Drosophila rolling pebbles: a multidomain protein required for myoblast fusion that recruits D-Titin in response to the myoblast attractant Dumbfounded.

The fusion of myoblasts leading to the formation of myotubes is an integral part of skeletal myogenesis in many organisms. In Drosophila, specialized founder myoblasts initiate fusion through expression of the receptor-like attractant Dumbfounded (Duf), which brings them into close contact with other myoblasts. Here, we identify Rols7, a gene expressed in founders, as an essential component for fusion during myotube formation. During fusion, Rols7 localizes in a Duf-dependent manner at membrane sites that contact other myoblasts. These sites are also enriched with D-Titin, which functions to maintain myotube structure and morphology. When Rols7 is absent or its localization is perturbed, the enrichment of D-Titin fails to occur. Rols7 integrates the initial event of myoblast attraction with the downstream event of myotube structural organization by linking Duf to D-Titin.     

Antisocial, an intracellular adaptor protein, is required for myoblast fusion in Drosophila.

Somatic muscle formation in Drosophila requires fusion of muscle founder cells with fusion-competent myoblasts. In a genetic screen for genes that control muscle development, we identified antisocial (ants), a gene that encodes an ankyrin repeat-, TPR repeat-, and RING finger-containing protein, required for myoblast fusion. In ants mutant embryos, founder cells and fusion-competent myoblasts are properly specified and patterned, but they are unable to form myotubes. ANTS, which is expressed specifically in founder cells, interacts with the cytoplasmic domain of Dumbfounded, a founder cell transmembrane receptor, and with Myoblast city, a cytoskeletal protein, both of which are also required for myoblast fusion. These findings suggest that ANTS functions as an intracellular adaptor protein that relays signals from Dumbfounded to the cytoskeleton during myoblast fusion.     

An interplay between two EGF-receptor ligands, Vein and Spitz, is required for the formation of a subset of muscle precursors in Drosophila.

Activation of the Drosophila EGF-receptor (DER) is spatially and temporally controlled by the release of its various ligands. DER and its ligand Spitz mediate the formation of specific somatic muscle precursors. We show that a second DER ligand, Vein, complements the activity of Spitz in the development of various somatic muscle precursors. In vn mutant embryos, the DER-dependent muscle precursors do not form in some of the segments. This phenotype is significantly enhanced in embryos carrying only one copy of wild type spitz. Our analysis suggests that Vein activation of DER differs qualitatively from that of Spitz in that it does not lead to the expression of the inhibitory protein Argos, possibly leading to a continuous activation of the DER signaling pathway.     

The actin nucleator WASp is required for myoblast fusion during adult Drosophila myogenesis

Myoblast fusion provides a fundamental, conserved mechanism for muscle fiber growth. We demonstrate here that the functional contribution of Wsp, the Drosophila homolog of the conserved actin nucleation-promoting factor (NPF) WASp, is essential for myoblast fusion during the formation of muscles of the adult fly. Disruption of Wsp function results in complete arrest of myoblast fusion in all muscles examined. Wsp activity during adult Drosophila myogenesis is specifically required for muscle cell fusion and is crucial both for the formation of new muscle fibers and for the growth of muscles derived from persistent larval templates. Although Wsp is expressed both in fibers and individual myoblasts, its activity in either one of these cell types is sufficient. SCAR, a second major Arp2/3 NPF, is also required during adult myoblast fusion. Formation of fusion-associated actin 'foci' is dependent on Arp2/3 complex function, but appears to rely on a distinct, unknown nucleator. The comprehensive nature of these requirements identifies Arp2/3-based branched actin polymerization as a universal mechanism underlying myoblast fusion.     

