REVIEW

Tendon development and musculoskeletal assembly: emergingroles for the extracellular matrixArul Subramanian and Thomas F. Schilling*

ABSTRACTTendons and ligaments are extracellular matrix (ECM)-rich structuresthat interconnect muscles and bones. Recent work has shown howtendon fibroblasts (tenocytes) interact with muscles via the ECM toestablish connectivity and strengthen attachments under tension.Similarly, ECM-dependent interactions between tenocytes andcartilage/bone ensure that tendon-bone attachments form with theappropriate strength for the force required. Recent studies have alsoestablished a close lineal relationship between tenocytes and skeletalprogenitors, highlighting the fact that defects in signals modulated bythe ECM can alter the balance between these fates, as occurs incalcifying tendinopathies associated with aging. The dynamic fine-tuning of tendon ECM composition and assembly thus gives rise tothe remarkable characteristics of this unique tissue type. Here, weprovide an overview of the functions of the ECM in tendon formationandmaturation that attempts to integrate findings from developmentalgenetics with those of matrix biology.

KEY WORDS: Tendon, Ligament, Tenocyte, Extracellular matrix

IntroductionTendons and ligaments are connective tissues that transmitmechanical forces between muscles and bones. Tendons attachmuscle to skeleton, whereas ligaments attach skeletal elements toeach other and stabilize skeletal joints. Vertebrates have evolved aremarkable variety of tendons and ligaments to accommodate theirdistinct modes of locomotion as well as their dramatic variations inbody size and strength. These range from broad sheets to highlyelastic cables, such as those of the Achilles’ tendon and the cruciateligaments of the knee. Because of their structural roles, injuries totendons and ligaments are extremely common and oftendebilitating. Thus, a fundamental question in musculoskeletalbiology is how these connective tissue structures develop in thecorrect locations and acquire the strength necessary to translatecontractions of muscles into skeletal movements.Both tendons and ligaments contain fibroblasts (termed tenocytes

and ligamentocytes, respectively) embedded in a uniqueextracellular matrix (ECM) that is composed mainly of collagenfibril arrays capable of withstanding incredibly strong tensile forces.These fibrils are crosslinked to one another and wrapped in a tendonsheath (Banos et al., 2008; Kannus et al., 1998; Ros et al., 1995).This basic structure is shared among tendons and ligaments, and thefibroblasts that produce the ECM develop from commonprogenitors with similar gene expression profiles (Juneja andVeillette, 2013; Sugimoto et al., 2013; Tozer and Duprez, 2005;Yang et al., 2013). However, each tendon or ligament differs in itsprecise ECM composition, size and strength (Birch et al., 2013).

How are these differences established? The answer to this questionhas important implications for understanding how diseases ordamage to tendons and ligaments arise and for developing bettertreatment strategies.

Despite their pivotal roles in musculoskeletal connectivity andfunctional stability, the mechanisms that control tendondevelopment have received much less attention than the processesof myogenesis or skeletogenesis. Only a handful of factors areknown to help specify tenocyte progenitor cells (TPCs) at muscleattachments, induce them to differentiate, and maintain and repairthem 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 inanimal models (e.g. fly, fish, chick and mouse embryos), inparticular those focusing on the formation of myotendinousjunctions (MTJs; the major sites of force transmission), haverevealed a crucial link between developing TPCs and the dynamicECM that surrounds these cells. Indeed, ECM proteins (e.g.collagens, laminins, thrombospondins) initially guide myofibers totheir sites of attachment, but also mediate signaling between TPCsand muscles, regulate the maturation of MTJs, and maintain tendonsin response to mechanical force (Kjaer, 2004; Schwartz et al., 2013;Snow and Henry, 2009). Here, we review recent genetic studies thathave identified crucial roles for the ECM in tendon development,and we discuss the emerging nexus between the transcriptionalcontrol of tenocyte differentiation and the organization of the ECMassociated with muscle fibers (myomatrix), MTJs and tendons(Fig. 1).

ECM production and regulation in developing tendonsCollagens (predominantly Col1a) constitute the bulk of maturemammalian tendon ECM and MTJs, whereas laminins (Lams) andmany other non-collagenous ECM components comprise theremainder (Kannus et al., 1998; Kannus, 2000; Kjaer, 2004;Birch et al., 2013; Thorpe et al., 2013). Which cells secrete theseECM proteins, how are they produced in the correct proportions,and how do they assemble?

The transcriptional regulation of ECM production by TPCsStrikingly, all of the key transcription factors known to function inTPC development directly regulate the transcription of genesencoding ECM proteins (Fig. 2A,B). The best studied of these isScleraxis (Scx), a basic helix-loop-helix transcription factor, and theearliest known marker of TPCs. Scx is first induced through theinterplay of sonic hedgehog (Shh) and fibroblast growth factor(FGF) signaling in the ‘syndetome’ compartment of somites (theregion of the sclerotome adjacent to the myotome) and bytransforming growth factor beta (TGFβ) signaling in the limbs ofmice at embryonic day (E) 10.5 (Schweitzer et al., 2001; Brent et al.,2003; Havis et al., 2014). In vitro, Scx overexpression is sufficient totransform mesenchymal stem cells (MSCs) and human embryonicstem cells (hESCs) into tenocytes (Fig. 2A) (Alberton et al., 2012;

Department of Developmental and Cell Biology, University of California, Irvine,Irvine, CA 92697-2300, USA.

*Author for correspondence (tschilli@uci.edu)

4191

© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT

Chen et al., 2012; Li et al., 2015). Thus, considerable effort has beenmade to elucidate the functions of Scx in the tenocyte lineage and toidentify its downstream targets.The loss of Scx (i.e. as in Scx−/− mutant mice) disrupts tenocyte

differentiation leading to atrophy of force-transmitting tendonsand a disorganized tendon ECM (Murchison et al., 2007). Theexpression of the major structural collagens, Col1a1, Col1a2,Col3a1 and Col14a1, is strongly reduced in Scx−/− mutants. Scxdirectly controls Col1a1 and Col1a2 transcription (Fig. 2B) (Espiraet al., 2009; Léjard et al., 2007). Numerous other tendon regulators,such as the glycoprotein tenomodulin (Tnmd), are also

downregulated in Scx−/− mutants (Murchison et al., 2007;Shukunami et al., 2006). At the ultrastructural level, the loss ofScx disrupts the sheaths that surround each fascicle of collagenfibrils as well as cellular processes, which normally encircle thefibrils (Murchison et al., 2007). These results demonstrate that Scxcontrols tendon ECM production, which is essential for effectiveforce transmission.

It should be noted that TPCs still develop in Scx−/− mutant mice,suggesting that other genes are required for the initial steps of TPCspecification. In Drosophila, the transcription factor Stripe (Sr)specifies TPCs in epidermal segment border cells (Volk andVijayRaghavan, 1994). Flies lacking Sr function fail to form TPCsand display disrupted muscle patterning and attachments, whereasSr overexpression transforms ectodermal progenitors into TPCs(Becker et al., 1997). Embryonic TPCs in mice express orthologsof Sr – Egr1 and Egr2 (Lejard et al., 2011) – and Egr1 is sufficientto induce Scx expression and specify MSCs as tenocytes in vitro(Guerquin et al., 2013). However, like Scx, both Egr1 andEgr2 appear to be dispensable for tenocyte specification, asEgr1−/−/Egr2−/− double mutant mice are viable. Instead theyregulate the tendon ECM and MTJ, binding to tendon-specificenhancer elements ofCol1a1 andCol1a2 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 tendoninjuries as adults (Guerquin et al., 2013). Thus, in contrast to flies,vertebrate Egrs function in tendon ECM production rather than TPCspecification.

Another potential TPC ‘specifier’ is the TALE family atypicalIroquois-like homeodomain protein Mohawk (Mkx). Like Scx andEgr1, Mkx can drive bone marrow-derived MSCs towards atenocyte fate in vitro (Liu et al., 2015; Otabe et al., 2015).However, in mouse embryos the expression of Mkx begins indeveloping tenocytes later than that of Scx or Egr1/2 (at E13.5-14.5), becoming restricted to tendon sheath cells and collateralligaments (which stabilize joints) in the limbs by E16.5 (Anderson

Laminin(Lam)

Fibronectin(Fn)

Collagen(Col)

Thrombospondin(Tsp)

Key

MyomatrixFn, Lam, Col, MMP,TIMP, Tsp

Tendon matrixSLRP, Col, Lam,Tsp, MMP, TIMP

MTJ

Musclefiber

Tenocyte

Fig. 1. Composition of the ECM surrounding muscle, tendon andmyotendinous junctions. A muscle fiber (green) secretes ECM componentsinto its surroundings (the myomatrix). Some of these components overlap withthose of the tendon ECM, which is secreted by tenocytes (red). Myomatrix isprimarily composed of Lam trimers and Fn. By contrast, the tendonmatrix is rich in Col1a trimers and thrombospondin pentamers. Themyotendinous junction (MTJ) is the narrow zone in which ECM components oftendon and muscle interact.

Shh, FGF,TGFβ(Axial) BMP

Scx Sox5/6/9

FGF (Mouse limb)

Scx Sox9

Runx2

ScxMkxTnmd

Scx

Egr1Egr2

Col1a1

Col1a1

Col1a2

Col1a2

Col3a1Col14a1

Tnmd

Tnmd

Tsp4 Mkx

Col1a1Col1a2TnmdFmod

Mkx

Sox6MyodRunx

ChM-1Col2a1Col9a2Col11a2AggCompRunx2Sox9 Runx2

Mmp9Mmp13Tram2

Egr1

Dcn

Tgfb2

BgnTgfbr2

A Tenocyte specification B Tendon-specific ECM gene regulation

C Skeletal-specific ECM gene regulation

MSC

TPCSPCTgfb2

TenocyteOsteoblast

Smad3 Smad3

Smad3

Fig. 2. Transcriptional regulation of tenocyte specification and tendon ECM production. (A) A mesenchymal stem cell (MSC, purple), the commonprogenitor for skeletal and tenocyte progenitors, becomes a skeletogenic progenitor cell (SPC, blue) if it is exposed to high levels of BMP signaling, then itexpresses Sox5/6/9 followed by Runx2 during its differentiation into an osteoblast. By contrast, anMSC becomes a tendon progenitor cell (TPC, pink) if it receiveshigh 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 theprogenitor 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 inmyogenic and skeletogenic progenitor formation. (C) Transcription factors expressed in skeletogenic progenitor cells (e.g. Sox9 and Runx2) directly regulate thetranscription of a distinct set of ECM target genes.

