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© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 4191-4204 doi:10.1242/dev.114777
REVIEW
Tendon development and musculoskeletal assembly: emerging
roles for the extracellular matrix
Arul Subramanian and Thomas F. Schilling*
Tendons and ligaments are extracellular matrix (ECM)-rich structures
that interconnect muscles and bones. Recent work has shown how
tendon fibroblasts (tenocytes) interact with muscles via the ECM to
establish connectivity and strengthen attachments under tension.
Similarly, ECM-dependent interactions between tenocytes and
cartilage/bone ensure that tendon-bone attachments form with the
appropriate strength for the force required. Recent studies have also
established a close lineal relationship between tenocytes and skeletal
progenitors, highlighting the fact that defects in signals modulated by
the ECM can alter the balance between these fates, as occurs in
calcifying tendinopathies associated with aging. The dynamic finetuning of tendon ECM composition and assembly thus gives rise to
the remarkable characteristics of this unique tissue type. Here, we
provide an overview of the functions of the ECM in tendon formation
and maturation that attempts to integrate findings from developmental
genetics with those of matrix biology.
KEY WORDS: Tendon, Ligament, Tenocyte, Extracellular matrix
Introduction
Tendons and ligaments are connective tissues that transmit
mechanical forces between muscles and bones. Tendons attach
muscle to skeleton, whereas ligaments attach skeletal elements to
each other and stabilize skeletal joints. Vertebrates have evolved a
remarkable variety of tendons and ligaments to accommodate their
distinct modes of locomotion as well as their dramatic variations in
body size and strength. These range from broad sheets to highly
elastic cables, such as those of the Achilles’ tendon and the cruciate
ligaments of the knee. Because of their structural roles, injuries to
tendons and ligaments are extremely common and often
debilitating. Thus, a fundamental question in musculoskeletal
biology is how these connective tissue structures develop in the
correct locations and acquire the strength necessary to translate
contractions of muscles into skeletal movements.
Both tendons and ligaments contain fibroblasts (termed tenocytes
and ligamentocytes, respectively) embedded in a unique
extracellular matrix (ECM) that is composed mainly of collagen
fibril arrays capable of withstanding incredibly strong tensile forces.
These fibrils are crosslinked to one another and wrapped in a tendon
sheath (Banos et al., 2008; Kannus et al., 1998; Ros et al., 1995).
This basic structure is shared among tendons and ligaments, and the
fibroblasts that produce the ECM develop from common
progenitors with similar gene expression profiles (Juneja and
Veillette, 2013; Sugimoto et al., 2013; Tozer and Duprez, 2005;
Yang et al., 2013). However, each tendon or ligament differs in its
precise ECM composition, size and strength (Birch et al., 2013).
Department of Developmental and Cell Biology, University of California, Irvine,
Irvine, CA 92697-2300, USA.
*Author for correspondence ([email protected])
How are these differences established? The answer to this question
has important implications for understanding how diseases or
damage to tendons and ligaments arise and for developing better
treatment strategies.
Despite their pivotal roles in musculoskeletal connectivity and
functional stability, the mechanisms that control tendon
development have received much less attention than the processes
of myogenesis or skeletogenesis. Only a handful of factors are
known to help specify tenocyte progenitor cells (TPCs) at muscle
attachments, induce them to differentiate, and maintain and repair
them in the adult (Aslan et al., 2008; Huang et al., 2015; Liu et al.,
2012, 2014; Schweitzer et al., 2010; Yang et al., 2013). Studies in
animal models (e.g. fly, fish, chick and mouse embryos), in
particular those focusing on the formation of myotendinous
junctions (MTJs; the major sites of force transmission), have
revealed a crucial link between developing TPCs and the dynamic
ECM that surrounds these cells. Indeed, ECM proteins (e.g.
collagens, laminins, thrombospondins) initially guide myofibers to
their sites of attachment, but also mediate signaling between TPCs
and muscles, regulate the maturation of MTJs, and maintain tendons
in response to mechanical force (Kjaer, 2004; Schwartz et al., 2013;
Snow and Henry, 2009). Here, we review recent genetic studies that
have identified crucial roles for the ECM in tendon development,
and we discuss the emerging nexus between the transcriptional
control of tenocyte differentiation and the organization of the ECM
associated with muscle fibers (myomatrix), MTJs and tendons
(Fig. 1).
ECM production and regulation in developing tendons
Collagens ( predominantly Col1a) constitute the bulk of mature
mammalian tendon ECM and MTJs, whereas laminins (Lams) and
many other non-collagenous ECM components comprise the
remainder (Kannus et al., 1998; Kannus, 2000; Kjaer, 2004;
Birch et al., 2013; Thorpe et al., 2013). Which cells secrete these
ECM proteins, how are they produced in the correct proportions,
and how do they assemble?
The transcriptional regulation of ECM production by TPCs
Strikingly, all of the key transcription factors known to function in
TPC development directly regulate the transcription of genes
encoding ECM proteins (Fig. 2A,B). The best studied of these is
Scleraxis (Scx), a basic helix-loop-helix transcription factor, and the
earliest known marker of TPCs. Scx is first induced through the
interplay of sonic hedgehog (Shh) and fibroblast growth factor
(FGF) signaling in the ‘syndetome’ compartment of somites (the
region of the sclerotome adjacent to the myotome) and by
transforming growth factor beta (TGFβ) signaling in the limbs of
mice at embryonic day (E) 10.5 (Schweitzer et al., 2001; Brent et al.,
2003; Havis et al., 2014). In vitro, Scx overexpression is sufficient to
transform mesenchymal stem cells (MSCs) and human embryonic
stem cells (hESCs) into tenocytes (Fig. 2A) (Alberton et al., 2012;
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MTJ
Myomatrix
Tendon matrix
Fn, Lam, Col, MMP,TIMP, Tsp
SLRP, Col, Lam,Tsp, MMP, TIMP
Tenocyte
Muscle
fiber
Key
Laminin
(Lam)
Fibronectin
(Fn)
Thrombospondin
(Tsp)
Collagen
(Col)
Fig. 1. Composition of the ECM surrounding muscle, tendon and
myotendinous junctions. A muscle fiber (green) secretes ECM components
into its surroundings (the myomatrix). Some of these components overlap with
those of the tendon ECM, which is secreted by tenocytes (red). Myomatrix is
primarily composed of Lam trimers and Fn. By contrast, the tendon
matrix is rich in Col1a trimers and thrombospondin pentamers. The
myotendinous junction (MTJ) is the narrow zone in which ECM components of
tendon and muscle interact.
Chen et al., 2012; Li et al., 2015). Thus, considerable effort has been
made to elucidate the functions of Scx in the tenocyte lineage and to
identify its downstream targets.
The loss of Scx (i.e. as in Scx −/− mutant mice) disrupts tenocyte
differentiation leading to atrophy of force-transmitting tendons
and a disorganized tendon ECM (Murchison et al., 2007). The
expression of the major structural collagens, Col1a1, Col1a2,
Col3a1 and Col14a1, is strongly reduced in Scx −/− mutants. Scx
directly controls Col1a1 and Col1a2 transcription (Fig. 2B) (Espira
et al., 2009; Léjard et al., 2007). Numerous other tendon regulators,
such as the glycoprotein tenomodulin (Tnmd), are also
A Tenocyte specification
downregulated in Scx −/− mutants (Murchison et al., 2007;
Shukunami et al., 2006). At the ultrastructural level, the loss of
Scx disrupts the sheaths that surround each fascicle of collagen
fibrils as well as cellular processes, which normally encircle the
fibrils (Murchison et al., 2007). These results demonstrate that Scx
controls tendon ECM production, which is essential for effective
force transmission.
