Scleraxis is required for maturation of tissue domains for proper integration of the musculoskeletal system

Yoshimoto, Yuki; Takimoto, Aki; Watanabe, Hitomi; Hiraki, Yuji; Kondoh, Gen; Shukunami, Chisa

doi:10.1038/srep45010

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Published: 22 March 2017

Scleraxis is required for maturation of tissue domains for proper integration of the musculoskeletal system

Yuki Yoshimoto¹,
Aki Takimoto²,
Hitomi Watanabe³,
Yuji Hiraki²,
Gen Kondoh³ &
…
Chisa Shukunami^1,2

Scientific Reports volume 7, Article number: 45010 (2017) Cite this article

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Abstract

Scleraxis (Scx) is a basic helix-loop-helix transcription factor that is expressed persistently in tendons/ligaments, but transiently in entheseal cartilage. In this study, we generated a novel Scx^Cre knock-in (KI) allele, by in-frame replacement of most of Scx exon 1 with Cre recombinase (Cre), to drive Cre expression using Scx promoter and to inactivate the endogenous Scx. Reflecting the intensity and duration of endogenous expression, Cre-mediated excision occurs in tendinous and ligamentous tissues persistently expressing Scx. Expression of tenomodulin, a marker of mature tenocytes and ligamentocytes, was almost absent in tendons and ligaments of Scx^Cre/Cre KI mice lacking Scx to indicate defective maturation. In homozygotes, the transiently Scx-expressing entheseal regions such as the rib cage, patella cartilage, and calcaneus were small and defective and cartilaginous tuberosity was missing. Decreased Sox9 expression and phosphorylation of Smad1/5 and Smad3 were also observed in the developing entheseal cartilage, patella, and deltoid tuberosity of Scx^Cre/Cre KI mice. These results highlighted the functional importance of both transient and persistent expression domains of Scx for proper integration of the musculoskeletal components.

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Introduction

During early stages of musculoskeletal development, scleraxis (Scx), a basic helix-loop-helix transcription factor, is expressed in the tendon/ligament cell population and the subpopulation of chondrogenic cells that contribute to establishment of chondro-tendinous/ligamentous junctions^1,2,3,4. Scx expression occurs only transiently in the chondrogenic lineage because it is rapidly downregulated during the early stages of chondrogenesis³. In contrast, Scx is persistently expressed in the tendon/ligament cell lineage, which also includes the diaphragm and cordae tendinae of the heart, during differentiation and maturation^5,6,7,8. Persistent expression of Scx, in the developing tendons and ligaments in vivo, was also visualised in ScxGFP reporter transgenic mice that faithfully recapitulate endogenous Scx-expressing domains under the control of the promoter/enhancer of mouse Scx^3,9. In Scx^−/− embryos, force-transmitting and intermuscular tendons are reported to be severely affected and are hypoplastic⁷. However, so far, other Scx-expressing domains have not been explored in detail yet.

Tenomodulin (Tnmd) is an excellent cell surface marker to evaluate differentiation and maturation of dense connective tissues, including tendons and ligaments^10,11,12,13. Scx positively regulates the expression of type I collagen (Col1) and Tnmd in tenocytes^6,7,14. Transcriptome analysis of mouse embryonic limb tendons also revealed that Tnmd is the second most differentially expressed gene of the top 100 genes differentially expressed between E11.5 and E14.5¹⁵. Studies on Tnmd null mice demonstrated that Tnmd is necessary for tenocyte proliferation and tendon maturation¹⁶. Cellular adhesion in the periodontal ligament is decreased on loss of Tnmd and enhanced on Tnmd overexpression¹⁷. In a rat rotator cuff healing model, fibroblast growth factor-2 enhanced tendon-to-bone healing, in which Tnmd expression was associated with formation of aligned collagen fibres in the repair tissue¹⁸. Loss of Tnmd results in reduced self-renewal and augmented senescence of tendon stem/progenitor cells¹⁹. Furthermore, forced expression of Scx resulted in the conversion of human bone marrow-derived mesenchymal stem cells into tendon progenitor cells that differentiate into Tnmd-expressing cells²⁰. Additionally in tenocytes, Tnmd expression is markedly upregulated by retroviral overexpression of Scx^6,21.

In this study, we report a novel Scx^Cre knock-in (KI) mouse line that was generated by in-frame replacement of most of the exon 1 on mouse Scx locus with Cre recombinase (Cre), to inactivate endogenous Scx and to drive Cre expression using the endogenous Scx promoter. In this established Scx^Cre KI line, Cre expression was detected using Rosa-CAG-LSL-tdTomato (Rosa-tdTomato) reporter mice expressing tandem dimer Tomato (tdTomato) by Cre-mediated recombination. A number of tdTomato-expressing cells of Scx^Cre/+; Rosa-tdTomato or Scx^Cre/Cre; Rosa-tdTomato mice were observed in tendons, ligaments, annulus fibrosus of intervertebral discs, and patella cartilage. Loss of Scx resulted in an almost absent expression of Tnmd in tendons, ligaments, and annulus fibrosus of intervertebral discs. Cartilaginous elements transiently expressing Scx were defective in Scx^Cre/Cre KI mice lacking Scx. Sox9 expression and phosphorylation of Smad1/5 and Smad 3 were markedly decreased in these Scx⁺ cartilaginous elements. Our results clearly demonstrated the functional importance of Scx in maturation of tissue domains that express Scx both persistently and transiently during development.

