Thrombospondin: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References
Gene name - Thrombospondin
Cytological map position-26F6-27A1
Function - ECM ligand
Symbol - Tsp
FlyBase ID: FBgn0031850
Genetic map position - 2L: 6,686,819..6,709,085 [-]
Classification - thrombospondin homolog
Cellular location - secreted
|Recent literature||Greenblatt Ben-El, R. T., Hassan, A. and Salzberg, A. (2017). Loss of thrombospondin reveals a possible role for the extracellular matrix in chordotonal cap cell elongation. Int J Dev Biol 61(3-4-5): 311-318. PubMed ID: 28621428
In the Drosophila larva, major proprioceptive input is provided to the brain by sub-epidermal stretch receptors called chordotonal organs (ChO). Similarly to the body wall muscle that needs to be attached on both of its sides to the larval exoskeleton in order to generate movement, the sensory unit of a ChO must be stably anchored to the cuticle on both of its sides in order to sense the relative displacement of body parts. Through an RNAi screen this study has identified thrombospondin (Tsp), a secreted calcium binding glycoprotein, as a critical component in the anchoring of ChOs to the cuticle. The Tsp protein starts to accumulate in the extracellular matrix (ECM) surrounding the ChO attachment cells towards the end of embryogenesis and that it becomes highly concentrated at the attachment junction during larval stages. In the absence of Tsp, the ChO's accessory cells fail to form a stable junction with their epidermal attachment cells and organ integrity is not maintained. Tsp is a known player in the establishment of the myotendinous junctions in both invertebrates and vertebrates. Thus, these findings extend the known similarities between muscle-attachment and ChO-attachment cells. In addition to its role in establishing the ChO attachment junctions, Tsp was found to affect ligament cell migration and cap cell elongation. Most interestingly, the Tsp protein was found to decorate the ChO cap cells along their entire length, suggesting that the elongated cap cells are supported by the ECM to which they attach via integrin-based, Tsp-dependent, adhesion plaques. The ECM enwrapping the cap cells is probably important for keeping the cap cells fasciculate and may also provide mechanical support that allows the extremely elongated cells to maintain tension.
Organogenesis of the somatic musculature in Drosophila is directed by the precise adhesion between migrating myotubes and their corresponding ectodermally derived tendon cells. Whereas the PS integrins mediate the adhesion between these two cell types, their extracellular matrix (ECM) ligands have been only partially characterized. This study shows that the ECM protein Thrombospondin (Tsp), produced by tendon cells, is essential for the formation of the integrin-mediated myotendinous junction. Tsp expression is induced by the tendon-specific transcription factor Stripe, and accumulates at the myotendinous junction following the association between the muscle and the tendon cell. In tsp mutant embryos, migrating somatic muscles fail to attach to tendon cells and often form hemiadherens junctions with their neighboring muscle cells, resulting in nonfunctional somatic musculature. Talin accumulation at the cytoplasmic faces of the muscles and tendons is greatly reduced, implicating Tsp as a potential integrin ligand. Consistently, purified Tsp C-terminal domain polypeptide mediates spreading of PS2 integrin-expressing S2 cells in a KGD- and PS2-integrin-dependent manner. A model is proposed in which the myotendinous junction is formed by the specific association of Tsp with multiple muscle-specific PS2 integrin receptors and a subsequent consolidation of the junction by enhanced tendon-specific production of Tsp secreted into the junctional space (Subramanian, 2007).
The development of functional musculature depends on the correct encounter and adhesion of muscles with their corresponding tendon cells. In Drosophila the hemiadherens junctions, formed on both sides of the myotendinous junction, mediate the adhesion between muscles and their corresponding tendon cells. The muscle-specific integrin heterodimer αPS2ßPS accumulates at the muscle counterpart of this junction, and binds to its specific extracellular matrix (ECM) ligand Tiggrin. Correspondingly, the tendon-specific integrin heterodimer αPS1ßPS accumulates at the tendon counterpart of the myotendinous junction, and is thought to associate with the laminin ligand. Both hemiadherens junctions on each cell type exhibit a symmetrical distribution, raising the possibility that, although each cell utilizes a distinct integrin heterodimer, the formation of the myotendinous junction is coordinated between the two cell types. In the absence of the common ßPS subunit (Myospheroid), muscles initially interact with tendon cells; however, following muscle contraction the muscles detach from the tendon cells and round up (the myospheroid phenotype). Notably, lack of the muscle-specific αPS2 subunit similarly leads to muscle detachment; by contrast, however, lack of the tendon-specific αPS1 (e.g., in the mew mutant embryos) does not lead to muscle detachment. mew mutant embryos hatch, suggesting that occupation of the muscle-specific αPS2ßPS by its ligand may be sufficient for the formation of embryonic myotendinous junctions. The αPS1 belongs to the laminin-binding type α family of receptors and binds to laminin. Drosophila laminin may consist of ß1 and ß2 subunits and either of two laminin α subunits. The αPS1 is thought to associate with laminin containing the LanA subunit (also known as α3,5), which when deleted does not exhibit significant muscle-tendon attachment defects. By contrast, lack of the laminin α1,2 (wing blister), which associates with the αPS2ßPS (Graner, 1998), results in a mild muscle-detachment phenotype (e.g., wing blister mutants) (Martin, 1999), pointing to the crucial function of the muscle-specific PS2 in the formation of the myotendinous junction (Subramanian, 2007).
Tiggrin, a Drosophila-specific ECM component, has been shown to associate with the muscle-specific αPS2ßPS integrin. However, homozygous tiggrin mutant embryos do form muscle-tendon junctions and the adult flies are only semilethal (Subramanian, 2007).
In addition to its role in the establishment of myotendinous junctions, integrin-mediated adhesion is essential for several biological processes, including dorsal closure, visceral mesoderm development and the development of the adult fly wing. Wing epithelial cells from the dorsal and ventral aspects of the wing form specialized integrin-mediated adherens junctions required for the development of the adult fly wing. At morphogenesis dorsal wing epithelial cells expressing αPS1ßPS are brought together with ventral cells that express αPS2ßPS. Adhesion between these two epithelial sheets of cells is presumably mediated by specific ECM ligands. Although the involvement of the laminin α1,2 (wing blister) has been described, ligand specificity of each of the PS integrin receptors in this context has yet to be elucidated (Subramanian, 2007).
