kon-tiki/perdido: Biological Overview | References
Gene name - kon-tiki/perdido
Synonyms - CG10275
Cytological map position- 36F5-36F5
Function - receptor
Keywords - mesoderm, myotube migration, muscle founder cell, tendon cell
Symbol - kon/perd
FlyBase ID: FBgn0032683
Genetic map position - 2L: 18,487,746..18,496,426 [-]
Classification - Laminin G domain, chondroitin sulfate proteoglycan (CSPG) repeats, and C-terminal PDZ binding domain
Cellular location - transmembrane
|Recent literature||Losada-Perez, M., Harrison, N. and Hidalgo, A.
(2016). Molecular mechanism of
central nervous system repair by the Drosophila NG2 homologue
kon-tiki. J Cell Biol [Epub ahead of print]. PubMed ID:
Neuron glia antigen 2 (NG2)-positive glia are repair cells that proliferate upon central nervous system (CNS) damage, promoting functional recovery. However, repair is limited because of the failure of the newly produced glial cells to differentiate. It is a key goal to discover how to regulate NG2 to enable glial proliferation and differentiation conducive to repair. Drosophila has an NG2 homologue called kon-tiki (kon), of unknown CNS function. This study shows that kon promotes repair and identifies the underlying mechanism. Crush injury up-regulates kon expression downstream of Notch. Kon in turn induces glial proliferation and initiates glial differentiation by activating glial genes and prospero (pros). Two negative feedback loops with Notch and Pros allow Kon to drive the homeostatic regulation required for repair. By modulating Kon levels in glia, CNS repair could be prevented or promoted. Thus, the functional links between Kon, Notch, and Pros are essential for, and can drive, repair. Analogous mechanisms could promote CNS repair in mammals.
Directed cell migration and target recognition are critical for the development of both the nervous and muscular systems. Molecular mechanisms that control these processes in the nervous system have been intensively studied, whereas those that act during muscle development are still largely uncharacterized. This study identified a transmembrane protein, Kon-tiki (Kon, FlyBase term Perdido), that mediates myotube target recognition in the Drosophila embryo. Kon is expressed in a specific subset of myotubes and is required autonomously for these myotubes to recognize their tendon cell targets and to establish a stable connection. Kon is enriched at myotube tips during targeting and signals through the intracellular adaptor Dgrip in a conserved molecular pathway. Forced overexpression of Kon stimulates muscle motility. It is proposed that Kon promotes directed myotube migration and transduces a target-derived signal that initiates the formation of a stable connection (Schnorrer, 2007).
The same gene CG10275 was identified by Estrada (2007) and termed perdido ('lost' in Spanish). perdido is expressed in a subset of embryonic founder myoblasts, and is essential for Drosophila muscles to find their attachment sites. It encodes a conserved single-pass transmembrane cell adhesion protein that contains laminin globular extracellular domains and a small intracellular domain with a PDZ binding consensus sequence at the C-terminus. In vivo visualization of muscle guidance in both perd mutant embryos and dsRNA-injected embryos show that some ventral muscles fail to reach their attachment sites and instead form rounded multinucleated myotubes. Genetic interaction experiments done with a newly developed RNA interference assay combined with in vivo visualization of muscle development suggest that perd may be a ligand for the laminin binding αPS1-βPS integrin heterodimer, which is expressed in the tendon cells. Genetic and biochemical evidence indicates that perd is necessary to localize the muscle guidance PDZ protein DGrip to the plasma membrane. These results suggest that Perd forms an essential protein complex for muscle guidance by engaging the myotube with the tendon cell via an extracellular interaction with the tendon integrin complex and by an intracellular interaction with DGrip. The function of perd in muscle pathfinding resembles the role of its vertebrate orthologs NG2/MCSP in cell migration in the nervous system (Estrada, 2007).
The development of several different organ systems requires migrating precursor cells to locate, recognize, and connect to specific target cells. A striking and intensively studied example of this process occurs during nervous system development, as the axons and dendrites of differentiating neurons seek out their respective synaptic partners. Muscle cells face a similar challenge in finding, identifying, and attaching to their target cells -- the tendon cells that connect to bone (in vertebrates) or epidermis (in invertebrates). Just as the establishment of correct neuronal wiring specificity is critical for neural function, so too is the precise connection specificity between muscles and tendon cells essential for normal muscular function. Yet, whereas great progress has been made over the past decade in defining the molecules and mechanisms that mediate neuronal guidance and target recognition, those that mediate muscle guidance and target recognition are still poorly understood. Indeed, it is not even clear to what extent the superficially similar processes of neuronal and muscle targeting rely on shared or distinct molecular mechanisms (Schnorrer, 2007).
