Moleskin is essential for the formation of the myotendinous junction in Drosophila

It is the precise connectivity between skeletal muscles and their corresponding tendon cells to form a functional myotendinous junction (MTJ) that allows for the force generation required for muscle contraction and organismal movement. The Drosophila MTJ is composed of secreted extracellular matrix (ECM) proteins deposited between integrin-mediated hemi-adherens junctions on the surface of muscle and tendon cells. In this paper, we have identified a novel, cytoplasmic role for the canonical nuclear import protein Moleskin (Msk) in Drosophila embryonic somatic muscle attachment. Msk protein is enriched at muscle attachment sites in late embryogenesis and msk mutant embryos exhibit a failure in muscle–tendon cell attachment. Although the muscle–tendon attachment sites are reduced in size, components of the integrin complexes and ECM proteins are properly localized in msk mutant embryos. However, msk mutants fail to localize phosphorylated focal adhesion kinase (pFAK) to the sites of muscle–tendon cell junctions. In addition, the tendon cell specific proteins Stripe (Sr) and activated mitogen-activated protein kinase (MAPK) are reduced in msk mutant embryos. Our rescue experiments demonstrate that Msk is required in the muscle cell, but not in the tendon cells. Moreover, muscle attachment defects due to loss of Msk are rescued by an activated form of MAPK or the secreted epidermal growth factor receptor (Egfr) ligand Vein. Taken together, these findings provide strong evidence that Msk signals non-autonomously through the Vein-Egfr signaling pathway for late tendon cell late differentiation and/or maintenance.     

WNT5 interacts with the Ryk receptors doughnut and derailed to mediate muscle attachment site selection in Drosophila melanogaster

In recent years a number of the genes that regulate muscle formation and maintenance in higher organisms have been identified. Studies employing invertebrate and vertebrate model organisms have revealed that many of the genes required for early mesoderm specification are highly conserved throughout evolution. Less is known about the molecules that mediate the steps subsequent to myogenesis, e. g. myotube guidance and attachment to tendon cells. We use the stereotypic pattern of the Drosophila embryonic body wall musculature in genetic approaches to identify novel factors required for muscle attachment site selection. Here, we show that Wnt5 is needed in this process. The lateral transverse muscles frequently overshoot their target attachment sites and stably attach at novel epidermal sites in Wnt5 mutant embryos. Restoration of WNT5 expression in either the muscle or the tendon cell rescues the mutant phenotype. Surprisingly, the novel attachment sites in Wnt5 mutants frequently do not express the Stripe (SR) protein which has been shown to be required for terminal tendon cell differentiation. A muscle bypass phenotype was previously reported for embryos lacking the WNT5 receptor Derailed (DRL). drl and Wnt5 mutant embryos also exhibit axon path finding errors. DRL belongs to the conserved Ryk receptor tyrosine kinase family which includes two other Drosophila orthologs, the Doughnut on 2 (DNT) and Derailed-2 (DRL-2) proteins. We generated a mutant allele of dnt and find that dnt, but not Drl-2, mutant embryos also show a muscle bypass phenotype. Genetic interaction experiments indicate that drl and dnt act together, likely as WNT5 receptors, to control muscle attachment site selection. These results extend previous findings that at least some of the molecular pathways that guide axons towards their targets are also employed for guidance of muscle fibers to their appropriate attachment sites.     

Exoskeleton anchoring to tendon cells and muscles in molting isopod crustaceans

Specialized mechanical connection between exoskeleton and underlying muscles in arthropods is a complex network of interconnected matrix constituents, junctions and associated cytoskeletal elements, which provides prominent mechanical attachment of the epidermis to the cuticle and transmits muscle tensions to the exoskeleton. This linkage involves anchoring of the complex extracellular matrix composing the cuticle to the apical membrane of tendon cells and linking of tendon cells to muscles basally. The ultrastructural arhitecture of these attachment complexes during molting is an important issue in relation to integument integrity maintenance in the course of cuticle replacement and in relation to movement ability. The aim of this work was to determine the ultrastructural organization of exoskeleton - muscles attachment complexes in the molting terrestrial isopod crustaceans, in the stage when integumental epithelium is covered by both, the newly forming cuticle and the old detached cuticle. We show that the old exoskeleton is extensively mechanically connected to the underlying epithelium in the regions of muscle attachment sites by massive arrays of fibers in adult premolt Ligia italica and in prehatching embryos and premolt marsupial mancas of Porcellio scaber. Fibers expand from the tendon cells, traverse the new cuticle and ecdysal space and protrude into the distal layers of the detached cuticle. They likely serve as final anchoring sites before exuviation and may be involved in animal movements in this stage. Tendon cells in the prehatching embryo and in marsupial mancas display a substantial apicobasally oriented transcellular arrays of microtubules, evidently engaged in myotendinous junctions and in apical anchoring of the cuticular matrix. The structural framework of musculoskeletal linkage is basically established in described intramarsupial developmental stages, suggesting its involvement in animal motility within the marsupium.     