4192

REVIEW Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT

et al., 2006). Mkx−/− mutant mice are viable, fertile, and formnormal tendons at first with no defects in Scx expression but laterexhibit reduced levels of Col1a1, Col1a2, Tnmd, fibromodulin(Fmod) and decorin (Dcn) as well as thinning of collagen fibrils (Itoet al., 2010; Kimura et al., 2011; Liu et al., 2010). Similar to Scx,Mkx can function as a transcriptional activator when complexedwith Smad2/3 to promote Col1a1, Col1a2, Tnmd and Dcnexpression as well as TGFβ2 expression in murine MSCs (Liuet 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 maturationand ECM production and might function, in part, to maintaintenocytes by preventing them from acquiring myogenic orskeletogenic fates.Both Scx and Mkx interact with Smad3, an essential

transcriptional mediator of TGFβ signaling, to regulate tendonECM 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 limbmesenchyme, initially form TPCs in the limbs but lose them byE14.5, suggesting a role for TGFβ signaling in tendon maintenance(Pryce et al., 2009). Tendon defects in Smad3−/− mutants are muchless severe, with transient reductions in Col1a1, Col1a2 and Tnmdexpression in their limbs. Like Mkx, Smad3 can also inhibit theexpression and activity of MyoD as well as that of skeletogenicfactors such as Runx2 in vitro (Fig. 2A, Fig. 3) (Alliston et al., 2001;Kang et al., 2005; Liu et al., 2001). However, unlike Mkx, whichrepresses MyoD transcription, Smad3 acts post-translationally bybinding E-box sites in MyoD and sequestering it away from itstargets (Chuang et al., 2014). These results hint at a dynamicnetwork involving TGFβ signaling, Scx and Mkx to achieve andmaintain 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 theproduction of tendon ECM and MTJ assembly and in therepression of other mesenchymal fates. This is important not onlyin the context of tenocyte development but also in the regulation ofMSCs, where the balance between these factors determines if a cellbecomes a TPC or a skeletogenic (or myogenic) progenitor(Fig. 2A). An attractive model is one in which Scx (in concertwith TGFβ signaling) shifts the fate of MSCs towards TPCs andinitiates tendon ECM production, whereas factors expressed later indevelopment, such as Mkx and Egr1, supplement the role of Scx byinducing the expression of ECM proteins as well as by repressingmyogenic and skeletogenic fates. Furthermore, signaling mediatedby mechanical forces upregulates expression of Scx, Mkx andSmad3, stimulating more ECM production, thereby providingpositive feedback for fine-tuning tendon strength, as we discussfurther below (Eliasson et al., 2008; Maeda et al., 2011).

ECM production and function during tenocyte morphogenesis andMTJ formationAlthough the migration of muscle progenitors has been well studiedin both invertebrates and vertebrates, very little is known about therole of ECM in morphogenesis of TPCs and the establishment of theMTJ. In Drosophila, myoblasts migrate to sites of attachment andinteract with tenocytes located at fixed sites at segment borders in theepidermis (Volk and VijayRaghavan, 1994; Schweitzer et al., 2010).These myoblasts recognize tenocytes through multiple signalsincluding Thrombospondin (Tsp) in the ECM, which binds muscleintegrins (Itgs) (Subramanian et al., 2007). Similarly, vertebratemyoblasts in the trunk and limbs elongate and attach via TPCs alreadylocalized to future muscle attachment sites. How do migratingvertebratemyofibers and TPCs interact, and how do these interactionsdiffer between the trunk, limb and head? In the chick, early progenitorpools of limb TPCs condense and split to form individual tendons(Kardon, 1998). By contrast, cranial TPCs that arise in the neural crestmigrate to the locations of future MTJs (Grenier et al., 2009; Noden,1988; Noden and Trainor, 2005). Trunk TPCs arise from thesyndetome of somites, whereas limb TPCs are thought to originatefrom lateral plate mesoderm (Brent et al., 2003; Kardon, 1998). These

A Tendon-independent phase

B Tendon-dependent phase

Tsp4Lama2Itg

Tgfβr2Alk

Col1a1Col1a2

FACIT ColCol12a1

Fn

ECM

Smad

3

Scx, MkxEgr1, Egr2

ECM

Myoblast

Myoblast

TPC

TPC

ECM

Scx, Mkx

MyoDRunx2 Sox6,

Sox9

KeyTgfβ2

Fig. 3. Myoblast-tenocyte interactions and ECM production. (A,B)The formation of myotendinous junctions can be considered as a two-step process. In the initial tendon-independent phase (A) in vertebrates(shown here for zebrafish trunkmuscles), myoblasts (green) synthesize a‘pre-tendon’ECM that includes the integrin ligands Tsp4 and Lama2. ThisECM accumulates in the absence of TPCs (brown).Mechanotransduction coupled with TGFβ signaling (through Tgfβ2 andTgfβr2) leads to the Smad3-dependent expression of Scx and Mkx inTPCs, which in turn leads to the expression of tendon-selective ECMgenes. Smad3 and Mkx also repress the activity of MyoD, Sox9 andRunx2 to repress myogenic and skeletogenic fates during tenocytedifferentiation. A later tendon-dependent phase (B) relies on theproduction of ECM, particularly Col1a1, Col1a2, Col12a1 and Col14a1,by more mature TPCs, which extend processes into the ECM.

4193

REVIEW Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT

distinct embryonic origins and modes of tendon morphogenesis raisethe question of where the patterning information for muscleconnectivity lies, within the TPCs or within the musclesthemselves? Chick-quail chimera studies suggests that, at least forcranial and limb muscles, the TPCs and tendon matrix determine thepattern of attachments (Kardon, 1998; Kieny and Chevallier, 1979;Noden, 1988). Do muscles and the myomatrix play any role in TPCformation or maintenance? In the limbs of chick embryos in whichmuscle progenitors have been surgically removed, tenocytes initiallydevelop in the correct locations but later degenerate (Kardon, 1998).Limb tendons also degenerate inMyoD−/− and Pax3−/− mutant micethat lack the entire limbmusculature (Bonnin et al., 2005; Brent et al.,2005). Cranial tendons inmyod1−/−/myf5−/− double mutant zebrafishshow similar defects (Chen and Galloway, 2014). These resultssuggest a dependence on muscles and the myomatrix for tenocytemaintenance but not specification. As discussed below, thesephenotypes could be due to a lack of mechanical forces transmittedvia the ECM.What are the roles of specific ECM components of the myomatrix

or tendon ECM during the initial establishment of contact betweenmyoblasts and TPCs at attachment sites? Studies of axial muscles inthe zebrafish trunk have provided insights into this process (SnowandHenry, 2009). These muscles attach to intersegmental boundaries(ISBs) during embryogenesis before the appearance of TPCs. ISBsare anatomically distinct from later tendons, but contain many of thesame ECM components and serve analogous functions in bearing theforces ofmuscle contraction. During this ‘tendon-independent’ phaseof development (Fig. 3A), fibronectin (Fn) and laminin-alpha2(Lama2) in the myomatrix are highly enriched at ISBs as themyoblasts elongate and are required for embryonic muscleattachments (Koshida et al., 2005; Snow et al., 2008). Mammalianmuscles also require Fn and Lam for migration and attachment(Turner et al., 1983; Bajanca et al., 2006; Vaz et al., 2012). Fn andLam bind to Itg and dystrophin/dystroglycan complexes on musclecell surfaces. In zebrafish, this leads to localized phosphorylation offocal adhesion kinase (pFAK; Ptk2ab – Zebrafish InformationNetwork) at the ends of myofibers where they attach to ISBs, whichstabilizes myotome boundaries (Bassett et al., 2003; Henry et al.,2005; Parsons et al., 2002; Snow et al., 2008). TPCs only appear lateralong the ISBs, where they contribute additional ECM to strengthenexisting 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, somiticmuscles attach via the myomatrix at ISBs prior to the appearance ofTPCs. 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 bymyoblasts and accumulates at ISBs prior to muscle attachment(Fig. 3A) (Subramanian and Schilling, 2014). Tsp4b maintainsmuscle attachments at the ISB, and its depletion leads to detachmentupon contraction. The transplantation of wild-type myoblasts intoTsp4b-deficient embryos locally rescues muscle attachments atISBs, consistent with a role for Tsp4b in the myomatrix during thetendon-independent phase of attachment (Subramanian andSchilling, 2014). Interestingly, tsp4b mRNA abruptly disappearsfrom differentiating myofibers as they attach, suggesting a feedbackmechanism that regulates tsp4b transcription. Mammalianmyoblasts also express Tsp4, and human TSP4 (THBS4)expression increases in pathological conditions such as Duchennemuscular dystrophy and alpha-sarcoglycanopathies (Chen et al.,

2000; Jelinsky et al., 2010). Thus, Tsp4 in the myomatrix helpsmuscles attach in the absence of TPCs and might also facilitatesubsequent tendon maintenance and response to damage.

ECM and collagen fibril assembly during tendon and MTJ maturationAs MTJs mature, tenocytes secrete the bulk of the MTJ/tendonECM, particularly the many proteins and proteoglycans that makeup the core functional units, the collagen fibrils (Fig. 3B). Theseinteract with one another and align into groups of fibrils or fasciclesduring progressive phases of muscle attachment (Fig. 4). Thisfibrillar organization is essential for tendons to bear the stress ofmuscle contraction and prevent bone detachment (avulsionfractures) by controlling force distribution (Birch et al., 2013; Panet al., 2013; Schwartz et al., 2013; Pingel et al., 2014). The fibrillarnetwork of proteins includes: (1) core force-transmitting, structuralcollagens (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 distributeforces efficiently and reduce friction (Figs 3, 4) (Wang et al., 2012;Dunkman et al., 2013). Although the integration of structuralcollagens into fibrils has been well documented, recent studies haveprovided insight into the functions of scaffolding proteins andspecialized collagens during fibril assembly. These studies suggestthat similar to the early ECM at developing MTJs, ECM proteins ofthe maturing tendon provide continuous feedback in response tomechanical force (Choi et al., 2014; Popov et al., 2015; Wall andBanes, 2005; Zhang and Wang, 2010).

Col12a1, Col14a1 and Col22a1 belong to the class of fibril-associated collagens with interrupted triple helices (FACITs), whichlocalize to muscle attachments in avian and mouse tendons, and arealso expressed in human tendon fibroblasts (Fig. 3B; Fig. 4) (Kochet al., 2004;Wälchli et al., 1994). Studies of FACITs at zebrafish ISBshave been informative for understanding their functions as MTJsmature. In zebrafish, muscles first express Col12a1 and Col22a1 atlarval stages and these progressively align into the orthogonal fibrilarrays of mature MTJ/tendon ECM (Charvet et al., 2011, 2013).Col12a1 is expressed earlier than Col22a1 and colocalizes withLama2 throughout muscle fiber attachment (Bader et al., 2009).Col22a1 maintains attachments under tension, and its expressionincreases in tendinopathies (see Box 1) in humans (Charvet et al.,2013; Jelinsky et al., 2011). Recently, mutations in the humanCOL12A1 gene that disrupt COL12A1 secretion have been linked to aform ofBethlemmyopathy (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 inhuman tendinopathies (Bönnemann, 2011; Jelinsky et al., 2011). Inmice, Col12a1 interacts with tenascin C (Tnc) and helps crosslinkother collagens during fibril maturation by interacting with Dcn (Veitet al., 2006). Transmission electron microscopy studies also suggestthat 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 FACITproteins are highly conserved regulators of tendon ultrastructure andelasticity.