Tendon cells are specified in the Drosophila ectoderm as a result of the activity of the tendon-specific transcription factor Stripe. Embryos mutant for stripe do not develop normal tendon cells. Conversely, ectopic expression of Stripe leads to ectopic development of tendon cells. Thrombospondin was recovered in a search for genes that are regulated by the tendon-specific transcription factor Stripe (Subramanian, 2007).
Thrombospondins (Tsps) are a family of extracellular matrix proteins that mediate cell-cell and cell-matrix interactions by binding membrane receptors, extracellular matrix proteins and cytokines (Adams, 2001; Lawler, 2000). In vertebrates there are five tsp genes expressed in various tissues, including the brain (TSP1 and Tsp2), bones (Tsp5) and tendons (Tsp4). Tsp1 and Tsp2 are closely related trimeric proteins that share the same set of structural and functional domains. Tsp4 and Tsp5 are pentameric and differ from Tsp1 and Tsp2 in their domain arrangement. All Tsps share a typical C-terminal domain (CTD) that contains epidermal growth factor (EGF)-like repeats, and a Ca-binding domain. The N-terminal domain contains additional conserved regions including the laminin G-like domain (which is not present in Tsp5) (Adams, 2004). Drosophila tsp is encoded by a single gene that is spliced into four variants, among which only one (TspA) contains the conserved CTD, which in addition to the EGF repeats and Ca-binding domains also includes a putative integrin-binding KGD motif. The N-terminal domain contains a conserved heparin-binding domain and putative integrin-binding motifs RGD and KGD. Drosophila Tsp (Adams, 2003) is closest in structure to vertebrate Tsp-5/COMP, which is expressed mainly in cartilage and certain other connective tissues and has a role in chondrocyte attachment, differentiation and cartilage ECM assembly (Subramanian, 2007).
A wide range of functions has been attributed to the different Tsps, including a role in platelet aggregation, inflammatory response, regulation of angiogenesis during wound healing, and tumor growth (Adams, 2004). Tsp1 and Tsp2 have been described (Christopherson, 2005) as astrocyte-secreted components that promote synapse formation in the CNS (Subramanian, 2007).
The large isoform of Drosophila Tsp has been shown to form pentamers and exhibits heparin-binding activity. Its major sites of expression in the embryo are the muscle attachment sites, and also the precursors of the longitudinal visceral muscles. In larval stages (Adams, 2003) it is expressed in wing imaginal discs (Subramanian, 2007).
This study reports that Drosophila Tsp is a key ECM component that is required for muscle-specific adhesion to tendon cells. In tsp mutant embryos muscles fail to attach to tendon cells, and often aggregate and form ectopic integrin-mediated junctions with neighboring muscles. This leads to nonfunctional somatic musculature and embryonic lethality. In the embryo, Tsp is required for integrin-mediated adhesion as measured by Talin-specific accumulation. Furthermore, Tsp can functionally bind to αPS2ßPS-integrins; the purified CTD of Tsp mediates PS2 integrin-dependent cell spreading in a KGD- and PS2-dependent manner (Subramanian, 2007).
Taken together, these results suggest a model whereby Tsp produced by tendon cells is required for muscle-specific adhesion to tendons by binding the muscle-specific αPS2ßPS integrin receptors, and a subsequent consolidation of the junction by enhanced tendon-specific production of Tsp secreted into the junctional space (Subramanian, 2007).
It is suggested that the dynamics of myotendinous junction formation involve the following sequential steps. (1) When the myotube is very close to the tendon cell, Tsp secreted continuously from the tendon cell associates with the muscle leading edge and binds to the muscle-specific αPS2ßPS integrin receptors. Because Drosophila Tsp forms pentamers, each pentamer potentially associates with several PS2 receptors, leading to accumulation of αPS2ßPS receptors at the myotube leading edge. This association triggers integrin-mediated adhesion and Talin accumulation at the cytoplasmic tail of the PS2 integrin receptors. (2) Tsp may bind to the tendon surfaces through an unknown ligand. (3) Stripe levels in the tendon cell are elevated following the establishment of the muscle-tendon junction, because of Vein-EGF receptor (EGFR) signaling. Stripe induces the elevation of Tsp levels, creating a positive feedback loop that encourages further secretion and accumulation of Tsp at the junction site, strengthening the myotendinous junction (Subramanian, 2007).
The KGD site in the CTD of Tsp was shown to trigger PS2 integrin-dependent cell spreading. This sequence had been shown to bind certain types of vertebrate integrin receptors (Scarborough, 1993). The N-terminal domain of Tsp contains an additional KGD site, and an RGD site, both implicated in integrin-binding activity. These sites may also contribute to the binding of the PS2 muscle-specific integrins. Therefore, each Tsp pentamer contains multiple binding sites for PS2 integrin receptors, and thus may induce receptor aggregation at the muscle leading edge. It remains to be determined whether Tsp is capable of binding to PS1 integrins or other receptors expressed by the tendon cell (Subramanian, 2007).
Whether Tsp functions as an integrin ligand in other tissues (e.g., midgut, salivary gland, dorsal closure and the wing epithelium) is yet to be elucidated. Phenotypic analysis of the tsp8R mutant embryos did not reveal a major phenotype in the gut, CNS or dorsal closure. Similarly, tsp8R mutant clones induced at the larvae stage did not result with wing blisters as in integrin-induced clones. Although mutants for the tsp8R allele did not show staining with the anti-Tsp antibody, it is still possible that residual Tsp activity is retained in the mutants because of the activity of the other TSP isoforms (which were not affected by the deletion of the EP excision at the CTD). In addition, maternal tsp transcripts were detected that may partially rescue the zygotic tsp phenotype in the early developmental stages (Subramanian, 2007).