Integrins modulate the Egfr signaling pathway to regulate tendon cell differentiation in the Drosophila embryo

Changes in the extracellular matrix (ECM) govern the differentiation of many cell types during embryogenesis. Integrins are cell matrix receptors that play a major role in cell-ECM adhesion and in transmitting signals from the ECM inside the cell to regulate gene expression. In this paper, it is shown that the PS integrins are required at the muscle attachment sites of the Drosophila embryo to regulate tendon cell differentiation. The analysis of the requirements of the individual alpha subunits, alphaPS1 and alphaPS2, demonstrates that both PS1 and PS2 integrins are involved in this process. In the absence of PS integrin function, the expression of tendon cell-specific genes such as stripe and beta1 tubulin is not maintained. In addition, embryos lacking the PS integrins also exhibit reduced levels of activated MAPK. This reduction is probably due to a downregulation of the Epidermal Growth Factor receptor (Egfr) pathway, since an activated form of the Egfr can rescue the phenotype of embryos mutant for the PS integrins. Furthermore, the levels of the Egfr ligand Vein at the muscle attachment sites are reduced in PS mutant embryos. Altogether, these results lead to a model in which integrin-mediated adhesion plays a role in regulating tendon cell differentiation by modulating the activity of the Egfr pathway at the level of its ligand Vein.     

Active muscle migration during insect metamorphosis

The R1 abdominal retractor muscles of the insect Tenebrio molitor change position during the course of metamorphosis. These muscles detach from the epidermal tendon cells at their anterior ends, and migrate in a posterior direction, parallel to the body axis, to form completely new attachments shortly before adult emergence. Movement is preceded by the loss of sarcomere structure, and the muscles migrate in a partially dedifferentiated condition, closely accompanied by satellite cells and haemocytes. Movement appears to result from the extension of muscle processes towards the epidermis posterior to the larval attachment sites, which contact reciprocal processes extended from the epidermis. Contacts at the new posterior sites are then reinforced, and relinquished at the anterior. This cycle is subsequently repeated. It is envisaged that migration ceases when the muscles encounter a contour in the epidermal gradient known to specify the position of the adult muscle attachment sites. This positional information may be encoded in the epidermal basal lamina. The muscles then redifferentiate, with concurrent differentiation of new epidermal tendon cells. Development of adult muscle attachments appears to require reciprocal morphogenetic interactions between muscle and epidermis.     

Prepattern genes and signaling molecules regulate stripe expression to specify Drosophila flight muscle attachment sites

In Drosophila, muscles attach to epidermal tendon cells specified by the gene stripe (sr). Flight muscle attachment sites are prefigured on the wing imaginal disc by sr expression in discrete domains. We describe the mechanisms underlying the specification of these domains of sr expression. We show that the concerted activities of the wingless (wg), decapentaplegic (dpp) and Notch (N) signaling pathways, and the prepattern genes pannier (pnr) and u-shaped (ush) establish domains of sr expression. N is required for initiation of sr expression. pnr is a positive regulator of sr, and is inhibited by ush in this function. The Wg signal differentially influences the formation of different sr domains. These results identify the multiple regulatory elements involved in the positioning of Drosophila flight muscle attachment sites.     

The Drosophila wing hearts consist of syncytial muscle cells that resemble adult somatic muscles

In Drosophila, hemolymph circulation in the wings is accomplished by a pair of wing hearts located in the thorax. The embryonic progenitors of these organs were only recently discovered and found to belong to the cardiac mesoderm. In this study, the functional morphology and the structure of mature organs were studied by light and electron microscopy to characterize the tissues arising from this new set of progenitors. Each wing heart consists of 7-8 muscle cells providing the pumping force, a thin layer of non-contractile mononucleated cells separating the muscle cells from the body cavity, and acellular suspending strands opposing the muscle contractions. The muscle cells are multinucleated syncytia attached to the cuticle via epidermal tendon cells. They have central nuclei and sarcomeres with discontinuous Z-discs, A-bands, and I-bands, whereas H-bands and M-bands are indiscernible. From 9 to 11 actin filaments surround each myosin filament. Mitochondria are abundantly interspersed between myofibrils and accumulated in characteristic outpockets of the plasma membrane. The analysis revealed that the wing heart muscles resemble in their ultrastructure and their mode of attachment adult somatic muscles. This suggests that, despite their origin in the cardiac mesoderm, wing heart progenitors are functionally related to somatic adult muscle precursors.     