It should be noted that TPCs still develop in Scx −/− mutant mice,
suggesting that other genes are required for the initial steps of TPC
specification. In Drosophila, the transcription factor Stripe (Sr)
specifies TPCs in epidermal segment border cells (Volk and
VijayRaghavan, 1994). Flies lacking Sr function fail to form TPCs
and display disrupted muscle patterning and attachments, whereas
Sr overexpression transforms ectodermal progenitors into TPCs
(Becker et al., 1997). Embryonic TPCs in mice express orthologs
of Sr – Egr1 and Egr2 (Lejard et al., 2011) – and Egr1 is sufficient
to induce Scx expression and specify MSCs as tenocytes in vitro
(Guerquin et al., 2013). However, like Scx, both Egr1 and
Egr2 appear to be dispensable for tenocyte specification, as
Egr1 −/−/Egr2 −/− double mutant mice are viable. Instead they
regulate the tendon ECM and MTJ, binding to tendon-specific
enhancer elements of Col1a1 and Col1a2 that are also bound by Scx
(Fig. 2B) (Léjard et al., 2007, 2011; Guerquin et al., 2013). Egr1 −/−
mutant mice also downregulate Tnmd and are slow to heal tendon
injuries as adults (Guerquin et al., 2013). Thus, in contrast to flies,
vertebrate Egrs function in tendon ECM production rather than TPC
specification.
Another potential TPC ‘specifier’ is the TALE family atypical
Iroquois-like homeodomain protein Mohawk (Mkx). Like Scx and
Egr1, Mkx can drive bone marrow-derived MSCs towards a
tenocyte fate in vitro (Liu et al., 2015; Otabe et al., 2015).
However, in mouse embryos the expression of Mkx begins in
developing tenocytes later than that of Scx or Egr1/2 (at E13.514.5), becoming restricted to tendon sheath cells and collateral
ligaments (which stabilize joints) in the limbs by E16.5 (Anderson
B Tendon-specific ECM gene regulation
MSC
BMP
Shh, FGF,TGFβ
(Axial)
Smad3
Scx
FGF (Mouse limb)
SPC
Sox5/6/9
Scx
TPC
Scx
Egr1
Egr2
Sox9
Col1a1
Col1a2
Tnmd
Col3a1
Col14a1
Smad3
Tsp4
Col1a1
Col1a2
Tgfb2
Tnmd
Bgn
Tgfbr2
Tgfb2
Col1a1
Col1a2
Tnmd
Fmod
Dcn
Mkx
Sox6
Myod
Runx
Smad3
Mkx
C Skeletal-specific ECM gene regulation
Scx Mkx
Tnmd
Egr1
Osteoblast
Tenocyte
Sox9
Runx2
Mmp9
Mmp13
Tram2
Fig. 2. Transcriptional regulation of tenocyte specification and tendon ECM production. (A) A mesenchymal stem cell (MSC, purple), the common
progenitor for skeletal and tenocyte progenitors, becomes a skeletogenic progenitor cell (SPC, blue) if it is exposed to high levels of BMP signaling, then it
expresses Sox5/6/9 followed by Runx2 during its differentiation into an osteoblast. By contrast, an MSC becomes a tendon progenitor cell (TPC, pink) if it receives
high levels of Shh, FGF and TGFβ signaling, then expresses scleraxis (Scx) followed by Mkx, Egr1 and Tnmd during its transition into a tenocyte. The fate of the
progenitor cells is determined by the level of Sox9 and Scx. The plasticity of progenitor cell fate at this stage is represented by the double-headed gray arrow.
(B) The transcription factors involved in tenocyte specification also regulate the transcription of genes encoding ECM proteins. Direct (black) and indirect (gray)
transcriptional target genes regulated by Scx, Egr1, Egr2 and Mkx in TPCs are indicated. Note that Mkx also represses the expression of factors involved in
myogenic and skeletogenic progenitor formation. (C) Transcription factors expressed in skeletogenic progenitor cells (e.g. Sox9 and Runx2) directly regulate the
transcription of a distinct set of ECM target genes.
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Runx2
ChM-1
Col2a1
Col9a2
Col11a2
Agg
Comp
Runx2
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A Tendon-independent phase
E
EC
CM
M
TPC
Smad
3
Myoblast
Scx, Mkx
MyoD
Runx2
EC
B Tendon-dependent phase
Sox6,
Sox9
M
Fig. 3. Myoblast-tenocyte interactions and ECM production. (A,B)
The formation of myotendinous junctions can be considered as a twostep process. In the initial tendon-independent phase (A) in vertebrates
(shown here for zebrafish trunk muscles), myoblasts (green) synthesize a
‘pre-tendon’ ECM that includes the integrin ligands Tsp4 and Lama2. This
ECM accumulates in the absence of TPCs (brown).
Mechanotransduction coupled with TGFβ signaling (through Tgfβ2 and
Tgfβr2) leads to the Smad3-dependent expression of Scx and Mkx in
TPCs, which in turn leads to the expression of tendon-selective ECM
genes. Smad3 and Mkx also repress the activity of MyoD, Sox9 and
Runx2 to repress myogenic and skeletogenic fates during tenocyte
differentiation. A later tendon-dependent phase (B) relies on the
production of ECM, particularly Col1a1, Col1a2, Col12a1 and Col14a1,
by more mature TPCs, which extend processes into the ECM.
TPC
Myoblast
Scx, Mkx
Egr1, Egr2
Key
Fn
Itg
Col1a1
Col1a2
Tgfβr2
Alk
Tsp4
Lama2
Tgfβ2
et al., 2006). Mkx −/− mutant mice are viable, fertile, and form
normal tendons at first with no defects in Scx expression but later
exhibit reduced levels of Col1a1, Col1a2, Tnmd, fibromodulin
(Fmod) and decorin (Dcn) as well as thinning of collagen fibrils (Ito
et al., 2010; Kimura et al., 2011; Liu et al., 2010). Similar to Scx,
Mkx can function as a transcriptional activator when complexed
with Smad2/3 to promote Col1a1, Col1a2, Tnmd and Dcn
expression as well as TGFβ2 expression in murine MSCs (Liu
et al., 2015). However, at other promoters it interacts with the Sin3A/
histone deacetylase (HDAC) complex to repress gene expression,
including that of key myogenic factors such as MyoD (Myod1),
Sox6 and the cartilage determinant Sox9 (Fig. 2B) (Anderson et al.,
2009; Anderson et al., 2012; Berthet et al., 2013; Chuang et al.,
2014). Thus, like Scx and Egr1/2, Mkx controls tendon maturation
and ECM production and might function, in part, to maintain
tenocytes by preventing them from acquiring myogenic or
skeletogenic fates.
Both Scx and Mkx interact with Smad3, an essential
transcriptional mediator of TGFβ signaling, to regulate tendon
ECM production (Berthet et al., 2013; Hosokawa et al., 2010;
Katzel et al., 2011; Oka et al., 2008; Pryce et al., 2009).
