|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 21 February 2007
doi: 10.1242/dev.000406
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot
76100, Israel.
2 Department of Molecular/Cell Biology, University of Arizona, Tucson, AZ
85721-0001, USA.
Author for correspondence (e-mail:
lgvolk{at}weizmann.ac.il)
Accepted 23 January 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Myotendinous junction, Thrombospondin, Integrin, Muscle, Tendon, Drosophila
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Brower,
2003; Brown,
2000). The muscle-specific integrin heterodimer 
PS2ßPS
accumulates at the muscle counterpart of this junction, and binds to its
specific extracellular matrix (ECM) ligand Tiggrin
(Bunch et al., 1998;
Fogerty et al., 1994).
Correspondingly, the tendon-specific integrin heterodimer PS1ßPS
accumulates at the tendon counterpart of the myotendinous junction, and is
thought to associate with the laminin ligand
(Gotwals et al., 1994;
Prokop et al., 1998). 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, muscles
initially interact with tendon cells; however, following muscle contraction
the muscles detach from the tendon cells and round up (the
myospheroid phenotype) (Bokel and
Brown, 2002). Notably, lack of the muscle-specific PS2
subunit similarly leads to muscle detachment
(Brown, 1994); by contrast,
however, lack of the tendon-specific PS1 (e.g. in the mew
mutant embryos) does not lead to muscle detachment
(Brower et al., 1995).
mew mutant embryos hatch, suggesting that occupation of the
muscle-specific PS2ßPS by its ligand may be sufficient for the
formation of embryonic myotendinous junctions. The PS1 belongs to the
laminin-binding type family of receptors and binds to laminin
(Gotwals et al., 1994).
Drosophila laminin may consist of ß1 and ß2 subunits and
either of two laminin subunits
(Garrison et al., 1991;
MacKrell et al., 1993). The
PS1 is thought to associate with laminin containing the LanA
subunit (also known as 3,5), which when deleted does not exhibit
significant muscle-tendon attachment defects
(Prokop et al., 1998). By
contrast, lack of the laminin 1,2, which associates with the
PS2ßPS (Graner et al.,
1998), results in a mild muscle-detachment phenotype (e.g.
wing blister mutants) (Martin et
al., 1999), pointing to the crucial function of the
muscle-specific PS2 in the formation of the myotendinous junction.
Tiggrin, a Drosophila-specific ECM component, has been shown to
associate with the muscle-specific PS2ßPS integrin. However,
homozygous tiggrin mutant embryos do form muscle-tendon junctions and
the adult flies are only semilethal (Bunch
et al., 1998; Fogerty et al.,
1994).
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 (Brabant et al.,
1998; Brower et al.,
1995; Devenport and Brown,
2004; Leptin et al.,
1989; Martin-Bermudo et al.,
1999; Narasimha and Brown,
2004; Stark et al.,
1997). 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 PS1ßPS are brought together with ventral cells
that express PS2ßPS. Adhesion between these two epithelial sheets
of cells is presumably mediated by specific ECM ligands. Although the
involvement of the laminin 1,2 (wing blister) has been
described, ligand specificity of each of the PS integrin receptors in this
context has yet to be elucidated.
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
(Frommer et al., 1996).
Conversely, ectopic expression of Stripe leads to ectopic development of
tendon cells (Becker et al.,
1997; Vorbruggen and Jackle,
1997). In a search for genes that are regulated by the
tendon-specific transcription factor Stripe, we recovered Drosophila
Thrombospondin.
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 and Lawler, 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 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 (Adams et al.,
2003).
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 and Lawler, 2004).
Recently, Tsp1 and Tsp2 were described as astrocyte-secreted components that
promote synapse formation in the CNS
(Christopherson et al.,
2005).
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 it is expressed in wing
imaginal discs (Adams et al.,
2003).
Here we report 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, we show that Tsp can functionally bind to
PS2ßPS-integrins as the purified CTD of Tsp mediates PS2
integrin-dependent cell spreading in a KGD- and PS2-dependent manner.
