscle. We show that the localization of Mhcl is dependent on the presence of Rols7, and we postulate that Mhcl acts at the FuRMAS as an actin motor protein. We further show that Mhcl deficient embryos develop a wild-type musculature. We thus propose that Mhcl functions redundantly to other myosin heavy chains in myoblasts. Lastly, we found that the protein is detectable adjacent to the sarcomeric Z-discs, suggesting an additional function in mature muscles.

The syncytial visceral and somatic musculature develops independently of ß3-Tubulin during Drosophila embryogenesis, while maternally supplied ß1-Tubulin is stable until the early steps of myoblast fusion.

Microtubules are necessary for fusion and elongation of vertebrate muscle cells. In Drosophila, several isoforms of ß-Tubulin, the functional subunit of microtubules, are expressed in different tissues of the developing embryo, while solely the ß3-Tubulin isoform is detected in large amounts during differentiation of the somatic and visceral musculature. Here we show the unexpected result that all mesodermal tissues develop correctly in ß3-Tubulin loss of function mutants. Furthermore, we show that ß2-Tubulin transcripts are not detectable in embryos and an exceptional zygotic ß1-Tubulin expression in ß3-Tubulin mutants cannot be observed. Nevertheless, a maternally contributed ß1-Tubulin-GFP fusion protein (from protein trap collection, Buszczak et al., 2007, Genetics 175, 1505-1531) acts in a dominant negative way, disturbing embryonic development from early stages on. This effect can be observed to the same extent in a zygotic ß3-Tubulin mutant situation. Our results indicate that the maternally supplied ß1-Tubulin based microtubule network is sufficient for myoblast fusion, myotube elongation and sarcomere formation both during visceral and somatic muscle development in Drosophila embryogenesis.

Drosophila Swiprosin-1/EFHD2 accumulates at the prefusion complex stage during Drosophila myoblast fusion.

In the Drosophila embryo, transient cell adhesion during myoblast fusion is known to lead to the formation of fusion-restricted myogenic-adhesive structures (FuRMASs). Here, we report that within these FuRMASs, a Drosophila homologue of human and mouse swiprosins (EF-hand-domain-containing proteins) is expressed, which we named Drosophila Swiprosin-1 (Drosophila Swip-1). Drosophila Swip-1 is highly conserved and is closely related to the calcium-binding proteins swiprosin-1 and swiprosin-2 that have a role in the immune system in humans and mice. Our study shows that Drosophila Swip-1 is also expressed in corresponding cells of the Drosophila immune system. During myoblast fusion, Drosophila Swip-1 accumulates transiently in the foci of fusion-competent myoblasts (FCMs). Both the EF-hand and the coiled-coil domain of Drosophila Swip-1 are required to localise the protein to these foci. The formation of Drosophila Swip-1 foci requires successful cell adhesion between FCMs and founder cells (FCs) or growing myotubes. Moreover, Drosophila Swip-1 foci were found to increase in number in sing(22) mutants, which arrest myoblast fusion after prefusion complex formation. By contrast, Drosophila Swip-1 foci are not significantly enriched in blow(2) and kette(J4-48) mutants, which stop myogenesis beyond the prefusion complex stage but before plasma membrane merging. Therefore, we hypothesise that Drosophila Swip-1 participates in the breakdown of the prefusion complex during the progression of myoblast fusion.

Myoblast fusion in Drosophila melanogaster is mediated through a fusion-restricted myogenic-adhesive structure (FuRMAS).

During myogenesis in Drosophila embryos, a prominent adhesive structure is formed between precursor cells and fusion-competent myoblasts (fcms). Here, we show that Duf/Kirre and its interaction partners Rols7 (found in founder myoblasts and growing myotubes) and Sns (found in fcms) are organized in a ring-structure at the contact points of fcms with precursor cells, while cytoskeletal components like F-actin and Titin are centered in this ring in both cell types. The cytoplasmic protein Blow colocalizes with the actin plugs in fcms after cell adhesion. Furthermore, the requirement of additional as yet unidentified components was demonstrated by using mammalian C2C12 myoblasts. In this study, we propose that the fusion-restricted myogenic-adhesive structure (FuRMAS) is pivotal in linking cell adhesion as well as local F-actin assembly and dynamics to downstream events that ultimately lead to plasma membrane fusion. Moreover, we suggest that the FuRMAS may restrict the area of membrane breakdown.

Blown fuse regulates stretching and outgrowth but not myoblast fusion of the circular visceral muscles in Drosophila.