Other proteins involved in fibril assembly include the smallleucine-rich proteoglycans (SLRPs) such as Dcn (Fig. 4), Fmod,biglycan and lumican. These are found in relatively small amountsin tendons, yet loss-of-function mutations in the genes encodingthese proteins disrupt collagen fibrillogenesis (Chakravarti, 2002;Corsi et al., 2002; Zhang et al., 2006). SLRPs mainly crosslink

4194

REVIEW Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT

Tsp4

Fn

Dcn

Myo

tube

Itg

Myo

tube

Cartilage Cells

Myo

tube

Lam

DMD complex

FACIT collagen

Col1a1Col1a2 trimer

Tsp4

Fn

Dcn

Myo

tube

Itg

Cartilage cells

Myo

tube

Cartilage cells

Myo

tube

Lam

DMD complex

Tenocyte

FACIT collagen

Col1a1Col1a2 trimer

A Early attachment phase

B Mid-attachment phase

C Late attachment phase

Tenocyte

Key

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 attachmentphase, myoblasts (green) first extend towards a cartilage condensation (blue) and reorganize the local ECM by secreting Tsp4 (red), which interacts with Fn andLam. 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 andcartilage 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. Themagnified view illustrates howCol1a1 trimers begin to align perpendicular to skeletal cells (enthesis, dark blue and orange). Dystrophin (DMD) complexes appearon muscle surfaces. (C) In the final late attachment phase, collagen fibrils become crosslinked into a lattice, with tenocytes (red cells) extending processes tosurround fibrils, and entheses chondrifying (purple). The magnified view shows Col1a1 trimers becoming crosslinked by FACIT collagens and surrounded bytenocyte (red) processes, stabilizing the ECM and its interactions with Itgs on muscle and cartilage cells.

4195

REVIEW Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT

collagen fibrils, but they also appear to cross-regulate one another’stranscription through as yet unknown mechanisms (Yoon andHalper, 2005; Zhang et al., 2006; Dunkman et al., 2013). This mightinvolve feedback regulation of Scx and Mkx expression through thetendon ECM, and recent studies have suggested that Dcn and Fmodare 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 thedynamics of fibril ultrastructure feedback on ECM production tomodify tendon strength continuously.Tendon ECM and collagen fibrils are also continuously

remodeled in response to mechanical forces, at least around thecircumference 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 moderatemechanical forces show an increase in collagen fibril diameter,though diameter decreases with excessive mechanical force (DeAlmeida et al., 2010; Pingel et al., 2014). This remodeling occursprimarily through the activities of matrix metalloproteinases(MMPs) and their corresponding tissue inhibitors (TIMPs), aswell as disintegrin and metalloprotease with thrombospondinrepeats (ADAMTS) proteases (Bedi et al., 2010; Gotoh et al.,2013; Jones et al., 2006; Maeda et al., 2013). Almost all of the 23MMPs and 19 ADAMTS proteins known to be expressed invertebrates are detectable in adult tendon tissue and play a varietyof both positive and negative roles in establishing a functional MTJand tendon ECM (Davis et al., 2013; Spanoudes et al., 2014).MMPs are zinc-dependent endopeptidases that bind and unwindthe triple helix of collagen monomers. Collagenases such as Mmp1target the structural collagens for degradation, whereas gelatinasessuch as Mmp2 and Mmp9 and membrane-bound MMPs, such asMmp14, degrade smaller network collagens. Importantly, recentstudies have shown that the expression and activity of MMPs areregulated 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 tendonrepair (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 repairMultiple signaling pathways involving the ECM influence both theformation of muscle attachments and the maturation of tenocytes.Important players in muscle cells include Itgs and dystrophin, whichinteract with ECM components at the MTJ. Furthermore, in bothmuscles and tendons, mechanical forces are thought to have a role indynamic remodeling of the ECM.

Integrin signalingMany collagens and laminins at developing MTJs directly bind Itgcomplexes that are present in the membranes of muscle cells andtenocytes (Fig. 5) (Docheva et al., 2014; Mayer et al., 1997; Panet al., 2013; Rooney et al., 2006, 2012). In muscle, these continuousstructural links between ECM proteins, the sarcolemma and thecytoskeleton, maintain fiber integrity and modulate adhesion andgene expression. Not surprisingly, several types of humantendinopathies (see Box 1) are associated with changes in theexpression of Itgs and their ligands (Bönnemann, 2011; Jelinskyet al., 2011; Schessl et al., 2006). It is thus important to determinethe specific roles of Itg signaling duringMTJ maturation and tendonrepair after injury.

Different Itg heterodimer combinations lend specificity fordifferent ligands. For example, laminins (Lama2, Lama4) bindItga7/b1 in themuscle basementmembrane (Fig. 5) (Yurchencoet al.,2004; Durbeej, 2010; Carmignac and Durbeej, 2012), and defects inLAMA2 have been associated with merosin-deficient musculardystrophy in humans (Tomé et al., 1994; Rooney et al., 2012).Furthermore, mutations that disrupt genes encoding crosslinkingcollagens (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? InDrosophila, Sr promotes the transcription of the single flythrombospondin (Tsp) gene in tenocytes. Tsp binds αPS2 (If)/βPS(Mys) Itg heterodimers on fly muscle and tendon cell surfaces andpatterns muscle attachments (Chanana et al., 2007; Subramanianet al., 2007). Vertebrates have at least five thrombospondins, withdiverse functions in cell migration, vasculogenesis, wound healingand 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 laterby TPCs (Fig. 3B), and is essential for muscle attachment(Subramanian and Schilling, 2014). Accordingly, zebrafishembryos depleted of Tsp4b have defects in laminin localization andFAKphosphorylation (indicating reduced Itg signaling) at developingISBs, and their muscles detach under tension. These findings suggestthat, in zebrafish, Tsp4 plays key roles in organizing the ECM of bothmuscle and tendon, particularly those components essential for Itgsignaling. This requirement for Tsp4 is at least partially conserved, asTsp4−/− mutant mice show defects in ECM deposition in developingtendons (Fig. 4) (Frolova et al., 2014; Subramanian and Schilling,2014). Tsp4 and other subtype B thrombospondins form pentamersthat directly bind to collagens, laminins and other ECM proteins(Hauser et al., 1995). Surprisingly, human TSP4 proteinmicroinjected into the ECM surrounding Tsp4b-deficient myofibersin zebrafish localizes to ISBs and locally rescues laminin localization,Itg signaling and muscle attachments, suggesting that Tsp4 couldfunction as a scaffold for other ECMproteins during their assembly atmuscle attachments. Consistent with this model, the ability ofmicroinjected zebrafish tsp4b mRNA to rescue Tsp4b-deficientattachments requires Itg binding and pentamerization; mutation ofeither the Itg-binding (KGD) domain or the pentamerization (CQAC)domain of Tsp4b disrupts its localization to tendons and eliminates itsability to rescue muscle attachments in Tsp4b-deficient larvae(Subramanian and Schilling, 2014). These results, along with apotentially conserved requirement for Tsp4 in mice, suggest thatTSP4 defects could contribute to human tendinopathies, highlightingTSP4 as an attractive therapeutic target for strengthening the tendonECM. They also highlight the close association between the structuraland signaling roles of the ECM.

Box 1. TendinopathiesThe term ‘tendinopathy’ refers to a diverse set of tendon disorders(overlapping in some cases both in genetic and molecular terms withmyopathies) that are caused either by genetic mutations in ECMcomponents of MTJs or by mechanical stress that leads to tendonECM damage. Hallmarks of tendinopathies include COL1A1, COL1A2,COL4A1 and COL4A2 overexpression, fibril disorganization, increasedcollagen crosslinking, reduced tissue inhibition of MMPs (i.e. TIMPactivity), and elevated expression of MMP2, MMP14 and MMP19 as wellas of versican, biglycan and Dcn (Jelinsky et al., 2011; Parkinson et al.,2011; Dunkman et al., 2013; Zhou et al., 2014). Recent studies have alsoidentified COL6A1/2/3 and COL12A1 as genes underlying one form ofhuman ‘myotendinopathy’, which affects both myomatrix structure andtendon matrix structure (Bönnemann, 2011; Pan et al., 2013).

4196

REVIEW Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT

TGFβ signalingTGFβ signaling provides another striking example of the relationshipbetween ECM structure and signaling in tendons. Genetic studies inmice have revealed crucial roles for TGFβ at multiple steps in tendondevelopment, maturation, maintenance and repair (Figs 2, 3, 5).Removing the functions of both TGFβ2 and TGFβ3 ligands, or ofTGFβr2, eliminates most if not all differentiated tendons, whereasexogenous TGFβ is sufficient to induce the expression of Scxand Col1a1 (Fig. 2A) (Chuang et al., 2014; Pryce et al., 2009).Tgfb2−/−/Tgfb3−/− mutant mice lose Scx expression in TPCsbetween E11.5 and E12.5, suggesting that TGFβ is required forTPC maintenance (Pryce et al., 2009). Signaling through TGFβr2phosphorylates Smad2 and Smad3, which translocate to the nucleusand activate target genes, thereby maintaining differentiatedtenocytes (Figs 3 and 5). Mouse Smad3−/− mutants have reducedtendon tensile strength and increased spacing between collagenfascicles 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 thatduring tenocyte differentiation, Mkx activates the expression ofTGFβ in differentiating MSCs (Fig. 5) (Liu et al., 2015). TGFβs aresecreted bound to latent TGFβ-binding proteins (LTBPs), whichform part of the large latency complex (LLC) in the ECM (Wipffet al., 2007; Maeda et al., 2011). They are also secreted along withlatency-associated peptides (LAPs), which block association withTGFβ receptors, and along with other proteins of the LLC, theybecome incorporated into ECM via interactions between LTBPs andFn, fibrillin or Dcn (Isogai et al., 2003; Rifkin, 2005; Farhat et al.,2012). In this manner, TGFβs are stored in the ECM and must bereleased from the LLC and LAPs in order to be ‘activated’ andavailable to interact with cognate receptors (Horiguchi et al., 2012).Activation may occur by release of TGFβ stimulated by shearingforces, LLC degradation by proteases, interactions with Itgs throughRGD motifs on LTBPs themselves, or by the activity of Mmp2,

Mmp9 and Bmp1 proteases (Munger and Sheppard, 2011). Fewstudies have addressed these mechanisms of TGFβ activationspecifically for the ECM of tendons or MTJs. A recenttranscriptomic analysis of Scx-expressing tenocytes from mouselimbs reveals that both TGFβ and MAPK signaling are stronglyupregulated, but that only TGFβ upregulates Tsp2, Tsp4 and LTBPcomponents of the ECM and promotes the tenocyte cell fate (Haviset al., 2014).

The correct regulation of TGFβ activity is crucial not only fortendon development but also for healing injured tendons. Injurieslead to excessive release of TGFβ owing to mechanical force-mediated activation of TGFβ (discussed below) and can causefibrotic scarring of the tendon, thereby disrupting its function(Farhat et al., 2015). In one model, elevated TGFβ mightoveractivate MMPs, which in turn promotes further release ofactive TGFβ from the ECM as well as activating expression ofScx and Mkx and driving further ECM production.

Mechanical forces and signalingAs alluded to throughout this Review, tenocytes actively sensemechanical force, leading to changes in gene expression,cytoskeletal organization and ECM secretion (Fig. 5) (Banoset al., 2008; Maeda et al., 2009, 2013). Such feedback must beextremely important for a tissue that constantly adjusts its stiffnessto changing loads. It depends, at least in part, on signaling throughgap junctional complexes localized to tenocyte processes (Kruegeland Miosge, 2010; Maeda et al., 2012). Indeed, rats subjected torunning on treadmills have increased Tnmd and Col1a1 expressionas well as TPC proliferation (Eliasson et al., 2009; Zhang andWang,2013). Elevated secretion of both Col4a1 and Col6a1 is also seen indeveloping chick tendons under stress, and this alters thecrosslinking of fibrils (Marturano et al., 2014), thereby fine-tuning tendon strength and promoting repair (Bailey et al., 1998;Willett et al., 2010).What are the molecular mechanisms underlyingthese cellular responses?