Taken together, our results suggest a model whereby Tsp produced by tendon
cells is required for muscle-specific adhesion to tendons by binding the
muscle-specific PS2ßPS integrin receptors, and a subsequent
consolidation of the junction by enhanced tendon-specific production of Tsp
secreted into the junctional space.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cloning of Tsp cDNA for expression in flies and bacteria
Expressed sequence tags (ESTs) used for creating tsp cDNA were
GH27479 (Open Biosystems, USA) and AT07402 (Drosophila Genomics
Resource Center).
uas-tsp-AD was prepared by fusing the ESTs GH27479 (encoding the N-terminal domain) and AT07402 (encoding the CTD) in frame and cloning the fused product into a pUAST vector. In order to fuse the ESTs two rounds of PCR were performed. In the first PCR, the respective ESTs were amplified individually with the following primers:
GH27479: forward primer 5'-ATGAATTGGACGCGCGTG-3' and reverse primer 5'-CCTGAGGGGCACGGTGTG-3'. The reverse primer has partial sequence similarity with the 5' region of AT07402.
AT07402: forward primer 5'-CACACCGTGCCCCTCAGGCGCCCAGTGCCTCCAGGT-3' and reverse primer 5'-TCAGTCCTGCAACTCCAC-3' (tspA-R-cDNA). The products of the first round of PCRs were ligated and used as templates for PCR. Here the forward primer was the same as that used to amplify GH27479, whereas the reverse primer was a different one with an EcoRI linker to facilitate later cloning processes - 5'-GGAATTCCGGAATGGTTTGAAGAGTGCG-3' (tspA-EcoRI-R). The fused PCR product (tspAD) was cloned into pUAST at the EcoRI site to yield the uas-tspAD clone. The sequences of the various plasmids were verified by sequencing.
The cDNA GH27479 was cloned into the pGEX vector to produce a GST fusion peptide. The protein was expressed and purified according to standard protocols.
The Gateway Cloning Technology (Invitrogen Life Technologies, USA) was used to produce the purified CTD protein fused with His tag. The CTD encoding sequence was amplified for five cycles using the forward primer 5'-AAAAAGCAGGCTTCTTTAACTTTAAGAAGGAGATATAACTATGCATCACCATCACCATCACTCTGCCCAGTGCCTCCAGGTT-3', which contains part of the recombination sequence and the 6x His tag sequence in the 5' region, and the reverse primer 5'-AGAAAGCTGGGTCTCAGTCCTGCAACTCCACCTTC-3' that consists of the recombination sequence at the 5' region of the insert. The product from the above PCR was used as a template for a regular PCR reaction with the forward primer 5'-GGGGACAAGTTTGTACAAAAGGGGACAAGTTTGTACAAAAAAGCAGGC-3', which completes the homologous recombination sequence at the 5' end of the insert, and the reverse primer 5'-GGGGACCACTTTGTACAAGAGGGGACCACTTTGTACAAGAAAGCTGGG-3', which completes the homologous recombination at the 3' end. This PCR product was used to produce an entry clone in pDONR-201, by recombination. The clone was confirmed by sequencing and was used to transfer the insert into the pDEST14 expression vector. The Tsp-His tag protein was expressed and purified according to the protocols of the Gateway Expression System and Qiagen's Ni-NTA agarose bead system. The purified proteins were concentrated and diluted in PBS containing 2 mM CaCl2 three times. Protein concentration was determined by comparison of Coomassie Blue staining on a standard protein gel. Bovine serum albumin (BSA) was used as a concentration standard.
Staining of embryos
Antibody staining was performed essentially as described previously
(Ashburner, 1989). Primary
antibodies used were anti-Myosin heavy chain (MHC) (P. Fisher, Stony Brook,
NY), anti-Stripe, anti-Tsp (produced in our laboratory), anti-PSß,
anti-Talin (N. Brown, University of Cambridge, Cambridge) and anti-Tiggrin (J.
and L. Fessler, UCLA). Secondary antibodies included Cy3, fluoresceine or
horseradish peroxidase (HRP)-conjugated anti-guinea pig, anti-rat, anti-rabbit
or anti-mouse (Jackson, USA).
