Cleaved Slit directs embryonic muscles

The formation of functional musculoskeletal system relies on proper connectivity between muscles and their corresponding tendon cells. In Drosophila, larval muscles are born during early embryonic stages, and elongate toward tendons that are embedded within the ectoderm in later. The Slit/Robo signaling pathway had been implicated in the process of muscle elongation toward tendons. Here we discuss our recent findings regarding the critical contribution of Slit cleavage for immobilization and stabilization of the Slit signal on the tendon cells. Slit cleavage produces 2 polypeptides, the N-terminal Slit-N, which is extremely stable, undergoes oligomerization, and associates with the tendon cell surfaces, and the C-terminal Slit-C, which rapidly degrades. Slit cleavage leads to immobilization of Slit signaling on tendons, leading to a short-range repulsion, which eventually arrest further muscle elongation. Robo2, which is co-expressed with Slit by the tendon cells facilitates Slit cleavage. This activity does not require the cytoplasmic signaling domain of Robo2. We suggest that Robo2-dependent Slit cleavage, and the formation of Slit-N oligomers on the tendon cell surfaces direct muscle elongation, and provide a stop signal for the approaching muscle, through binding to Robo and Robo3 receptors expressed by the muscles.     

Cleaved Slit directs embryonic muscles

The formation of functional musculoskeletal system relies on proper connectivity between muscles and their corresponding tendon cells. In Drosophila, larval muscles are born during early embryonic stages, and elongate toward tendons that are embedded within the ectoderm in later. The Slit/Robo signaling pathway had been implicated in the process of muscle elongation toward tendons. Here we discuss our recent findings regarding the critical contribution of Slit cleavage for immobilization and stabilization of the Slit signal on the tendon cells. Slit cleavage produces 2 polypeptides, the N-terminal Slit-N, which is extremely stable, undergoes oligomerization, and associates with the tendon cell surfaces, and the C-terminal Slit-C, which rapidly degrades. Slit cleavage leads to immobilization of Slit signaling on tendons, leading to a short-range repulsion, which eventually arrest further muscle elongation. Robo2, which is co-expressed with Slit by the tendon cells facilitates Slit cleavage. This activity does not require the cytoplasmic signaling domain of Robo2. We suggest that Robo2-dependent Slit cleavage, and the formation of Slit-N oligomers on the tendon cell surfaces direct muscle elongation, and provide a stop signal for the approaching muscle, through binding to Robo and Robo3 receptors expressed by the muscles.     

Functional dissection of drosophila capricious: its novel roles in neuronal pathfinding and selective synapse formation

Drosophila Capricious (CAPS) is a transmembrane protein with leucine-rich repeat (LRR) motifs, expressed on small subsets of neurons and muscles, including muscle 12 and the motoneurons that innervate it (muscle 12 MNs). Panmuscle ectopic expression of CAPS alters the target specificity of muscle 12 MNs, indicating that CAPS can function in muscles as a target recognition molecule. In this study, we first examined the effect of ectopic panneural expression of CAPS on the motoneuronal circuit. We found that panneural expression of CAPS alters the pathfinding of muscle 12 MNs. The defect appeared to be caused by changes in the steering behavior of muscle 12 MNs at a specific choice point along their pathway to the target muscle. These results revealed a novel function of CAPS in axon pathfinding. We then performed deletion analyses of CAPS. We expressed CAPS lacking the intracellular domain in all neurons or in all muscles, and studied their ability to induce the pathfinding and targeting phenotypes. We found that the function of muscularly expressed CAPS in target recognition is intracellular domain dependent, whereas the function of neurally expressed CAPS in pathfinding is not, suggesting that CAPS may function in neurons and muscles in a different manner. The requirement of the intracellular domain for the function of muscularly expressed CAPS suggests the presence of a signaling event within muscle cells that is essential for selective synapse formation.     

Follow the fatty brick road: lipid signaling in cell migration

Lysophospholipids play important roles in the migration of lymphocytes, smooth muscle cells and germ cells in vertebrates and invertebrates. In vertebrates, the migratory responses are mediated by specific G-protein-coupled receptors. These are expressed in both migrating lymphocyte and smooth muscle cells, and in their surrounding cells. In Drosophila germ cell migration, lipid phosphatases also act in both the surrounding and the migrating cells. In all three scenarios, the contributions of these genes in the stationary and migrating cells are being teased apart by genetic studies and direct observation, with exciting results.     