The body muscles of the Drosophila embryo provide an ideal model system for a genetic approach to the problem of muscle targeting. As in vertebrates, the musculature of the Drosophila embryo is highly stereotyped, with uniquely identifiable muscles connecting to specific attachment sites. A total of 30 muscles form in each of the abdominal hemisegments A2-A7, with each muscle having its characteristic size, shape, and epidermal attachment sites. These muscles are single multinucleated cells, and their development can be readily followed by live imaging. Thus, this system offers the opportunity to use genetic and imaging methods to explore muscle targeting in vivo with single-cell resolution (Schnorrer, 2007).
Drosophila muscle cells develop from two types of myoblasts: founder cells and fusion-competent myoblasts (FCMs). Each muscle has a single founder cell, which is thought to determine the characteristic features of the muscle. These founder cells can be defined by the expression of specific combinations of transcription factors, which give each founder cell, and hence each muscle, its unique identity. The FCMs, on the other hand, appear to be more generic in nature, potentially fusing with any myotube. They contribute material rather than identity to the growing myotube (Schnorrer, 2007).
Muscle migration proceeds in three phases. (1) The founder cells migrate relative to each other to assume the approximate position in which the muscle will form. (2) FCMs begin to fuse with the founder cells, forming polarized myotubes with a highly dynamic leading edge at each end. These dynamic tips resemble the growth cones of neurons and appear to have a similar function in directing migration toward specific target cells. (3) Each myotube tip recognizes its specific attachment site and establishes a stable connection. These attachment sites are the tendon cells, specialized epidermal cells located along intersegmental borders and also within segments (Schnorrer, 2007).
How do myotubes locate and recognize their specific tendon cell targets? An attractive model, based on the present understanding of neuronal wiring specificity, is that individual myotubes and tendon cells may express distinct sets of surface or secreted proteins that constitute a molecular recognition system. If this is the case, then what are these molecules, how do they function in myotube targeting, and are they the same as or different than those that operate in the nervous system? To date, only two molecular systems for muscle guidance have been identified, both initially defined by their roles in neuronal wiring. Slit and its Robo family receptors repel both axons and myotubes away from the midline, and may later attract some of the same myotubes to their intersegmental attachment sites. Similarly, the Derailed (Drl) receptor helps some commissural axons to choose the right pathway across the midline and some myotubes to select the right intrasegmental attachment sites. Thus, at least some of the mechanisms involved in axon guidance and targeting are also utilized during muscle development (Schnorrer, 2007).
A less biased approach is to screen directly for genes involved in muscle development, and indeed this is the only way to identify factors that do not also act in neurons. A mutagenesis screen resulted in the identification of the gene kon-tiki (kon), which is essential for promoting directed migration and target recognition of a subset of ventral myotubes. kon mutations do not affect embryonic nervous system development, indicating that muscles and neurons use distinct as well as shared guidance and recognition molecules. The Kon protein is a large transmembrane protein that concentrates at muscle tips. The cytoplasmic domain of Kon contains a PDZ-binding motif and interacts with the PDZ-domain protein Dgrip -- a cytoplasmic adaptor previously shown to function in targeting of these myotubes. Both Kon and Dgrip are highly conserved, and they appear to define an ancient evolutionary molecular pathway that mediates specific muscle targeting (Schnorrer, 2007).
The kon gene was localized, using SNP-on-chip technology, to a position proximal to 36A10 on chromosome 2L. The location of kon was further refined by deficiency mapping: Df(2L)TW137 and Df(2L)M36-S5 delete kon but Df(2L)Exel8083 and Df(2L)Exel6041 do not. This placed kon within the 116 kb interval from 18,451 to 18,567 kb, a region that includes seven annotated genes. Of these genes, CG10275 seemed a strong candidate for kon, based on its size and its predicted product. Indeed, when all of the annotated exons of CG10275 from kon heterozygous adults were sequenced, single-nucleotide substitutions were found in eight of the nine alleles. Five of these alleles are associated with nonsense mutations; the other three are missense mutations in conserved domains (Schnorrer, 2007).
The computational annotation of kon was experimentally refined to include two additional exons which were identified by 5' RACE on embryonic cDNA. The kon mRNA thus includes a total of 12 exons with a total length of 8.3 kb. It is predicted to encode a transmembrane protein of 2381 amino acids, including an N-terminal signal sequence, a large extracellular region, a single membrane-spanning segment, and a cytoplasmic region of 159 amino acids. The predicted extracellular region is composed of 2 lamininG domains followed by 15 chondroitin sulfate proteoglycan (CSPG) repeats, which are structurally related to cadherin domains. The intracellular region lacks any known protein domain, but includes at the C terminus a predicted binding site for PDZ-domain-containing proteins (Schnorrer, 2007).
