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Movement of the vertebrate body is supported by the connection of muscle, tendon and bone. Each skeletal muscle in the vertebrate body has a unique shape and attachment site; however, the mechanism that ensures reproducible muscle patterning is incompletely understood. In this study, we conducted targeted cell ablation using scleraxis (Scx)-Cre to examine the role of Scx-lineage cells in muscle morphogenesis and attachment in mouse embryos. We found that muscle bundle shapes and attachment sites were significantly altered in embryos with Scx-lineage cell ablation. Muscles in the forelimb showed impaired bundle separation and limb girdle muscles distally dislocated from their insertion sites. Scx-lineage cells were required for post-fusion myofiber morphology, but not for the initial segregation of myoblasts in the limb bud. Furthermore, muscles could change their attachment site, even after formation of the insertion. Lineage tracing suggested that the muscle patterning defect was primarily attributed to the reduction of tendon/ligament cells. Our study demonstrates an essential role of Scx-lineage cells in the reproducibility of skeletal muscle attachment, in turn revealing a previously unappreciated tissue–tissue interaction in musculoskeletal morphogenesis.

Neural crest cells (NCCs), a multipotent stem cell population, contribute to cardiac development as a source of the outflow septum, vascular smooth muscle and semilunar valves. However, genetic programs underlying lineage diversification of cardiac NCCs remain largely unknown. Using single-cell (sc) and spatial transcriptomics, we demonstrate multiple NCC subpopulations with distinct gene expression signatures; smooth muscle(-like), non-muscle mesenchymal, and Schwann cell progenitor/melanoblast-like cells. Integrative scRNA-seq and scATAC-seq analyses predict lineage trajectories starting from immature NCCs, which bifurcate into smooth muscle(-like) and non-muscle mesenchymal lineages in association with hierarchical transcription factor networks. Combinatory analyses with Cre-mediated genetic lineage tracing characterize intermediate NCCs at the bifurcation as Sox9+/Scx+ tendon and cartilage progenitor-like cells with genetic programs, some of which are common to skeletal tissues whereas others are unique to cardiac NCCs. These findings provide a basis for understanding the roles of NCCs in cardiac development and pathogenesis particularly associated with calcification.

How mechanical stress affects physical performance via tendons is not fully understood. Piezo1 is a mechanosensitive ion channel, and E756del PIEZO1 was recently found as a gain-of-function variant that is common in individuals of African descent. We generated tendon-specific knock-in mice using R2482H Piezo1 , a mouse gain-of-function variant, and found that they had higher jumping abilities and faster running speeds than wild-type or muscle-specific knock-in mice. These phenotypes were associated with enhanced tendon anabolism via an increase in tendon-specific transcription factors, Mohawk and Scleraxis, but there was no evidence of changes in muscle. Biomechanical analysis showed that the tendons of R2482H Piezo1 mice were more compliant and stored more elastic energy, consistent with the enhancement of jumping ability. These phenotypes were replicated in mice with tendon-specific R2482H Piezo1 replacement after tendon maturation, indicating that PIEZO1 could be a target for promoting physical performance by enhancing function in mature tendon. The frequency of E756del PIEZO1 was higher in sprinters than in population-matched nonathletic controls in a small Jamaican cohort, suggesting a similar function in humans. Together, this human and mouse genetic and physiological evidence revealed a critical function of tendons in physical performance, which is tightly and robustly regulated by PIEZO1 in tenocytes.

The musculoskeletal system is integrated by tendons that are characterized by the expression of scleraxis (Scx), a functionally important transcription factor. Here, we newly developed a tenocyte induction method using induced pluripotent stem cells established from ScxGFP transgenic mice by monitoring fluorescence, which reflects a dynamic differentiation process. Among several developmentally relevant factors, transforming growth factor-beta 2 (TGF-β2) was the most potent inducer for differentiation of tenomodulin-expressing mature tenocytes. Single-cell RNA sequencing (scRNA-seq) revealed 11 distinct clusters, including mature tenocyte population and tenogenic differentiation trajectory, which recapitulated the in vivo developmental process. Analysis of the scRNA-seq dataset highlighted the importance of retinoic acid (RA) as a regulatory pathway of tenogenic differentiation. RA signaling was shown to have inhibitory effects on entheseal chondrogenic differentiation as well as TGF-β2-dependent tenogenic/fibrochondrogenic differentiation. The collective findings provide a new opportunity for tendon research and further insight into the mechanistic understanding of the differentiation pathway to a tenogenic fate.