The role of Lbx1 in migration of muscle precursor cells

The homeobox gene Lbx1 is expressed in migrating hypaxial muscle precursor cells during development. These precursors delaminate from the lateral edge of the dermomyotome and form distinct streams that migrate over large distances, using characteristic paths. The targets of migration are limbs, septum transversum and the floor of the first branchial arch where the cells form skeletal muscle of limbs and shoulders, diaphragm and hypoglossal cord, respectively. We used gene targeting to analyse the function of Lbx1 in the mouse. Myogenic precursor cells delaminate from the dermomyotome in Lbx1 mutants, but migrate in an aberrant manner. Most critically affected are migrating cells that move to the limbs. Precursor cells that reach the dorsal limb field are absent. In the ventral limb, precursors are present but distributed in an abnormal manner. As a consequence, at birth some muscles in the forelimbs are completely lacking (extensor muscles) or reduced in size (flexor muscles). Hindlimb muscles are affected strongly, and distal limb muscles are more affected than proximal ones. Other migrating precursor cells heading towards the floor of the first branchial arch move along the appropriate path in Lbx1 mutants. However, these cells migrate less efficiently and reduced numbers of precursors reach their distal target. At birth, the internal lingual muscle is therefore reduced in size. We suggest that Lbx1 controls the expression of genes that are essential for the recognition or interpretation of cues that guide migrating muscle precursors and maintain their migratory potential.     

Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit

Recognition of the large secreted protein Slit by receptors of the Robo family provides fundamental signals in axon guidance and other developmental processes. In Drosophila, Slit-Robo signalling regulates midline crossing and the lateral position of longitudinal axon tracts. We report the functional dissection of Drosophila Slit, using structure analysis, site-directed mutagenesis and in vitro assays. The N-terminal region of Slit consists of a tandem array of four independently folded leucine-rich repeat (LRR) domains, connected by disulphide-tethered linkers. All three Drosophila Robos were found to compete for a single highly conserved site on the concave face of the second LRR domain of Slit. We also found that this domain is sufficient for biological activity in a chemotaxis assay. Other Slit activities may require Slit dimerisation mediated by the fourth LRR domain. Our results show that a small portion of Slit is able to induce Robo signalling and indicate that the distinct functions of Drosophila Robos are encoded in their divergent cytosolic domains.     

Slit coordinates cardiac morphogenesis in Drosophila

Slit is a secreted guidance cue that conveys repellent or attractive signals from target and guidepost cells. In Drosophila, responsive cells express one or more of three Robo receptors. The cardial cells of the developing heart express both Slit and Robo2. This is the first report of coincident expression of a Robo and its ligand. In slit mutants, cardial cell alignment, polarization and uniform migration are disrupted. The heart phenotype of robo2 mutants is similar, with fewer migration defects. In the guidance of neuronal growth cones in Drosophila, there is a phenotypic interaction between slit and robo heterozygotes, and also with genes required for Robo signaling. In contrast, in the heart, slit has little or no phenotypic interaction with Robo-related genes, including Robo2, Nck2, and Disabled. However, there is a strong phenotypic interaction with Integrin genes and their ligands, including Laminin and Collagen, and intracellular messengers, including Talin and ILK. This indicates that Slit participates in adhesion or adhesion signaling during heart development.     

Singling out Drosophila tendon cells: a dialogue between two distinct cell types.

The precise match between somatic muscles and their epidermal attachment cells is achieved through a continuous dialogue between these two cell types. Whereas tendon cells direct myotube migration and final patterning, the muscles are essential for the maintenance of the fate of tendon cells. The Drosophila neuregulin-like ligand, Vein, and its receptor, the epidermal growth factor receptor (Egfr), are critical components in the inductive signaling process that takes place between muscles and tendon cells. Additional gene products that relay the Vein-Egfr effect in Drosophila are conserved in the vertebrate neuregulin-mediated cascade. This review describes genetic and molecular aspects of the muscle-tendon inductive processes in Drosophila, and compares them with the relevant mechanisms in the vertebrate embryo.     

