RESEARCH ARTICLE 1269
Development 134, 1269-1278 (2007) doi:10.1242/dev.000406
Thrombospondin-mediated adhesion is essential for the
formation of the myotendinous junction in Drosophila
Arul Subramanian1,*, Bess Wayburn1,*, Thomas Bunch2 and Talila Volk1,†
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. We show 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 PS2integrin-dependent manner. We propose a model 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.
INTRODUCTION
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 (Bokel and Brown,
2002; Brower, 2003; Brown, 2000). The muscle-specific integrin
heterodimer ␣PS2␤PS 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 ␣PS1␤PS 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 ␤PS 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 musclespecific ␣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 ␣PS2␤PS by its ligand may be
sufficient for the formation of embryonic myotendinous junctions.
The ␣PS1 belongs to the laminin-binding type ␣ family of receptors
1
Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100,
Israel. 2Department of Molecular/Cell Biology, University of Arizona, Tucson, AZ
85721-0001, USA.
*These authors contributed equally to this work
†
Author for correspondence (e-mail: [email protected])
Accepted 23 January 2007
and binds to laminin (Gotwals et al., 1994). Drosophila laminin may
consist of ␤1 and ␤2 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 muscletendon attachment defects (Prokop et al., 1998). By contrast, lack of
the laminin ␣1,2, which associates with the ␣PS2␤PS (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 ␣PS2␤PS 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
␣PS1␤PS are brought together with ventral cells that express
␣PS2␤PS. 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
DEVELOPMENT
KEY WORDS: Myotendinous junction, Thrombospondin, Integrin, Muscle, Tendon, Drosophila
1270 RESEARCH ARTICLE
MATERIALS AND METHODS
Fly strains
The following gal4 lines were used: engrailed-gal4, patched-gal4, 69Bgal4, Df(2L)BSC9 (uncovering tsp), Df(3)DG4 (uncovering stripe),
mysXG43 and sr155 (obtained from the Bloomington Drosophila Stock
Center). tsp8R was produced by P-element-mediated transposition from
P{Epgy2}TspEY00724. UAS-tspA was produced in our laboratory.
For the rescue experiments the following lines were created:
tsp8R/Cyo(BB);sr-gal4/TM6(BB), tsp8R/Cyo(BB);mef2-gal4/TM6(BB) and
tsp8R/Cyo(BB);UAS-tspA/TM6(BB). The first or second fly strains were
crossed with the third strain.
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 Nterminal 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 6⫻ 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-PS␤, 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.
DEVELOPMENT
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 ␣PS2␤PS-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 ␣PS2␤PS integrin receptors,
and a subsequent consolidation of the junction by enhanced tendonspecific production of Tsp secreted into the junctional space.
Development 134 (7)
Thrombospondin is essential for the myotendinous junction
RESEARCH ARTICLE 1271
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 pHS␤PS
together with either pHS␣PS2m8, pHS␣PS2m8-LOF or pHS␣PS1 (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 ␣PS2m8LOF 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).
For cell spreading, 96-well tissue culture plates were coated overnight at
4°C with approximately 30-40 mg/ml C-terminal Tsp polypeptide or the
mutant C-terminal Tsp polypeptide KGD>LGE in PBS+0.3 mM CaCl2, then
blocked with 20% dried milk in PBS+0.3 mM CaCl2 for 1 hour at room
temperature, and washed three times with PBS+0.3 mM CaCl2. Transiently
transfected cells were diluted to 2⫻105 per ml in M3 medium, lacking serum
but containing 2 mg/ml BSA, and allowed to spread on the coated wells for
1 hour. At this time the cells were fixed and observed for cell spreading using
a Nikon phase-contrast microscope (Nikon Diaphot-TMD). Results are
expressed as the mean and standard error of four experiments. For each
experiment three fields of cells, each containing more than 100 cells, were
scored.
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 ␮g 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 ␮g/␮l. This would
indicate a concentration of approximately 150 ␮g/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).
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 1213 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).
DEVELOPMENT
RESULTS
The distribution of Tsp changes following the
formation of the myotendinous junction
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 et al., 2003). 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
Fig. 1. Tsp distribution changes following the formation of the
myotendinous junction. Wild-type embryos at stage 12-13 (A-D) or
stage 16 (E-H) labeled for Thrombospondin (A,E, Tsp, red), Stripe (B,F,
marking all tendon cells, blue) and Myosin heavy chain (C,G, MHC,
marking all somatic muscles, green) are shown, together with their
merged images (D,H). Arrowheads mark the distribution of Tsp prior to
muscle-tendon binding, whereas arrows mark the accumulation of Tsp
at the myotendinous junction following muscle binding.