Results

Establishment of Scx^Cre KI mice

On mouse chromosome 15, Scx gene, consisting of two exons, is located within the third intron of block of proliferation 1 (Bop1), which is transcribed in the opposite orientation^9,22. The targeting strategy placed Cre expression under the control of Scx promoter and led to the inactivation of Scx allele by in-frame replacement of most of its exon 1, which encodes for most of the coding region, including a b-HLH region. The linearised Scx^CreNeo targeting vector (Fig. 1a) was electroporated into KY1.1 embryonic stem (ES) cell line (C57BL/6J × 129S6/SvEvTac)²³. ES cell clone with the correct targeting event was microinjected into blastocysts to obtain chimeric mice. We generated heterozygous mice by crossing these chimeras with C57BL/6 wild type mice and confirmed the successful germ line transmission of Scx^CreNeo targeted allele. Heterozygous Scx^CreNeo/+ KI mice were viable, whereas homozygous Scx^{CreNeo/CreNeo} KI mice were embryonic lethal. This is consistent with the previous report that conventional Scx knockout mice die around embryonic day (E)10.5²⁴. To obtain Scx^Cre/+ KI mice, we deleted the flippase (FLP) recombinase target (FRT)-flanked neomycin-resistance (Neo) cassette by mating Scx^CreNeo/+ KI mice with mice expressing FLP recombinase (FLPe)²⁵. The correctly targeted event of Scx^Cre/+ KI mice was confirmed by PCR of tail tip DNA and Southern blot analysis (Fig. 1b,c). Expression of Scx was not detected in a Scx^Cre/Cre KI mouse embryo at E12.5 (Fig. 1d). Thus, we successfully disrupted endogenous Scx by inserting a Cre in-frame into the ATG start site of mouse Scx gene.

**Figure 1: Targeting strategy to generate *Scx*^Cre KI mice.**

Cre-mediated recombination in Scx^Cre KI; Rosa-tdTomato

Cre-mediated recombination was examined by crossing Scx^Cre/+ KI mice with Rosa-tdTomato reporter mice²⁶. Although endogenous expression of Scx is detectable around E9.5, Cre-mediated tdTomato expression in Scx^Cre/+; Rosa-tdTomato was first detected around E16.5 (data not shown). In the hind limbs of Scx^Cre/+; Rosa-tdTomato neonates, we detected tdTomato expression in the Achilles tendon and in tendons and ligaments associated with knee joint and heel (Fig. 2a–c). Tail tendons of Scx^Cre/+; Rosa-tdTomato neonates were also positive for tdTomato (Fig. 2d). Moreover, at postnatal day (P)14, more intense expression of tdTomato was observed in body tendons and ligaments (Fig. 2e,f,g). In the trunk of 2-week-old Scx^Cre/+; Rosa-tdTomato mice, intense tdTomato expression was observed in tendons of longissimus muscle (Fig. 2e), central tendon of diaphragm (Fig. 2f), and costal tendons (Fig. 2g). In the hindlimb of Scx^Cre/+; Rosa-tdTomato neonates, tdTomato-positive cells were found in patella ligament, anterior and posterior cruciate ligaments, quadriceps femoris tendon, patella cartilage, femur cartilage, and meniscus (Fig. 2h–j). In the intervertebral region of Scx^Cre/+; Rosa-tdTomato neonates, tdTomato expression was detected in the outer annulus fibrosus on the ventral side (Fig. 2i). In the hindlimb of Scx^Cre/Cre; Rosa-tdTomato neonates, the number of tdTomato-positive cells in patella cartilage increased (Fig. 2k). Mosaic Cre-mediated recombination could have occurred because of heterogeneity in the endogenous Scx mRNA levels and time window of Scx expression in the Scx-expressing cells.