Visualization of embryos was performed with a Bio-Rad Radiance 2100 confocal system coupled with a Nikon Eclipse TE300 microscope.
Cell culture, spreading and flow cytometry
Drosophila S2 cells were maintained and transformed as described
(Jannuzi et al., 2002). Cells
were transiently transfected with the selectable marker bearing plasmid pH8CO
and integrin subunit expressing plasmid pHSßPS together with either
pHSPS2m8, pHSPS2m8-LOF or pHSPS1
(Bunch et al., 1992;
Baker et al., 2002;
Li et al., 1998).
PS2m8-LOF produces a mutant PS2m8 subunit (222-224 YWQ>AWA)
that is impaired in ligand binding (Baker
et al., 2002; Irie et al.,
1995). Following transfection, cells were grown for 2 days in
selection medium containing methotrexate. Expression of PS2 integrins was
similar for both wild type and the PS2m8-LOF as determined by staining
with the PS2-specific monoclonal antibody CF.2C7
(Brower et al., 1984) followed
by flow cytometry [mean fluorescence intensities (MFI) were 320±5 and
316±14, respectively. For untransformed S2 cells MFI levels averaged
46]. High levels of PS1 integrin expression in the PS1-transfected cells was
confirmed by staining with the PS1-specific antibody DK1A4
(Brower et al., 1984) followed
by flow cytometry (MFI was 1173±87; for untransformed S2 cells MFI
levels averaged 110).
|
Polyacrylamide electrophoresis of fusion proteins
Wild type (KGD) and mutant (LGE) CTD fusion proteins were electrophoresed
on a 4-15% gradient polyacrylamide gel together with 1.0, 0.5 and 0.25 µg
BSA and prestained Precision Plus Protein All Blue standards (Bio-Rad). The
gel was then stained with Coomassie Blue. Each CTD concentration appears to be
between 0.1 and 0.2 µg/µl. This would indicate a concentration of
approximately 150 µg/ml. A 1/4 dilution of this was used for the cell
spreading assays. Preliminary observations indicated that less spreading was
supported by a further 1/4 dilution, whereas no additional spreading was seen
if the CTD preparations were used without dilution (T.B., unpublished).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). We have raised an antibody to Tsp and used it 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 (Fig. 3B). 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 (labeled with Stripe)
(Fig. 1). 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, we examined the distribution of
Tsp 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
(Fig. 2, arrowheads).
Nevertheless, we still detect higher accumulation of Tsp at the muscle edges,
suggesting partial integrin-independent association with the muscle cells
(Fig. 2, arrow). We have initially recovered Tsp 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 (A.S., unpublished). Further analysis showed that in stripe mutant embryos Tsp protein is still detected, possibly because of earlier Stripe-independent transcriptional input (Fig. 2E,F). 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 (Fig. 2G-I). Because Stripe expression is greatly upregulated following muscle-tendon interaction, it is assumed that Stripe-dependent Tsp induction is linked to muscle-tendon interaction. We conclude that Tsp distribution is dynamic and correlates with the biogenesis of adherens junction formation.
|
To assess the contribution of Tsp to the assembly of somatic musculature, we analyzed the phenotype of tsp mutant embryos. The initial myotube fusion and the migration of muscles towards tendon cells appear normal in the tsp mutant embryos (Fig. 4). 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 (Fig. 5). 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 (see Fig. 6), 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 (Fig. 5G), but not following overexpression of TspA in the muscles using the mef-2-gal4 driver (Fig. 5H). This suggests that the tendon-specific expression of Tsp is essential for its function.
To test whether the somatic muscles in the tsp mutant embryos are capable of forming integrin-mediated adherens junctions, we stained the embryos 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 (Fig. 6). However, we detected ectopic integrin staining at various locations (Fig. 6, arrowheads), 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 (Fig. 6, brackets and arrow). 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 (Fig. 6J, arrowhead), in contrast to wild-type embryos (Fig. 6E, arrowhead).
|
PS2ßPS 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 (Fig. 7, arrows).