In higher vertebrates, cephalic neural crest cells (NCCs) form craniofacial skeleton by differentiating into chondrocytes and osteoblasts. A subpopulation of cephalic NCCs, cardiac NCCs (CNCCs), migrates to the heart. However, CNCCs mostly do not yield skeletogenic derivatives, and the molecular mechanisms of this fate restriction remain elusive. We identify a disintegrin and metalloprotease 19 (Adam19) as a position-specific fate regulator of NCCs. Adam19-depleted mice abnormally form NCC-derived cartilage in their hearts through the upregulation of Sox9 levels in CNCCs. Moreover, NCC-lineage-specific Sox9-overexpressing mice recapitulate CNCC chondrogenesis. In vitro experiments show that Adam19 mediates the cleavage of bone morphogenic protein (BMP) type I receptor Alk2 (Acvr1), whereas pharmacogenetic approaches reveal that Adam19 inhibits CNCC chondrogenesis by suppressing the BMP-Sox9 cascade, presumably through processing Alk2. These findings suggest a metalloprotease-dependent mechanism attenuating cellular responsiveness to BMP ligands, which is essential for both the positional restriction of NCC skeletogenesis and normal heart development., 心臓内での軟骨形成をおさえる仕組みを解明 --プロテアーゼが細胞の軟骨分化を防ぐ--. 京都大学プレスリリース. 2019-10-21.

Background:The effects of fibroblast growth factor 2 (FGF-2) on healing after surgical repair of chronic rotator cuff (RC) tears remain unclear.Hypothesis:FGF-2 enhances tenogenic healing response, leading to biomechanical and histological improvement of repaired chronic RC tears in rats.Study Design:Controlled laboratory study.Methods:Adult male Sprague-Dawley rats (n = 117) underwent unilateral surgery to refix the supraspinatus tendon to its insertion site 3 weeks after detachment. Animals were assigned to either the FGF-2 group or a control group. The effects of FGF-2 were assessed via biomechanical tests at 3 weeks after detachment and at 6 and 12 weeks postoperatively and were assessed histologically and immunohistochemically for proliferating cell nuclear antigen and mesenchymal stem cell (MSC)–related markers at 2, 6, and 12 weeks postoperatively. The expression of tendon/enthesis-related markers, including SRY-box 9 (Sox9), scleraxis (Scx), and tenomodulin (Tnmd), were assessed by real-time reverse transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. The effect of FGF-2 on comprehensive gene expressions at the healing site was evaluated by microarray analysis.Results:The FGF-2 group showed a significant increase in mechanical strength at 6 and 12 weeks compared with control; the FGF-2 group also showed significantly higher histological scores at 12 weeks than control, indicating the presence of more mature tendon-like tissue. At 12 weeks, Scx and Tnmd expression increased significantly in the FGF-2 group, whereas no significant differences in Sox9 were found between groups over time. At 2 weeks, the percentage of positive cells expressing MSC-related markers increased in the FGF-2 group. Microarray analysis at 2 weeks after surgery showed that the expression of several growth factor genes and extracellular matrix–related genes was influenced by FGF-2 treatment.Conclusion:FGF-2 enhanced the formation of tough tendon-like tissues including an increase in Scx- or Tnmd-expressing cells at 12 weeks after surgical repair of chronic RC tears. The increase in mesenchymal progenitors and the changes in gene expression upon FGF-2 treatment in the early phase of healing appear to be related to a certain favorable microenvironment for tenogenic healing response of chronic RC tears.Clinical Relevance:These findings may provide advantages in therapeutic strategies for patients with RC tears.