**Figure 2: Cre-mediated recombination in tendons, ligaments, and diaphragm of *Scx*^Cre/+; *Rosa-tdTomato* mice.**

Tendon deficiency and skeletal abnormalities in Scx^Cre/Cre KI neonates

Heterozygous Scx^Cre/+ KI mice were viable, fertile, and displayed no apparent developmental defects (Fig. 3a,c,e,g). Consistent with previous findings⁷, morphological defects in force-transmitting and intermuscular tendons were evident at birth, in Scx^Cre/Cre KI neonates (Fig. 3b,d,f,h). The diaphragm of Scx^Cre/Cre KI neonates is functional and permits normal breathing. The forelimb autopod of Scx^Cre/Cre KI neonates was locked in a dorsal flexure (Fig. 4b), compared to that of Scx^Cre/+ KI neonates (Fig. 4a). The deltoid tuberosity (DT) and tibial tuberosity observed in Scx^Cre/+ KI neonates (Fig. 4a,k,m) were missing in Scx^Cre/Cre KI neonates (Fig. 4b,l,n). The rib cage, transverse process of lumbar, patella cartilage, and entheseal cartilage of calcaneus of Scx^Cre/Cre KI neonates were smaller than that of Scx^Cre/+ KI neonates (Fig. 4c–j).

**Figure 3: Tendon deficiency in *Scx*^Cre/Cre KI mice.**

**Figure 4: Skeletal abnormalities in *Scx*^Cre/Cre KI mice.**

Defective maturation of tendons, ligaments, and annulus fibrosus of intervertebral discs in Scx^Cre/Cre KI neonates

Tnmd is related to a cartilage-derived angiogenesis inhibitor gene product, chondromodulin (Chmd)²⁷, and is a marker of mature tenocytes and ligamentocytes^10,11,28. Tnmd expression is positively regulated by Scx⁶. Scx was heterogeneously expressed in the developing leg tendons (Fig. 5a), whereas uniform expression of Tnmd was observed (Fig. 5b). Higher levels of Scx expression in the tendons were detected near the myotendinous junction (Fig. 5a). We confirmed inactivation of endogenous Scx expression in Scx^Cre/Cre at E15.5 by in situ hybridisation (Fig. 5c,d). As previously reported⁷, in Scx null mice, gene expression of Tnmd and type XIV collagen was not detectable in forelimb tendons. However, during postnatal growth, Tnmd expression in other dense connective tissues, such as ligaments and annulus fibrosus of intervertebral discs, has not been evaluated yet. Thus, we investigated localisation of Col1 and Tnmd in tendons and ligaments of Scx^Cre/+ and Scx^Cre/Cre KI mice, by in situ hybridisation (Fig. 5e–h) and double immunostaining (Figs 6 and 7). In the hindlimb of a Scx^Cre/+ neonate, tendons and ligaments were positive for Col1 and Tnmd (Fig. 6a,c,e,g). In the knee joint of a homozygous Scx^Cre/Cre KI neonate, patella ligament, anterior and posterior cruciate ligaments, and anchoring tendons were positive for Col1, but Tnmd expression was almost absent (Fig. 6b,d). Similarly, in the heel of a homozygous Scx^Cre/Cre KI neonate, tendons and ligaments, including the Achilles and extensor digitorum longus tendons, were positive for Col1 but negative for Tnmd (Fig. 6f,h). The meniscus was positive for Col1 but negative for Tnmd at E16.5, despite finding tdTomato positive cells in the meniscus of Scx^Cre/+; Rosa-tdTomato mice (Figs 2j and 6i,j). The outer annulus fibrosus of intervertebral discs in Scx^Cre/+ KI neonates and the tendinous region of the diaphragm in wild type mice were also positive for Col1 and Tnmd (Fig. 7a,c,e), but Tnmd expression was almost absent in that of Scx^Cre/Cre KI mice (Fig. 7b,d,f). These results suggest that loss of Scx causes defective maturation of tendons, ligaments, and outer annulus fibrosus of intervertebral discs.

**Figure 5: Expression of *Tnmd, Scx*, and *Col1a1* in *Scx*^Cre/Cre KI mice.**

**Figure 6: Decreased expression of Tnmd in tendons and ligaments of *Scx*^Cre/Cre KI mice.**

**Figure 7: Decreased expression of Tnmd in annulus fibrosus of intervertebral disc and diaphragm of *Scx*^Cre/Cre KI mice.**

Defective maturation of knee joint of Scx^Cre/Cre KI mice

Tnmd expression, observed in cruciate ligaments of Scx^Cre/+; Rosa-tdTomato mice, was missing in Scx^Cre/Cre; Rosa-tdTomato mice (Fig. 8a,b). In knee joints of 2-week-old Scx^Cre/+ KI mice, tendons and ligaments developed well (Fig. 8c), whereas defective maturation was observed in knee joints of homozygous Scx^Cre/Cre KI mice (Fig. 8d). The size of the patella was also significantly small and entheseal cartilage was defective in a Scx^Cre/Cre KI mouse (Fig. 8d). To analyse endochondral ossification in the patella, immunostaining using antibodies against type II collagen (Col2), osteocalcin (Ocn), and sclerostin was performed. In a Scx^+/+ mouse, a central part of the patella was vascularised to be replaced by bone stained with Ocn, but Col2-positive staining indicates the presence of cartilaginous matrix (Fig. 8e,f,h,i). At this stage, the patella of a Scx^Cre/Cre KI mouse was Col2-positive without any indication of vascularisation and ossification (Fig. 8g,j), suggesting a delay in the endochondral ossification of the patella. In the entheseal region of patella tendon and ligament of a Scx^+/+ mouse, sclerostin-positive cells were observed in Col2- and Ocn-positive calcified fibrocartilage (Fig. 8e,f,h,i). Col2-positive unmineralised fibrocartilage was also present at an adjacent region in patella tendon and ligament of a Scx^+/+ mouse (Fig. 8f). Such a structure was not observed in the Scx^Cre/Cre KI mouse. Taken together, Scx is also required for maturation of tendons, ligaments, and the patella.