Accordingly, Tgfb2−/−/Tgfb3−/− conditional double mutant mice,
or conditional mutant mice lacking Tgfβr2 receptors in limb
mesenchyme, initially form TPCs in the limbs but lose them by
E14.5, suggesting a role for TGFβ signaling in tendon maintenance
(Pryce et al., 2009). Tendon defects in Smad3−/− mutants are much
less severe, with transient reductions in Col1a1, Col1a2 and Tnmd
expression in their limbs. Like Mkx, Smad3 can also inhibit the
expression and activity of MyoD as well as that of skeletogenic
factors such as Runx2 in vitro (Fig. 2A, Fig. 3) (Alliston et al., 2001;
Kang et al., 2005; Liu et al., 2001). However, unlike Mkx, which
represses MyoD transcription, Smad3 acts post-translationally by
binding E-box sites in MyoD and sequestering it away from its
targets (Chuang et al., 2014). These results hint at a dynamic
network involving TGFβ signaling, Scx and Mkx to achieve and
maintain the tenocyte fate.
In summary, to date no single factor fits the bill as being
both necessary and sufficient for TPC specification. Rather,
transcriptional regulators of tenocytes share functions in the
production of tendon ECM and MTJ assembly and in the
repression of other mesenchymal fates. This is important not only
in the context of tenocyte development but also in the regulation of
MSCs, where the balance between these factors determines if a cell
becomes a TPC or a skeletogenic (or myogenic) progenitor
(Fig. 2A). An attractive model is one in which Scx (in concert
with TGFβ signaling) shifts the fate of MSCs towards TPCs and
initiates tendon ECM production, whereas factors expressed later in
development, such as Mkx and Egr1, supplement the role of Scx by
inducing the expression of ECM proteins as well as by repressing
myogenic and skeletogenic fates. Furthermore, signaling mediated
by mechanical forces upregulates expression of Scx, Mkx and
Smad3, stimulating more ECM production, thereby providing
positive feedback for fine-tuning tendon strength, as we discuss
further below (Eliasson et al., 2008; Maeda et al., 2011).
ECM production and function during tenocyte morphogenesis and
MTJ formation
Although the migration of muscle progenitors has been well studied
in both invertebrates and vertebrates, very little is known about the
role of ECM in morphogenesis of TPCs and the establishment of the
MTJ. In Drosophila, myoblasts migrate to sites of attachment and
interact with tenocytes located at fixed sites at segment borders in the
epidermis (Volk and VijayRaghavan, 1994; Schweitzer et al., 2010).
These myoblasts recognize tenocytes through multiple signals
including Thrombospondin (Tsp) in the ECM, which binds muscle
integrins (Itgs) (Subramanian et al., 2007). Similarly, vertebrate
myoblasts in the trunk and limbs elongate and attach via TPCs already
localized to future muscle attachment sites. How do migrating
vertebrate myofibers and TPCs interact, and how do these interactions
differ between the trunk, limb and head? In the chick, early progenitor
pools of limb TPCs condense and split to form individual tendons
(Kardon, 1998). By contrast, cranial TPCs that arise in the neural crest
migrate to the locations of future MTJs (Grenier et al., 2009; Noden,
1988; Noden and Trainor, 2005). Trunk TPCs arise from the
syndetome of somites, whereas limb TPCs are thought to originate
from lateral plate mesoderm (Brent et al., 2003; Kardon, 1998). These
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FACIT Col
Col12a1
distinct embryonic origins and modes of tendon morphogenesis raise
the question of where the patterning information for muscle
connectivity lies, within the TPCs or within the muscles
themselves? Chick-quail chimera studies suggests that, at least for
cranial and limb muscles, the TPCs and tendon matrix determine the
pattern of attachments (Kardon, 1998; Kieny and Chevallier, 1979;
Noden, 1988). Do muscles and the myomatrix play any role in TPC
formation or maintenance? In the limbs of chick embryos in which
muscle progenitors have been surgically removed, tenocytes initially
develop in the correct locations but later degenerate (Kardon, 1998).
Limb tendons also degenerate in MyoD−/− and Pax3−/− mutant mice
that lack the entire limb musculature (Bonnin et al., 2005; Brent et al.,
2005). Cranial tendons in myod1−/−/myf5−/− double mutant zebrafish
show similar defects (Chen and Galloway, 2014). These results
suggest a dependence on muscles and the myomatrix for tenocyte
maintenance but not specification. As discussed below, these
phenotypes could be due to a lack of mechanical forces transmitted
via the ECM.
What are the roles of specific ECM components of the myomatrix
or tendon ECM during the initial establishment of contact between
myoblasts and TPCs at attachment sites? Studies of axial muscles in
the zebrafish trunk have provided insights into this process (Snow and
Henry, 2009). These muscles attach to intersegmental boundaries
(ISBs) during embryogenesis before the appearance of TPCs. ISBs
are anatomically distinct from later tendons, but contain many of the
same ECM components and serve analogous functions in bearing the
forces of muscle contraction. During this ‘tendon-independent’ phase
of development (Fig. 3A), fibronectin (Fn) and laminin-alpha2
(Lama2) in the myomatrix are highly enriched at ISBs as the
myoblasts elongate and are required for embryonic muscle
attachments (Koshida et al., 2005; Snow et al., 2008). Mammalian
muscles also require Fn and Lam for migration and attachment
(Turner et al., 1983; Bajanca et al., 2006; Vaz et al., 2012). Fn and
Lam bind to Itg and dystrophin/dystroglycan complexes on muscle
cell surfaces. In zebrafish, this leads to localized phosphorylation of
focal adhesion kinase (pFAK; Ptk2ab – Zebrafish Information
Network) at the ends of myofibers where they attach to ISBs, which
stabilizes myotome boundaries (Bassett et al., 2003; Henry et al.,
2005; Parsons et al., 2002; Snow et al., 2008). TPCs only appear later
along the ISBs, where they contribute additional ECM to strengthen
existing attachments in what we refer to here as a ‘tendon-dependent’
phase (Fig. 3B) (Charvet et al., 2011, 2013; Chen and Galloway,
2014; Subramanian and Schilling, 2014). Thus, in zebrafish, somitic
muscles attach via the myomatrix at ISBs prior to the appearance of
TPCs. This may help explain how other types of attachments, such as
‘fleshy insertions’ of mammalian muscles, develop.
Another ECM protein recently shown to be crucial for tendon
development is thrombospondin 4 (Tsp4; also known as Thbs4).
Like Fn and Lam, zebrafish Tsp4b is initially produced by
myoblasts and accumulates at ISBs prior to muscle attachment
(Fig. 3A) (Subramanian and Schilling, 2014). Tsp4b maintains
muscle attachments at the ISB, and its depletion leads to detachment
upon contraction. The transplantation of wild-type myoblasts into
Tsp4b-deficient embryos locally rescues muscle attachments at
ISBs, consistent with a role for Tsp4b in the myomatrix during the
tendon-independent phase of attachment (Subramanian and
Schilling, 2014). Interestingly, tsp4b mRNA abruptly disappears
from differentiating myofibers as they attach, suggesting a feedback
mechanism that regulates tsp4b transcription. Mammalian
myoblasts also express Tsp4, and human TSP4 (THBS4)
expression increases in pathological conditions such as Duchenne
muscular dystrophy and alpha-sarcoglycanopathies (Chen et al.,
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2000; Jelinsky et al., 2010). Thus, Tsp4 in the myomatrix helps
muscles attach in the absence of TPCs and might also facilitate
subsequent tendon maintenance and response to damage.