ScxMkx

Col1a1Col1a2Col3a1Col6a1Col12a1Col14a1Tgfb2TnmdFmodTsp4

Egr1Egr2

Mechanical stress

TGFβLLC

Lama2,Lama4 Tsp4b ItgTgfβrTGFβ

Actin

filaments

Actin filaments

ECM

Col1a FACIT Col

Muscle Tenocyte

ECM Col22a1Muscle

contraction

Smad3

Smad

3Sm

ad3

Key

Fig. 5. Model for ECM-mediated feedback frommechanical force and its effects on tenocyte gene expression.A tenocyte (orange) synthesizes the tendonECM, including Lama2, Lama4 (brown), Tsp4b (red pentagons), Col1a (blue) and FACIT Col (purple), all of which signal through Itg receptors (dark blue) onmuscle and tenocyte cell surfaces in response to mechanical stress (gray arrows). In addition, stress causes the ECM to release TGFβ (yellow) from the TGFβlarge latent complex (LLC) (gray dotted arrows). Itg and TGFβ signaling in tenocytes feedback to regulate Scx-, Egr1/2- and Mkx-induced transcription (dashedarrows) 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 ECMgenes. The muscle fiber also contributes to the tendon matrix by secreting FACIT Col22a1.

4197

REVIEW Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT

TGFβ signaling is one such mechanosensitive pathway thatcould control the response of tendons to force. Mechanical forcecauses 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 intenocytes, 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 toforce also upregulates ITGA1 and ITGA2 expression in humanTPC cultures (Popov et al., 2015). Tenocytes elevate expressionof Tgfb1, Tgfbr2 and Smad7 in response to injury in mice(Guerquin et al., 2013). Thus, one attractive model is that forcetriggers TGFβ signaling leading to increased expression of Scxand Mkx, which in turn activates TGFβ expression, creating apositive-feedback loop that leads secondarily to remodeling/strengthening of ECM (Fig. 5) (Liu et al., 2015). Notably, TGFβsignaling in response to mechanical forces also controls MMPexpression during tendon repair (Katzel et al., 2011; Farhat et al.,2015). Interestingly, injured tendons in Egr1−/− mutant mice failto 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 bonefractures (Davis et al., 2013; Pan et al., 2013; Schwartz et al.,2013; Zhang et al., 2006). How fibrils physically anchor to cellsand how the dynamics of these anchors are regulated under loadremain unclear. Biopsies of human Achilles’ tendons have shownthat fibrils buckle in overloaded tendons (Pingel et al., 2014). Intenocytes cultured in collagen gels, mechanical force translatesinto cytoskeletal force through non-muscle myosin II bound toactin fibrils associated with focal adhesion complexes, which inturn associate with Itgs and other ECM receptors. The chemicalinhibition of myosin II function (using blebbistatin) reducescytoskeletal traction forces and leads to ECM remodeling (Maedaet al., 2013). Thus, in addition to inducing transcriptionalchanges, mechanical force can stimulate changes in cytoskeletaltension 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 afterinjury. For example, tenocytes in silicone micropillar gels elevateMMP expression levels in response to gel deformation (Maedaet al., 2013). In addition, humans show dramatic increases in thelevels of MMP2, MMP9 and MMP14 in adult tendons followingendurance exercise, suggesting that these proteins aidMTJ repair inresponse to mechanotransduction (Rullman et al., 2009). Thiseffect depends on the timing of loading as, in cultured tendonfascicles, cells upregulate MMP2 and MMP13 after very shortcycles of loading, but downregulate MMP1 after longer cycles(Maeda et al., 2009, 2013). Similarly, in vitro studies ofmechanicalloading onmouse tenocytes have shown that, whereas low levels ofshear force lead to upregulation of Col1a and Tmnd, increasing theforce leads to upregulation of Runx2 and Sox9 (Zhang and Wang,2015). Understanding these dynamic responses to mechanicalstimulation might lead to improved therapeutic interventions; thesystemic inhibition of MMPs, for example, can reduce fibroticscarring of muscle ECM (Farhat et al., 2015). MMP expression isalso regulated by TGFβs (Yu and Stamenkovic, 2000; Ge andGreenspan, 2006; Farhat et al., 2015). In turn, MMP2, MMP9 andBMP1 proteases might specifically digest LLC and release activeTGFβ, establishing a positive-feedback loop that could help fine-tune MMP levels, both during normal tendon function and inresponse to tendon injury or exercise (Fig. 5).

ECM functions at tendon-bone attachmentsSo far, we have emphasized the ECM associated with tendon-muscle attachments. But tendons also attach, at their other ends, tocartilage/bone under a unique set of mechanical constraints. Manytendons insert into bony protrusions known as osteotendinousjunctions or ‘entheses’, such as the deltoid tuberosity on thehumerus (Fig. 6A). A characteristic structural feature of entheses isthe presence of ‘fibrocartilage’, a tissue with physical propertiessomewhere in between cartilage and tendon. Within an enthesis, arapid transition from a more bone-like cellular and ECM structure toa more cartilage-like (less rigid) structure in regions closer to thetendon is observed. In addition, a unique mineralization gradientforms from the bony front to the point of tendon insertion, the widthof which is constant with corresponding changes in cellular density(Schwartz et al., 2012). The enthesis ECM also shows more Dcnand Bgn localized to the tendon side, whereas Col2a1, Col9, Col10and aggrecan localize to the bony side (Thomopoulos et al., 2003).These gradual transitions in ECM and rigidity are essential for theproper transmission of contractile forces to the bone to preventavulsion fractures (Zelzer et al., 2014).

In the appendicular skeleton, entheses are established throughspecialized contours and protuberances on bones called eminences.Recent genetic studies in mice have begun to elucidate the signalsthat control the formation of these structures (Blitz et al., 2009,2013; Zelzer et al., 2014). In mouse embryos, eminencedevelopment coincides with the formation of muscle/tendonattachments, suggesting that attachments impose physical changeson the bone. However, mouse mutants that lack muscles, such assplotch delayed (spd) or muscular dysgenesis (mdg) mutants, stillform eminences like the deltoid tuberosity on the humerus, despitesevere joint fusions (Fig. 6B) (Blitz et al., 2009).

Perhaps not surprisingly, TGFβ signaling also plays importantroles in the formation of entheses. Loss of TGFβ signaling throughablation of Tgfβr2 in limb mesenchyme eliminates eminences in thelimb, though this might be due to a broader or earlier role for TGFβsignaling in skeletal/tendon progenitors (Fig. 6B) (Blitz et al., 2009,2013). Instead, evidence is building to suggest that the more crucialinducers of eminences are other members of the TGFβ superfamily,namely bone morphogenetic proteins (BMPs). The conditionaldeletion in mice of Bmp4 specifically in tenocytes using an Scx:Credriver completely eliminates eminences (Blitz et al., 2009). Pulse-chase labeling studies show that eminences form from secondaryfields of Scx/Sox9 co-expressing cells that lie immediately adjacentto major skeletal condensations (Fig. 6B) (Blitz et al., 2013); thesecells form in mutants lacking BMP signaling, but do notdifferentiate. Early Sox9 expression is observed in both theskeletal condensation and the eminence, but the subset of thesecells that express Scx are delayed in expression of Col2a1, therebyrestricting chondrogenesis to the developing enthesis. Scx-expressing cells in developing entheses express Bmp4 and this islost in tendons of Scx−/−mutant mice, which lack entheses. Thus, anattractive hypothesis is that Bmp4 secreted by tenocytes inducesenthesis formation in adjacent skeletogenic mesenchyme (Fig. 6B).Interestingly, Sox9-expressing secondary fields of eminenceprogenitors still form in Bmpr1a−/− mutant mice but neverdifferentiate, indicating a role for BMP signaling in maturationrather than specification. Taken together, these studies establish anearly phase of eminence/enthesis development, which isindependent of muscle development, and suggest that Scx-drivenBmp4 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

4198

REVIEW Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT

and chondrocytes is an emerging theme both during tendondevelopment and in MSCs (Fig. 2A) (Asou et al., 2002; Soedaet al., 2010; Sugimoto et al., 2013). The close lineal relationshipbetween tenocytes and chondrocytes/osteocytes (Fig. 2A) and theirregulation at entheses also may help explain the ectopic ossificationof tendons (see Box 2) that occurs normally in some species as well

as during aging and disease (Magne and Bougault, 2015; Zhang andWang, 2015).

Finally, and not surprisingly, entheses are also extremelysensitive to mechanical forces. Mechanical forces have long beenknown to be important for the development of the bones to whichmuscles attach, as well as for the maintenance of skeletogenic cell

Wild type spd mutant

Gdf5

Col2a1

Col2a1Col2a1

Col2a1

Col2bCol2b

Bmp4 lof in limb mesenchyme

Tgfβr2 lof in limb andSox9 lof in tenocytesWild type

Sox9Col2a

Primary field

Secondary field

Enthesis

Gdf5-expressing joint cells

Col2b-expressing cartilage cells at articular surface

Col2a-expressing cartilage cells

A

B

Scx+/Bmp+ TPCs

Sox9high,Scxlow enthesis cells

Scxhigh,Sox9low enthesis cells Tenocytes Sox9+ cells that do

not express Col2a

Tendon

Muscle

Fig. 6. ECM functions at tendon-bone attachments. (A) Diagrams illustrating changes in cartilage at the developing humero-ulnar joint of the mouseforelimb 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 lossof muscles in spd mutants leads to loss of Gdf5 expression, disorganized Col2b+ interzone cells and joint fusion. (B) Diagrams illustrating changes in cartilageand tenocytes at a developing eminence. The primary field contains cells that form chondrocytes within the developing bone, whereas the secondary fieldconsists 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 muscleinsertion 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 ofthe 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.

4199

REVIEW Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT

populations (Shwartz et al., 2013). The paralysis of specific musclesleads to bone defects at sites of attachment and loss of jointprogenitors (Kahn et al., 2009). The tendon ECM plays vital roles inthese adaptations of muscles and bones to mechanical loading(Kjaer, 2004).