Drosophila mind bomb2 is required for maintaining muscle integrity and survival

We report that the Drosophila mind bomb2 (mib2) gene is a novel regulator of muscle development. Unlike its paralogue, mib1, zygotic expression of mib2 is restricted to somatic and visceral muscle progenitors, and their respective differentiated musculatures. We demonstrate that in embryos that lack functional Mib2, muscle detachment is observed beginning in mid stage 15 and progresses rapidly, culminating in catastrophic degeneration and loss of most somatic muscles by stage 17. Notably, the degenerating muscles are positive for apoptosis markers, and inhibition of apoptosis in muscles prevents to a significant degree the muscle defects. Rescue experiments with Mib1 and Neuralized show further that these E3 ubiquitin ligases are not capable of ameliorating the muscle mutant phenotype of mib2. Our data suggest strongly that mib2 is involved in a novel Notch- and integrin-independent pathway that maintains the integrity of fully differentiated muscles and prevents their apoptotic degeneration.     

The notch signaling pathway is required to specify muscle progenitor cells in Drosophila.

Organization and function of the Notch signaling pathway in Drosophila are best understood with respect to its role in the process of selection of neural progenitor cells. However, there is evidence that, besides neurogenesis, the Notch signaling pathway is involved in several other developmental processes, one of which is the selection of muscle progenitor cells. Thus, the number of these cells is increased in neurogenic mutants, and it has been proposed that muscle progenitor cells are selected from clusters of equivalent cells expressing genes of the achaete-scute gene complex (AS-C). Here, I present evidence for the participation of additional elements of the Notch signaling pathway in myogenesis. Gal4 mediated expression of a Notch variant, E(spl) and Hairless shows that the selection of muscle progenitor cells obeys principles apparently identical to those acting at the selection of neural progenitor cells.     

Hedgehog signaling is required for commitment but not initial induction of slow muscle precursors

In the embryonic mouse retina, retinoic acid (RA) is unevenly distributed along the dorsoventral axis: RA-rich zones in dorsal and ventral retina are separated by a horizontal RA-poor stripe that contains the RA-inactivating enzyme CYP26A1. To explore the developmental role of this arrangement, we studied formation of the retina and its projections in Cyp26a1 null-mutant mice. Expression of several dorsoventral markers was not affected, indicating that CYP26A1 is not required for establishing the dorsoventral retina axis. Analysis of the mutation on a RA-reporter mouse background confirmed, as expected, that the RA-poor stripe was missing in the retina and its projections at the time when the optic axons first grow over the diencephalon. A day later, however, a gap appeared both in retina and retinofugal projections. As explanation, we found that CYP26C1, another RA-degrading enzyme, had emerged centrally in a narrower domain within the RA-poor stripe. While RA applications increased retinal Cyp26a1 expression, they slightly reduced Cyp26c1. These observations indicate that the two enzymes function independently. The safeguard of the RA-poor stripe by two distinct enzymes during later development points to a role in maturation of a significant functional feature like an area of higher visual acuity that develops at its location.     

Drosophila SPD-2 is an essential centriole component required for PCM recruitment and astral-microtubule nucleation

SPD-2 is a C. elegans centriolar protein required for both centriole duplication and pericentriolar material (PCM) recruitment [1-4]. SPD-2 is conserved in Drosophila (DSpd-2) and is a component of the fly centriole [5-7]. The analysis of a P element-induced hypomorphic mutation has shown that DSpd-2 is primarily required for PCM recruitment at the sperm centriole but is dispensable for both centriole duplication and aster formation [5]. Here we show that null mutations carrying early stop codons in the DSpd-2 coding sequence suppress astral microtubule (MT) nucleation in both neuroblasts (NBs) and spermatocytes. These mutations also disrupt proper Miranda localization in dividing NBs, as previously observed in mutants lacking astral MTs [8-10]. Spermatocyte analysis revealed that DSpd-2 is enriched at both the centrioles and the PCM and is required for the maintenance of cohesion between the two centrioles but not for centriole duplication. We found that DSpd-2 localization at the centrosome requires the wild-type activity of Asl but is independent of the function of D-PLP, Cnn, gamma-tubulin, DGrip91, and D-TACC. Conversely, DSpd-2 mutants displayed normal centrosomal accumulations of Asl and D-PLP, strongly reduced amounts of Cnn, gamma-tubulin, and DGrip91, and diffuse localization of D-TACC. These results indicate that DSpd-2 functions in a very early step of the PCM recruitment pathway.     