ECM and collagen fibril assembly during tendon and MTJ maturation
As MTJs mature, tenocytes secrete the bulk of the MTJ/tendon
ECM, particularly the many proteins and proteoglycans that make
up the core functional units, the collagen fibrils (Fig. 3B). These
interact with one another and align into groups of fibrils or fascicles
during progressive phases of muscle attachment (Fig. 4). This
fibrillar organization is essential for tendons to bear the stress of
muscle contraction and prevent bone detachment (avulsion
fractures) by controlling force distribution (Birch et al., 2013; Pan
et al., 2013; Schwartz et al., 2013; Pingel et al., 2014). The fibrillar
network of proteins includes: (1) core force-transmitting, structural
collagens ( particularly Col1a1, Col1a2, Col2a1 and Col3a1); (2)
scaffolding proteins [e.g. Tsp2 (Thbs2), Tsp4, Comp, Lama2]; and
(3) specialized crosslinking collagens (e.g. Col6a1, Col12a1,
Col14a1 and Col22a1) and various crosslinking factors [e.g. Dcn,
Fmod, biglycan (Bgn)], which hold fibrils together to distribute
forces efficiently and reduce friction (Figs 3, 4) (Wang et al., 2012;
Dunkman et al., 2013). Although the integration of structural
collagens into fibrils has been well documented, recent studies have
provided insight into the functions of scaffolding proteins and
specialized collagens during fibril assembly. These studies suggest
that similar to the early ECM at developing MTJs, ECM proteins of
the maturing tendon provide continuous feedback in response to
mechanical force (Choi et al., 2014; Popov et al., 2015; Wall and
Banes, 2005; Zhang and Wang, 2010).
Col12a1, Col14a1 and Col22a1 belong to the class of fibrilassociated collagens with interrupted triple helices (FACITs), which
localize to muscle attachments in avian and mouse tendons, and are
also expressed in human tendon fibroblasts (Fig. 3B; Fig. 4) (Koch
et al., 2004; Wälchli et al., 1994). Studies of FACITs at zebrafish ISBs
have been informative for understanding their functions as MTJs
mature. In zebrafish, muscles first express Col12a1 and Col22a1 at
larval stages and these progressively align into the orthogonal fibril
arrays of mature MTJ/tendon ECM (Charvet et al., 2011, 2013).
Col12a1 is expressed earlier than Col22a1 and colocalizes with
Lama2 throughout muscle fiber attachment (Bader et al., 2009).
Col22a1 maintains attachments under tension, and its expression
increases in tendinopathies (see Box 1) in humans (Charvet et al.,
2013; Jelinsky et al., 2011). Recently, mutations in the human
COL12A1 gene that disrupt COL12A1 secretion have been linked to a
form of Bethlem myopathy (Bushby et al., 2014; Schessl et al., 2006);
other forms of this myopathy are caused by mutations in COL6A1,
COL6A2 and COL6A3, which also show elevated expression in
human tendinopathies (Bönnemann, 2011; Jelinsky et al., 2011). In
mice, Col12a1 interacts with tenascin C (Tnc) and helps crosslink
other collagens during fibril maturation by interacting with Dcn (Veit
et al., 2006). Transmission electron microscopy studies also suggest
that Col12a1 forms complexes with structural collagens (e.g. Col1a1,
Col1a2), as well as other scaffolding and crosslinking proteins (Dcn,
Fmod, thrombospondins) (Font et al., 1996). Thus the FACIT
proteins are highly conserved regulators of tendon ultrastructure and
elasticity.
Other proteins involved in fibril assembly include the small
leucine-rich proteoglycans (SLRPs) such as Dcn (Fig. 4), Fmod,
biglycan and lumican. These are found in relatively small amounts
in tendons, yet loss-of-function mutations in the genes encoding
these proteins disrupt collagen fibrillogenesis (Chakravarti, 2002;
Corsi et al., 2002; Zhang et al., 2006). SLRPs mainly crosslink
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M
Myyo
otu
tubbee
A Early attachment phase
Cartilage cells
Myotube
B Mid-attachment phase
Tenocyte
Cartilage
Cartilage Cells
cells
Myo
tube
C Late attachment phase
Tenocyte
Tsp4
Dcn
Itg
Fn
Lam
FACIT
collagen
Col1a1
Col1a2
trimer
DMD
complex
Fig. 4. Maturation and assembly of the tendon ECM. Diagram illustrating progressive changes in the ECM at an MTJ as it matures. (A) In the early attachment
phase, myoblasts (green) first extend towards a cartilage condensation (blue) and reorganize the local ECM by secreting Tsp4 (red), which interacts with Fn and
Lam. A magnified view (right) of the boxed area illustrates how Tsp4 pentamers assemble Fn, Lam and Dcn and facilitate binding to Itgs on both muscle and
cartilage cell surfaces, thereby promoting adhesion. (B) Following this, in the mid-attachment phase, linear collagen fibrils (Col1a1 trimers, dark blue) form,
tenocytes (red) invade, and Sox9+/Scx+ progenitors (dark blue and orange) become detected at the future attachment site on the cartilage, the enthesis. The
magnified view illustrates how Col1a1 trimers begin to align perpendicular to skeletal cells (enthesis, dark blue and orange). Dystrophin (DMD) complexes appear
on muscle surfaces. (C) In the final late attachment phase, collagen fibrils become crosslinked into a lattice, with tenocytes (red cells) extending processes to
surround fibrils, and entheses chondrifying ( purple). The magnified view shows Col1a1 trimers becoming crosslinked by FACIT collagens and surrounded by
tenocyte (red) processes, stabilizing the ECM and its interactions with Itgs on muscle and cartilage cells.
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The term ‘tendinopathy’ refers to a diverse set of tendon disorders
(overlapping in some cases both in genetic and molecular terms with
myopathies) that are caused either by genetic mutations in ECM
components of MTJs or by mechanical stress that leads to tendon
ECM damage. Hallmarks of tendinopathies include COL1A1, COL1A2,
COL4A1 and COL4A2 overexpression, fibril disorganization, increased
collagen crosslinking, reduced tissue inhibition of MMPs (i.e. TIMP
activity), and elevated expression of MMP2, MMP14 and MMP19 as well
as of versican, biglycan and Dcn (Jelinsky et al., 2011; Parkinson et al.,
2011; Dunkman et al., 2013; Zhou et al., 2014). Recent studies have also
identified COL6A1/2/3 and COL12A1 as genes underlying one form of
human ‘myotendinopathy’, which affects both myomatrix structure and
tendon matrix structure (Bönnemann, 2011; Pan et al., 2013).
collagen fibrils, but they also appear to cross-regulate one another’s
transcription through as yet unknown mechanisms (Yoon and
Halper, 2005; Zhang et al., 2006; Dunkman et al., 2013). This might
involve feedback regulation of Scx and Mkx expression through the
tendon ECM, and recent studies have suggested that Dcn and Fmod
are regulated by Mkx (Fig. 2B) (Ito et al., 2010; Liu et al., 2010;
Alberton et al., 2012). These results point to a system by which the
dynamics of fibril ultrastructure feedback on ECM production to
modify tendon strength continuously.