The transmembrane protein Perdido interacts with Grip and integrins to mediate myotube projection and attachment in the Drosophila embryo

The molecular mechanisms underlying muscle guidance and formation of myotendinous junctions are poorly understood both in vertebrates and in Drosophila. We have identified a novel gene that is essential for Drosophila embryonic muscles to form proper projections and stable attachments to epidermal tendon cells. Loss-of-function of this gene - which we named perdido (perd)-results in rounded, unattached muscles. perd is expressed prior to myoblast fusion in a subset of muscle founder cells, and it encodes a conserved single-pass transmembrane cell adhesion protein that contains laminin globular extracellular domains and a small intracellular domain with a C-terminal PDZ-binding consensus sequence. Biochemical experiments revealed that the Perd intracellular domain interacts directly with one of the PDZ domains of the Glutamate receptor interacting protein (Grip), another factor required for formation of proper muscle projections. In addition, Perd is necessary to localize Grip to the plasma membrane of developing myofibers. Using a newly developed, whole-embryo RNA interference assay to analyze genetic interactions, perd was shown to interact not only with Grip but also with multiple edematous wings, which encodes one subunit of the alpha PS1-beta PS integrin expressed in tendon cells. These experiments uncovered a previously unrecognized role for the alpha PS1-beta PS integrin in the formation of muscle projections during early stages of myotendinous junction development. We propose that Perd regulates projection of myotube processes toward and subsequent differentiation of the myotendinous junction by priming formation of a protein complex through its intracellular interaction with Grip and its transient engagement with the tendon cell-expressed laminin-binding alpha PS1-beta PS integrin.     

Slit and Robo control cardiac cell polarity and morphogenesis

Basic aspects of heart morphogenesis involving migration, cell polarization, tissue alignment, and lumen formation may be conserved between Drosophila and humans, but little is known about the mechanisms that orchestrate the assembly of the heart tube in either organism. The extracellular-matrix molecule Slit and its Robo-family receptors are conserved regulators of axonal guidance. Here, we report a novel role of the Drosophila slit, robo, and robo2 genes in heart morphogenesis. Slit and Robo proteins specifically accumulate at the dorsal midline between the bilateral myocardial progenitors forming a linear tube. Manipulation of Slit localization or its overexpression causes disruption in heart tube alignment and assembly, and slit-deficient hearts show disruptions in cell-polarity marker localization within the myocardium. Similar phenotypes are observed when Robo and Robo2 are manipulated. Rescue experiments suggest that Slit is secreted from the myocardial progenitors and that Robo and Robo2 act in myocardial and pericardial cells, respectively. Genetic interactions suggest a cardiac morphogenesis network involving Slit/Robo, cell-polarity proteins, and other membrane-associated proteins. We conclude that Slit and Robo proteins contribute significantly to Drosophila heart morphogenesis by guiding heart cell alignment and adhesion and/or by inhibiting cell mixing between the bilateral compartments of heart cell progenitors and ensuring proper polarity of the myocardial epithelium.     

GAPs in Slit-Robo signaling

Neuronal migration requires the integration of a number of diverse environmental cues and subsequent translation to specific responses such as directed cytoskeletal remodeling. Accurate knowledge of the signal transduction pathways linking activation of surface receptors to actin dynamics is necessary in order to understand the regulation of such processes. Activation of the Roundabout (Robo) receptor mediates a repulsive response in certain pioneering axons and migratory neurons. Recently, Wong et al have described a signaling link between Robo activation and specific GTPase activity that appears to regulate neuronal migration. A novel family of GTPase regulators, responsive to Slit-Robo engagement, has been identified and convincingly shown to alter the migration of neuronal cells. This study not only delineates a specific signaling route from guidance receptors to directed neuronal movement, but also offers clues towards potential regulatory mechanisms that ensure specificity of the Slit-Robo response.     