An additional relevant ECM component at the myotendinous junction is laminin. Laminin α1,2 (encoded by wing blister) is required for the formation of the myotendinous junction (Martin, 1999). Laminin α1,2 contains an RGD sequence and also binds to the PS2 integrins (Graner, 1998), demonstrating the crucial role of these receptors in the formation of the myotendinous junctions. It is possible that laminin containing the laminin α1,2 subunit associates with Tsp in the myotendinous junctional space. Both laminin and Tsp carry a heparin-binding domain and it is possible that they interact indirectly through a putative heparin-containing proteoglycan. Because no changes in laminin distribution was detected following overexpression of Tsp (using anti-laminin antibody), it is not thought that there is any direct Tsp-laminin interaction. The heparan sulfate glycoprotein Syndecan is produced by the muscle cells. In syndecan mutant embryos the somatic muscle pattern is defective, a phenotype that is attributed to an effect of Syndecan on Slit distribution and function. However, Syndecan at the muscle cell membrane may contribute to a putative indirect interaction between Tsp and laminin through its heparin-containing domain. Such interaction may enhance the accumulation of ECM components such as Tsp and laminin at the myotendinous junction. In support of this hypothesis, vertebrate Tsp has been shown to bind Syndecan at its CTD (Adams, 2004). However, syndecan homozygous mutant embryos do not exhibit alterations in Tsp distribution, arguing against a central role for Syndecan in Tsp distribution. Nevertheless, it remains possible that another heparin domain-containing protein functions to promote Tsp and laminin deposition at the myotendinous junction (Subramanian, 2007).
It is considered that the Stripe-dependent positive feedback that upregulates tsp transcription contributes significantly to the establishment of the myotendinous junction. Previous studies have shown that muscle-tendon interactions form a signaling center, which is initiated by muscle-dependent Vein secretion and accumulation at the myotendinous junction. Vein activates the EGFR pathway in the tendon cell, leading to a significant elevation of the transcription factor Stripe. This study shows that Stripe induces upregulation of Tsp. Taking these results together, it is suggested that the initial formation of the hemiadherens junction creates a self-auto-regulatory nucleation center, which leads to additional deposition of Tsp and possibly other ECM components. These, in turn, gradually strengthen the hemiadherence junction formed between the muscle and the tendon cell (Subramanian, 2007).
Vertebrate Thrombospondins are essential for a variety of biological activities, including cell adhesion, migration, angiogenesis, etc. This work reveals an intriguing similarity between the role of Tsp in the formation of the myotendinous junction and the role of vertebrate Tsp1 and Tsp2 in the induction of synapses. It was shown that Tsp provided by oligodendrocytes is a potent inducer of synapse formation on the dendrites of cultured neurons (Christopherson, 2005). Although these synapses are not electrically active, the Tsp-induced synapses exhibit typical synaptic ultra-structures. The biogenesis of the myotendinous junction carries several similarities to the biogenesis of synapses, including the mutual crosstalk between the two cell types involved and the gradual formation of the junction at both cell membranes involved (Subramanian, 2007).
In summary, this analysis of Tsp function reveals the molecular dynamics and biogenesis of the myotendinous junction. A similar scenario may unfold during Tsp-dependent synapse formation in the development of vertebrate embryos (Subramanian, 2007).
The isolated oligomerisation domain of TSP-5/COMP has been demonstrated to self-assemble into a five-stranded alpha-helix. To establish the oligomerisation status of D-TSP experimentally and in view of the very high predicted molecular mass of a pentamer of the full-length polypeptide (591,240 Da), the equivalent region of D-TSP was expressed in His-tagged form in Escherichia coli and analysed by Tricine-PAGE under reducing or non-reducing conditions. The reduced protein resolved at the calculated molecular mass, 7.9 kDa, whereas the non-reduced material resolved at an apparent molecular mass of 38 kDa, which corresponded closely to the predicted molecular mass of a pentamer (39.5 kDa) (Adams, 2003).
The properties of full-length recombinant D-TSP were examined in comparison to other ECM molecules. Secreted, V5His6-tagged D-TSP resolved as two bands with apparent molecular masses of 135 kDa and 150 kDa under reducing conditions. The lower band may result from proteolytic susceptibility, as has been noted for mammalian TSPs. Under non-reducing conditions, D-TSP resolved as a single band of apparent molecular mass 600 kDa that was clearly larger than the TSP-5/COMP pentamer of 500 kDa (Adams, 2003).
To characterise native D-TSP, polyclonal antisera were raised to a C-terminal epitope. The affinity-purified immunoglobulins specifically detected proteins of approximately 125,000 Da in 0-16 hour embryos or soft tissue extracts of adult fly bodies under reducing conditions. A presumed proteolytic fragment of around 60,000 Da was also prominent in the fly body. Both bands were competed by the immunising peptide. The secretion of D-TSP by several D. melanogaster cell lines was investigated and it was found that the protein was low or undetectable in the medium of Kc-167 cells and S2 cells, but was readily detectable in the medium of clone 8 imaginal disc cells. To determine whether D-TSP was deposited into ECM, clone 8 cells were grown to high density and extracts made under conditions that preserve assembled, insoluble ECM while removing the cell layer. D-TSP was not extracted by 20 mM NH4OH, but was solubilised by 2 M or 8 M urea. These results demonstrate that endogenous secreted D-TSP becomes associated with ECM in cultured cells. With regard to the production of the 60,000 Da band detected in adult flies, it was noted that minor bands of similar molecular mass were detected in the long-term cell cultures (Adams, 2003).
To localise endogenous matrix-associated D-TSP, non-permeabilised clone 8 cells were stained with the affinity-purified IgG to D-TSP. D-TSP was specifically detected in a non-fibrillar, pericellular location around the cells. The intensity of staining was non-uniform, with clusters of cells showing bright staining and others little or no staining. Staining was blocked in the presence of the immunizing peptide. Pre-treatment of the cultures with 2 M urea abolished the staining completely (Adams, 2003).
Although the amino-terminal domain of insect TSPs did not correspond to the laminin-G-like fold predicted for TSPs 1-4, the presence of canonical heparin-binding motifs in the N-terminal domains of Drosophila and Anopheles TSPs suggested that insect TSPs might also have heparin-binding activity. To test this, conditioned medium from cells expressing recombinant D-TSP was precleared on glutathione-Sepharose beads, then incubated with heparin-Sepharose and washed in buffers with increasing concentration of NaCl. D-TSP bound specifically to heparin-Sepharose and binding activity was detectable even after elution with 0.6 M NaCl. According to normalization by scanning densitometry, 65% was retained in the presence of 0.6 M NaCl and 34% was retained in the presence of 1 M NaCl. To relate the activity to the amino-terminal domain, the same experiment was carried out with a truncated protein (designated P341), consisting of the amino-terminal domain, coiled-coil domain and first EGF-like domain of D-TSP with a His-tag was prepared. This protein resolved as a monomer of 43 kDa under reducing conditions. P341 bound specifically to heparin-Sepharose and the interaction was stable to elution by 1 M NaCl. According to normalisation by scanning densitometry, 75% of the bound material was resistant to elution with 0.6 M NaCl and 45% was retained in the presence of 1 M NaCl. Endogenous D-TSP secreted by clone 8 cells also bound to heparin effectively. These results identify D-TSP, and its amino-terminal domain, as a strong heparin-binding protein (Adams, 2003).