Tendon ECM and collagen fibrils are also continuously
remodeled in response to mechanical forces, at least around the
circumference of a tendon fascicle (Frolova et al., 2014;
Heinemeier et al., 2012; Herchenhan et al., 2013; Kjaer, 2004;
Pingel et al., 2014). TPCs in culture subjected to moderate
mechanical forces show an increase in collagen fibril diameter,
though diameter decreases with excessive mechanical force (De
Almeida et al., 2010; Pingel et al., 2014). This remodeling occurs
primarily through the activities of matrix metalloproteinases
(MMPs) and their corresponding tissue inhibitors (TIMPs), as
well as disintegrin and metalloprotease with thrombospondin
repeats (ADAMTS) proteases (Bedi et al., 2010; Gotoh et al.,
2013; Jones et al., 2006; Maeda et al., 2013). Almost all of the 23
MMPs and 19 ADAMTS proteins known to be expressed in
vertebrates are detectable in adult tendon tissue and play a variety
of both positive and negative roles in establishing a functional MTJ
and tendon ECM (Davis et al., 2013; Spanoudes et al., 2014).
MMPs are zinc-dependent endopeptidases that bind and unwind
the triple helix of collagen monomers. Collagenases such as Mmp1
target the structural collagens for degradation, whereas gelatinases
such as Mmp2 and Mmp9 and membrane-bound MMPs, such as
Mmp14, degrade smaller network collagens. Importantly, recent
studies have shown that the expression and activity of MMPs are
regulated by signals activated in response to mechanical forces,
such as Itg and Tgfβ (Yu and Stamenkovic, 2000; Farhat et al.,
2015). MMP misregulation also occurs upon tendon inflammation,
and recent studies suggest that MMP inhibition can improve tendon
repair (Bedi et al., 2010; Jelinsky et al., 2011; Farhat et al., 2012;
Davis et al., 2013).
ECM-mediated signaling during tendon and MTJ formation,
maturation and repair
Multiple signaling pathways involving the ECM influence both the
formation of muscle attachments and the maturation of tenocytes.
Important players in muscle cells include Itgs and dystrophin, which
interact with ECM components at the MTJ. Furthermore, in both
muscles and tendons, mechanical forces are thought to have a role in
dynamic remodeling of the ECM.
4196
Many collagens and laminins at developing MTJs directly bind Itg
complexes that are present in the membranes of muscle cells and
tenocytes (Fig. 5) (Docheva et al., 2014; Mayer et al., 1997; Pan
et al., 2013; Rooney et al., 2006, 2012). In muscle, these continuous
structural links between ECM proteins, the sarcolemma and the
cytoskeleton, maintain fiber integrity and modulate adhesion and
gene expression. Not surprisingly, several types of human
tendinopathies (see Box 1) are associated with changes in the
expression of Itgs and their ligands (Bönnemann, 2011; Jelinsky
et al., 2011; Schessl et al., 2006). It is thus important to determine
the specific roles of Itg signaling during MTJ maturation and tendon
repair after injury.
Different Itg heterodimer combinations lend specificity for
different ligands. For example, laminins (Lama2, Lama4) bind
Itga7/b1 in the muscle basement membrane (Fig. 5) (Yurchenco et al.,
2004; Durbeej, 2010; Carmignac and Durbeej, 2012), and defects in
LAMA2 have been associated with merosin-deficient muscular
dystrophy in humans (Tomé et al., 1994; Rooney et al., 2012).
Furthermore, mutations that disrupt genes encoding crosslinking
collagens (e.g. COL12A1 in the case of Ehlers–Danlos syndrome),
which bind Itga1/b1 or Itga2/b1, cause widespread defects in skin,
bones and tendons (Mayer et al., 1997; Zou et al., 2014).
Which other Itg ligands control MTJ formation and maturation? In
Drosophila, Sr promotes the transcription of the single fly
thrombospondin (Tsp) gene in tenocytes. Tsp binds αPS2 (If)/βPS
(Mys) Itg heterodimers on fly muscle and tendon cell surfaces and
patterns muscle attachments (Chanana et al., 2007; Subramanian
et al., 2007). Vertebrates have at least five thrombospondins, with
diverse functions in cell migration, vasculogenesis, wound healing
and cancer (Adams and Lawler, 2004; Bornstein et al., 2004;
Kyriakides et al., 1999; Mustonen et al., 2012). Among these,
zebrafish Tsp4b is first secreted by myoblasts (Fig. 3A) and later
by TPCs (Fig. 3B), and is essential for muscle attachment
(Subramanian and Schilling, 2014). Accordingly, zebrafish
embryos depleted of Tsp4b have defects in laminin localization and
FAK phosphorylation (indicating reduced Itg signaling) at developing
ISBs, and their muscles detach under tension. These findings suggest
that, in zebrafish, Tsp4 plays key roles in organizing the ECM of both
muscle and tendon, particularly those components essential for Itg
signaling. This requirement for Tsp4 is at least partially conserved, as
Tsp4 −/− mutant mice show defects in ECM deposition in developing
tendons (Fig. 4) (Frolova et al., 2014; Subramanian and Schilling,
2014). Tsp4 and other subtype B thrombospondins form pentamers
that directly bind to collagens, laminins and other ECM proteins
(Hauser et al., 1995). Surprisingly, human TSP4 protein
microinjected into the ECM surrounding Tsp4b-deficient myofibers
in zebrafish localizes to ISBs and locally rescues laminin localization,
Itg signaling and muscle attachments, suggesting that Tsp4 could
function as a scaffold for other ECM proteins during their assembly at
muscle attachments. Consistent with this model, the ability of
microinjected zebrafish tsp4b mRNA to rescue Tsp4b-deficient
attachments requires Itg binding and pentamerization; mutation of
either the Itg-binding (KGD) domain or the pentamerization (CQAC)
domain of Tsp4b disrupts its localization to tendons and eliminates its
ability to rescue muscle attachments in Tsp4b-deficient larvae
(Subramanian and Schilling, 2014). These results, along with a
potentially conserved requirement for Tsp4 in mice, suggest that
TSP4 defects could contribute to human tendinopathies, highlighting
TSP4 as an attractive therapeutic target for strengthening the tendon
ECM. They also highlight the close association between the structural
and signaling roles of the ECM.