Dynamin and endocytosis are required for the fusion of osteoclasts and myoblasts

Cell–cell fusion is an evolutionarily conserved process that leads to the formation of multinucleated myofibers, syncytiotrophoblasts and osteoclasts, allowing their respective functions. Although cell–cell fusion requires the presence of fusogenic membrane proteins and actin-dependent cytoskeletal reorganization, the precise machinery allowing cells to fuse is still poorly understood. Using an inducible knockout mouse model to generate dynamin 1– and 2–deficient primary osteoclast precursors and myoblasts, we found that fusion of both cell types requires dynamin. Osteoclast and myoblast cell–cell fusion involves the formation of actin-rich protrusions closely associated with clathrin-mediated endocytosis in the apposed cell. Furthermore, impairing endocytosis independently of dynamin also prevented cell–cell fusion. Since dynamin is involved in both the formation of actin-rich structures and in endocytosis, our results indicate that dynamin function is central to the osteoclast precursors and myoblasts fusion process, and point to an important role of endocytosis in cell–cell fusion.     

Twinfilin is required for actin-dependent developmental processes in Drosophila.

The actin cytoskeleton is essential for cellular remodeling and many developmental and morphological processes. Twinfilin is a ubiquitous actin monomer-binding protein whose biological function has remained unclear. We discovered and cloned the Drosophila twinfilin homologue, and show that this protein is ubiquitously expressed in different tissues and developmental stages. A mutation in the twf gene leads to a number of developmental defects, including aberrant bristle morphology. This results from uncontrolled polymerization of actin filaments and misorientation of actin bundles in developing bristles. In wild-type bristles, twinfilin localizes diffusively to cytoplasm and to the ends of actin bundles, and may therefore be involved in localization of actin monomers in cells. We also show that twinfilin and the ADF/cofilin encoding gene twinstar interact genetically in bristle morphogenesis. These results demonstrate that the accurate regulation of size and dynamics of the actin monomer pool by twinfilin is essential for a number of actin-dependent developmental processes in multicellular eukaryotes.     

The heat shock protein 83 (Hsp83) is required for Raf-mediated signalling in Drosophila.

The heat shock protein Hsp90 has been shown to associate with various cellular signalling proteins such as steroid hormone receptors, src-like kinases and the serine/threonine kinase Raf. While the interaction between steroid hormone receptors and Hsp90 appears to be essential for ligand binding and activation of the receptors, the role of Hsp90 in Raf activation is less clear. We have identified mutations in the hsp83 gene, the Drosophila homologue of hsp90, in a search for dominant mutations that attenuate signalling from Raf in the developing eye. The mutations result in single amino acid substitutions in the Hsp83 protein and cause a dominant-negative effect on the function of the wild-type protein. We show that both wild-type and mutant forms of Hsp83 bind to the activated Drosophila Raf but the mutant Hsp83 protein causes a reduction in the kinase activity of Raf. Our results indicate that Hsp83 is essential for Raf function in vivo.     

Partitioning of N-ethylmaleimide-sensitive fusion (NSF) protein function in Drosophila melanogaster: dNSF1 is required in the nervous system, and dNSF2 is required in mesoderm

The N-ethylmaleimide-sensitive fusion protein (NSF) promotes the fusion of secretory vesicles with target membranes in both regulated and constitutive secretion. While it is thought that a single NSF may perform this function in many eukaryotes, previous work has shown that the Drosophila genome contains two distinct NSF genes, dNSF1 and dNSF2, raising the possibility that each plays a specific secretory role. To explore this possibility, we generated mutations in the dNSF2 gene and used these and novel dNSF1 loss-of-function mutations to analyze the temporal and spatial requirements and the degree of functional redundancy between dNSF1 and dNSF2. Results of this analysis indicate that dNSF1 function is required in the nervous system beginning at the adult stage of development and that dNSF2 function is required in mesoderm beginning at the first instar larval stage of development. Additional evidence suggests that dNSF1 and dNSF2 may play redundant roles during embryonic development and in the larval nervous system. Ectopic expression studies demonstrate that the dNSF1 and dNSF2 gene products can functionally substitute for one another. These results indicate that the Drosophila NSF proteins exhibit similar functional properties, but have evolved distinct tissue-specific roles.     