The correct assembly of the myotendinous junction (MTJ) is crucial for proper muscle function. In Drosophila, this junction comprises hemi-adherens junctions that are formed upon arrival of muscles at their corresponding tendon cells. The MTJ mainly comprises muscle-specific alphaPS2betaPS integrin receptors and their tendon-derived extracellular matrix ligand Thrombospondin (Tsp). Reported here is the identification and functional analysis of a novel tendon-derived secreted protein named Slowdown (Slow). Homozygous slow mutant larvae exhibit muscle or tendon rupture, sluggish larval movement, partial lethality, and the surviving adult flies are unable to fly. These defects result from improper assembly of the embryonic MTJ. In slow mutants, Tsp prematurely accumulates at muscle ends, the morphology of the muscle leading edge changes and the MTJ architecture is aberrant. Slow was found to form a protein complex with Tsp. This complex is biologically active and capable of altering the morphology and directionality of muscle ends. This analysis implicates Slow as an essential component of the MTJ, crucial for ensuring muscle and tendon integrity during larval locomotion (Gilsohn, 2010).
When migrating muscles reach their target tendons they reorganize their leading edge in order to arrest migration and form the integrin-mediated MTJ. The possible molecular link between the arrest of muscle migration and the formation of the MTJ has not yet been characterized. The present study analyzes these processes, revealing the function of the novel gene product Slow, a Drosophila ortholog of vertebrate Egfl7, as an important modulator of integrin-mediated adhesion (Gilsohn, 2010).
The phenotype of muscle/tendon rupture observed in slow mutant larvae is unique and does not resemble that of mutants for the muscle- or tendon-specific integrins, or for their specific ligands Tsp, Laminin or Tiggrin, in which the typical phenotype is muscle detachment from tendon cells and muscle cell rounding following initial muscle contraction in the embryo. Despite the localization of Slow at the MTJ, its deletion leads to phenotypes similar to those caused by mutations affecting either the cytoskeletal arrangement of mature muscles. These unique phenotypes enabled identification of a novel and crucial aspect of MTJ construction, namely the correct assembly of the integrin receptors and their ECM ligand Tsp at the surfaces of the muscle ends (Gilsohn, 2010).
It is suggested that the defect in slow mutants, manifested by the rupture of both muscles and tendons, might be explained by two mechanisms, which are not mutually exclusive. First, the lack of Slow may result in an aberrant arrangement of the ECM material deposited between the muscle and the tendon, which becomes too rigid and compact; therefore, the mechanical stress imposed by muscle contraction would lead to muscle or tendon rupture. Second, the fine architecture of the muscle-tendon hemi-adherens junction is aberrant, leading to an unequal distribution of mechanical forces upon muscle contraction, resulting in sporadic muscle or tendon rupture. Ultrastructure electron microscopy analysis of the larval muscle-tendon junction did not reveal significant changes in the arrangement of the electron-dense material deposited between the two cell types. Thus, the possiblity if favored that muscle/tendon rupture occurs due to aberrant formation of the MTJ and the unequal distribution of mechanical forces occurring following muscle contraction (Gilsohn, 2010).
When the muscle leading edge reaches the tendon cell, it must undergo morphological changes prior to the establishment of the hemi-adherens junction with the ECM ligand(s) provided by the tendon cell. Should junction formation precede the smoothening and widening of the muscle leading edge, it might lead to the formation of an MTJ with aberrant morphology, in which the integrin receptors are not homogenously distributed and the muscle surfaces are rough. Such a scenario is consistent with observation that in slow mutant embryos, Tsp and muscle-specific integrins prematurely accumulate at the muscle leading edge prior to its arrival at the tendon cell. It was also shown, both in live embryos and fixed material, that at a later developmental stage the muscle leading edge in slow mutant embryos does not spread, resulting in a narrow contact area. These two processes may be interconnected so that the premature accumulation of Tsp and integrin leads to the abnormal pointed morphology of the muscle leading edge. These changes then lead to abnormal MTJ architecture and eventually to defective muscle function, resulting in muscle/tendon rupture and lethality. Additional support for this model comes from the observation that the sole overexpression of αPS2βPS integrin at an early stage of muscle migration leads to a muscle phenotype that is reminiscent of slow, i.e., an altered morphology of the muscle ends and torn muscles at the larval stage (Gilsohn, 2010).
It is therefore suggested that Slow activity allows the muscle leading edges to correctly spread along the surfaces of the tendon cells in order to maximize the contact surface area and to enable the gradual and homogenous distribution of integrins along the entire surfaces of the contact area (Gilsohn, 2010).
Whether the formation of the Slow-Tsp complex (observed in S2 cells) is directly linked to the premature accumulation of Tsp and integrin at the muscle leading edge is not clear at this stage. It is possible that the KGD domain in Tsp, which is required both for integrin binding and for association with Slow, is masked by Slow, attenuating the Tsp-integrin association with this site; thus, in the absence of Slow, Tsp-integrin interaction occurs prematurely by the association of the muscle integrin with this site. Alternatively, Slow-Tsp association at the KGD site may facilitate the association of the integrin receptors with the alternative RGD site located at the N-terminal region of Tsp, and this might be important for proper Tsp-integrin interaction. Experiments could not distinguish between these possibilities. In vitro spreading assays of αPS2βPS-expressing S2 cells showed that Slow reduces integrin-dependent cell spreading on a Tsp matrix. This favors the possibility that Slow attenuates integrin-Tsp binding (Gilsohn, 2010).
Importantly, the genetic interaction found between slow and inflated indicates that in the absence of Slow, the muscle-specific integrin functions less efficiently in mediating proper muscle function. This supports the possibility that Slow regulates integrin-dependent MTJ formation by allowing gradual accumulation of αPS2βPS integrin at the MTJ, and that reducing integrin levels further worsens MTJ construction (Gilsohn, 2010).