DEVELOPMENT
Integrin signaling
Box 1. Tendinopathies
REVIEW
Development (2015) 142, 4191-4204 doi:10.1242/dev.114777
Mechanical stress
ECM
ECM Col22a1
Sm
ad3
Muscle
contraction
Actin
filaments
Smad3
Tenocyte
Egr1
Egr2
Scx
Mkx
Smad3
Muscle
Col1a1
Col1a2
Col3a1
Col6a1
Col12a1
Col14a1
Tgfb2
Tnmd
Fmod
Tsp4
Actin
filaments
Key
Lama2,
Lama4
Tsp4b
TGFβ
Tgfβr
Itg
TGFβ
LLC
Col1a
FACIT
Col
Fig. 5. Model for ECM-mediated feedback from mechanical force and its effects on tenocyte gene expression. A tenocyte (orange) synthesizes the tendon
ECM, including Lama2, Lama4 (brown), Tsp4b (red pentagons), Col1a (blue) and FACIT Col ( purple), all of which signal through Itg receptors (dark blue) on
muscle and tenocyte cell surfaces in response to mechanical stress (gray arrows). In addition, stress causes the ECM to release TGFβ (yellow) from the TGFβ
large latent complex (LLC) (gray dotted arrows). Itg and TGFβ signaling in tenocytes feedback to regulate Scx-, Egr1/2- and Mkx-induced transcription (dashed
arrows) of the same Itg ligands as well as of other ECM components to modulate tendon stiffness. Smad3 also interacts with Scx and Mkx to activate target ECM
genes. The muscle fiber also contributes to the tendon matrix by secreting FACIT Col22a1.
TGFβ signaling provides another striking example of the relationship
between ECM structure and signaling in tendons. Genetic studies in
mice have revealed crucial roles for TGFβ at multiple steps in tendon
development, maturation, maintenance and repair (Figs 2, 3, 5).
Removing the functions of both TGFβ2 and TGFβ3 ligands, or of
TGFβr2, eliminates most if not all differentiated tendons, whereas
exogenous TGFβ is sufficient to induce the expression of Scx
and Col1a1 (Fig. 2A) (Chuang et al., 2014; Pryce et al., 2009).
Tgfb2−/−/Tgfb3−/− mutant mice lose Scx expression in TPCs
between E11.5 and E12.5, suggesting that TGFβ is required for
TPC maintenance (Pryce et al., 2009). Signaling through TGFβr2
phosphorylates Smad2 and Smad3, which translocate to the nucleus
and activate target genes, thereby maintaining differentiated
tenocytes (Figs 3 and 5). Mouse Smad3 −/− mutants have reduced
tendon tensile strength and increased spacing between collagen
fascicles as well as reduced Mkx and increased Mmp9 expression
(Berthet et al., 2013; Katzel et al., 2011).
Interestingly, the most likely source of TGFβ ligand at muscle
attachments is the ECM. A recent in vitro study has shown that
during tenocyte differentiation, Mkx activates the expression of
TGFβ in differentiating MSCs (Fig. 5) (Liu et al., 2015). TGFβs are
secreted bound to latent TGFβ-binding proteins (LTBPs), which
form part of the large latency complex (LLC) in the ECM (Wipff
et al., 2007; Maeda et al., 2011). They are also secreted along with
latency-associated peptides (LAPs), which block association with
TGFβ receptors, and along with other proteins of the LLC, they
become incorporated into ECM via interactions between LTBPs and
Fn, fibrillin or Dcn (Isogai et al., 2003; Rifkin, 2005; Farhat et al.,
2012). In this manner, TGFβs are stored in the ECM and must be
released from the LLC and LAPs in order to be ‘activated’ and
available to interact with cognate receptors (Horiguchi et al., 2012).
Activation may occur by release of TGFβ stimulated by shearing
forces, LLC degradation by proteases, interactions with Itgs through
RGD motifs on LTBPs themselves, or by the activity of Mmp2,
Mmp9 and Bmp1 proteases (Munger and Sheppard, 2011). Few
studies have addressed these mechanisms of TGFβ activation
specifically for the ECM of tendons or MTJs. A recent
transcriptomic analysis of Scx-expressing tenocytes from mouse
limbs reveals that both TGFβ and MAPK signaling are strongly
upregulated, but that only TGFβ upregulates Tsp2, Tsp4 and LTBP
components of the ECM and promotes the tenocyte cell fate (Havis
et al., 2014).
The correct regulation of TGFβ activity is crucial not only for
tendon development but also for healing injured tendons. Injuries
lead to excessive release of TGFβ owing to mechanical forcemediated activation of TGFβ (discussed below) and can cause
fibrotic scarring of the tendon, thereby disrupting its function
(Farhat et al., 2015). In one model, elevated TGFβ might
overactivate MMPs, which in turn promotes further release of
active TGFβ from the ECM as well as activating expression of
Scx and Mkx and driving further ECM production.
Mechanical forces and signaling
As alluded to throughout this Review, tenocytes actively sense
mechanical force, leading to changes in gene expression,
cytoskeletal organization and ECM secretion (Fig. 5) (Banos
et al., 2008; Maeda et al., 2009, 2013). Such feedback must be
extremely important for a tissue that constantly adjusts its stiffness
to changing loads. It depends, at least in part, on signaling through
gap junctional complexes localized to tenocyte processes (Kruegel
and Miosge, 2010; Maeda et al., 2012). Indeed, rats subjected to
running on treadmills have increased Tnmd and Col1a1 expression
as well as TPC proliferation (Eliasson et al., 2009; Zhang and Wang,
2013). Elevated secretion of both Col4a1 and Col6a1 is also seen in
developing chick tendons under stress, and this alters the
crosslinking of fibrils (Marturano et al., 2014), thereby finetuning tendon strength and promoting repair (Bailey et al., 1998;
Willett et al., 2010). What are the molecular mechanisms underlying
these cellular responses?
4197
DEVELOPMENT
TGFβ signaling
TGFβ signaling is one such mechanosensitive pathway that
could control the response of tendons to force. Mechanical force
causes release of TGFβ1 from LTBPs in the ECM (Fig. 5)
(Maeda et al., 2011; Wipff et al., 2007). Under normal loads,
TGFβ signaling through Smad2/3 maintains Scx expression in
tenocytes, whereas excessive loading disrupts TGFβ signaling,
damages the ECM and leads to tenocyte cell death in mice
(Maeda et al., 2009, 2010, 2011). TGFβ signaling in response to
force also upregulates ITGA1 and ITGA2 expression in human
TPC cultures (Popov et al., 2015). Tenocytes elevate expression
of Tgfb1, Tgfbr2 and Smad7 in response to injury in mice
(Guerquin et al., 2013). Thus, one attractive model is that force
triggers TGFβ signaling leading to increased expression of Scx
and Mkx, which in turn activates TGFβ expression, creating a
positive-feedback loop that leads secondarily to remodeling/
strengthening of ECM (Fig. 5) (Liu et al., 2015). Notably, TGFβ
signaling in response to mechanical forces also controls MMP
expression during tendon repair (Katzel et al., 2011; Farhat et al.,
2015). Interestingly, injured tendons in Egr1 −/− mutant mice fail
to upregulate TGFβ and Scx or to repair their tendons efficiently
(Guerquin et al., 2013).
The unique collagen fibril organization of tendons allows them
to bear the stress of muscle contraction and prevents bone
fractures (Davis et al., 2013; Pan et al., 2013; Schwartz et al.,
2013; Zhang et al., 2006). How fibrils physically anchor to cells
and how the dynamics of these anchors are regulated under load
remain unclear. Biopsies of human Achilles’ tendons have shown
that fibrils buckle in overloaded tendons (Pingel et al., 2014). In
tenocytes cultured in collagen gels, mechanical force translates
into cytoskeletal force through non-muscle myosin II bound to
actin fibrils associated with focal adhesion complexes, which in
turn associate with Itgs and other ECM receptors. The chemical
inhibition of myosin II function (using blebbistatin) reduces
cytoskeletal traction forces and leads to ECM remodeling (Maeda
et al., 2013). Thus, in addition to inducing transcriptional
changes, mechanical force can stimulate changes in cytoskeletal
tension that reverberate back to the ECM to alter its ultrastructure.