1272 RESEARCH ARTICLE
Development 134 (7)
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.
Tsp is required for muscle binding to tendon cells
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 (FlyBase annotation
data). We have induced a mutation in the tsp locus (tsp8R) 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. We mapped the
imprecise excision using PCR with primers flanking the insertion
site and 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 (Fig. 3). 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 (Fig. 3C).
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 ␤PS. The typical integrin-positive bands
DEVELOPMENT
Fig. 2. Tsp levels are reduced in myospheroid mutant and are induced by Stripe. (A-D) Mutant embryos lacking functional ␤PS (mysXG43)
and labeled with Tsp (A, red), Stripe (B, blue) and Myosin heavy chain (C, MHC, green) are shown. The merged image is shown in D. Arrows
indicate sites where muscles are still associated with tendon cells, and Tsp is higher. Arrowheads indicate sites where muscles have detached, and
Tsp is significantly low. (E,F) stripe mutant embryos stained for Tsp (E, green) and MHC (red). F is the merged image. Tsp is still detected in these
embryos. (G-I) Embryos overexpressing Stripe using the engrailed-gal4 driver, stained for Tsp (G, red) and anti-Stripe (H, green). Their merged image
is shown in I. Tsp is highly induced by Stripe.
Thrombospondin is essential for the myotendinous junction
RESEARCH ARTICLE 1273
Fig. 3. The CTD is deleted in tsp8R
mutation. Upper panel: a scheme
depicting the genomic structure of the
tsp gene (upper draw) and the location
of the EP element as well as the deletion
obtained by its excision (lower draw). A
domain map of the TSP protein as well as
the truncated protein obtained after the
imprecise excision lacking the conserved
CTD, Tsp repeats and four out of six EGF
domains is shown. Lower panel: the
merged image of wild-type embryo (A),
embryo homozygous for Df(2L)BSC9,
which deletes tsp locus (B), and embryo
homozygous for the P-excision tsp8R (C)
stained for Tsp (red) and Myosin heavy
chain (MHC) (green). Note the loss of Tsp
staining in Df(2L)BSC9 and in tsp8R
mutant embryos.
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 musclespecific 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 integrinmediated 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 ␤PS-integrin staining and is consistent with the loss of
DEVELOPMENT
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 ␤PS 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 ␤PS 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).
The muscle-muscle junctions detected by the staining for ␤PS
may utilize the Tiggrin ligand to assemble the ␣PS2␤PS integrins
on both sides of the hemiadherens junction. Staining with antiTiggrin 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
appropriate myotendinous junction. Thus, in the absence of
functional Tsp, individual myotubes fail to form integrin-mediated
adherens junction with tendon cells.
Tsp promotes cell spreading in a KGD- and PS2dependent manner
The results so far are consistent with a model where the tendondependent Tsp promotes adhesion of the muscles by binding to the
␣PS2␤PS 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
␮g/ml to coat tissue culture plates. S2 cells expressing either
␣PS2m8␤PS integrin, a mutated ␣PS2m8 or ␣PS1␤PS 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
␣PS2m8␤PS 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 ␣PS1␤PS 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
Fig. 4. Muscle migration at stage 14 is normal in tsp mutant
embryos. Wild-type (A-C) or tsp8R mutant (D-F) embryos at stage 14
stained for Stripe (A,D, red) and Myosin heavy chain (B,E, MHC, green).
C,F are their corresponding merged images. Muscle migration and
arrangement is normal in the mutant at this stage.
Development 134 (7)
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
␣PS2␤PS integrin receptors with the KGD site that is included in
the CTD of Tsp.
DISCUSSION
The formation of a functional myotendinous junction is essential for
the normal function of the somatic musculature and the viability of
hatched larvae. In Drosophila embryos this junction develops
following the migration of myotubes towards their corresponding
tendon cells. The signal that promotes muscles to form the junction
with tendon cells, as well as the mechanism governing mutual
induction of the shared junction sites, is yet to be elucidated. Here,
Fig. 5. Muscle rounding and aggregation are observed in tsp8R
mutant embryos at stage 16. Wild-type (A-C) or tsp8R mutant (D-F)
embryos at stage 16 stained for Stripe (A,D, red) and Myosin heavy
chain (B,E, MHC, green) and their corresponding merged images (C,F).
The somatic muscles round up (arrowheads in E,F indicate rounding of
the ventral-longitudinal muscles) and tend to form aggregates (arrows
in E). (G,H) tsp8R mutant embryos overexpressing TspA driven by stripegal4 (G) or by mef-2-gal4 (H) double-labeled with MHC (green) and
with Tsp (red).