The ectopic expression of Tsp and Slow led to clear changes in the somatic muscle pattern, as well as to altered morphology of the muscle leading edge. Upon arrival at the ectopic expression site of Tsp and Slow, the leading edge of several muscles displayed a pointed morphology directed towards the ectopic expression site. In other cases, muscles arrived at their respective tendons, although their leading edge conformation was also altered to a narrower edge. This result further suggests a role for Slow in regulating the shape of muscle ends and emphasizes the biological significance of Tsp-Slow complex formation. Ectopic integrin accumulation was barely detected at the muscle edges, which terminated at the ectopic Tsp-Slow sites, supporting the notion that Slow attenuates integrin-Tsp association and providing an explanation for the premature integrin and Tsp accumulation in the absence of Slow (Gilsohn, 2010).
Taken together, these results demonstrate that Slow modulates the interaction between Tsp and integrin to impose the correct MTJ architecture, although the exact mechanism of Slow action at the molecular level requires further analysis (Gilsohn, 2010).
Vertebrate Egfl7 is highly expressed in endothelial cells during embryonic stages and after injury, and is downregulated in most fully differentiated blood vessels of adult tissue. Egfl7 knockout in zebrafish and mice leads to aberrant blood vessel formation, resulting in severe hemorrhages throughout the body and potential lethality. Recently, this phenotype was attributed to the deletion of the Mir126 regulatory microRNA, which resides within intronic sequences of Egfl7. Therefore, the unique role of Egfl7 in blood vessel formation remains to be further clarified. Significantly, Mir126 is not included within intronic sequences of Drosophila slow (Gilsohn, 2010).
In summary, the results demonstrate a unique and novel function for the Drosophila Egfl7 ortholog Slow in coordinating the morphological changes that occur at the muscle leading edge following its arrival at the tendon cell and in the establishment of the MTJ. The link between Slow and Tsp might be highly relevant to blood vessel development in vertebrates, which, in addition to Egfl7, is also characterized by expression of Tsp1 and Tsp2, representing a potential general molecular paradigm for Slow/Egfl7 activity (Gilsohn, 2010).
To obtain information on the expression of Drosophila TSP in relation to cell differentiation, the spatial expression of the D-TSP gene during embryogenesis was examined. Transcripts were first detected immediately after egg deposition, suggesting a maternal contribution. During the blastoderm stage, the message disappeared gradually and no message was observed during gastrulation. During germband extension, transcripts appeared again in a patch at the anterior dorsal side. This patch persisted throughout late germband extension and a repetitive pattern became apparent in the ectoderm and mesoderm. This pattern did not change during germband retraction, and at the retracted stage strong transcription was observed in a segmental pattern that corresponded to the future apodeme cells and a subset of myoblasts. Anteriorly, RNA expression was observed in the dorsal pharygneal myoblasts. At stage 15, strong expression persisted in most apodeme cells and in a subset of muscle cells. In embryos close to hatching, most cells of apodemal origin (i.e. intersegmental apodemes and intrasegmental apodemes 1 and 2) showed transcription of D-TSP. Interestingly, transcription of D-TSP was readily detectable in imaginal discs, in particular in the wing disc, where strongest expression was found in the presumptive wing notum and at low levels on the presumptive wing itself. Transcripts were also detected in several areas of the leg disc (Adams, 2003).
Previous studies showed that tsp mRNA is detected in segmental stripes that correspond to the muscle attachment sites from stage 11 of embryonic development (Adams, 2003). An antibody was raised to Tsp and used to correlate the distribution of Tsp protein with the process of somatic muscle development. The specificity of the antibody is demonstrated by its lack of reactivity with embryos homozygous for Df(2L)BSC9, which removes the tsp gene. At stage 12-13, prior to the formation of muscle-tendon junctions, Tsp protein is detected as scattered dots around the Stripe-expressing tendon cells. These dots appear to be external to the tendon cells, as judged by their distance from the tendon cell. At stage 16, following the establishment of the myotendinous junction, Tsp becomes highly concentrated at the muscle-tendon junction sites, as deduced from its localization between the muscle edges and the tendon cell nucleus. The change in Tsp distribution is consistent with a dynamic process, where following the formation of the myotendinous junction, Tsp associates preferentially with this site. To investigate further the relationship between Tsp distribution and muscle-tendon adherens junction formation, the distribution of Tsp was examined in myospheroid (mysXG43) mutant embryos lacking functional integrins. In mys mutant embryos, the muscles migrate normally towards their attachment sites and the muscle pattern appears normal at stage 14-15 of embryonic development. However, at stage 16, when the muscles initiate their contraction, they pull away from the attachment sites and become rounded. Tsp distribution in mysxg43 embryos at stage 16 is not as concentrated as in wild-type embryos and often appears as scattered dots, similarly to stage 12-13 wild-type embryos. Nevertheless, higher accumulation of Tsp is still detected at the muscle edges, suggesting partial integrin-independent association with the muscle cells (Subramanian, 2007).
Tsp was initially recovered in a microarray screen for genes that are downstream of Stripe by comparing the gene expression profile of embryos overexpressing Stripe in the ectoderm with that of wild-type embryos. Further analysis showed that in stripe mutant embryos Tsp protein is still detected, possibly because of earlier Stripe-independent transcriptional input. Importantly, overexpression of Stripe using the engrailed-gal4 driver leads to a significant induction of Tsp expression, confirming the microarray results and the ability of Stripe to induce Tsp expression. Because Stripe expression is greatly upregulated following muscle-tendon interaction, it is assumed that Stripe-dependent Tsp induction is linked to muscle-tendon interaction. It is concluded that Tsp distribution is dynamic and correlates with the biogenesis of adherens junction formation (Subramanian, 2007).
The genomic organization of the tsp gene suggests that the gene produces four splice variants, of which only TspA includes the conserved CTD and the Tsp type-3 repeats. A mutation in the tsp locus (tsp8R) was induced by imprecise excision of an EP element inserted within the tsp coding region, 847 nucleotides 5' to the stop codon of TspA. Although flies homozygous for precise excisions of this EP element are viable, the imprecise excision leads to embryonic lethality. The imprecise excision was mapped using PCR with primers flanking the insertion site and it was found that it removes 3457 nucleotides within the tsp gene. The deletion results in the putative production of a truncated protein, which lacks the conserved CTD, the Tsp type-3 repeats and four out of six EGF repeats. The gene on the 3' flank of the tsp gene (CG11327) is not affected. Staining of the tsp8R homozygous mutants with the anti-Tsp antibody (raised against the N-terminal domain) revealed that the truncated Tsp protein is not detected in these embryos (Subramanian, 2007).