MMPs have also been shown to modulate ECM structure in
response to mechanical cues during MTJ maturation and after
injury. For example, tenocytes in silicone micropillar gels elevate
MMP expression levels in response to gel deformation (Maeda
et al., 2013). In addition, humans show dramatic increases in the
levels of MMP2, MMP9 and MMP14 in adult tendons following
endurance exercise, suggesting that these proteins aid MTJ repair in
response to mechanotransduction (Rullman et al., 2009). This
effect depends on the timing of loading as, in cultured tendon
fascicles, cells upregulate MMP2 and MMP13 after very short
cycles of loading, but downregulate MMP1 after longer cycles
(Maeda et al., 2009, 2013). Similarly, in vitro studies of mechanical
loading on mouse tenocytes have shown that, whereas low levels of
shear force lead to upregulation of Col1a and Tmnd, increasing the
force leads to upregulation of Runx2 and Sox9 (Zhang and Wang,
2015). Understanding these dynamic responses to mechanical
stimulation might lead to improved therapeutic interventions; the
systemic inhibition of MMPs, for example, can reduce fibrotic
scarring of muscle ECM (Farhat et al., 2015). MMP expression is
also regulated by TGFβs (Yu and Stamenkovic, 2000; Ge and
Greenspan, 2006; Farhat et al., 2015). In turn, MMP2, MMP9 and
BMP1 proteases might specifically digest LLC and release active
TGFβ, establishing a positive-feedback loop that could help finetune MMP levels, both during normal tendon function and in
response to tendon injury or exercise (Fig. 5).
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Development (2015) 142, 4191-4204 doi:10.1242/dev.114777
ECM functions at tendon-bone attachments
So far, we have emphasized the ECM associated with tendonmuscle attachments. But tendons also attach, at their other ends, to
cartilage/bone under a unique set of mechanical constraints. Many
tendons insert into bony protrusions known as osteotendinous
junctions or ‘entheses’, such as the deltoid tuberosity on the
humerus (Fig. 6A). A characteristic structural feature of entheses is
the presence of ‘fibrocartilage’, a tissue with physical properties
somewhere in between cartilage and tendon. Within an enthesis, a
rapid transition from a more bone-like cellular and ECM structure to
a more cartilage-like (less rigid) structure in regions closer to the
tendon is observed. In addition, a unique mineralization gradient
forms from the bony front to the point of tendon insertion, the width
of which is constant with corresponding changes in cellular density
(Schwartz et al., 2012). The enthesis ECM also shows more Dcn
and Bgn localized to the tendon side, whereas Col2a1, Col9, Col10
and aggrecan localize to the bony side (Thomopoulos et al., 2003).
These gradual transitions in ECM and rigidity are essential for the
proper transmission of contractile forces to the bone to prevent
avulsion fractures (Zelzer et al., 2014).
In the appendicular skeleton, entheses are established through
specialized contours and protuberances on bones called eminences.
Recent genetic studies in mice have begun to elucidate the signals
that control the formation of these structures (Blitz et al., 2009,
2013; Zelzer et al., 2014). In mouse embryos, eminence
development coincides with the formation of muscle/tendon
attachments, suggesting that attachments impose physical changes
on the bone. However, mouse mutants that lack muscles, such as
splotch delayed (spd) or muscular dysgenesis (mdg) mutants, still
form eminences like the deltoid tuberosity on the humerus, despite
severe joint fusions (Fig. 6B) (Blitz et al., 2009).
Perhaps not surprisingly, TGFβ signaling also plays important
roles in the formation of entheses. Loss of TGFβ signaling through
ablation of Tgfβr2 in limb mesenchyme eliminates eminences in the
limb, though this might be due to a broader or earlier role for TGFβ
signaling in skeletal/tendon progenitors (Fig. 6B) (Blitz et al., 2009,
2013). Instead, evidence is building to suggest that the more crucial
inducers of eminences are other members of the TGFβ superfamily,
namely bone morphogenetic proteins (BMPs). The conditional
deletion in mice of Bmp4 specifically in tenocytes using an Scx:Cre
driver completely eliminates eminences (Blitz et al., 2009). Pulsechase labeling studies show that eminences form from secondary
fields of Scx/Sox9 co-expressing cells that lie immediately adjacent
to major skeletal condensations (Fig. 6B) (Blitz et al., 2013); these
cells form in mutants lacking BMP signaling, but do not
differentiate. Early Sox9 expression is observed in both the
skeletal condensation and the eminence, but the subset of these
cells that express Scx are delayed in expression of Col2a1, thereby
restricting chondrogenesis to the developing enthesis. Scxexpressing cells in developing entheses express Bmp4 and this is
lost in tendons of Scx−/− mutant mice, which lack entheses. Thus, an
attractive hypothesis is that Bmp4 secreted by tenocytes induces
enthesis formation in adjacent skeletogenic mesenchyme (Fig. 6B).
Interestingly, Sox9-expressing secondary fields of eminence
progenitors still form in Bmpr1a−/− mutant mice but never
differentiate, indicating a role for BMP signaling in maturation
rather than specification. Taken together, these studies establish an
early phase of eminence/enthesis development, which is
independent of muscle development, and suggest that Scx-driven
Bmp4 signaling non-autonomously regulates their formation
(Fig. 6B) (Blitz et al., 2013; Murchison et al., 2007; Pryce et al.,
2009). Such coordinated expression of Scx and Sox9 in tenocytes
DEVELOPMENT
REVIEW
REVIEW
Development (2015) 142, 4191-4204 doi:10.1242/dev.114777
A
spd mutant
Wild type
Col2b
Gdf5
Col2a1
Col2b
Col2a1
Col2a1
Gdf5-expressing
joint cells
B
Col2a1
Col2b-expressing
cartilage cells at
articular surface
Col2a-expressing
cartilage cells
Tgfβr2 lof in limb and
Sox9 lof in tenocytes
Wild type
Bmp4 lof in
limb mesenchyme
Enthesis
Secondary
field
Primary
field
Sox9
Col2a
Tendon
Muscle
Sox9high,Scxlow
enthesis cells
Scxhigh,Sox9low
enthesis cells
Tenocytes
Sox9+ cells that do
not express Col2a
Fig. 6. ECM functions at tendon-bone attachments. (A) Diagrams illustrating changes in cartilage at the developing humero-ulnar joint of the mouse
forelimb in wild-type embryos (left) and in splotch delayed (spd, Pax3) mutants (right), which lack muscles. Proliferating chondrocytes express Col2a1 (light blue),
whereas cells forming at the edges of the joint express Col2b (dark blue), and cells in the joint interzone secrete Gdf5 (green) into the joint region. The loss
of muscles in spd mutants leads to loss of Gdf5 expression, disorganized Col2b+ interzone cells and joint fusion. (B) Diagrams illustrating changes in cartilage
and tenocytes at a developing eminence. The primary field contains cells that form chondrocytes within the developing bone, whereas the secondary field
consists of Sox9-positive progenitor cells that lie outside of the primary field. In wild-type embryos, three different subsets of Scx-expressing cells at a muscle
insertion site of a developing long bone are found: Sox9+/Scx+, Scx+ or Scx+/Bmp4+. Loss of Tgfβr2 in limb mesenchyme or of Sox9 in tenocytes leads to a loss of
the Sox9/Scx co-expressing and Sox9-expressing population in the secondary field, but not other tenocytes. Loss of Bmp4 signaling leads to a loss of both Sox9+/
Scx+ and Scx+ populations in the secondary field. Dotted lines outline primary field. Dashed lines outline secondary field. lof, loss of function.
and chondrocytes is an emerging theme both during tendon
development and in MSCs (Fig. 2A) (Asou et al., 2002; Soeda
et al., 2010; Sugimoto et al., 2013). The close lineal relationship
between tenocytes and chondrocytes/osteocytes (Fig. 2A) and their
regulation at entheses also may help explain the ectopic ossification
of tendons (see Box 2) that occurs normally in some species as well
as during aging and disease (Magne and Bougault, 2015; Zhang and
Wang, 2015).