SummaryThe elaborate movement of the vertebrate body is supported by the precise connection of muscle, tendon and bone. Each of the >600 distinct skeletal muscles in the human body has unique attachment sites; however, the mechanism through which muscles are reproducibly attached to designated partner tendons during embryonic development is incompletely understood. We herein show that Screlaxis-positive tendon cells have an essential role in correct muscle attachment in mouse embryos. Specific ablation of Screlaxis-positive cells resulted in dislocation of muscle attachment sites and abnormal muscle bundle morphology. Step-by-step observation of myogenic cell lineage revealed that post-fusion myofibers, but not migrating myoblasts, require tendon cells for their morphology. Furthermore, muscles could change their attachment site, even after the formation of the insertion. Our study demonstrated an essential role of tendon cells in the reproducibility and plasticity of skeletal muscle patterning, in turn revealing a novel tissue-tissue interaction in musculoskeletal morphogenesis.Graphical abstract

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2+ pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1+ mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues., Highlighted Article: Analysis of conditional and temporal knockout mice reveals roles of Ebf3 in the differentiation of lateral plate mesenchymal cells towards pre-osteoblasts during sternum ossification at the boundary of the lateral and somitic mesoderm.

The intervertebral disc (IVD) degeneration is thought to be closely related to ingrowth of new blood vessels. However, the impact of anti‐angiogenic factors in the maintenance of IVD avascularity remains unknown. Tenomodulin (Tnmd) is a tendon/ligament‐specific marker and anti‐angiogenic factor with abundant expression in the IVD. It is still unclear whether Tnmd contributes to the maintenance of IVD homeostasis, acting to inhibit vascular ingrowth into this normally avascular tissue. Herein, we investigated whether IVD degeneration could be induced spontaneously by the absence of Tnmd. Our results showed that Tnmd was expressed in an age‐dependent manner primarily in the outer annulus fibrous (OAF) and it was downregulated at 6 months of age corresponding to the early IVD degeneration stage in mice. Tnmd knockout (Tnmd − / −) mice exhibited more rapid progression of age‐related IVD degeneration. These signs include smaller collagen fibril diameter, markedly lower compressive stiffness, reduced multiple IVD‐ and tendon/ligament‐related gene expression, induced angiogenesis, and macrophage infiltration in OAF, as well as more hypertrophic‐like chondrocytes in the nucleus pulposus. In addition, Tnmd and chondromodulin I (Chm1, the only homologous gene to Tnmd) double knockout (Tnmd − / − Chm1 − / −) mice displayed not only accelerated IVD degeneration, but also ectopic bone formation of IVD. Lastly, the absence of Tnmd in OAF‐derived cells promoted p65 and matrix metalloproteinases upregulation, and increased migratory capacity of human umbilical vein endothelial cells. In sum, our data provide clear evidences that Tnmd acts as an angiogenic inhibitor in the IVD homeostasis and protects against age‐related IVD degeneration. Targeting Tnmd may represent a novel therapeutic strategy for attenuating age‐related IVD degeneration., Cartoon highlighting the hallmarks of Tnmd−/− intervertebral disc (IVD) phenotype. Tnmd−/− mice exhibited more rapid progression of age‐related IVD degeneration. These signs include lower disc height index, smaller collagen fibrils with lower compressive stiffness in the outer annulus fibrosus, reduced multiple IVD‐ and tendon/ligament‐related gene expression, induced angiogenesis and macrophage infiltration in outer annulus fibrous, as well as more hypertrophic‐like chondrocytes in the nucleus pulposus.

A multipotent cell population co-expressing a basic-helix-loop-helix transcription factor scleraxis (Scx) and SRY-box 9 (Sox9) has been shown to contribute to the establishment of entheses (tendon attachment sites) during mouse embryonic development. The present study aimed to investigate the involvement of Scx+/Sox9+cells in the postnatal formation of fibrocartilaginous entheses and in the healing process after injury, usingScxGFPtransgenic mice. We demonstrate that Scx+/Sox9+cells are localized in layers at the insertion site during the postnatal formation of fibrocartilaginous entheses of supraspinatus tendon until postnatal 3 weeks. Further, these cells were rarely seen at postnatal 6 weeks, when mature fibrocartilaginous entheses were formed. Furthermore, we investigated the involvement of Scx+/Sox9+cells in the healing process after supraspinatus tendon enthesis injury, comparing the responses of 20- and 3-week-old mice. In the healing process of 20-week-old mice with disorganized fibrovascular tissue in response to injury, a small number of Scx+/Sox9+cells transiently appeared from 1 week after injury, but they were rarely seen at 4 weeks after injury. Meanwhile, in 3-week-old mice, a thin layer of fibrocartilaginous tissue with calcification was formed at healing enthesis at 4 weeks after injury. From 1 to 2 weeks after injury, more Scx+/Sox9+cells, widely distributed at the injured site, were seen compared with the 20-week-old mice. At 4 weeks after injury, these cells were located near the surface of the recreated fibrocartilaginous layer. This spatiotemporal localization pattern of Scx+/Sox9+cells at the injured enthesis in our 3-week-old mouse model was similar to that in postnatal fibrocartilaginous enthesis formation. These findings indicate that Scx+/Sox9+cells may have a role as entheseal progenitor-like cells during postnatal maturation of fibrocartilaginous entheses and healing after injury in a manner similar to that seen in embryonic development.