Conclusions and perspectivesVertebrate tendons begin life similar to skeletal progenitors in theembryo, but rapidly establish unique identities and tissueorganization, in large part through interactions with and assemblyof the tendon ECM. This involves: (1) essential transcription factorsexpressed in tenocytes, such as Scx, Mkx and Egr1, that drive theexpression of ECM proteins; (2) ECM components such as lamininsthat help establish muscle attachments in the absence of tenocytesand control tenocyte morphogenesis; (3) ECM components such asTsp4 that drive the assembly of collagen fibrils at MTJs throughtheir interactions with other ECM proteins and Itgs on muscle cellsurfaces; and (4) the maintenance and repair of these ECMcomponents in response to mechanical forces. In this Review, wehave highlighted recent genetic studies that have provided insightsinto the molecular mechanisms underlying these differentprocesses.The discovery of Scx and Mkx has provided inroads into

understanding the tendon/ligament gene regulatory program andrevealed the close relationship between tenocytes and skeletallineages. Scx is the earliest known marker of TPCs, and itsoverexpression transforms MSCs into tenocytes (Alberton et al.,2012), whereas Sox9 overexpression converts MSCs intocartilage (Fig. 2A) (Takimoto et al., 2012). However, unlikeSox9, which when eliminated leads to a failure to form cartilage,loss-of-function Scx or Mkx mutations in mice do not eliminatetenocytes, and mutants are viable although they have severemusculoskeletal deformities. This implies that tendondevelopment involves multiple, partially redundant transcriptionfactors and cell-cell signals, each playing a unique role inbuilding and maintaining the complex network of ECM proteinsat muscle attachments.These studies also highlight the fact that the stability of muscle

attachments is not pre-established during development butevolves through constant adaptation of the ECM to changing

mechanical load. This occurs regionally within each tendon fromits muscle origin to its bony insertion. Understanding the tendon/ligament gene regulatory program thus requires knowledge ofhow this dynamic network of ECM proteins self-assembles andfeeds back upon transcriptional regulators in response tomechanical forces.

The musculoskeletal system and its associated ECM have alsoevolved to suit dramatically different modes of locomotion, feedingstrategies and body sizes of different vertebrates. Changes in theinterconnections between individual muscles and bones underliemany of the evolutionary differences between species. For example,mandibles of fish (Malawi cichlids), birds (Darwin’s finches) anddogs can have very different functional morphologies depending ontheir feeding strategies, which notably have all been linked tochanges in BMP signaling (Abzhanov et al., 2004; Albertson et al.,2005; Schoenebeck et al., 2012). These reflect coordinated changesin the regulation of cranial neural crest cells that form not only thecraniofacial skeleton, but also cranial TPCs during evolution.Indeed, recent studies grafting neural crest cells between quail andduck embryos reveal that some distinct craniofacial morphologieshave evolved through changes in cell-intrinsic mechanisms. Jawcartilage differentiation, marked by Runx2 expression, occursearlier in the duck embryo, whereas tendon differentiation, markedby Scx expression, occurs earlier in the quail (Tokita and Schneider,2009). How these species-specific differences in timing of skeletaland tendon differentiation reflect changes in ECM organization andin mechanical stress to suit their adaptive functions is an importantarea for future investigation.

Understanding the genetic control of the development of tendons,their integration into the musculoskeletal system and their ECMorganization also has important implications for regenerativemedicine. Current efforts to treat tendon injuries or diseases focuseither on ameliorating inflammation or driving MSCs towards thetenocyte fate with hopes of stem cell therapy (Yang et al., 2013).Both approaches leave out the crucial role of establishing theappropriate ECM for a specific tendon type and the loads that itneeds to bear. Attaining the correct strength also relies on correctcellular responses to mechanical forces, which are transducedthrough the ECM. Thus, it is important to consider the ECM and itsfunctions going forward in efforts to improve diagnosis and therapydesign for tendon disorders.

AcknowledgementsWe thank members of the Schilling lab and anonymous reviewers for critical readingof the manuscript.

Competing interestsThe authors declare no competing or financial interests.

FundingThe authors were supported by National Institutes of Health awards [R21 AR62792and R01 DE13828 to T.F.S.]. Deposited in PMC for release after 12 months.

ReferencesAbzhanov, A., Protas, M., Grant, B. R., Grant, P. R. and Tabin, C. J. (2004). Bmp4

and morphological variation of beaks in Darwin’s finches. Science 305,1462-1465.

Adams, J. C. and Lawler, J. (2004). The thrombospondins. Int. J. Biochem. CellBiol. 36, 961-968.

Alberton, P., Popov, C., Pragert, M., Kohler, J., Shukunami, C., Schieker, M. andDocheva, D. (2012). Conversion of human bone marrow-derived mesenchymalstem cells into tendon progenitor cells by ectopic expression of scleraxis. StemCells Dev. 21, 846-858.

Albertson, R. C., Streelman, J. T., Kocher, T. D. and Yelick, P. C. (2005).Integration and evolution of the cichlid mandible: the molecular basis of alternatefeeding strategies. Proc. Natl. Acad. Sci. USA 102, 16287-16292.

Box 2. Ectopic tendon calcificationEctopic ossification of tendons occurs normally in some species and isalso observed during aging and in disease (Magne and Bougault, 2015;Zhang and Wang, 2015). Spondyloarthritis, for example, is aninflammatory enthesitis (Weinreb et al., 2014) that can lead to ectopicossification spreading from the bone to the tendon or ligament. Ectopicossification is also seen in calcifying tendinopathies, a commonconsequence of aging affecting as many as 1 in 5 adults over 50 yearsof age. Tendon ossification appears to be caused, in part, by tenocyte-dependent degradation of the tendon ECM (Magne and Bougault, 2015)as well as altered responses to mechanical forces and BMP/Smadsignaling (Rui et al., 2013). The genetic inactivation of two smallproteoglycans (Bgn and Fmod) of the ECM of cultured mouse TPCsleads to ectopic activation of BMP signaling and tendon ossification (Biet al., 2007). Mechanical stretching of TPCs in vitro can also lead to BMPupregulation and abnormal ossification (Zhang and Wang, 2013, 2015).Surprisingly, BMP signaling oscillates in a circadian manner, and thesecycles are deregulated in arrhythmic mutant mice, which correlates withincreased tendon ossification (Yeung et al., 2014) Thus, tendons andligaments are really tissues living on the edge with respect to their ECMcomposition and cellular constituents, presumably owing to their extremeresponsiveness to feedback through mechanical force.

4200

REVIEW Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT

Alliston, T., Choy, L., Ducy, P., Karsenty, G. and Derynck, R. (2001). TGF-beta-induced repression of CBFA1 by Smad3 decreases cbfa1 and osteocalcinexpression and inhibits osteoblast differentiation. EMBO J. 20, 2254-2272.

Anderson, D. M., Arredondo, J., Hahn, K., Valente, G., Martin, J. F., Wilson-Rawls, J. and Rawls, A. (2006). Mohawk is a novel homeobox gene expressed inthe developing mouse embryo. Dev. Dyn. 235, 792-801.

Anderson, D. M., Beres, B. J., Wilson-Rawls, J. and Rawls, A. (2009). Thehomeobox gene Mohawk represses transcription by recruiting the sin3A/HDACco-repressor complex. Dev. Dyn. 238, 572-580.

Anderson, D. M., George, R., Noyes, M. B., Rowton, M., Liu, W., Jiang, R., Wolfe,S. A., Wilson-Rawls, J. and Rawls, A. (2012). Characterization of the DNA-binding properties of the Mohawk homeobox transcription factor. J. Biol. Chem.287, 35351-35359.

Aslan, H., Kimelman-Bleich, N., Pelled, G. andGazit, D. (2008). Molecular targetsfor tendon neoformation. J. Clin. Invest. 118, 439-444.

Asou, Y., Nifuji, A., Tsuji, K., Shinomiya, K., Olson, E. N., Koopman, P. andNoda, M. (2002). Coordinated expression of scleraxis and Sox9 genesduring embryonic development of tendons and cartilage. J. Orthop. Res. 20,827-833.

Bader, H. L., Keene, D. R., Charvet, B., Veit, G., Driever, W., Koch, M. andRuggiero, F. (2009). Zebrafish collagen XII is present in embryonic connectivetissue sheaths (fascia) and basement membranes. Matrix Biol. 28, 32-43.

Bailey, A. J., Paul, R. G. and Knott, L. (1998). Mechanisms of maturation andageing of collagen. Mech. Ageing Dev. 106, 1-56.

Bajanca, F., Luz, M., Raymond, K., Martins, G. G., Sonnenberg, A., Tajbakhsh,S., Buckingham, M. and Thorsteinsdottir, S. (2006). Integrin alpha6beta1-laminin interactions regulate early myotome formation in the mouse embryo.Development 133, 1635-1644.

Banos, C. C., Thomas, A. H. and Kuo, C. K. (2008). Collagen fibrillogenesis intendon development: current models and regulation of fibril assembly. BirthDefects Res. C. Embryo Today 84, 228-244.

Bassett, D. I., Bryson-Richardson, R. J., Daggett, D. F., Gautier, P., Keenan,D. G. and Currie, P. D. (2003). Dystrophin is required for the formation of stablemuscle attachments in the zebrafish embryo. Development 130, 5851-5860.

Becker, S., Pasca, G., Strumpf, D., Min, L. and Volk, T. (1997). Reciprocalsignaling between Drosophila epidermal muscle attachment cells and theircorresponding muscles. Development 124, 2615-2622.

Bedi, A., Kovacevic, D., Hettrich, C., Gulotta, L. V., Ehteshami, J. R., Warren,R. F. and Rodeo, S. A. (2010). The effect of matrix metalloproteinase inhibition ontendon-to-bone healing in a rotator cuff repair model. J. Shoulder Elbow Surg. 19,384-391.

Berthet, E., Chen, C., Butcher, K., Schneider, R. A., Alliston, T. andAmirtharajah, M. (2013). Smad3 binds Scleraxis and Mohawk and regulatestendon matrix organization. J. Orthop. Res. 31, 1475-1483.

Bi, Y., Ehirchiou, D., Kilts, T. M., Inkson, C. A., Embree, M. C., Sonoyama, W.,Li, L., Leet, A. I., Seo, B.-M., Zhang, L. et al. (2007). Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche. Nat. Med. 13,1219-1227.

Birch, H. L., Thorpe, C. T. and Rumian, A. P. (2013). Specialization of extracellularmatrix for function in tendons and ligaments. Muscles. Ligaments Tendons J. 3,12-22.

Blitz, E., Viukov, S., Sharir, A., Shwartz, Y., Galloway, J. L., Pryce, B. A.,Johnson, R. L., Tabin, C. J., Schweitzer, R. and Zelzer, E. (2009). Bone ridgepatterning during musculoskeletal assembly is mediated through SCX regulationof Bmp4 at the tendon-skeleton junction. Dev. Cell 17, 861-873.

Blitz, E., Sharir, A., Akiyama, H. and Zelzer, E. (2013). Tendon-bone attachmentunit is formed modularly by a distinct pool of Scx- and Sox9-positive progenitors.Development 140, 2680-2690.

Bonnemann, C. G. (2011). The collagen VI-related myopathies: muscle meets itsmatrix. Nat. Rev. Neurol. 7, 379-390.

Bonnin, M.-A., Laclef, C., Blaise, R., Eloy-Trinquet, S., Relaix, F., Maire, P. andDuprez, D. (2005). Six1 is not involved in limb tendon development, but isexpressed in limb connective tissue under Shh regulation. Mech. Dev. 122,573-585.

Bornstein, P., Agah, A. and Kyriakides, T. R. (2004). The role of thrombospondins1 and 2 in the regulation of cell–matrix interactions, collagen fibril formation, andthe response to injury. Int. J. Biochem. Cell Biol. 36, 1115-1125.

Brent, A. E., Schweitzer, R. and Tabin, C. J. (2003). A somitic compartment oftendon progenitors. Cell 113, 235-248.

Brent, A. E., Braun, T. and Tabin, C. J. (2005). Genetic analysis of interactionsbetween the somitic muscle, cartilage and tendon cell lineages during mousedevelopment. Development 132, 515-528.