Terminal tendon cell differentiation requires the glide/gcm complex

Locomotion relies on stable attachment of muscle fibres to their target sites, a process that allows for muscle contraction to generate movement. Here, we show that glide/gcm and glide2/gcm2, the fly glial cell determinants, are expressed in a subpopulation of embryonic tendon cells and required for their terminal differentiation. By using loss-of-function approaches, we show that in the absence of both genes, muscle attachment to tendon cells is altered, even though the molecular cascade induced by stripe, the tendon cell determinant, is normal. Moreover, we show that glide/gcm activates a new tendon cell gene independently of stripe. Finally, we show that segment polarity genes control the epidermal expression of glide/gcm and determine, within the segment, whether it induces glial or tendon cell-specific markers. Thus, under the control of positional cues, glide/gcm triggers a new molecular pathway involved in terminal tendon cell differentiation, which allows the establishment of functional muscle attachment sites and locomotion.     

“Importin” signaling roles for import proteins: The function of Drosophila importin-7 (DIM-7) in muscle-tendon signaling

The formation of a mature myotendinous junction (MTJ) between a muscle and its site of attachment is a highly regulated process that involves myofiber migration, cell-cell signaling, and culminates with the stable adhesion between the adjacent muscle-tendon cells. Improper establishment or maintenance of muscle-tendon attachment sites results in a decrease in force generation during muscle contraction and progressive muscular dystrophies in vertebrate models. Many studies have demonstrated the important role of the integrins and integrin-associated proteins in the formation and maintenance of the MTJ. We recently demonstrated that moleskin (msk), the gene that encodes for Drosophila importin-7 (DIM-7), is required for the proper formation of muscle-tendon adhesion sites in the developing embryo. Further studies demonstrated an enrichment of DIM-7 to the ends of muscles where the muscles attach to their target tendon cells. Genetic analysis supports a model whereby msk is required in the muscle and signals via the secreted epidermal growth factor receptor (Egfr) ligand Vein to regulate tendon cell maturation. These data demonstrate a novel role for the canonical nuclear import protein DIM-7 in establishment of the MTJ.     

“Importin” signaling roles for import proteins

The formation of a mature myotendinous junction (MTJ) between a muscle and its site of attachment is a highly regulated process that involves myofiber migration, cell-cell signaling, and culminates with the stable adhesion between the adjacent muscle-tendon cells. Improper establishment or maintenance of muscle-tendon attachment sites results in a decrease in force generation during muscle contraction and progressive muscular dystrophies in vertebrate models. Many studies have demonstrated the important role of the integrins and integrin-associated proteins in the formation and maintenance of the MTJ. We recently demonstrated that moleskin (msk), the gene that encodes for Drosophila importin-7 (DIM-7), is required for the proper formation of muscle-tendon adhesion sites in the developing embryo. Further studies demonstrated an enrichment of DIM-7 to the ends of muscles where the muscles attach to their target tendon cells. Genetic analysis supports a model whereby msk is required in the muscle and signals via the secreted epidermal growth factor receptor (Egfr) ligand Vein to regulate tendon cell maturation. These data demonstrate a novel role for the canonical nuclear import protein DIM-7 in establishment of the MTJ.     

Two Independent Functions of Collier/Early B Cell Factor in the Control of Drosophila Blood Cell Homeostasis

Blood cell production in the Drosophila hematopoietic organ, the lymph gland, is controlled by intrinsic factors and extrinsic signals. Initial analysis of Collier/Early B Cell Factor function in the lymph gland revealed the role of the Posterior Signaling Center (PSC) in mounting a dedicated cellular immune response to wasp parasitism. Further, premature blood cell differentiation when PSC specification or signaling was impaired, led to assigning the PSC a role equivalent to the vertebrate hematopoietic niche. We report here that Collier is expressed in a core population of lymph gland progenitors and cell autonomously maintains this population. The PSC contributes to lymph gland homeostasis by regulating blood cell differentiation, rather than by maintaining core progenitors. In addition to PSC signaling, switching off Collier expression in progenitors is required for efficient immune response to parasitism. Our data show that two independent sites of Collier/Early B Cell Factor expression, hematopoietic progenitors and the PSC, achieve control of hematopoiesis.