The immune system evolved to efficiently eradicate invading bacteria and terminate inflammation through balancing inflammatory and regulatory T-cell responses. In autoimmune arthritis, pathogenic TH17 cells induce bone destruction and autoimmune inflammation. However, whether a beneficial function of T-cell-induced bone damage exists is unclear. Here, we show that bone-damaging T cells have a critical function in the eradication of bacteria in a mouse model of periodontitis, which is the most common infectious disease. Bacterial invasion leads to the generation of specialized TH17 cells that protect against bacteria by evoking mucosal immune responses as well as inducing bone damage, the latter of which also inhibits infection by removing the tooth. Thus, bone-damaging T cells, which may have developed to stop local infection by inducing tooth loss, function as a double-edged sword by protecting against pathogens while also inducing skeletal tissue degradation., IL-17-producing T cells are protective against infection, but the authors of this article previously showed that these cells also contribute to inflammatory bone destruction. Here they show in the context of periodontitis that microbiota-driven Th17-mediated bone destruction may actually be a physiological rather than a pathological process, as associated tooth loss prevents dissemination of oral bacteria.

During tooth movement in orthodontic treatment, bone formation and resorption occur on the tension and compression sides of the alveolar bone, respectively. Although the bone formation activity increases in the periodontal ligament (PDL) on the tension side, the PDL itself is not ossified and maintains its homeostasis, indicating that there are negative regulators of bone formation in the PDL. Our previous report suggested that scleraxis (Scx) has an inhibitory effect on ossification of the PDL on the tension side through the suppression of calcified extracellular matrix formation. However, the molecular biological mechanisms of Scx-modulated inhibition of ossification in the tensioned PDL are not fully understood. The aim of the present study is to clarify the inhibitory role of Scx in osteoblast differentiation of PDL cells and its underlying mechanism. Our in vivo experiment using a mouse experimental tooth movement model showed that Scx expression was increased during early response of the PDL to tensile force. Scx knockdown upregulated expression of alkaline phosphatase, an early osteoblast differentiation marker, in the tensile force-loaded PDL cells in vitro. Transforming growth factor (TGF)-β1-Smad3 signaling in the PDL was activated by tensile force and inhibitors of TGF-β receptor and Smad3 suppressed the tensile force-induced Scx expression in PDL cells. Tensile force induced ephrin A2 (Efna2) expression in the PDL and Efna2 knockdown upregulated alkaline phosphatase expression in PDL cells under tensile force loading. Scx knockdown eliminated the tensile force-induced Efna2 expression in PDL cells. These findings suggest that the TGF-β1-Scx-Efna2 axis is a novel molecular mechanism that negatively regulates the tensile force-induced osteoblast differentiation of PDL cells.

Summary Tendons and ligaments are dense fibrous bands of connective tissue that integrate musculoskeletal components in vertebrates. Tendons connect skeletal muscles to the bone and function as mechanical force transmitters, whereas ligaments bind adjacent bones together to stabilize joints and restrict unwanted joint movement. Fibroblasts residing in tendons and ligaments are called tenocytes and ligamentocytes, respectively. Tenomodulin (Tnmd) is a type II transmembrane glycoprotein that is expressed at high levels in tenocytes and ligamentocytes, and is also present in periodontal ligament cells and tendon stem/progenitor cells. Tnmd is related to chondromodulin-1 (Chm1), a cartilage-derived angiogenesis inhibitor, and both Tnmd and Chm1 are expressed in the CD31 − avascular mesenchyme. The conserved C-terminal hydrophobic domain of these proteins, which is characterized by the eight Cys residues to form four disulfide bonds, may have an anti-angiogenic function. This review highlights the molecular characterization and function of Tnmd, a specific marker of tendons and ligaments.