Circular visceral muscles of Drosophila are binuclear syncytia arising from fusion of two different kinds of myoblasts: a circular visceral founder cell and one visceral fusion-competent myoblast. In contrast to fusion leading to the somatic body-wall musculature, myoblast fusion for the circular visceral muscles does not result in massive syncytia but instead in syncytia interconnected with multiple cytoplasmic bridges, which differentiate into large web-shaped muscles. Here, we show that these syncytial circular visceral muscles build a gut-enclosing network with the interwoven longitudinal visceral muscles. At the ultrastructural level, during circular visceral myoblast fusion and the first step of somatic myoblast fusion prefusion complexes and electron-dense plaques were not detectable which was surprising as these structures are characteristic for the second step of somatic myoblast fusion. Moreover, we demonstrate that Blown fuse (Blow), a cytoplasmic protein essential for the second step of somatic myoblast fusion, plays a different role in circular visceral myogenesis. Blow is known to be essential for progression beyond the prefusion complex in the somatic mesoderm; however, analysis of blow mutants established that it has a restricted role in stretching and outgrowth of the syncytia in the circular visceral muscles. Furthermore, we also found that in the visceral mesoderm, Blow is expressed in both the fusion-competent myoblasts and circular visceral founders, while expression in the somatic mesoderm is initially restricted to fusion-competent myoblasts. We also demonstrate that different enhancer elements in the first intron of blow are responsible for this distinct expression pattern. Thus, we propose a model for Blow in which this protein is involved in at least two clearly differing processes during Drosophila muscle formation, namely somatic myoblast fusion on the one hand and stretching and outgrowth of circular visceral muscles on the other.

Drosophila rolling pebbles colocalises and putatively interacts with alpha-Actinin and the Sls isoform Zormin in the Z-discs of the sarcomere and with Dumbfounded/Kirre, alpha-Actinin and Zormin in the terminal Z-discs.

The rolling pebbles gene of Drosophila encodes two proteins, one of which, Rols7, is essential for myoblast fusion. In addition, Rols 7 is expressed during myofibrillogenesis and in the mature muscles. Here it overlaps with alpha-Actinin (alpha-Actn) and the N-terminus of D-Titin/Kettin/Zormin in the Z-line of the sarcomeres. In the attachment sites of the somatic muscles, Rols7 and the immunoglobulin superfamily protein Dumbfounded/Kin of irreC (Duf/Kirre) colocalise. As Duf/Kirre is detectable only transiently, it may be involved in establishing the first contact of the outgrowing muscle fiber to the epidermal attachment site. We propose that Rols7 and Duf/Kirre link the terminal Z-disc to the cell membrane by direct interaction. This is supported by the fact that in yeast two hybrid assays the tetratricopeptide repeat E (TPR E) of Rols7 shows interaction with the intracellular domain of Duf/Kirre. The colocalisation of Rols7 with alpha-Actn and with D-Titin/Kettin/Zormin in the Z-dics is reflected in interactions with different domains of Rols7 in this assay. In summary, these data show that besides the role in myoblast fusion, Rols7 is a scaffold protein during myofibrillogenesis and in the Z-line of the sarcomere as well as in the terminal Z-disc linking the muscle to the epidermal attachment sites.

In Drosophila melanogaster, the rolling pebbles isoform 6 (Rols6) is essential for proper Malpighian tubule morphology.

During myoblast fusion, cell-cell recognition along with cell migration and adhesion are essential biological processes. The factors involved in these processes include members of the immunoglobulin superfamily like Sticks and stones (Sns), Dumbfounded (Duf) and Hibris (Hbs), SH3 domain-containing adaptor molecules like Myoblast city (Mbc) and multidomain proteins like Rolling pebbles (Rols). For rolling pebbles, two differentially expressed transcripts have been defined (rols7 and rols6). However, to date, only a muscle fusion phenotype has been described and assigned to the lack of the mesoderm-specific expressed rols7 transcript. Here, we show that a loss of the second rolling pebbles transcript, rols6, which is expressed from the early bud to later embryonic stages during Malpighian tubule (MpT) development, leads to an abnormal MpT morphology that is not due to defects in cell determination or proliferation but to aberrant morphogenesis. In addition, when Myoblast city or Rac are knocked out, a similar phenotype is observed. Myoblast city and Rac are essentially involved in the development of the somatic muscles and were proposed to be interaction partners of Rols7. Because of the predicted structural similarities of the Rols7 and Rols6 proteins, we argue that genetic interaction of rols6, mbc and rac might lead to proper MpT morphology. We also propose that these interactions result in stable cell connections due to rearrangement of the cytoskeleton.