**Figure 8: Hypoplastic formation of patella and ligaments in knee joint of *Scx*^Cre/Cre KI mice.**

Decreased Sox9 expression and phosphorylation of Smad1/5 and Smad3 in the developing entheseal cartilage, patella, and deltoid tuberosity of Scx^Cre/Cre KI mice

Entheseal cartilage around the tendon/ligament attachment sites and sesamoid bones such as patella are derived from Scx/Sox9 double positive progenitors^2,4. Blitz et al. reported that activation of TGF-β and BMP signaling is required for specification and differentiation of these progenitors, respectively^4,29. Thus, we examined how loss of Scx affects expression of Sox9 and activation of these signaling pathways in the developing forelimb and hindlimb of heterozygotes and homozygotes at E13.5. Activation of TGF-β and BMP signaling pathways was monitored by phosphorylation of Smad1/5 and Smad3 that are intracellular downstream mediators³⁰. As shown in Fig. 9, mature chondrocytes and progenitor cells of the patella and the deltoid tuberosity were visualized as Chmd positive (green) and Sox9 positive regions (red), respectively. In a Scx^Cre/Cre KI mouse, Sox9 expression was markedly decreased in the developing patella and entheseal cartilage (Fig. 9b,h), compared with that in a Scx^Cre/+ KI mouse (Fig. 9a,g). Sox9 positive progenitors in the developing deltoid tuberosity of a Scx^Cre/+ KI mouse (Fig. 9g) were absent in a Scx^Cre/Cre KI mouse (Fig. 9h). Similarly, phosphorylation of Smad1/5 and Smad3 observed in a Scx^Cre/+ KI mouse (Fig. 9c,e,i,k) was markedly decreased especially in the prospective patella and deltoid tuberosity of a Scx^Cre/Cre KI mouse (Fig. 9d,f,j,l).

**Figure 9: Decreased Sox9 expression and phosphorylation of Smad1/5 and Smad3 in the developing patella and deltoid tuberosity of *Scx*^Cre/Cre KI mouse.**

Gene expression of Col14a1, Decorin, Mohawk, Tnmd and Egr1 in Scx^Cre/Cre KI mice

In addition, we investigated the expression of other tendon-associated genes such as Col14a1, Decorin (Dcn), Mohawk (Mkx), Tnmd, and Early growth response 1 (Egr1) in Scx^Cre/Cre KI embryos at E15.5. Col14a1 is a member of the fibril-associated collagens with interrupted triple helices (FACIT) collagen family³¹. Dcn is a small leucine-rich proteoglycan involved in regulating collagen fibrillogenesis³² and Mkx is a family member of atypical homeobox genes that are expressed in developing tendons^33,34. Egr1 is reported to be a transcription factor regulating the expression of Col1a1 in tendon development³⁵. In a Scx^Cre/Cre KI mouse, Col14a1 expression was almost absent in the developing triceps brachii tendons (Fig. 10a,b). In a wild type mouse, we found expression of Dcn in the developing tendon, dermis, and soft connective tissues (Fig. 10c), which are consistent with previous findings³². In Scx^Cre/Cre KI embryos, we found that Dcn expression was almost absent in triceps brachii tendons (Fig. 10d) and also found generally weak expression of Mkx (Fig. 10e,f). Around the pelvic bone and lumbar at E15.5, Tnmd expressing tendons were also positive for Egr1 (Fig. 10g,i). In Scx^Cre/Cre KI embryos, expression of Tnmd and Egr1 was lost and decreased in theses tendons, respectively (Fig. 10h,j). These findings suggest that loss of Scx affects the expression of genes related to tendon maturation.