A previously identified enhancer 10 kb upstream of the Aggrecan (Acan) gene (UE) can drive cartilage specific reporter expression in vivo. Here, we report that the paralogous transcription factors PAX1 and PAX9 differentially drive UE, depending on the presence or absence of SOX9-driven transactivation. In the developing vertebral column, PAX1/9 expression was inversely correlated with Acan expression. Moreover, PAX1/9 was co-expressed with SOX9/5/6 in the intervertebral mesenchyme and the inner annulus fibrosus (AF), and with SOX9 in the outer AF. Significant Acan upregulation was observed during chondrification of Pax1-silenced AF cells, while, Acan was significantly downregulated by persistent expression of Pax1 in cartilage. Deletion of UE using CRISPR/Cas9 resulted in ~30% and ~40% reduction of Acan expression in cartilage and the AF, respectively. In the UE, PAX1/9 acts as weak transactivators through a PAX1/9-binding site partially overlapped with a SOX9-binding site. In the presence of SOX9, which otherwise drives robust Acan expression along with SOX5/6, PAX1/9 competes with SOX9 for occupancy of the binding site, resulting in reduced transactivation of Acan. Coimmunoprecipitation revealed the physical interaction of Pax1 with SOX9. Thus, transactivation of the UE is differentially regulated by concerted action of PAX1/9, SOX9, and SOX5/6 in a context-dependent manner.

Tendons and ligaments are dense fibrous connective tissues mainly composed of type I collagen, aligned in highly ordered arrays along the axis of the tendon and ligament. The enthesis is defined as the attachment site of a tendon, ligament, joint capsule, or fascia to bone. During morphogenesis, the cell population co-expressing Scleraxis(Scx)and the SRY-box containing gene 9(Sox9)contributes to the formation of fibrocartilaginous entheses. Scx regulates tendon and ligament maturation, while Sox9 is a key regulatory factor for cartilage formation. The considerable mechanical forces transmitted through the enthesis and avascular properties of the tissue make it more prone to injuries and degenerative changes. Thus, integration of tendons or ligaments with bone following surgical repair remains a clinical challenge. In this review, we summarize the current knowledge regarding the formation, maintenance, damage, and repair of fibrocartilaginous entheses, focusing on the rotator cuff tendon-to-bone attachment sites.

Tenomodulin (Tnmd) is a type II transmembrane glycoprotein predominantly expressed in tendons and ligaments. We found that scleraxis (Scx), a member of the Twist-family of basic helix-loop-helix transcription factors, is a transcriptional activator of Tnmd expression in tenocytes. During embryonic development, Scx expression preceded that of Tnmd. Tnmd expression was nearly absent in tendons and ligaments of Scx-deficient mice generated by transcription activator-like effector nucleases-mediated gene disruption. Tnmd mRNA levels were dramatically decreased during serial passages of rat tenocytes. Scx silencing by small interfering RNA significantly suppressed endogenous Tnmd mRNA levels in tenocytes. Mouse Tnmd contains five E-box sites in the ~1-kb 5′-flanking region. A 174-base pair genomic fragment containing a TATA box drives transcription in tenocytes. Enhancer activity was increased in the upstream region (−1030 to −295) of Tnmd in tenocytes, but not in NIH3T3 and C3H10T1/2 cells. Preferential binding of both Scx and Twist1 as a heterodimer with E12 or E47 to CAGATG or CATCTG and transactivation of the 5′-flanking region were confirmed by electrophoresis mobility shift and dual luciferase assays, respectively. Scx directly transactivates Tnmd via these E-boxes to positively regulate tenocyte differentiation and maturation.

Scoliosis is a three-dimensional rotation of the spine that is defined as lateral curvature with a Cobb angle greater than 10 degrees. About 2–3% of the global population is affected by scoliosis, and more than 80% of scoliosis are caused by unknown factors (idiopathic). Adolescent idiopathic scoliosis is the most common type of scoliosis and occurs in children over 10 years, showing a female predominance. Of scoliosis patients, 10% have curve progression requiring medical interventions such as bracing and surgery. Scoliosis research has been delayed due to the genetic complexity and a lack of relevant animal models for functional studies; however, significant breakthroughs of scoliosis study have recently been made using zebrafish. The zebrafish is a powerful tool, owing to easy genetic manipulation and a natural susceptibility to spinal curvature. Here, we summarize the utility of zebrafish as a model system for human scoliosis.