The large Tiggrin dots observed between the longitudinal muscles are
consistent with defects in the muscle-tendon interaction, as the tendon cells
are arranged as a line of cells at this region, leading to the subsequent line
of Tiggrin (and integrin) staining. We conclude 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.
Integrin-mediated adherens junctions are greatly reduced in tsp mutant embryos
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 (Brown et al.,
2002). 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 (Fig.
8, brackets). 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.
|
|
PS2ßPS
integrin receptors. To test this model directly, we cloned the CTD 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 PS2m8ßPS integrin, a mutated PS2m8 or
PS1ßPS receptors were plated on these cultured dishes and the
percentage of spreaded cells was determined.
Tsp CTD induces a significant elevation in the number of
PS2m8ßPS 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
(Fig. 9, upper panel).
Similarly, cells expressing a mutated PS2m8 did not induce cell
spreading on the CTD and behaved like the control cells where no ligand was
added. Cells expressing PS1ßPS 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. We were not able to produce
efficient amounts of the Tsp N-terminal domain, presumably because of its
instability.
These experiments are consistent with a direct binding of
PS2ßPS integrin receptors with the KGD site that is included in
the CTD of Tsp.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
PS2ßPS integrin receptors. Because Drosophila Tsp forms
pentamers, each pentamer potentially associates with several PS2 receptors,
leading to accumulation of PS2ßPS 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
(Yarnitzky et al., 1997).
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.
We showed that the KGD site in the CTD of Tsp triggers PS2
integrin-dependent cell spreading. This sequence had been shown to bind
certain types of vertebrate integrin receptors
(Scarborough et al., 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.
|
|
An additional relevant ECM component at the myotendinous junction is
laminin. Laminin 1,2 (encoded by wing blister) is required for
the formation of the myotendinous junction
(Martin et al., 1999). Laminin
1,2 contains an RGD sequence and also binds to the PS2 integrins
(Graner et al., 1998),
demonstrating the crucial role of these receptors in the formation of the
myotendinous junctions. It is possible that laminin containing the laminin
1,2 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 we do not detect changes in laminin distribution following
overexpression of Tsp (using anti-laminin antibody), we do not consider there
to be any direct Tsp-laminin interaction (data not shown). 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
(Steigemann et al., 2004).
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 and Lawler, 2004).
However, syndecan homozygous mutant embryos do not exhibit
alterations in Tsp distribution (data not shown), 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.
We consider 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 (Yarnitzky et
al., 1997). We show that Stripe induces upregulation of Tsp.
Taking these results together, we suggest 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.
Vertebrate Thrombospondins are essential for a variety of biological
activities, including cell adhesion, migration, angiogenesis, etc. Our 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 et al., 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.
In summary, our 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.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Adams, J. C. (2001). Thrombospondins: multifunctional regulators of cell interactions. Annu. Rev. Cell Dev. Biol. 17,25 -51.[CrossRef][Medline]
Adams, J. C. and Lawler, J. (2004). The thrombospondins. Int. J. Biochem. Cell Biol. 36,961 -968.[CrossRef][Medline]
Adams, J. C., Monk, R., Taylor, A. L., Ozbek, S., Fascetti, N., Baumgartner, S. and Engel, J. (2003). Characterisation of Drosophila thrombospondin defines an early origin of pentameric thrombospondins. J. Mol. Biol. 328,479 -494.[CrossRef][Medline]
Ashburner, M. (1989). Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Baker, S. E., Lorenzen, J. A., Miller, S. W., Bunch, T. A.,
Jannuzi, A. L., Ginsberg, M. H., Perkins, L. A. and Brower, D. L.
(2002). Genetic interaction between integrins and moleskin, a
gene encoding a Drosophila homolog of importin-7.
Genetics 162,285
-296.