Bushby, K. M. D., Collins, J. and Hicks, D. (2014). Collagen type VI myopathies.Adv. Exp. Med. Biol. 802, 185-199.

Carmignac, V. and Durbeej, M. (2012). Cell-matrix interactions in muscle disease.J. Pathol. 226, 200-218.

Chakravarti, S. (2002). Functions of lumican and fibromodulin: lessons fromknockout mice. Glycoconj. J. 19, 287-293.

Chanana, B., Graf, R., Koledachkina, T., Pflanz, R. and Vorbruggen, G. (2007).AlphaPS2 integrin-mediated muscle attachment in Drosophila requires the ECMprotein Thrombospondin. Mech. Dev. 124, 463-475.

Charvet, B., Malbouyres, M., Pagnon-Minot, A., Ruggiero, F. and Le Guellec, D.(2011). Development of the zebrafish myoseptum with emphasis on themyotendinous junction. Cell Tissue Res. 346, 439-449.

Charvet, B., Guiraud, A., Malbouyres, M., Zwolanek, D., Guillon, E., Bretaud, S.,Monnot, C., Schulze, J., Bader, H. L., Allard, B. et al. (2013). Knockdown ofcol22a1 gene in zebrafish induces a muscular dystrophy by disruption of themyotendinous junction. Development 140, 4602-4613.

Chen, J. W. and Galloway, J. L. (2014). The development of zebrafish tendon andligament progenitors. Development 141, 2035-2045.

Chen, Y.-W., Zhao, P., Borup, R. and Hoffman, E. P. (2000). Expression profilingin the muscular dystrophies: identification of novel aspects of molecularpathophysiology. J. Cell Biol. 151, 1321-1336.

Chen, X., Yin, Z., Chen, J. I., Shen, W. l., Liu, H. H., Tang, Q., Fang, Z., Lu, L. R.,Ji, J. and Ouyang, H. W. (2012). Force and scleraxis synergistically promote thecommitment of human ES cells derived MSCs to tenocytes. Sci. Rep. 2, 977.

Choi, W. J., Park, M. S., Park, K. H., Courneya, J.-P., Cho, J. S., Schon, L. C. andLee, J. W. (2014). Comparative analysis of gene expression in normal anddegenerative human tendon cells: effects of cyclic strain. Foot Ankle Int. 35,1045-1056.

Chuang, H.-N., Hsiao, K.-M., Chang, H.-Y., Wu, C.-C. and Pan, H. (2014). Thehomeobox transcription factor Irxl1 negatively regulates MyoD expression andmyoblast differentiation. FEBS J. 281, 2990-3003.

Corsi, A., Xu, T., Chen, X.-D., Boyde, A., Liang, J., Mankani, M., Sommer, B.,Iozzo, R. V., Eichstetter, I., Robey, P. G., et al. (2002). Phenotypic effects ofbiglycan deficiency are linked to collagen fibril abnormalities, are synergized bydecorin deficiency, and mimic Ehlers-Danlos-like changes in bone and otherconnective tissues. J. Bone Miner. Res. 17, 1180-1189.

Davis, M. E., Gumucio, J. P., Sugg, K. B., Bedi, A. and Mendias, C. L. (2013).MMP inhibition as a potential method to augment the healing of skeletal muscleand tendon extracellular matrix. J. Appl. Physiol. 115, 884-891.

De Almeida, F. M., Tomiosso, T. C., Biancalana, A., Mattiello-Rosa, S. M., Vidal,B. C., Gomes, L. and Pimentel, E. R. (2010). Effects of stretching onmorphological and biochemical aspects of the extracellular matrix of the ratcalcaneal tendon. Cell Tissue Res. 342, 97-105.

Docheva, D., Popov, C., Alberton, P. and Aszodi, A. (2014). Integrin signaling inskeletal development and function. Birth Defects Res. C Embryo Today Rev. 102,13-36.

Dunkman, A. A., Buckley, M. R., Mienaltowski, M. J., Adams, S. M., Thomas,S. J., Satchell, L., Kumar, A., Pathmanathan, L., Beason, D. P., Iozzo, R. V.et al. (2013). Decorin expression is important for age-related changes in tendonstructure and mechanical properties. Matrix Biol. 32, 3-13.

Durbeej, M. (2010). Laminins. Cell Tissue Res. 339, 259-268.Eliasson, P., Fahlgren, A. and Aspenberg, P. (2008). Mechanical load and BMP

signaling during tendon repair: a role for follistatin? Clin. Orthop. Relat. Res. 466,1592-1597.

Eliasson, P., Andersson, T. and Aspenberg, P. (2009). Rat Achilles tendonhealing: mechanical loading and gene expression. J. Appl. Physiol. 107, 399-407.

Espira, L., Lamoureux, L., Jones, S. C., Gerard, R. D., Dixon, I. M. C. andCzubryt, M. P. (2009). The basic helix–loop–helix transcription factor scleraxisregulates fibroblast collagen synthesis. J. Mol. Cell. Cardiol. 47, 188-195.

Farhat, Y. M., Al-Maliki, A. A., Chen, T., Juneja, S. C., Schwarz, E. M., O’Keefe,R. J. and Awad, H. A. (2012). Gene expression analysis of the pleiotropic effectsof TGF-β1 in an in vitro model of flexor tendon healing. PLoS ONE 7, e51411.

Farhat, Y. M., Al-Maliki, A. A., Easa, A., O’Keefe, R. J., Schwarz, E. M. and Awad,H. A. (2015). TGF-β1 suppresses plasmin and MMP activity in flexor tendon cellsvia PAI-1: implications for scarless flexor tendon repair. J. Cell Physiol. 230,318-326.

Font, B., Eichenberger, D., Rosenberg, L. M. and Van Der Rest, M. (1996).Characterization of the interactions of type XII collagen with two smallproteoglycans from fetal bovine tendon, decorin and fibromodulin. Matrix Biol.15, 341-348.

Frolova, E. G., Drazba, J., Krukovets, I., Kostenko, V., Blech, L., Harry, C.,Vasanji, A., Drumm, C., Sul, P., Jenniskens, G. J. et al. (2014). Control oforganization and function of muscle and tendon by thrombospondin-4.Matrix Biol.37, 35-48.

Ge, G. and Greenspan, D. S. (2006). BMP1 controls TGFβ1 activation via cleavageof latent TGFβ-binding protein. J. Cell Biol. 175, 111-120.

Gotoh, M., Mitsui, Y., Shibata, H., Yamada, T., Shirachi, I., Nakama, K., Okawa,T., Higuchi, F. and Nagata, K. (2013). Increased matrix metalloprotease-3 geneexpression in ruptured rotator cuff tendons is associated with postoperativetendon retear. Knee Surg. Sports Traumatol. Arthrosc. 21, 1807-1812.

Grenier, J., Teillet, M.-A., Grifone, R., Kelly, R. G. and Duprez, D. (2009).Relationship between neural crest cells and cranial mesoderm during headmuscle development. PLoS ONE 4, e4381.

Guerquin, M.-J., Charvet, B., Nourissat, G., Havis, E., Ronsin, O., Bonnin, M.-A.,Ruggiu, M., Olivera-Martinez, I., Robert, N., Lu, Y. et al. (2013). Transcription

4201

REVIEW Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT

factor EGR1 directs tendon differentiation and promotes tendon repair. J. Clin.Invest. 123, 3564-3576.

Hauser, N., Paulsson, M., Kale, A. and DiCesare, P. (1995). Tendonextracellular matrix contains pentameric thrombospondin-4 (TSP-4). FEBS Lett.368, 307-310.

Havis, E., Bonnin, M.-A., Olivera-Martinez, I., Nazaret, N., Ruggiu, M.,Weibel, J.,Durand, C., Guerquin, M.-J., Bonod-Bidaud, C., Ruggiero, F. et al. (2014).Transcriptomic analysis of mouse limb tendon cells during development.Development 141, 3683-3696.

Heinemeier, K. M., Skovgaard, D., Bayer, M. L., Qvortrup, K., Kjaer, A., Kjaer,M., Magnusson, S. P. and Kongsgaard, M. (2012). Uphill running improves ratAchilles tendon tissue mechanical properties and alters gene expression withoutinducing pathological changes. J. Appl. Physiol. 113, 827-836.

Henry, C. A., McNulty, I. M., Durst, W. A., Munchel, S. E. and Amacher, S. L.(2005). Interactions between muscle fibers and segment boundaries in zebrafish.Dev. Biol. 287, 346-360.

Herchenhan, A., Bayer, M. L., Svensson, R. B., Magnusson, S. P. and Kjaer, M.(2013). In vitro tendon tissue development from human fibroblasts demonstratescollagen fibril diameter growth associated with a rise in mechanical strength. Dev.Dyn. 242, 2-8.

Horiguchi, M., Ota, M. and Rifkin, D. B. (2012). Matrix control of transforminggrowth factor-β function. J. Biochem. 152, 321-329.

Hosokawa, R., Oka, K., Yamaza, T., Iwata, J., Urata, M., Xu, X., Bringas, P.,Nonaka, K. and Chai, Y. (2010). TGF-beta mediated FGF10 signaling incranial neural crest cells controls development of myogenic progenitor cellsthrough tissue–tissue interactions during tongue morphogenesis. Dev. Biol. 341,186-195.

Huang, A. H., Lu, H. H. and Schweitzer, R. (2015). Molecular regulation of tendoncell fate during development. J. Orthop. Res. 33, 800-812.

Isogai, Z., Ono, R. N., Ushiro, S., Keene, D. R., Chen, Y., Mazzieri, R.,Charbonneau, N. L., Reinhardt, D. P., Rifkin, D. B. and Sakai, L. Y. (2003).Latent transforming growth factor beta-binding protein 1 interacts with fibrillin andis a microfibril-associated protein. J. Biol. Chem. 278, 2750-2757.

Ito, Y., Toriuchi, N., Yoshitaka, T., Ueno-Kudoh, H., Sato, T., Yokoyama, S.,Nishida, K., Akimoto, T., Takahashi, M., Miyaki, S. et al. (2010). The Mohawkhomeobox gene is a critical regulator of tendon differentiation. Proc. Natl. Acad.Sci. USA 107, 10538-10542.

Jelinsky, S. A., Archambault, J., Li, L. and Seeherman, H. (2010). Tendon-selective genes identified from rat and human musculoskeletal tissues. J. Orthop.Res. 28, 289-297.

Jelinsky, S. A., Rodeo, S. A., Li, J., Gulotta, L. V., Archambault, J. M. andSeeherman, H. J. (2011). Regulation of gene expression in human tendinopathy.BMC Musculoskelet. Disord. 12, 86.

Jones, G. C., Corps, A. N., Pennington, C. J., Clark, I. M., Edwards, D. R.,Bradley, M. M., Hazleman, B. L. and Riley, G. P. (2006). Expression profiling ofmetalloproteinases and tissue inhibitors of metalloproteinases in normal anddegenerate human achilles tendon. Arthritis Rheum. 54, 832-842.

Juneja, S. C. and Veillette, C. (2013). Defects in tendon, ligament, and enthesis inresponse to genetic alterations in key proteoglycans and glycoproteins: a review.Arthritis 2013, 154812.