Kon is closely related to the NG2/CSPG4 and the 'similar to CSPG4' family proteins in vertebrates. These proteins share all of the extracellular domains present in Drosophila Kon, with 21%-25% identity and 39%-46% similarity in the lamininG and CSPG domains. The short cytoplasmic region is less well conserved, with the notable exception of the invariable QYWV sequence of the PDZ-binding motif. The in vivo functions of these Kon relatives in other species are unknown (Schnorrer, 2007).
NG2/CSPG4 is linked to chondroitin sulfate (CS) in human, mouse, and rat. However, the serine-999 residue that carries this modification in the rat homolog (Stallcup, 2001; see
Stallcup and Ng2 for subsequent studies) is not conserved in Drosophila Kon. To test whether Drosophila Kon contains a CS moiety linked to other residues, endogenous or overexpressed Kon protein were immunoprecipitated from embryos, and the electrophoretic mobility of Kon was compared with and without prior treatment with chondroitinase ABC. In both cases, a single sharp band of about 250-300 kD was observed. It is concluded that most Drosophila Kon protein is not modified by CS (Schnorrer, 2007).
kon mRNA is first detected at stage 10, possibly in the longitudinal visceral muscle precursors. These cells arise at the posterior of the embryo and migrate anteriorly to form muscles around the developing gut. More importantly, in the body wall muscles high expression was detected in the ventral-longitudinal myotubes in wild-type embryos at stage 14, the stage at which the first defects become apparent in these myotubes in kon mutant embryos. Taken together, these phenotypic, expression, and molecular data suggest that Kon might be a transmembrane receptor for a guidance or targeting cue provided by specific tendon cells -- a signal that may be transduced intracellularly through Kon's PDZ-binding motif (Schnorrer, 2007).
In each abdominal hemisegment of the Drosophila embryo, 30 muscles connect at their ends to distinct tendon cells in the epidermis. How does each muscle find and recognize its specific tendon cell targets? Myotubes are molecularly distinct before they begin to migrate, they generally migrate directly toward their targets, and they are unperturbed if other muscles are genetically ablated. Thus, it appears that each myotube is endowed with the ability to independently locate and recognize its specific target cells. Kon confers this ability on a specific subclass of myotubes (Schnorrer, 2007).
kon was identified based on the aberrant muscle patterns in kon mutant embryos: the ventral-longitudinal muscles VL1-4 do not connect to their target cells, but most other muscles appear to attach correctly. Time-lapse analysis of these mutants indicates that kon function is required for targeting of the VL myotubes. The directed migration of these myotubes toward the target is also reduced. Nevertheless, filopodia do frequently touch their target cells, but seem indifferent to them. General myotube motility appears normal, and if the VL myotubes are misrouted ventrally (as in slit mutants), then kon function is not required for their migration toward the ventral midline. Hence, kon function is required specifically for these myotubes to recognize their targets. The VL myotubes do not connect to potential alternative targets in kon mutants, such as the intrasegmental attachment sites of the VA muscles. This distinguishes kon from both slit and derailed (drl). Specific myotubes are misrouted in both slit and drl mutants, yet in these mutants the misrouted myotubes are still able to connect to tendon cells (possibly selecting alternate sites according to an unknown hierarchy of preferences). Thus, defective guidance alone does not preclude tendon cell attachment, although it may lead to the selection of the wrong targets. It is therefore concluded that kon function is specifically required for the VL muscles to recognize and attach to their tendon cell targets (Schnorrer, 2007).
Misexpression of Kon in other muscles does not redirect them to other targets. This is perhaps not surprising, since misexpression of many different axon guidance receptors -- such as Frazzled, Robo, and Ptp69d -- similarly does not lead to axonal misrouting. Presumably, in muscles as in neurons, guidance and targeting are controlled by a suite of factors acting in concert. It was however found that overexpression of Kon results in excessive filopodial activity, which persists even at the late stages during which muscles normally cease filopodial activity and establish stable contacts. This gain-of-function phenotype is consistent with the interpretation of the loss-of-function phenotype, namely, that Kon functions to promote migration until the correct connection is established. If too much Kon is present, this putative 'stop' or targeting signal might be overridden (Schnorrer, 2007).
What is the molecular mechanism underlying Kon function in myotube targeting? It was postulate that Kon is a receptor for a ligand produced by specific tendon cells, and that it transduces this signal intracellularly to modulate cytoskeletal dynamics at the myotube tip. This model is based on the following observations. (1) Kon is a single-pass transmembrane protein that localizes to myotube tips during the targeting steps. (2) It functions cell-autonomously. A VL1 myotube that expresses Kon can still target correctly in an embryo that otherwise completely lacks Kon protein. (3) Even if the rescued VL1 fails to attach in one segment, those in adjacent segments can still attach correctly. This argues against alternative models in which Kon might mediate homophilic adhesion across segment boundaries. (4) Kon protein accumulates in juxtaposition to tendon cells, as might be expected if tendon cells express a binding partner for Kon. (5) Full Kon function requires its cytoplasmic domain, including the C-terminal PDZ-binding motif (Schnorrer, 2007).