Roundabout 2 regulates migration of sensory neurons by signaling in trans

BACKGROUND: Roundabout (Robo) receptors and their ligand Slit are important regulators of axon guidance and cell migration. The development of Drosophila embryonic sense organs provides a neuronal migration paradigm where the in vivo roles of Slit and Robo can be assayed using genetics. RESULTS: Here we show that Slit-Robo signaling controls migration of Drosophila larval sensory neurons that are part of the Chordotonal (Cho) stretch receptor organs. We used live imaging to show that abdominal Cho organs normally migrate ventrally during development, whereas thoracic Cho organs do not. Robo2 overexpression in cis (in the sensory neurons) or in trans (on neighboring visceral mesoderm) transforms abdominal organs to a thoracic morphology and position by blocking migration, while loss of Slit-Robo signaling produces a reverse transformation in which thoracic organs migrate ectopically. Rescue and tissue-specific knockout experiments indicate that trans signaling by Robo2 contributes to the normal positioning of the thoracic Cho organs. The differential positioning of Cho organs between the thorax and abdomen is known to be regulated by Hox genes, and we show that the essential Hox cofactor Homothorax, represses Robo2 expression in the abdominal visceral mesoderm. CONCLUSIONS: Our results suggest that segment-specific neuronal migration patterns are directed through a novel signaling complex (the "Slit sandwich") in which Robo2 on the thoracic visceral mesoderm binds to Slit and presents it to Robo receptors on Cho neurons. The differential positioning of Cho organs between thorax and abdomen may be determined by Hox gene-mediated repression of robo2.     

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.     

“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.     

The N-terminal and transmembrane domains of Commissureless are necessary for its function and trafficking within neurons

Commissureless (Comm) is a novel transmembrane molecule necessary both for commissural axons to cross the midline of the Drosophila central nervous system and normal synaptogenesis. Comm is able to reduce cell surface levels of Roundabout (Robo), a receptor for the midline repellent Slit, on commissural axons and unknown inhibitors of synaptogenesis expressed on muscle cells. Comm is expressed dynamically and is found at the cell surface and within intracellular vesicles. Comm can bind Robo and when the proteins are co-expressed Robo is found co-localised with Comm intracellularly. Here we show that the ability of Comm to localise intracellularly and hence regulate Robo surface levels requires sequences in both the N-terminal and transmembrane domains. We also show that Comm can dimerise via its N-terminal domain. Furthermore, absence of the Comm N-terminal and transmembrane regions results in the protein being restricted to the neuron soma.     

Chimeric axon guidance receptors: the cytoplasmic domains of slit and netrin receptors specify attraction versus repulsion.

Frazzled (Fra) is the DCC-like Netrin receptor in Drosophila that mediates attraction; Roundabout (Robo) is a Slit receptor that mediates repulsion. Both ligands are expressed at the midline; both receptors have related structures and are often expressed by the same neurons. To determine if attraction versus repulsion is a modular function encoded in the cytoplasmic domain of these receptors, we created chimeras carrying the ectodomain of one receptor and the cytoplasmic domain of the other and tested their function in transgenic Drosophila. Fra-Robo (Fra's ectodomain and Robo's cytoplasmic domain) functions as a repulsive Netrin receptor; neurons expressing Fra-Robo avoid the Netrin-expressing midline and muscles. Robo-Fra (Robo's ectodomain and Fra's cytoplasmic domain) is an attractive Slit receptor; neurons and muscle precursors expressing Robo-Fra are attracted to the Slit-expressing midline.     

Compartmentalization of visual centers in the Drosophila brain requires Slit and Robo proteins

Brain morphogenesis depends on the maintenance of boundaries between populations of non-intermingling cells. We used molecular markers to characterize a boundary within the optic lobe of the Drosophila brain and found that Slit and the Robo family of receptors, well-known regulators of axon guidance and neuronal migration, inhibit the mixing of adjacent cell populations in the developing optic lobe. Our data suggest that Slit is needed in the lamina to prevent inappropriate invasion of Robo-expressing neurons from the lobula cortex. We show that Slit protein surrounds lamina glia, while the distal cell neurons in the lobula cortex express all three Drosophila Robos. We examine the function of these proteins in the visual system by isolating a novel allele of slit that preferentially disrupts visual system expression of Slit and by creating transgenic RNA interference flies to inhibit the function of each Drosophila Robo in a tissue-specific fashion. We find that loss of Slit or simultaneous knockdown of Robo, Robo2 and Robo3 causes distal cell neurons to invade the lamina, resulting in cell mixing across the lamina/lobula cortex boundary. This boundary disruption appears to lead to alterations in patterns of axon navigation in the visual system. We propose that Slit and Robo-family proteins act to maintain the distinct cellular composition of the lamina and the lobula cortex.