Kahn, J., Shwartz, Y., Blitz, E., Krief, S., Sharir, A., Breitel, D. A., Rattenbach, R.,Relaix, F., Maire, P., Rountree, R. B. et al. (2009). Muscle contraction isnecessary to maintain joint progenitor cell fate. Dev. Cell 16, 734-743.

Kang, J. S., Alliston, T., Delston, R. and Derynck, R. (2005). Repression of Runx2function by TGF-beta through recruitment of class II histone deacetylases bySmad3. EMBO J. 24, 2543-2555.

Kannus, P. (2000). Structure of the tendon connective tissue. Scand. J. Med. Sci.Sports 10, 312-320.

Kannus, P., Jozsa, L., Jarvinen, T. A. H., Jarvinen, T. L. N., Kvist, M., Natri, A.and Jarvinen, M. (1998). Location and distribution of non-collagenous matrixproteins in musculoskeletal tissues of rat. Histochem. J. 30, 799-810.

Kardon, G. (1998). Muscle and tendon morphogenesis in the avian hind limb.Development 125, 4019-4032.

Katzel, E. B., Wolenski, M., Loiselle, A. E., Basile, P., Flick, L. M., Langstein,H. N., Hilton, M. J., Awad, H. A., Hammert, W. C. and O’Keefe, R. J. (2011).Impact of Smad3 loss of function on scarring and adhesion formation duringtendon healing. J. Orthop. Res. 29, 684-693.

Kieny, M. and Chevallier, A. (1979). Autonomy of tendon development in theembryonic chick wing. J. Embryol. Exp. Morphol. 49, 153-165.

Kimura, W., Machii, M., Xue, X., Sultana, N., Hikosaka, K., Sharkar, M. T. K.,Uezato, T., Matsuda, M., Koseki, H. and Miura, N. (2011). Irxl1 mutant miceshow reduced tendon differentiation and no patterning defects in musculoskeletalsystem development. Genesis 49, 2-9.

Kjaer, M. (2004). Role of extracellular matrix in adaptation of tendon and skeletalmuscle to mechanical loading. Physiol. Rev. 84, 649-698.

Koch, M., Schulze, J., Hansen, U., Ashwodt, T., Keene, D. R., Brunken, W. J.,Burgeson, R. E., Bruckner, P. and Bruckner-Tuderman, L. (2004). A novelmarker of tissue junctions, collagen XXII. J. Biol. Chem. 279, 22514-22521.

Koshida, S., Kishimoto, Y., Ustumi, H., Shimizu, T., Furutani-Seiki, M.,Kondoh, H. and Takada, S. (2005). Integrin alpha5-dependent fibronectin

accumulation for maintenance of somite boundaries in zebrafish embryos. Dev.Cell 8, 587-598.

Kruegel, J. and Miosge, N. (2010). Basement membrane components arekey players in specialized extracellular matrices. Cell. Mol. Life Sci. 67,2879-2895.

Kyriakides, T. R., Leach, K. J., Hoffman, A. S., Ratner, B. D. and Bornstein, P.(1999). Mice that lack the angiogenesis inhibitor, thrombospondin 2, mount analtered foreign body reaction characterized by increased vascularity. Proc. Natl.Acad. Sci. USA 96, 4449-4454.

Lejard, V., Brideau, G., Blais, F., Salingcarnboriboon, R., Wagner, G., Roehrl,M. H. A., Noda, M., Duprez, D., Houillier, P. and Rossert, J. (2007). Scleraxisand NFATc regulate the expression of the pro-alpha1(I) collagen gene in tendonfibroblasts. J. Biol. Chem. 282, 17665-17675.

Lejard, V., Blais, F., Guerquin, M.-J., Bonnet, A., Bonnin, M.-A., Havis, E.,Malbouyres, M., Bidaud, C. B., Maro, G., Gilardi-Hebenstreit, P. et al. (2011).EGR1 and EGR2 involvement in vertebrate tendon differentiation. J. Biol. Chem.286, 5855-5867.

Li, Y., Ramcharan, M., Zhou, Z., Leong, D. J., Akinbiyi, T., Majeska, R. J. andSun, H. B. (2015). The role of scleraxis in fate determination of mesenchymalstem cells for tenocyte differentiation. Sci. Rep. 5, 13149.

Liu, D., Black, B. L. and Derynck, R. (2001). TGF-β inhibits muscle differentiationthrough functional repression of myogenic transcription factors by Smad3.GenesDev. 15, 2950-2966.

Liu, W., Watson, S. S., Lan, Y., Keene, D. R., Ovitt, C. E., Liu, H., Schweitzer, R.and Jiang, R. (2010). The atypical homeodomain transcription factor Mohawkcontrols tendon morphogenesis. Mol. Cell. Biol. 30, 4797-4807.

Liu, C.-F., Aschbacher-Smith, L., Barthelery, N. J., Dyment, N., Butler, D. andWylie, C. (2012). Spatial and temporal expression of molecular markers and cellsignals during normal development of the mouse patellar tendon. Tissue Eng. A18, 598-608.

Liu, H., Zhu, S., Zhang, C., Lu, P., Hu, J., Yin, Z., Ma, Y., Chen, X. andOuYang, H.(2014). Crucial transcription factors in tendon development and differentiation:their potential for tendon regeneration. Cell Tissue Res. 356, 287-298.

Liu, H., Zhang, C., Zhu, S., Lu, P., Zhu, T., Gong, X., Zhang, Z., Hu, J., Yin, Z.,Heng, B. C. et al. (2015). Mohawk promotes the tenogenesis of mesenchymalstem cells through activation of the TGFβ signaling pathway. Stem Cells 33,443-455.

Maeda, E., Shelton, J. C., Bader, D. L. and Lee, D. A. (2009). Differential regulationof gene expression in isolated tendon fascicles exposed to cyclic tensile strain invitro. J. Appl. Physiol. 106, 506-512.

Maeda, E., Fleischmann, C., Mein, C. A., Shelton, J. C., Bader, D. L. and Lee,D. A. (2010). Functional analysis of tenocytes gene expression in tendon fasciclessubjected to cyclic tensile strain. Connect. Tissue Res. 51, 434-444.

Maeda, T., Sakabe, T., Sunaga, A., Sakai, K., Rivera, A. L., Keene, D. R., Sasaki,T., Stavnezer, E., Iannotti, J., Schweitzer, R. et al. (2011). Conversion ofmechanical force into TGF-β-mediated biochemical signals. Curr. Biol. 21,933-941.

Maeda, E., Ye, S., Wang, W., Bader, D. L., Knight, M. M. and Lee, D. A. (2012).Gap junction permeability between tenocytes within tendon fascicles issuppressed by tensile loading. Biomech. Model. Mechanobiol. 11, 439-447.

Maeda, E., Sugimoto, M. and Ohashi, T. (2013). Cytoskeletal tension modulatesMMP-1 gene expression from tenocytes onmicropillar substrates. J. Biomech. 46,991-997.

Magne, D. and Bougault, C. (2015). What understanding tendon cell differentiationcan teach us about pathological tendon ossification. Histol. Histopathol. 30,901-910.

Marturano, J. E., Xylas, J. F., Sridharan, G. V., Georgakoudi, I. and Kuo, C. K.(2014). Lysyl oxidase-mediated collagen crosslinks may be assessed as markersof functional properties of tendon tissue formation. Acta Biomater. 10, 1370-1379.

Mayer, U., Saher, G., Fassler, R., Bornemann, A., Echtermeyer, F., von derMark, H., Miosge, N., Poschl, E. and von der Mark, K. (1997). Absence ofintegrin alpha 7 causes a novel form of muscular dystrophy. Nat. Genet. 17,318-323.

Munger, J. S. and Sheppard, D. (2011). Cross talk among TGF-β signalingpathways, integrins, and the extracellular matrix.Cold Spring Harb. Perspect. Biol.3, a005017.

Murchison, N. D., Price, B. A., Conner, D. A., Keene, D. R., Olson, E. N., Tabin,C. J. and Schweitzer, R. (2007). Regulation of tendon differentiation by scleraxisdistinguishes force-transmitting tendons from muscle-anchoring tendons.Development 134, 2697-2708.

Mustonen, E., Ruskoaho, H. and Rysa, J. (2012). Thrombospondin-4, tumournecrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14:Novel extracellular matrix modulating factors in cardiac remodelling.Ann. Med. 44,793-804.

Noden, D. M. (1988). Interactions and fates of avian craniofacial mesenchyme.Development 103 Suppl., 121-140.

Noden, D. M. and Trainor, P. A. (2005). Relations and interactions between cranialmesoderm and neural crest populations. J. Anat. 207, 575-601.

Oka, K., Oka, S., Hosokawa, R., Bringas, P., Brockhoff, H. C., Nonaka, K. andChai, Y. (2008). TGF-beta mediated Dlx5 signaling plays a crucial role in osteo-

4202

REVIEW Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT

chondroprogenitor cell lineage determination during mandible development. Dev.Biol. 321, 303-309.

Otabe, K., Nakahara, H., Hasegawa, A., Matsukawa, T., Ayabe, F., Onizuka, N.,Inui, M., Takada, S., Ito, Y., Sekiya, I. et al. (2015). Transcription factor Mohawkcontrols tenogenic differentiation of bone marrow mesenchymal stem cells in vitroand in vivo. J. Orthop. Res. 33, 1-8.

Pan, T.-C., Zhang, R.-Z., Markova, D., Arita, M., Zhang, Y., Bogdanovich, S.,Khurana, T. S., Bonnemann, C. G., Birk, D. E. and Chu, M.-L. (2013).COL6A3 protein deficiency in mice leads to muscle and tendon defects similar tohuman collagen VI congenital muscular dystrophy. J. Biol. Chem. 288,14320-14331.

Parkinson, J., Samiric, T., Ilic, M. Z., Cook, J. and Handley, C. J. (2011).Involvement of proteoglycans in tendinopathy. J. Musculoskelet. NeuronalInteract. 11, 86-93.

Parsons, M. J., Pollard, S. M., Saude, L., Feldman, B., Coutinho, P., Hirst,E. M. A. andStemple, D. L. (2002). Zebrafishmutants identify an essential role forlaminins in notochord formation. Development 129, 3137-3146.

Pingel, J., Lu, Y., Starborg, T., Fredberg, U., Langberg, H., Nedergaard, A.,Weis, M., Eyre, D., Kjaer, M. and Kadler, K. E. (2014). 3-D ultrastructure andcollagen composition of healthy and overloaded human tendon: evidence oftenocyte and matrix buckling. J. Anat. 224, 548-555.

Popov, C., Burggraf, M., Kreja, L., Ignatius, A., Schieker, M. and Docheva, D.(2015). Mechanical stimulation of human tendon stem/progenitor cells results inupregulation of matrix proteins, integrins and MMPs, and activation of p38 andERK1/2 kinases. BMC Mol. Biol. 16, 1219.

Pryce, B. A., Watson, S. S., Murchison, N. D., Staverosky, J. A., Dunker, N. andSchweitzer, R. (2009). Recruitment and maintenance of tendon progenitors byTGFbeta signaling are essential for tendon formation. Development 136,1351-1361.

Rifkin, D. B. (2005). Latent transforming growth factor-beta (TGF-beta)binding proteins: orchestrators of TGF-beta availability. J. Biol. Chem. 280,7409-7412.