**Figure 10: Expression of genes related to tendon formation in *Scx*^Cre/Cre KI mice.**

Discussion

We successfully generated Scx^Cre KI mice that can be used as genetic tools to reveal in vivo function of Scx-expressing tissue domains integrating the musculoskeletal components. In Scx^Cre KI mice, Cre expression was driven by Scx promoter and endogenous Scx was inactivated. Cre-mediated recombination efficiency of Scx^Cre KI mice reflected the intensity and duration of endogenous Scx expression. Cre-mediated excision was mainly observed in domains with persistent Scx expression, such as tendons, ligaments, and annulus fibrosus of intervertebral discs. In homozygous Scx^Cre/Cre KI mice, Tnmd, a marker of mature tenocytes and ligamentocytes, was almost absent. In addition, other tendon markers such as Col14a1, Dcn, Mkx, and Egr1 were downregulated in homozygotes. Moreover, regardless of the short duration of endogenous Scx expression, defective maturation was observed in Scx-expressing domains that are involved in integration of the musculoskeletal components.

Scx is localised in the intron 3 region of Bop1 gene on chromosome 15. Bop1 and Scx are a pair of bidirectional overlapping coding genes related to cellular proliferation and differentiation, respectively^7,22. The first reported Scx knockout failed to form mesoderm and died during early stages of embryogenesis²⁴, probably because the neomycin phosphotransferase gene linked to phosphoglycerate kinase promoter (PGK-Neo) cassette affected the expression of Bop1. Later, Murchison et al. reported generation and characterisation of a conditional allele in the Scx locus with FRT-Neo cassette, by flanking the first exon, which includes most of the coding region, with loxP site⁷. As was the case with homozygotes of Scx knockout with a Neo allele, we too failed to obtain viable Scx^CreNeo homozygous pups, but we successfully established a new line of Scx^Cre KI mice by mating a Scx^CreNeo heterozygote with a flippase-expressing FLPe mouse. Homozygous Scx^Cre/Cre KI mice with tendon defects were viable and similar to Scx knockout mice generated by Murchison et al.⁷, suggesting that the replacement of most of the exon 1 by Cre does not cause embryonic lethality, unlike as observed on retention of PGK-Neo cassette in this genomic region.

Cre-mediated recombination in a Scx^Cre/+; Rosa-tdTomato mouse was less efficient than that in a ScxCre-L or a ScxCre-H transgenic mouse mated with a Rosa-tdTomato reporter mouse. Under the control of the endogenous Scx promoter/enhancer in KI mice, Cre expression gradually increased to reach levels sufficient for recombination, thus enabling Cre-mediated tdTomato expression not in the Scx⁺ progenitor cell population but in the differentiated cell populations of tendons, ligaments, the annulus fibrosus of the intervertebral discs, and patella cartilage. More tdTomato positive cells were observed in Scx^Cre/Cre; Rosa-tdTomato mice than Scx^Cre/+; Rosa-tdTomato, suggesting that the number of cells expressing Cre above the threshold increases in homozygotes due to a gene dosage effect. In the Cre-loxP system, recombination occurs only when Cre protein is accumulated above the recombination threshold. Mosaic and later Cre-mediated recombination is considered to be observed due to heterogeneity in the endogenous Scx mRNA levels and a narrow time window of Scx expression.

We previously reported that Scx positively regulates the expression of Tnmd in tenocytes both in vivo and in vitro⁶. Tnmd expression in Scx-positive cells in the periodontal ligament is also upregulated by lentiviral overexpression of Scx and downregulated by knockdown of endogenous Scx³⁶. In this study, immunostaining of Scx^Cre/Cre homozygotes with anti-Tnmd antibody revealed that Scx was necessary for the expression of Tnmd in most parts of the developing tendinous and ligamentous tissues. As previously reported, Tnmd acts as a positive regulator of postnatal tendon growth, maturation of collagen fibres, and cellular adhesion in the periodontal ligament¹⁷. Loss of Tnmd resulted in abated tenocyte proliferation leading to reduced tenocyte density, greater variation in collagen fibril diameters, and increase in maximal fibril diameters¹⁶. Thus, ablation of Tnmd expression in tendons, ligaments, and annulus fibrosus of intervertebral discs of homozygous KI mice suggests defective phenotypes in these tissues, other than the previously reported defects in force-transmitting and intermuscular tendons.

In the spinal column, an intervertebral disc lies between adjacent vertebrae and acts as a shock absorber. Intervertebral discs consist of inner and outer annulus fibrosus of sclerotome origin and notochord-derived nucleus pulposus. The inner annulus fibrosus has cartilaginous matrix associated with Col2 fibres, whereas the outer annulus fibrosus has thick multiple layers of dense connective tissue containing predominantly Col1. We previously reported that Scx⁺/Sox9⁺ progenitor population gives rise to both inner and outer annulus fibrosus and that Sox9 in this population is indispensable for the formation of inner annulus fibrosus². In this study, we found that Tnmd expression in the outer annulus fibrosus was missing in Scx^Cre/Cre KI neonates. Thus, we demonstrated, for the first time, that Scx is necessary for the maturation of outer annulus fibrosus.