DEVELOPMENT
1274 RESEARCH ARTICLE
we show that the ECM protein Thrombospondin, produced and
secreted by the tendon cells, is essential for muscle adhesion to the
tendon cell through association with the muscle-specific PS2
Fig. 6. tsp mutant muscles interact with neighboring muscles and
not with tendon cells. Wild-type embryos (A-E) and tsp mutant
embryos (F-J) labeled for ␤PS integrin (A,F, red) and Myosin heavy chain
(B,G, MHC, green) are shown as well as their merged images (C,H).
Arrowheads in F and H indicate sites of ectopic muscle-muscle
interactions. The bracket in F and H indicates the sites of attachment of
the lateral transverse muscles, showing significantly reduced PS␤
staining at the muscle-tendon attachment sites of two lateral transverse
muscles and positive PS␤ staining when the third lateral transverse
muscle is attached to other muscles (arrowheads in F,H). (D-J) High
magnification of the ventral-longitudinal muscles of embryos stained
for ␤PS (red, D-J), MHC (green, D,I) and Stripe (blue, D-J). Arrowhead in
I and J shows ␤PS staining between the ventral-longitudinal muscle that
is not associated with tendons; the arrowhead in D and E shows the
parallel normal association of ␤PS staining with tendons in wild type.
RESEARCH ARTICLE 1275
integrin receptors. Moreover, we provide a model explaining the
biogenesis of the junction and its temporal and spatial regulation.
Based on our results, we suggest 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 ␣PS2␤PS
integrin receptors. Because Drosophila Tsp forms pentamers, each
pentamer potentially associates with several PS2 receptors, leading
to accumulation of ␣PS2␤PS 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
Fig. 7. Tiggrin accumulates at the muscle-muscle junction sites of
tsp mutant embryos. Wild-type embryos (A-C) or tsp mutant embryos
(D-F) labeled for Tiggrin (A,D, red) and Myosin heavy chain (B,E, MHC,
green), and their corresponding merged images (C,F). Arrows indicate
sites of muscle-muscle interactions.
DEVELOPMENT
Thrombospondin is essential for the myotendinous junction
1276 RESEARCH ARTICLE
Development 134 (7)
Fig. 8. Talin levels are significantly reduced in tsp mutant
embryos. Wild-type embryos (A-C) or tsp mutant embryos (D-F)
labeled for Talin (A,B, red) and Myosin heavy chain (B,E, MHC, green)
are shown as well as merged images (C,F). The brackets indicate the
location of the attachment sites of the three lateral transverse muscles
that do not stain for Talin in the tsp mutant. Talin is still detected in sites
of muscle-muscle attachments.
Fig. 9. PS2 integrin-expressing S2
cells spread on purified C-terminal
Tsp polypeptide. (A) S2 cells lacking or
expressing PS2m8 integrins were plated
on purified Tsp CTD polypeptide (CTD
KGD), Tsp CTD polypeptide with a
mutation in the putative integrinbinding site KGD>LGE (CTD LGE), or on
no ligand (Christopherson et al., 2005).
(B) Coomassie Blue staining of purified
Tsp CTD polypeptide, wild type (CTD)
and the KGD>LGE mutant protein
(CTD*). (C) S2 cell spreading as seen by
phase microscopy; arrowheads show
non-spread (left) versus spread (right)
cells. (D) Spreading of cells expressing
PS2m8 integrins, PS2m8 integrins
containing a mutation that severely
impairs ligand binding (mutPS2m8), or
PS1 integrins plated on purified Tsp CTD
polypeptide or no ligand. The
percentage of spread cells is shown.
Numbers represent the mean and
standard error of three experiments.
DEVELOPMENT
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.
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. Our 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, we detected maternal tsp transcripts that
may partially rescue the zygotic tsp phenotype in the early
developmental stages.
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 heparincontaining proteoglycan. Because we do not detect changes in
laminin distribution following overexpression of Tsp (using antilaminin antibody), we do not consider there to be any direct Tsplaminin 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.
We thank N. Brown, J. and L. Fessler and G. Vorbruggen for providing
antibodies and fly strains, and the Bloomington Stock Center for various fly
lines. We thank E. Schjechter, S. Shwarzbaum and members of the Volk
laboratory for critical reading of the manuscript. This work was supported by a
grant from the Minerva Foundation (T.V.) and NIH grant 5 R01 GM042474 (D.
Brower supporting T.B.).
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