Rooney, J. E., Welser, J. V., Dechert, M. A., Flintoff-Dye, N. L., Kaufman, S. J.and Burkin, D. J. (2006). Severe muscular dystrophy in mice that lack dystrophinand alpha7 integrin. J. Cell Sci. 119, 2185-2195.

Rooney, J. E., Knapp, J. R., Hodges, B. L., Wuebbles, R. D. and Burkin, D. J.(2012). Laminin-111 protein therapy reduces muscle pathology and improvesviability of a mouse model of merosin-deficient congenital muscular dystrophy.Am. J. Pathol. 180, 1593-1602.

Ros, M. A., Rivero, F. B., Hinchliffe, J. R. and Hurle, J. M. (1995).Immunohistological and ultrastructural study of the developing tendons of theavian foot. Anat. Embryol. 192, 483-496.

Rui, Y. F., Lui, P. P. Y., Wong, Y. M., Tan, Q. and Chan, K. M. (2013). Altered fate oftendon-derived stem cells isolated from a failed tendon-healing animal model oftendinopathy. Stem Cells Dev. 22, 1076-1085.

Rullman, E., Norrbom, J., Stromberg, A., Wågsater, D., Rundqvist, H., Haas, T.and Gustafsson, T. (2009). Endurance exercise activates matrixmetalloproteinases in human skeletal muscle. J. Appl. Physiol. 106, 804-812.

Schessl, J., Zou, Y. and Bonnemann, C. G. (2006). Congenital musculardystrophies and the extracellular matrix. Semin. Pediatr. Neurol. 13, 80-89.

Schoenebeck, J. J., Hutchinson, S. A., Byers, A., Beale, H. C., Carrington, B.,Faden, D. L., Rimbault, M., Decker, B., Kidd, J. M., Sood, R. et al. (2012).Variation of BMP3 contributes to dog breed skull diversity. PLoS Genet. 8,e1002849.

Schwartz, A. G., Pasteris, J. D., Genin, G. M., Daulton, T. L. and Thomopoulos,S. (2012). Mineral distributions at the developing tendon enthesis. PLoS ONE 7,e48630.

Schwartz, A. G., Lipner, J. H., Pasteris, J. D., Genin, G. M. and Thomopoulos, S.(2013). Muscle loading is necessary for the formation of a functional tendonenthesis. Bone 55, 44-51.

Schweitzer, R., Chyung, J. H., Murtaugh, L. C., Brent, A. E., Rosen, V., Olson,E. N., Lassar, A. and Tabin, C. J. (2001). Analysis of the tendon cell fate usingScleraxis, a specific marker for tendons and ligaments. Development 128,3855-3866.

Schweitzer, R., Zelzer, E. and Volk, T. (2010). Connecting muscles to tendons:tendons and musculoskeletal development in flies and vertebrates. Development137, 2807-2817.

Shukunami, C., Takimoto, A., Oro, M. and Hiraki, Y. (2006). Scleraxis positivelyregulates the expression of tenomodulin, a differentiation marker of tenocytes.Dev. Biol. 298, 234-247.

Shwartz, Y., Blitz, E. and Zelzer, E. (2013). One load to rule them all: mechanicalcontrol of the musculoskeletal system in development and aging. Differentiation86, 104-111.

Snow, C. J. and Henry, C. A. (2009). Dynamic formation of microenvironments atthe myotendinous junction correlates with muscle fiber morphogenesis inzebrafish. Gene Expr. Patterns 9, 37-42.

Snow, C. J., Peterson, M. T., Khalil, A. and Henry, C. A. (2008). Muscledevelopment is disrupted in zebrafish embryos deficient for fibronectin. Dev. Dyn.237, 2542-2553.

Soeda, T., Deng, J. M., de Crombrugghe, B., Behringer, R. R., Nakamura, T.and Akiyama, H. (2010). Sox9-expressing precursors are the cellular origin ofthe cruciate ligament of the knee joint and the limb tendons. Genesis 48,635-644.

Spanoudes, K., Gaspar, D., Pandit, A. and Zeugolis, D. I. (2014). The biophysical,biochemical, and biological toolbox for tenogenic phenotype maintenance in vitro.Trends Biotechnol. 32, 474-482.

Subramanian, A. and Schilling, T. F. (2014). Thrombospondin-4 controls matrixassembly during development and repair of myotendinous junctions. eLife 2014,e02372.

Subramanian, A., Wayburn, B., Bunch, T. and Volk, T. (2007). Thrombospondin-mediated adhesion is essential for the formation of the myotendinous junction inDrosophila. Development 134, 1269-1278.

Sugimoto, Y., Takimoto, A., Akiyama, H., Kist, R., Scherer, G., Nakamura, T.,Hiraki, Y. and Shukunami, C. (2013). Scx+/Sox9+ progenitors contribute tothe establishment of the junction between cartilage and tendon/ligament.Development 140, 2280-2288.

Takimoto, A., Oro, M., Hiraki, Y. and Shukunami, C. (2012). Direct conversion oftenocytes into chondrocytes by Sox9. Exp. Cell Res. 318, 1492-1507.

Thomopoulos, S.,Williams, G. R., Gimbel, J. A., Favata, M. and Soslowsky, L. J.(2003). Variation of biomechanical, structural, and compositional properties alongthe tendon to bone insertion site. J. Orthop. Res. 21, 413-419.

Thorpe, C. T., Birch, H. L., Clegg, P. D. and Screen, H. R. C. (2013). The role of thenon-collagenous matrix in tendon function. Int. J. Exp. Pathol. 94, 248-259.

Tokita, M. and Schneider, R. A. (2009). Developmental origins of species-specificmuscle pattern. Dev. Biol. 331, 311-325.

Tome, F. M., Evangelista, T., Leclerc, A., Sunada, Y., Manole, E., Estournet, B.,Barois, A., Campbell, K. P. and Fardeau, M. (1994). Congenital musculardystrophy with merosin deficiency. C. R. Acad. Sci. III. 317, 351-357.

Tozer, S. and Duprez, D. (2005). Tendon and ligament: development, repair anddisease. Birth Defects Res. C. Embryo Today 75, 226-236.

Turner, D. C., Lawton, J., Dollenmeier, P., Ehrismann, R. and Chiquet, M.(1983). Guidance of myogenic cell migration by oriented deposits of fibronectin.Dev. Biol. 95, 497-504.

Vaz, R., Martins, G. G., Thorsteinsdottir, S. and Rodrigues, G. (2012).Fibronectin promotes migration, alignment and fusion in an in vitro myoblast cellmodel. Cell Tissue Res. 348, 569-578.

Veit, G., Hansen, U., Keene, D. R., Bruckner, P., Chiquet-Ehrismann, R.,Chiquet, M. and Koch, M. (2006). Collagen XII interacts with avian tenascin-Xthrough its NC3 domain. J. Biol. Chem. 281, 27461-27470.

Volk, T. and VijayRaghavan, K. (1994). A central role for epidermal segment bordercells in the induction of muscle patterning in the Drosophila embryo.Development120, 59-70.

Walchli, C., Koch, M., Chiquet, M., Odermatt, B. F. and Trueb, B. (1994). Tissue-specific expression of the fibril-associated collagens XII and XIV. J. Cell Sci. 107,669-681.

Wall, M. E. and Banes, A. J. (2005). Early responses to mechanical load in tendon:Role for calcium signaling, gap junctions and intercellular communication.J. Musculoskelet. Neuronal Interact. 5, 70-84.

Wang, J. H.-C., Guo, Q. and Li, B. (2012). Tendon biomechanics andmechanobiology – a minireview of basic concepts and recent advancements.J. Hand Ther. 25, 133-141.

Weinreb, J. H., Sheth, C., Apostolakos, J., McCarthy, M. B., Barden, B., Cote,M. P. and Mazzocca, A. D. (2014). Tendon structure, disease, and imaging.Muscles. Ligaments Tendons J. 4, 66-73.

Willett, T. L., Labow, R. S., Aldous, I. G., Avery, N. C. and Lee, J. M. (2010).Changes in collagen with aging maintain molecular stability after overload:evidence from an in vitro tendon model. J. Biomech. Eng. 132, 031002.

Wipff, P.-J., Rifkin, D. B., Meister, J.-J. and Hinz, B. (2007). Myofibroblastcontraction activates latent TGF-beta1 from the extracellular matrix. J. Cell Biol.179, 1311-1323.

Yang, G., Rothrauff, B. B. and Tuan, R. S. (2013). Tendon and ligamentregeneration and repair: clinical relevance and developmental paradigm. BirthDefects Res. C. Embryo Today 99, 203-222.

Yeung, C.-Y. C., Gossan, N., Lu, Y., Hughes, A., Hensman, J. J., Bayer, M. L.,Kjær, M., Kadler, K. E. and Meng, Q.-J. (2014). Gremlin-2 is a BMP antagonistthat is regulated by the circadian clock. Sci. Rep. 4, 5183.

Yoon, J. H. and Halper, J. (2005). Tendon proteoglycans: biochemistry andfunction. J. Musculoskelet. Neuronal Interact. 5, 22-34.

Yu, Q. and Stamenkovic, I. (2000). Cell surface-localized matrix mealloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion andangiogenesis. Genes Dev. 14, 163-176.

Yurchenco, P. D., Amenta, P. S. and Patton, B. L. (2004). Basement membraneassembly, stability and activities observed through a developmental lens. MatrixBiol. 22, 521-538.

Zelzer, E., Blitz, E., Killian, M. L. and Thomopoulos, S. (2014). Tendon-to-boneattachment: from development to maturity. Birth Defects Res. C Embryo Today102, 101-112.

4203

REVIEW Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT

Zhang, J. and Wang, J. H. C. (2010). Mechanobiological response of tendon stemcells: implications of tendon homeostasis and pathogenesis of tendinopathy.J. Orthop. Res. 28, 639-643.

Zhang, J. andWang, J. H.-C. (2013). The effects of mechanical loading on tendons– an in vivo and in vitro model study. PLoS ONE 8, e71740.

Zhang, J. and Wang, J. H.-C. (2015). Moderate exercise mitigates the detrimentaleffects of aging on tendon stem cells. PLoS ONE 10, e0130454.

Zhang, G., Ezura, Y., Chervoneva, I., Robinson, P. S., Beason, D. P., Carine,E. T., Soslowsky, L. J., Iozzo, R. V. and Birk, D. E. (2006). Decorin regulates

assembly of collagen fibrils and acquisition of biomechanical properties duringtendon development. J. Cell Biochem. 98, 1436-1449.

Zhou, B., Zhou, Y. and Tang, K. (2014). An overview of structure, mechanicalproperties, and treatment for age-related tendinopathy. J. Nutr. Health Aging 18,441-448.

Zou, Y., Zwolanek, D., Izu, Y., Gandhy, S., Schreiber, G., Brockmann, K.,Devoto, M., Tian, Z., Hu, Y., Veit, G. et al. (2014). Recessive and dominantmutations in COL12A1 cause a novel EDS/myopathy overlap syndrome inhumans and mice. Hum. Mol. Genet. 23, 2339-2352.

4204

REVIEW Development (2015) 142, 4191-4204 doi:10.1242/dev.114777

DEVELO

PM

ENT