Scx is also transiently expressed in Sox9-expressing entheseal and sesamoid cartilage during early stages of chondrogenic differentiation, as previously reported^1,3,37. This is consistent with our previous findings from lineage analysis using ScxCre transgenic mice that entheseal and patella chondroprogenitors were positive for Scx³. In the Scx-expressing domain, mice lacking Sox9 lose the cartilaginous domain that contributes to the establishment of tendon/ligament attachment sites^2,4. In this study, we found that expression of Sox9 in Scx⁺/Sox9⁺ chondroprogenitors is markedly decreased. Even though Scx expression in Scx⁺/Sox9⁺ chondroprogenitors is only transient, loss of Scx caused defective formation of cartilaginous elements arising from the Scx⁺/Sox9⁺ progenitor population. Moreover, activation of BMP and TGF-β signalling pathways in these cartilaginous elements was diminished as evidenced by decreased phosphorylation of Smad1/5 and Smad3. These findings indicate that transient expression of Scx in chondroprogenitors is functionally important in the process of entheseal cartilage formation.

Muscle contractions in utero generate mechanical forces that are essential for normal embryonic development, through modulation of cell signalling and gene expression. As reported previously, Scx is the mechanical stress responsive gene that is upregulated in the periodontal ligament in response to tensile force and maintains its fibrogenic state by inhibiting mineralisation³⁶. Conversely, removal of tensile force in the tendon results in a decrease in Scx expression³⁸. The severe defective phenotypes, initially in force-transmitting and intermuscular tendons and later in ligaments of Scx^Cre/Cre KI mice lacking Scx, raised the possibility that Scx expression in response to mechanical force during embryonic and postnatal growth might be required for the proper development and structural maintenance of these tissues. Further investigation of target genes regulated by tensile force-responsive Scx may reveal how Scx participates in the regulation of formation and maintenance of tendons and ligaments during development and growth, in the presence or absence of mechanical stress. Such studies are now underway.

Materials and Methods

Animals and embryos

Mice were purchased from Japan SLC (Shizuoka, Japan) or from Shimizu Laboratory Supplies (Kyoto, Japan). The generation and establishment of ScxGFP transgenic strains have been reported previously³. For analysis of Cre activity, Rosa-CAG-LSL-tdTomato (Rosa-tdTomato) mice obtained from Jackson laboratory were used²⁶. Cre-mediated recombination was monitored by red fluorescence of tdTomato expression driven by CAG promoter. All animal experimental protocols were approved by the Animal Care Committee of the Institute for Frontier Life and Medical Sciences, Kyoto University and the Committee of Animal Experimentation, Hiroshima University, and conformed to institutional guidelines for the study of vertebrates. All methods were carried out in accordance with relevant guidelines and regulations.

Generation of Scx^Cre KI mouse strain

The Scx^CreNeo targeting vector was designed to insert the coding region of Cre, along with a translational stop codon followed by FRT-flanked PGK-Neo cassette, in place of the ATG codon of Scx coding region, with simultaneous deletion of most of the Scx exon 1 (Fig. 1a). For construction of the targeting vector, a fused fragment, containing 706 bp Scx genomic region and Cre (706ScxCre)³, was cut with BglII from pBluescript SK (+) (Stratagene). 706ScxCre was inserted into the BamHI site of pPE7neoW-F2LF with PGK-Neo cassette flanked by two FRT sites, so that the PGK promoter was oriented in the opposite direction to the Scx promoter. The 486 bp Scx genomic fragment downstream of BglII site in the first exon and first intron was amplified by PCR using a forward primer with HindIII and BamHI sites (Scx-547F: 5′-AAGCTTGGATCCAGATCTGCACCTTCTGCCTCAG-3′) and a reverse primer with HindIII and NotI sites (Scx-intR2: 5′-AAGCTTGCGGCCGCGGAGGGGTAGTGGCAC-3′). The BamHI site within Scx-547F was introduced for genotyping using Southern blot analysis. The HindIII-digested amplified fragment was inserted into the HindIII site of pPE7neoW-F2LF. PGK-Neo cassette with homology arms was then used for modification of bacterial artificial chromosome clone (RP23-415D19, CHORI) by using Red/ET recombination system (Gene Bridges) to generate ScxBAC-CreNeo, in which Cre was introduced in-frame with ATG of Scx exon 1 and replaced with a 548 bp region. The approximately 0.5 kb homology arms, homologous to extreme 5′ and 3′ ends of the genomic region to be retrieved from ScxBAC-CreNeo, were amplified by PCR. The 5′ homology arm was amplified with forward primer (F1: 5′-AAGCTTCGTTTGCCATCCAGGTCATAACCC-3′) and reverse primer (R1: 5′-ATCGATCCTTGTAAACTACCCACTTGTACCTAAG-3′). The 3′ homology arm was amplified with forward primer (F2: 5′-ATCGATGACAGGCCAGGCCGTGTTCCTGTG-3′) and reverse primer (R2: 5′-CTCGAGCCACATGGCCACTTCACCTCCTTCCTAC-3′). The resultant amplified 5′ and 3′ homology arms were subcloned into HindIII-ClaI and ClaI-XhoI sites of pMCS-DTA, respectively. For generation of the Scx^CreNeo targeting vector, the modified genomic region with 1.7 kb short and 6.3 kb long arms was retrieved from ScxBAC-CreNeo and transferred to pMCS-DTA. The PacI-linearised Scx^CreNeo targeting vector was electroporated into KY1.1 ES cell line (F1 hybrid of C57BL/6J and 129S6/SvEvTac)²³. Total 151 ES cell clones were obtained by positive and negative selection and then screened by PCR using forward primer (F3: 5′-GGATCCTCCTGGGCCCACAGGTCATAG-3′) and reverse primer (Cre-R1: 5′-CTTGCGAACCTCATCACTCGTTGCATCG-3′) (Fig. 1a). We obtained one correctly targeted ES clone that was microinjected into the blastocysts of BDF1 hybrid to generate chimeric mice.