Expression of a voltage-dependent potassium current precedes fusion of human muscle satelite cells (myoblasts)

Using the whole-cell recording patch clamp tehnique in clonal cultures of human muscle satellite cells (SC), we studied a voltage-gated potassium current analogous to the delayed rectifier current (I_kdr) described in adult human skeletal muscle. This current was absent in proliferating SC cultured in a growth medium containing 15% serum, except when the SC approached the end of their replicative life (between 77 and 124 days in culture); at that time, approximately 50% of the SC possessed I_kdr In contrast, I_kdr was expressed within less than 4 days in approximately 70% of the SC cultured in a serum-free medium (SFM) and within 24 h in differetiating medium. We belive that I_kdr may be a characteristic feature of fusion-component SC and that it may be involved in the fusion process for the following resons: (1) after the transfer in differentiating medium, cultures of SC in which the expression of I_kdr was previously promoted by exposure to SFM were found to fuse immediately, without the initial 24 h lag time observed in control sister cultures; (2) in the latter “naive” SC, I_kdr was expressed during the first day in differentiating medium, before SC began to fuse; (3) most of the SC that did not fuse even after weeks of exposure to differentiating medium did not express I_kdr; (4) TEA, at a concentration of 3 mM, reduces the amplitude of I_kdr by 55% and the fusion index by 55–67%. © 1995 Wiley-Liss, Inc.     

Formin3 is required for assembly of the F-actin structure that mediates tracheal fusion in Drosophila

During tracheal development in Drosophila, some branches join to form a continuous luminal network. Specialized cells at the branch tip, called fusion cells, extend filopodia to make contact and become doughnut shaped to allow passage of the lumen. These morphogenetic processes accompany the highly regulated cytoskeletal reorganization of fusion cells. We identified the Drosophila formin3 (form3) gene that encodes a novel formin and plays a role in tracheal fusion. Formins are a family of proteins characterized by highly conserved formin homology (FH) domains. The formin family functions in various actin-based processes, including cytokinesis and cell polarity. During embryogenesis, form3 mRNA is expressed mainly in the tracheal system. In form3 mutant embryos, the tracheal fusion does not occur at some points. This phenotype is rescued by the forced expression of form3 in the trachea. We used live imaging of GFP-moesin during tracheal fusion to show that an F-actin structure that spans the adjoining fusion cells and mediates the luminal connection does not form at abnormal anastomosis sites in form3 mutants. These results suggested that Form3 plays a role in the F-actin assembly, which is essential for cellular rearrangement during tracheal fusion.     

The Wiskott-Aldrich syndrome protein (WASP) is essential for myoblast fusion in Drosophila

In higher organisms, mononucleated myoblasts fuse to form multinucleated myotubes. During this process, myoblasts undergo specific changes in cell morphology and cytoarchitecture. Previously, we have shown that the actin regulator Kette (Hem-2/Nap-1) is essential for myoblast fusion. In this study, we describe the role of the evolutionary conserved Wiskott-Aldrich syndrome protein that serves as a regulator for the Arp2/3 complex for myoblast fusion. By screening an EMS mutagenesis collection, we discovered a new wasp allele that does not complete fusion during myogenesis. Interestingly, this new wasp3D3-035 allele is characterized by a disruption of fusion after precursor formation. The molecular lesion in this wasp allele leads to a stop codon preventing translation of the CA domain. Usually, the WASP protein exerts its function through the Arp2/3-interacting CA domain. Accordingly, a waspDeltaCA that is expressed in a wild-type background acts as dominant-negative during the fusion process. Furthermore, we show that the myoblast fusion phenotype of kette mutant embryos can be suppressed by reducing the gene dose of wasp3D3-035. Thus, Kette antagonizes WASP function during myoblast fusion.