To assess the contribution of Tsp to the assembly of somatic musculature, the phenotype of tsp mutant embryos was analyzed. The initial myotube fusion and the migration of muscles towards tendon cells appear normal in the tsp mutant embryos. However, a large proportion of the somatic muscles of stage 16 tsp8R mutant embryos are rounded. In addition, the muscles do not extend between their corresponding Stripe-expressing tendon cells, as in wild-type embryos. This phenotype is detected in all the muscle types at stage 16, although some variability exists between the distinct muscles, and the phenotype is more severe in embryos at late stage 16. No significant difference was detected between the phenotype of direct muscle-tendon junctions, e.g. the lateral transverse muscles, and indirect junctions, e.g. ventral-lateral muscles. The residual association of the muscles with tendons in tsp mutants may reflect the redundant function of laminin (Wing blister), which may contribute, although to a lesser extent, to muscle-tendon interaction. In addition, maternal tsp, which may still be present at this stage, may contribute to the residual muscle-tendon association. The rounded muscle phenotype is reminiscent of the mys mutant embryos, suggesting that in tsp mutant embryos the association of somatic muscles with tendon cells may be abrogated. The overall pattern of tendon cells was slightly aberrant, as deduced from the Stripe expression pattern, presumably reflecting the aberrant somatic muscle pattern. Essentially, a similar phenotype was observed in embryos trans-heterozygous for tsp8R and Df(2L)BSC9, which uncovers the entire tsp gene, suggesting that tsp8R represents a severe mutant allele of tsp. Importantly, the muscle phenotype of the tsp8R mutant embryos is rescued by overexpressing TspA in tendon cells using the stripe-gal4 driver, but not following overexpression of TspA in the muscles using the mef-2-gal4 driver. This suggests that the tendon-specific expression of Tsp is essential for its function (Subramanian, 2007).
To test whether the somatic muscles in the tsp mutant embryos are capable of forming integrin-mediated adherens junctions, the embryos were stained for integrin ßPS. The typical integrin-positive bands were still detected in each segment of the tsp mutant embryo, corresponding to the ends of the ventral and dorsal longitudinal muscles. However, ectopic integrin staining was detected at various locations, which appeared to correspond to regions of muscle-muscle interactions. This phenotype was detected in all tsp mutant embryos, in at least one segment. Importantly, the edges of each of the lateral transverse muscles, which normally interact with a single tendon cell, exhibited a large reduction in integrin staining except when the lateral transverse muscle was associated with a neighboring muscle cell. The positive staining of integrin at sites overlapping muscle-muscle interactions, as well as the rounding up of some of the muscles, raised the possibility that the somatic muscles of tsp mutant embryos bind primarily with neighboring muscle cells and not with tendon cells, and the relative 'normal' staining of ßPS integrin in the tsp mutant embryos represents sites of muscle-muscle-dependent adherens junctions. Indeed, a lateral view of tsp mutant embryos shows that in some cases ßPS observed at the ends of the muscles is not coupled to Stripe-expressing tendon cells, in contrast to wild-type embryos (Subramanian, 2007).
The muscle-muscle junctions detected by the staining for ßPS may utilize the Tiggrin ligand to assemble the αPS2ßPS integrins on both sides of the hemiadherens junction. Staining with anti-Tiggrin revealed that in tsp mutant embryos Tiggrin accumulation is not observed as stripes but rather in dots, and often Tiggrin-positive dots are detected in ectopic sites. The large Tiggrin dots observed between the longitudinal muscles are consistent with defects in the muscle-tendon interaction, since the tendon cells are arranged as a line of cells at this region, leading to the subsequent line of Tiggrin (and integrin) staining (Subramanian, 2007).
It is concluded that the somatic muscles in tsp mutant embryos fail to form junctions with tendon cells, but are still capable of forming integrin-mediated junctions with neighboring muscles, presumably using Tiggrin as an ECM ligand for the muscle-specific integrin (Subramanian, 2007).
The abnormal pattern of the somatic muscles in the tsp mutant embryo raised the possibility that the muscle-tendon integrin-mediated adhesion is defective in the mutant embryos. A hallmark of appropriate integrin-mediated adhesion is the accumulation of Talin at the cytoplasmic face of the hemiadherens junction, where it binds directly to the integrin cytoplasmic domain, modulating its ligand affinity and recruiting actin microfilaments to this site. A significant reduction of accumulated Talin levels in tsp mutant embryos is observed. Whereas Talin is still detected at the sites of muscle-muscle junctions, it was entirely missing at sites where individual muscles would normally form junctions with single tendon cells, in particular at the junction sites formed between the lateral transverse muscles and their corresponding tendon cells. The lack of Talin at these sites corresponds with the lack of ßPS-integrin staining and is consistent with the loss of appropriate myotendinous junction. Thus, in the absence of functional Tsp, individual myotubes fail to form integrin-mediated adherens junction with tendon cells (Subramanian, 2007).
The results so far are consistent with a model where the tendon-dependent Tsp promotes adhesion of the muscles by binding to the αPS2ßPS integrin receptors. To test this model directly, the CTD was cloned into a Histidine-tag expression vector. In addition, a mutated CTD (CTD*) where the KGD site was mutated into LGE was similarly produced. Both CTDs were produced in bacteria, purified on Ni-NTA agarose beads, concentrated, and diluted in PBS containing 0.3 mM CaCl2. The purified proteins were used at ~40 µg/ml to coat tissue culture plates. S2 cells expressing either αPS2m8ßPS integrin, a mutated αPS2m8 or αPS1ßPS receptors were plated on these cultured dishes and the percentage of spreaded cells was determined (Subramanian, 2007).
Tsp CTD induces a significant elevation in the number of αPS2m8ßPS cells spreading relative to a control where no ligand was added. Importantly, the mutated CTD (CTD-LGE) did not induce cell spreading and was similar to the no-ligand control. Similarly, cells expressing a mutated αPS2m8 did not induce cell spreading on the CTD and behaved like the control cells where no ligand was added. Cells expressing αPS1ßPS did not show a specific elevation in the number of cells spreading on the CTD. The relatively low percentage of spread cells on the Tsp-CTD may reflect a partial reconstitution of the Tsp-CTD produced and purified from bacteria. Large amounts of the Tsp N-terminal domain could not be produced, presumably because of its instability (Subramanian, 2007).