Genotyping

Mice were genotyped using tail tip genomic DNA by PCR or Southern blot analysis. The wild type Scx allele was detected with forward primer (F4: 5′-CTGGTGGGTGAGGCCTGTGGC-3′) and reverse primer (R3: 5′-GAGTCTGTGTCCCAAGGTATG-3′). The Scx^Cre allele was detected with forward primer (Cre-F1: 5′-TCCAATTTACTGACCGTACACCAA-3′) and reverse primer (Cre-R2: 5′-CCTGATCCTGGCAATTTCGGCTA-3′; Fig. 1b). For Southern blot analysis, 10 μg genomic DNA was digested with HindIII and the resulting DNA fragments were separated via 0.7% agarose gel electrophoresis. After transfer onto Nytran membranes with Turboblotter system (Schleicher and Schuell), hybridisation was performed using probes labelled with [α-³²P]- deoxycytidine triphosphate (dCTP) (PerkinElmer).

Northern blot analysis

Total RNA was extracted from wild type and Scx^Cre/+ KI embryos at E12.5. Fifteen micrograms of the total RNA was denatured with 6% formaldehyde, fractionated using 1% agarose gel electrophoresis, and transferred onto Nytran membranes with Turboblotter system. A specific cDNA probe for Scx was labelled with [α-³²P]- dCTP. Hybridisations were performed as previously described^6,21.

Histology

Scx^Cre/+; Rosa-tdTomato and Scx^Cre/Cre; Rosa-tdTomato neonates were fixed in 4% paraformaldehyde dissolved in phosphate-buffered saline (PFA/PBS) at 4 °C for 3 h, immersed in 20% sucrose/PBS, frozen, and cryosectioned to a thickness of 8 μm. After washing with PBS, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Cre-mediated tdTomato expression was detected with a fluorescent microscope. For histological evaluation of knee joint during postnatal growth, paraffin sections prepared from 2-week-old Scx^Cre/+ and Scx^Cre/Cre KI mice were rehydrated and stained with haematoxylin and eosin. The images were captured under the Leica DMRXA microscope equipped with the Leica DC500 camera (Leica Microsystems).

Immunostaining

Scx^Cre/+KI, Scx^Cre/Cre KI, Scx^Cre/+; Rosa-tdTomato, and Scx^Cre/Cre; Rosa-tdTomato neonates were immersed in 20% sucrose/PBS, frozen, and cryosectioned to a thickness of 8 μm. Sections were fixed in ice-cold acetone prior to double immunostaining of Chmd/Col1, Chmd/Tnmd, and myosin heavy chain (MHC)/Tnmd. For double immunostaining of Chmd and Sox9, p-Smad1/5, or p-Smad3, Scx^Cre/+ and Scx^Cre/Cre KI embryos were fixed in 4% PFA/PBS at 4 °C for 3 h, immersed in 20% sucrose/PBS, frozen, and cryosectioned to a thickness of 6 μm. For undecalcified sections, 4-week-old Scx^+/+ and Scx^Cre/Cre KI mice were anesthetised to perfuse with 4% PFA/PBS containing 20% sucrose. The legs were dissected, fixed in 4% PFA/PBS containing 20% sucrose for 3 h, embedded in SCEM (Leica Microsystems), frozen, and cryosectioned to a thickness of 4 μm, according to Kawamoto’s film method³⁹. The sections were decalcified with 0.25 M EDTA/PBS for 60 min and processed for immunostaining. After washing with PBS, the sections were incubated with 2% skim milk in PBS for 20 min and then incubated overnight at 4 °C with primary antibodies diluted with 2% skim milk in PBS. The sections were incubated with appropriate secondary antibodies conjugated with Alexa Fluor 488 or 594 (Life Technologies) and washed again with PBS. The primary antibodies used were anti-Tnmd (diluted 1:1000)^2,12, anti-Col1 (1:500; Rockland), anti-Chmd (diluted 1:500; Cosmo Bio), anti-Col2 (diluted 1:500; Rockland), anti-Ocn (1:800; TaKaRa), anti-sclerostin (1:500; R&D), anti-MHC (1:400; sigma), anti-Sox9 (1:800; Millipore), anti-p-Smad1/5 (1:50; Cell Signaling), and anti-p-Smad3 (1:250; Rockland). Nuclei were counterstained with DAPI. Images were captured using either a Leica DMRXA microscope equipped with a Leica DC500 camera (Leica Microsystems) or an OLYMPUS IX70 microscope equipped with an OLYMPUS DP80 camera (OLYMPUS).