Becker, S., Pasca, G., Strumpf, D., Min, L. and Volk, T. (1997). Reciprocal signaling between Drosophila epidermal muscle attachment cells and their corresponding muscles. Development 124,2615 -2622.[Abstract]
Bokel, C. and Brown, N. H. (2002). Integrins in development: moving on, responding to, and sticking to the extracellular matrix. Dev. Cell 3,311 -321.[CrossRef][Medline]
Brabant, M. C., Fristrom, D., Bunch, T. A., Baker, S. E. and Brower, D. L. (1998). The PS integrins are required for a regulatory event during Drosophila wing morphogenesis. Ann. N. Y. Acad. Sci. 857,99 -109.[CrossRef][Medline]
Brower, D. L. (2003). Platelets with wings: the maturation of Drosophila integrin biology. Curr. Opin. Cell Biol. 15,607 -613.[CrossRef][Medline]
Brower, D. L., Wilcox, M., Piovant, M., Smith, R. J. and Reger,
L. A. (1984). Related cell-surface antigens expressed with
positional specificity in Drosophila imaginal discs. Proc.
Natl. Acad. Sci. USA 81,7485
-7489.
Brower, D. L., Bunch, T. A., Mukai, L., Adamson, T. E., Wehrli, M., Lam, S., Friedlander, E., Roote, C. E. and Zusman, S. (1995). Nonequivalent requirements for PS1 and PS2 integrin at cell attachments in Drosophila: genetic analysis of the alpha PS1 integrin subunit. Development 121,1311 -1320.[Abstract]
Brown, N. H. (1994). Null mutations in the alpha PS2 and beta PS integrin subunit genes have distinct phenotypes. Development 120,1221 -1231.[Abstract]
Brown, N. H. (2000). Cell-cell adhesion via the ECM: integrin genetics in fly and worm. Matrix Biol. 19,191 -201.[CrossRef][Medline]
Brown, N. H., Gregory, S. L., Rickoll, W. L., Fessler, L. I., Prout, M., White, R. A. and Fristrom, J. W. (2002). Talin is essential for integrin function in Drosophila. Dev. Cell 3,569 -579.[CrossRef][Medline]
Bunch, T. A., Salatino, R., Engelsgjerd, M. C., Mukai, L., West, R. F. and Brower, D. L. (1992). Characterization of mutant alleles of myospheroid, the gene encoding the beta subunit of the Drosophila PS integrins. Genetics 132,519 -528.[Abstract]
Bunch, T. A., Graner, M. W., Fessler, L. I., Fessler, J. H., Schneider, K. D., Kerschen, A., Choy, L. P., Burgess, B. W. and Brower, D. L. (1998). The PS2 integrin ligand tiggrin is required for proper muscle function in Drosophila. Development 125,1679 -1689.[Abstract]
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.[CrossRef][Medline]
Devenport, D. and Brown, N. H. (2004).
Morphogenesis in the absence of integrins: mutation of both Drosophila beta
subunits prevents midgut migration. Development
131,5405
-5415.
Fogerty, F. J., Fessler, L. I., Bunch, T. A., Yaron, Y., Parker, C. G., Nelson, R. E., Brower, D. L., Gullberg, D. and Fessler, J. H. (1994). Tiggrin, a novel Drosophila extracellular matrix protein that functions as a ligand for Drosophila alpha PS2 beta PS integrins. Development 120,1747 -1758.[Abstract]
Frommer, G., Vorbruggen, G., Pasca, G., Jackle, H. and Volk, T. (1996). Epidermal egr-like zinc finger protein of Drosophila participates in myotube guidance. EMBO J. 15,1642 -1649.[Medline]
Garrison, K., MacKrell, A. J. and Fessler, J. H.
(1991). Drosophila laminin A chain sequence, interspecies
comparison, and domain structure of a major carboxyl portion. J.
Biol. Chem. 266,22899
-22904.
Gotwals, P. J., Fessler, L. I., Wehrli, M. and Hynes, R. O.
(1994). Drosophila PS1 integrin is a laminin receptor and differs
in ligand specificity from PS2. Proc. Natl. Acad. Sci.