Finally, and not surprisingly, entheses are also extremely
sensitive to mechanical forces. Mechanical forces have long been
known to be important for the development of the bones to which
muscles attach, as well as for the maintenance of skeletogenic cell
4199
DEVELOPMENT
Scx+/Bmp+
TPCs
REVIEW
Ectopic ossification of tendons occurs normally in some species and is
also observed during aging and in disease (Magne and Bougault, 2015;
Zhang and Wang, 2015). Spondyloarthritis, for example, is an
inflammatory enthesitis (Weinreb et al., 2014) that can lead to ectopic
ossification spreading from the bone to the tendon or ligament. Ectopic
ossification is also seen in calcifying tendinopathies, a common
consequence of aging affecting as many as 1 in 5 adults over 50 years
of age. Tendon ossification appears to be caused, in part, by tenocytedependent degradation of the tendon ECM (Magne and Bougault, 2015)
as well as altered responses to mechanical forces and BMP/Smad
signaling (Rui et al., 2013). The genetic inactivation of two small
proteoglycans (Bgn and Fmod) of the ECM of cultured mouse TPCs
leads to ectopic activation of BMP signaling and tendon ossification (Bi
et al., 2007). Mechanical stretching of TPCs in vitro can also lead to BMP
upregulation and abnormal ossification (Zhang and Wang, 2013, 2015).
Surprisingly, BMP signaling oscillates in a circadian manner, and these
cycles are deregulated in arrhythmic mutant mice, which correlates with
increased tendon ossification (Yeung et al., 2014) Thus, tendons and
ligaments are really tissues living on the edge with respect to their ECM
composition and cellular constituents, presumably owing to their extreme
responsiveness to feedback through mechanical force.
populations (Shwartz et al., 2013). The paralysis of specific muscles
leads to bone defects at sites of attachment and loss of joint
progenitors (Kahn et al., 2009). The tendon ECM plays vital roles in
these adaptations of muscles and bones to mechanical loading
(Kjaer, 2004).
Conclusions and perspectives
Vertebrate tendons begin life similar to skeletal progenitors in the
embryo, but rapidly establish unique identities and tissue
organization, in large part through interactions with and assembly
of the tendon ECM. This involves: (1) essential transcription factors
expressed in tenocytes, such as Scx, Mkx and Egr1, that drive the
expression of ECM proteins; (2) ECM components such as laminins
that help establish muscle attachments in the absence of tenocytes
and control tenocyte morphogenesis; (3) ECM components such as
Tsp4 that drive the assembly of collagen fibrils at MTJs through
their interactions with other ECM proteins and Itgs on muscle cell
surfaces; and (4) the maintenance and repair of these ECM
components in response to mechanical forces. In this Review, we
have highlighted recent genetic studies that have provided insights
into the molecular mechanisms underlying these different
processes.
The discovery of Scx and Mkx has provided inroads into
understanding the tendon/ligament gene regulatory program and
revealed the close relationship between tenocytes and skeletal
lineages. Scx is the earliest known marker of TPCs, and its
overexpression transforms MSCs into tenocytes (Alberton et al.,
2012), whereas Sox9 overexpression converts MSCs into
cartilage (Fig. 2A) (Takimoto et al., 2012). However, unlike
Sox9, which when eliminated leads to a failure to form cartilage,
loss-of-function Scx or Mkx mutations in mice do not eliminate
tenocytes, and mutants are viable although they have severe
musculoskeletal deformities. This implies that tendon
development involves multiple, partially redundant transcription
factors and cell-cell signals, each playing a unique role in
building and maintaining the complex network of ECM proteins
at muscle attachments.
These studies also highlight the fact that the stability of muscle
attachments is not pre-established during development but
evolves through constant adaptation of the ECM to changing
4200
mechanical load. This occurs regionally within each tendon from
its muscle origin to its bony insertion. Understanding the tendon/
ligament gene regulatory program thus requires knowledge of
how this dynamic network of ECM proteins self-assembles and
feeds back upon transcriptional regulators in response to
mechanical forces.
The musculoskeletal system and its associated ECM have also
evolved to suit dramatically different modes of locomotion, feeding
strategies and body sizes of different vertebrates. Changes in the
interconnections between individual muscles and bones underlie
many of the evolutionary differences between species. For example,
mandibles of fish (Malawi cichlids), birds (Darwin’s finches) and
dogs can have very different functional morphologies depending on
their feeding strategies, which notably have all been linked to
changes in BMP signaling (Abzhanov et al., 2004; Albertson et al.,
2005; Schoenebeck et al., 2012). These reflect coordinated changes
in the regulation of cranial neural crest cells that form not only the
craniofacial skeleton, but also cranial TPCs during evolution.
Indeed, recent studies grafting neural crest cells between quail and
duck embryos reveal that some distinct craniofacial morphologies
have evolved through changes in cell-intrinsic mechanisms. Jaw
cartilage differentiation, marked by Runx2 expression, occurs
earlier in the duck embryo, whereas tendon differentiation, marked
by Scx expression, occurs earlier in the quail (Tokita and Schneider,
2009). How these species-specific differences in timing of skeletal
and tendon differentiation reflect changes in ECM organization and
in mechanical stress to suit their adaptive functions is an important
area for future investigation.
Understanding the genetic control of the development of tendons,
their integration into the musculoskeletal system and their ECM
organization also has important implications for regenerative
medicine. Current efforts to treat tendon injuries or diseases focus
either on ameliorating inflammation or driving MSCs towards the
tenocyte fate with hopes of stem cell therapy (Yang et al., 2013).
Both approaches leave out the crucial role of establishing the
appropriate ECM for a specific tendon type and the loads that it
needs to bear. Attaining the correct strength also relies on correct
cellular responses to mechanical forces, which are transduced
through the ECM. Thus, it is important to consider the ECM and its
functions going forward in efforts to improve diagnosis and therapy
design for tendon disorders.
Acknowledgements
We thank members of the Schilling lab and anonymous reviewers for critical reading
of the manuscript.
Competing interests
The authors declare no competing or financial interests.
Funding
The authors were supported by National Institutes of Health awards [R21 AR62792
and R01 DE13828 to T.F.S.]. Deposited in PMC for release after 12 months.
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