In situ hybridisation

Antisense RNA probes for each gene were transcribed from linearised plasmids with a digoxygenin (DIG) RNA labelling kit (Roche) as previously described⁴⁰. For RNA probes, the cDNAs for Col1a1, Dcn, Mkx, and Egr1 were amplified by RT-PCR using the following primers (mCol1a1_F1: 5′-CCCTCAAGAGCCTGAGTCAGC-3′), (mCol1a1_R1: 5′-GTATTCGATGACTGTCTTGCCC-3′), (mDcn_F1: 5′-TGGTGCAGTGTTCTGATCTGG-3′), (mDcn_R1: 5′-GACTCACAGCCGAGTAGGAAGC-3′), (mfcMkx_F1: 5′-GCGGCCGCCCCCAATCATGAACACCATCG-3′),(mfcMkx_R1: 5′-GCGGCCGCTAGAAGCGCTGCACCAGTGGCA-3′), (mEgr1_F2: 5′-TCTCTTCTTACCCATCCCCAGTGC-3′), and (mEgr1_R2; 5′-TACTGAAATGATTTATCCAATACCATG-3′). RNA probes of Scx, and Tnmd were described previously^2,10. Mouse Col14a1 cDNA was also amplified using primers described previously⁷. For in situ hybridisation on frozen sections, mouse embryos were fixed with 4% PFA/PBS for 3 h, and treated in 20% sucrose before embedding in Tissue-Tek OCT compound (Sakura Finetek). Seven-micron-thick frozen sections of embedded embryos were prepared and postfixed with 4% PFA/PBS for 10 minutes at room temperature and carbethoxylated twice in 0.1% DEPC/PBS. Sections were treated in 5 × SSC, and hybridisation was performed at 58 °C with DIG-labelled antisense RNA probes. To detect DIG-labelled RNA probes, immunological detection was performed using an anti-DIG antibody conjugated with alkaline phosphatase (Anti-DIG-AP Fab fragment, Roche) and BM purple (Roche). Images were captured using an IX70 microscope equipped with a DP80 camera (OLYMPUS).

Skeletal preparation

Mouse neonates were dehydrated with ethanol. After removing skin and soft tissues, the neonates were stained with 0.015% Alcian blue 8GX (Sigma) and cleared with 2% potassium hydroxide. The neonates were then stained with 0.05% Alizarin Red S (Wako) in 1% KOH and cleared with 1% KOH.

Additional Information

How to cite this article: Yoshimoto, Y. et al. Scleraxis is required for maturation of tissue domains for proper integration of the musculoskeletal system. Sci. Rep. 7, 45010; doi: 10.1038/srep45010 (2017).

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Acknowledgements

We thank Ms. H. Sugiyama for her valuable secretarial help. This study was partly supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers JP26293395, JP26893164 and the Cooperative Research Program of Institute for Frontier Medical Sciences, Kyoto University, Japan.

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Authors and Affiliations

Department of Molecular Biology and Biochemistry, Division of Basic Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan
Yuki Yoshimoto & Chisa Shukunami
Laboratory of Cellular Differentiation, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan
Aki Takimoto, Yuji Hiraki & Chisa Shukunami
Laboratory of Animal Experiment for Regeneration, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan
Hitomi Watanabe & Gen Kondoh

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Yuki Yoshimoto
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Aki Takimoto
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Hitomi Watanabe
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Yuji Hiraki
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Contributions

Y.Y. and C.S. designed the study. Y.Y., A.T., H.W., and G.K. performed the experiments. Y.Y., Y.H., and C.S. analysed the results. Y.Y. and C.S prepared the manuscript.

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Correspondence to Chisa Shukunami.

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Yoshimoto, Y., Takimoto, A., Watanabe, H. et al. Scleraxis is required for maturation of tissue domains for proper integration of the musculoskeletal system. Sci Rep 7, 45010 (2017). https://doi.org/10.1038/srep45010

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Received: 20 September 2016
Accepted: 20 February 2017
Published: 22 March 2017
DOI: https://doi.org/10.1038/srep45010

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