These experiments are consistent with a direct binding of αPS2ßPS integrin receptors with the KGD site that is included in the CTD of Tsp (Subramanian, 2007).
During Drosophila embryogenesis, the attachment of somatic muscles to epidermal tendon cells requires heterodimeric PS-integrin proteins (α- and β-subunits). The α-subunits are expressed complementarily, either tendon cell- or muscle-specific, whereas the β-integrin subunit is expressed in both tissues. Mutations of β-integrin cause a severe muscle detachment phenotype, whereas α-subunit mutations have weaker or only larval muscle detachment phenotypes. Furthermore, mutations of extracellular matrix (ECM) proteins known to act as integrin binding partners have comparatively weak effects only, suggesting the presence of additional integrin binding ECM proteins required for proper muscle attachment. This study reports that mutations in the Drosophila gene thrombospondin (tsp) cause embryonic muscle detachment. tsp is specifically expressed in both developing and mature epidermal tendon cells. Its initial expression in segment border cells, the tendon precursors, is under the control of hedgehog-dependent signaling, whereas tsp expression in differentiated tendon cells depends on the transcription factor encoded by stripe. In the absence of tsp activity, no aspect of muscle pattern is affected, nor is formation of the initial contact between muscle and tendon cells or muscle-to-muscle attachments. However, when muscle contractions occur during late embryogenesis, muscles detach from the tendon cells. The Tsp protein is localized to the tendon cell ECM where muscles attach. Genetic interaction studies indicate that Tsp specifically interacts with the αPS2 integrin and that this interaction is needed to withstand the forces of muscle contractions at the tendon cells (Chanana, 2007).
Attachment of muscles to tendon cells critically depends on integrin activity. The integrin-like muscle detachment phenotype of the tsp mutants as well as the co-localization of Tsp and integrin proteins strongly suggest that Tsp plays a decisive role in the integrin-mediated cell adhesion process of muscles and tendon cells. In fact, Tsp was shown to encode a pentameric glycosylated protein that is part of the ECM (Adams, 2003). Thus, it could indeed function as a direct binding partner of the PS-integrins (Chanana, 2007).
In order to establish such a functional link between tsp and PS-integrins by genetic means, double mutant embryos were generated that carry either a strong loss of function allele for the αPS1 subunit of integrin due to the mewM6 mutation or a hypomorphic allele for the αPS2 subunit due to the ifB2 mutation in combination with only one wild-type copy of tsp (tspΔ6 and tspΔ79) (Chanana, 2007).
In comparison with wild-type embryos mewM6 mutants bearing two copies of tsp develop a normal muscle pattern with muscle detachment in only few segments. mewM6 mutant embryos that have only one wild-type copy of tsp develop a weak muscle detachment phenotype affecting a small number of longitudinal muscles. In contrast, ifB2 mutants bearing two copies of tsp developed a mild detachment phenotype in several segments, which was strongly enhanced, both with respect to the extent of detachment and penetrance in embryos with only one remaining wild-type copy of tsp. In these embryos, longitudinal, ventral as well as dorsal muscles were found to be detached, a phenomenon not observed in tsp mutant embryos. Furthermore, in mewM6 mutants lacking both wild-type copies of tsp, only a mild enhancement of the tsp mutant phenotype was observed. In contrast, ifB2 lacking both wild-type copies of tsp develop dramatic muscle pattern defects beyond an additive effect of the two individual mutant phenotypes. The enhancement of the muscle detachment phenotype of if mutants by the removal of one copy of tsp and the dramatic enhancement that affects even the set of muscles that are not affected in each of the single if or tsp mutants establishes an essential role of Tsp in the αPS2-dependent muscle attachment process. In contrast, the weak effects of the reduction of the tsp dose in hemizygous mewM6 mutants makes a prominent role of Tsp in αPS1-dependent muscle attachment rather unlikely (Chanana, 2007).
Vertebrate Tsp is a glycosylated protein that forms oligomers and is capable of interacting with both calcium and heparin (Adams, 2001). Furthermore, it has been shown to directly interact with the extracellular part of integrin proteins (Lawler, 1988). This interaction depends on a highly conserved RGB motif, which is characteristic of integrin binding proteins of the ECM (Chanana, 2007).Vertebrate genomes code for up to five Tsps (Adams, 2001), whereas the Drosophila genome contains only a single tsp-coding sequence (Adams, 2003), which, however, encodes two Tsp variants which differ in their carboxyterminal regions. Previous biochemical studies on Drosophila Tsp showed that the protein is secreted and able to form a pentameric structure as suggested by the molecular weight of the secreted native complex (Adams, 2003). At the sequence level, the conserved Drosophila Tsp contains all functionally characterized domains including the critical RGD motif required for integrin binding. In contrast to vertebrate Tsp, the RGD motif in Drosophila Tsp is positioned in the aminoterminal region, close to two BBXB sequence motifs known to bind to heparin, instead of the third Tsp/COMP domain (Adams, 2003). In addition, a KGD motif is observed in the third Tsp/COMP domain of Drosophila Tsp which was shown to serve also as an interaction motif for integrins (Ruoslahti, 1996). Drosophila Tsp contains therefore two RGD/KGD motifs that would allow direct binding of PS2, the integrin heterodimer that was previously found to associate with Tig, an interaction that was shown to be dependent on the presence of the RGD motif. Furthermore, an RGD motif is also required to mediate the interaction of PS2 with the laminin α-chain Wb (Chanana, 2007).
Drosophila tsp is expressed in all ectodermal tendon precursor cells, strongly enriched in those positioned at the segment border of the embryo. Furthermore, tsp is expressed in all differentiated tendon cells after muscle contact. Therefore, tsp is expressed in all cells that have previously been identified by the expression of stripe. stripe encodes an EGR-type Zn-finger transcription factor that is required for tendon cell differentiation. Like stripe, the initial expression of tsp is controlled by Hedgehog signaling at the segment borders and requires stripe activity only during the later stages when the tendon cells are already differentiated. These results suggest that the genes stripe and tsp are activated in parallel by Hh-dependent Ci activity, and that stripe activity maintains the expression of tsp during the later stages when Ci activity has ceased (Chanana, 2007).