The identity of the putative Kon ligand is unknown. Potential ligands have however been identified for vertebrate Kon family proteins. For example, NG2 binds to PDGF-AA and bFGF (Goretzki, 1999), to the kringle domains of plasminogen and angiostatin (Goretzki, 1999), to types V and VI collagen (Burg, 1996; Tillet, 1997), and to galectin-3 (Fukushi, 2004; Wen, 2006). The interaction with galectin-3 is thought to lead to integrin activation (Fukushi, 2004), which could potentially modulate a cell's migratory or adhesive properties. Although at least one of the two Drosophila galectins is expressed in migrating myotubes, a similar mechanism is unlikely to apply for Kon because Drosophila integrins are required for stable muscle attachment, not for migration or targeting. Determining the nature, localization, and function of this ligand is a major goal for future genetic and biochemical studies (Schnorrer, 2007).
Such approaches have however already allowed identification of a cytoplasmic partner for Kon: the PDZ protein Dgrip. Biochemically, the C-terminal PDZ-binding motif in Kon interacts in vitro with the seventh PDZ domain of Dgrip. Genetically, truncating Kon before the PDZ-binding domain and completely eliminating Dgrip result in quantitatively indistinguishable muscle defects. Similarly, the seventh PDZ domain of Dgrip was recently shown to be important to mediate Dgrip function, presumably through Kon. This Kon-Dgrip interaction is also conserved in vertebrates, since mouse NG2 binds to the seventh PDZ domain of mouse Grip1 or Grip2 in vitro and in vivo (Stegmuller, 2003). If Dgrip forms homomultimers through PDZ domains 4-6, as its vertebrate counterparts do, then this might facilitate the clustering of Kon proteins. Alternatively, or in addition, Dgrip might function as an adaptor to recruit other signaling components to the Kon-Dgrip complex. Vertebrate Grip1 is thought to perform this function for other transmembrane proteins, recruiting signaling proteins such as the RasGEF Grasp1 or other adaptors such as Liprin-α. Recently the broadly expressed transmembrane protein Echinoid (Ed) was identified as an binding partner for PDZ domains 1, 2, and 7 of Drosophila Dgrip. Although ed mutants do not display major muscle defects, the VL defects observed in Dgrip mutants are enhanced by removing one copy of ed, indicating that ed may modulate Dgrip activity, possibly by binding to PDZ domains 1 and 2 (Schnorrer, 2007).
While Dgrip clearly plays a critical role in Kon signaling, it is also noted that the complete loss of kon function results in a phenotype that is significantly stronger than that which results from the loss of Dgrip, or the C-terminal truncation of Kon. Thus, Kon can exert at least some function that is independent of its interaction with Dgrip. This might reflect the ability of Kon to activate alternative signaling pathways, possibly involving interactions with other transmembrane proteins. It is also possible that Kon might have a dual function as both a receptor and a ligand, with only the former involving Dgrip. Genetic data do not presently offer any insight into the molecular basis for this additional, Dgrip-independent function of Kon (Schnorrer, 2007).
The structures of Kon and Grip proteins, as well as their physical interactions, have been preserved over more than 600 million years of evolution. In Drosophila, this signaling pathway mediates targeting of myotubes during embryonic development. Functions of this pathway in other species are thus far unknown. In vertebrates, there are two subfamilies of Kon proteins, equally related to Drosophila Kon: the NG2/CSPG4 subfamily and the 'similar to CSPG4' subfamily. Most research has been focused on NG2 itself. In a striking parallel to Kon, NG2 is expressed in developing skeletal muscle (Stallcup, 2002). However, NG2 is more broadly expressed than Kon, including in other migrating cells such as developing neurons, glia, mesenchymal cells, and bone (Fukushi, 2003; Niehaus, 1999; Nishiyama, 1991; Stallcup, 2002), as well as a variety of melanomas and cancer cell lines (Stallcup, 2002). NG2 stimulates cell migration in vitro (Fang, 1999; Niehaus, 1999), and smooth muscle cells from NG2−/− mice display a reduced migratory response to PDGF-AA, a putative NG2 ligand (Grako, 1999). Thus, Kon family proteins may have an evolutionary ancient role in the regulation of cell migration. It is anticipated that further functional studies of NG2 or other Kon family proteins might reveal more specific roles for these proteins in muscle migration and targeting in vertebrates. Conversely, further genetic studies of muscle targeting in Drosophila may provide clues as to how this conserved signaling pathway contributes to cancer metastasis (Schnorrer, 2007).
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date revised: 8 October 2007
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