USA 91,11447
-11451.
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.
Irie, A., Kamata, T., Puzon-McLaughlin, W. and Takada, Y. (1995). Critical amino acid residues for ligand binding are clustered in a predicted beta-turn of the third N-terminal repeat in the integrin alpha 4 and alpha 5 subunits. EMBO J. 14,5550 -5556.[Medline]
Jannuzi, A. L., Bunch, T. A., Brabant, M. C., Miller, S. W., Mukai, L., Zavortink, M. and Brower, D. L. (2002). Disruption of C-Terminal cytoplasmic domain of bPS integrin subunit has dominant negative properties in developing Drosophila. Mol. Biol. Cell 4,1352 -1365.
Lawler, J. (2000). The functions of thrombospondin-1 and-2. Curr. Opin. Cell Biol. 12,634 -640.[CrossRef][Medline]
Leptin, M., Bogaert, T., Lehmann, R. and Wilcox, M. (1989). The function of PS integrins during Drosophila embryogenesis. Cell 56,401 -408.[CrossRef][Medline]
Li, X., Graner, M. W., Williams, E. L., Roote, C. E., Bunch, T. A. and Zusman, S. (1998). Requirements for the cytoplasmic domain of the alphaPS1, alphaPS2 and betaPS integrin subunits during Drosophila development. Development 125,701 -711.[Abstract]
MacKrell, A. J., Kusche-Gullberg, M., Garrison, K. and Fessler, J. H. (1993). Novel Drosophila laminin A chain reveals structural relationships between laminin subunits. FASEB J. 7,375 -381.[Abstract]
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.
Martin-Bermudo, M. D., Alvarez-Garcia, I. and Brown, N. H. (1999). Migration of the Drosophila primordial midgut cells requires coordination of diverse PS integrin functions. Development 126,5161 -5169.[Abstract]
Narasimha, M. and Brown, N. H. (2004). Novel functions for integrins in epithelial morphogenesis. Curr. Biol. 14,381 -385.[CrossRef][Medline]
Prokop, A., Martin-Bermudo, M. D., Bate, M. and Brown, N. H. (1998). Absence of PS integrins or laminin A affects extracellular adhesion, but not intracellular assembly, of hemiadherens and neuromuscular junctions in Drosophila embryos. Dev. Biol. 196,58 -76.[CrossRef][Medline]
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.
Stark, K. A., Yee, G. H., Roote, C. E., Williams, E. L., Zusman, S. and Hynes, R. O. (1997). A novel alpha integrin subunit associates with betaPS and functions in tissue morphogenesis and movement during Drosophila development. Development 124,4583 -4594.[Abstract]
Steigemann, P., Molitor, A., Fellert, S., Jackle, H. and Vorbruggen, G. (2004). Heparan sulfate proteoglycan syndecan promotes axonal and myotube guidance by slit/robo signaling. Curr. Biol. 14,225 -230.[CrossRef][Medline]
Vorbruggen, G. and Jackle, H. (1997). Epidermal
muscle attachment site-specific target gene expression and interference with
myotube guidance in response to ectopic stripe expression in the developing
Drosophila epidermis. Proc. Natl. Acad. Sci. USA
94,8606
-8611.
Yarnitzky, T., Min, L. and Volk, T. (1997). The
Drosophila neuregulin homolog Vein mediates inductive interactions between
myotubes and their epidermal attachment cells. Genes
Dev. 11,2691
-2700.
This article has been cited by other articles:
|
|
 |
|
  L. Zhang, Y. Zhang, and K. G. T. Hagen A Mucin-type O-Glycosyltransferase Modulates Cell Adhesion during Drosophila Development J. Biol. Chem., December 5, 2008; 283(49): 34076 - 34086. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Adams, A. A. Bentley, M. Kvansakul, D. Hatherley, and E. Hohenester Extracellular matrix retention of thrombospondin 1 is controlled by its conserved C-terminal region J. Cell Sci., March 15, 2008; 121(6): 784 - 795. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