Tsp is secreted from epidermal tendon cells and accumulates at the tendon cell matrix, a specific ECM to which the muscles attach. The functional characterisation of the newly generated tsp alleles, which fail to express detectable amounts of tsp transcript, showed that Tsp is necessary for the proper anchoring of muscles at the tendons cells. As observed with mutants affecting the β subunit and the αPS2 subunit of integrin, mys and if, respectively, the muscles were found to detach from their epidermal attachment sites once muscle contraction occurs. Thus, tsp activity is not required for any aspect of muscle pattern formation and/or muscle guidance as well as proper adherence to tendon cells but plays an essential role in maintaining the interconnection between muscles and tendons cells once contraction occurs. Although the muscle detachments are less pronounced than in mys or if mutants, the detachment phenotype of tsp mutants is by far stronger than the corresponding phenotypes that are caused by the loss of other ECM proteins, such as Tig, Wb and LanA, known to be integrin interaction partners. The strong and specific enhancement of the detachment phenotype of mutants that carry a weak if allele, in response to the loss of one or both tsp wild-type alleles strongly suggests that Tsp functions as an essential ECM binding partner of αPS2 encoded by if. The specificity of the genetic interaction shown in this study is consistent with the finding that binding to αPS2 requires an RGD motif as has been found in Tig and Wb as well. Mutations of either tig or wb cause weak muscle detachment phenotypes as observed with tsp mutants, suggesting a redundant αPS2 integrin interaction system in which ECM binding is provided by different partners and that each of them is required for the proper anchoring of the muscles. This conclusion is consistent with the finding that tig, wb and tsp mutants display a weaker phenotype than the if loss of function mutants. Based on the specific expression of tsp in both tendon cell precursors and differentiated tendon cells, which differs from the multiple expression sites of tig and wb, together with the strong enhancement of the muscle detachment phenotype in if and tsp double mutants, it appears likely that tsp is the crucial interaction partner of the αPS2 integrin subunit to provide proper anchoring of muscles to tendon cells. This proposal, and the relative contribution of each of the by now three different αPS2 integrin subunit binding proteins, can be tested once double and triple mutant combinations for all the genes involved and biochemical test systems become available (Chanana, 2007).
The following two reviews deal with Thrombospondins:
Bornstein, P. (2001). Thrombospondins as matricellular modulators of cell function. J. Clin. Invest. 107(8): 929-34. Medline abstract: 11306593
Murphy-Ullrich, J. E. (2001). The de-adhesive activity of matricellular proteins: is intermediate cell adhesion an adaptive state? J. Clin. Invest. 107(7): 785-790. Medline abstract: 11285293
Search PubMed for articles about Drosophila Thrombospondin
Search PubMed for articles about Drosophila Thrombospondin
Adams, J. C. (2001). Thrombospondins: multifunctional regulators of cell interactions. Annu. Rev. Cell Dev. Biol. 17: 25-51. Medline abstract: 11687483
Adams, J. C., et al. (2003). Characterisation of Drosophila thrombospondin defines an early origin of pentameric thrombospondins. J. Mol. Biol. 328(2): 479-94. Medline abstract: 12691755
Adams, J. C. and Lawler, J. (2004). The thrombospondins. Int. J. Biochem. Cell Biol. 36: 961-968. Medline abstract: 15094109
Adolph, K. W. (2001). A thrombospondin homologue in Drosophila melanogaster: cDNA and protein structure. Gene 269(1-2): 177-84. Medline abstract: 11376949
Bornstein, P. (2001). Thrombospondins as matricellular modulators of cell function. J. Clin. Invest. 107(8): 929-34. Medline abstract: 11306593
Chanana, B., Graf, R., Koledachkina, T., Pflanz, R. and Vorbruggen, G. (2007). αPS2 integrin-mediated muscle attachment in Drosophila requires the ECM protein Thrombospondin. Mech Dev. 124(6): 463-75. Medline abstract: 17482800
Christopherson, K. S., Ullian, E. M., Stokes, C. C., Mullowney, C. E., Hell, J. W., Agah, A., Lawler, J., Mosher, D. F., Bornstein, P. and Barres, B. A. (2005). Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. Cell 120: 421-433. Medline abstract: 15707899
Gilsohn, E. and Volk, T. (2010). Slowdown promotes muscle integrity by modulating integrin-mediated adhesion at the myotendinous junction. Development 137(5): 785-94. PubMed Citation: 20110313
Graner, M. W., Bunch, T. A., Baumgartner, S., Kerschen, A. and Brower, D. L. (1998). Splice variants of the Drosophila PS2 integrins differentially interact with RGD-containing fragments of the extracellular proteins tiggrin, ten-m, and D-laminin 2. J. Biol. Chem. 273: 18235-18241. Medline abstract: 9660786
Lawler, J., Weinstein, R. and Hynes, R. O. (1988). Cell attachment to thrombospondin: the role of ARG-GLY-ASP, calcium, and integrin receptors, J. Cell. Biol. 107: 2351-2361. Medline abstract: 2848850
Lawler, J. (2000). The functions of thrombospondin-1 and-2. Curr. Opin. Cell Biol. 12: 634-640. Medline abstract: 10978901
Martin, D., Zusman, S., Li, X., Williams, E. L., Khare, N., DaRocha, S., Chiquet-Ehrismann, R. and Baumgartner, S. (1999). wing blister, a new Drosophila laminin alpha chain required for cell adhesion and migration during embryonic and imaginal development. J. Cell Biol. 145: 191-201. Medline abstract: 10189378
Murphy-Ullrich, J. E. (2001). The de-adhesive activity of matricellular proteins: is intermediate cell adhesion an adaptive state? J. Clin. Invest. 107(7): 785-790. Medline abstract: 11285293
Ruoslahti, E. (2006). RGD and other recognition sequences for integrins, Annu. Rev. Cell. Dev. Biol. 12: 697-715. Medline abstract: 8970741
Scarborough, R. M., Naughton, M. A., Teng, W., Rose, J. W., Phillips, D. R., Nannizzi, L., Arfsten, A., Campbell, A. M. and Charo, I. F. (1993). Design of potent and specific integrin antagonists. Peptide antagonists with high specificity for glycoprotein IIb-IIIa. J. Biol. Chem. 268: 1066-1073. Medline abstract: 8419315
Subramanian, A., Wayburn, B., Bunch, T. and Volk, T. (2007). Thrombospondin-mediated adhesion is essential for the formation of the myotendinous junction in Drosophila. Development 134(7): 1269-78. Medline abstract: 17314133
date revised: 15 February 2011
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