Cellular FLICE-like inhibitory protein (c-FLIP, gene symbol CFLAR) was first identified as a negative regulator of death receptor-mediated apoptosis in mammals. To understand the ubiquity and diversity of the c-FLIP protein subfamily during evolution, c-FLIP orthologs were identified from a comprehensive range of vertebrates, including birds, amphibians, and fish, and were characterized by combining experimental and computational analysis. Predictions of three-dimensional protein structures and molecular phylogenetic analysis indicated that the conserved structural features of c-FLIP proteins are all derived from an ancestral caspase-8, although they rapidly diverged from the subfamily consisting of caspases-8, -10, and -18. The functional role of the c-FLIP subfamily members is nearly ubiquitous throughout vertebrates. Exogenous expression of non-mammalian c-FLIP proteins in cultured mammalian cells suppressed death receptor-mediated apoptosis, implying that all of these proteins possess anti-apoptotic activity. Furthermore, non-mammalian c-FLIP proteins induced NF-κB activation much like their mammalian counterparts. The CFLAR mRNAs were synthesized during frog and fish embryogenesis. Overexpression of a truncated mutant of c-FLIP in the Xenopus laevis embryos by mRNA microinjection caused thorax edema and abnormal constriction of the abdomen. Depletion of cflar transcripts in zebrafish resulted in developmental abnormalities accompanied by edema and irregular red blood cell flow. Thus, our results demonstrate that c-FLIP/CFLAR is conserved in both protein structure and function in several vertebrate species, and suggest a significant role of c-FLIP in embryonic development., Highlights • c-FLIP/CFLAR is specific to the vertebrate lineage. • A rapid evolutionary divergence of the c-FLIP subfamily members has occurred. • The pseudocatalytic triad is formed by evolutionarily conserved amino acids. • Vertebrate c-FLIP proteins work on both apoptotic inhibition and NF-κB activation. • c-FLIP proteins play a developmental role during embryogenesis of frog and fish.

Background: Fibroblast growth factor (FGF)–2 has the potential to enhance tendon-to-bone healing after rotator cuff (RC) injury. Hypothesis: FGF-2 stimulates tenogenic differentiation of progenitors to improve the biomechanical strength and histological appearance of repaired RCs in rats. Study Design: Controlled laboratory study. Methods: Adult male Sprague-Dawley rats (N = 156) underwent unilateral surgery to repair the supraspinatus tendon to insertion sites. The FGF-2-treated group (gelatin hydrogel containing 5 μg of FGF-2) and a control group (gelatin hydrogel only) were compared to investigate the effects of FGF-2 at 2, 4, 6, 8, and 12 weeks postoperatively. Biomechanical testing was performed at 6 and 12 weeks. Semiquantitative histological analysis and immunohistochemical analysis for the proliferating cell nuclear antigen (PCNA) were performed, and the expression of tendon-related markers, including Scleraxis ( Scx) and Tenomodulin ( Tnmd), was monitored by real-time reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. SRY-box containing gene 9 ( Sox9) expression was monitored by RT-PCR and immunohistochemical analysis. At 2 and 4 weeks, immunohistochemical analysis for mesenchymal stem cell (MSC) markers was also performed. Results: The FGF-2-treated group demonstrated a significant improvement in mechanical strength at 6 and 12 weeks and significantly higher histological scores than the control group at ≥4 weeks. The average incidence of PCNA-positive cells was significantly higher at 2 and 4 weeks, and more cells expressing MSC markers were detected at the insertion site in the FGF-2-treated group. The expression level of Scx increased significantly in the FGF-2-treated group from 4 to 8 weeks, while the Tnmd level increased significantly from 4 to 12 weeks postoperatively. The localization of Tnmd overlapped with the locations of reparative tissues accompanying collagen fibers with an aligned orientation. Sox9 expression was significantly upregulated at 4 weeks in the FGF-2-treated group. Conclusion: FGF-2 promotes growth of the tenogenic progenitor cells, which participate in tendon-to-bone healing, resulting in biomechanical and histological improvement of the repaired RC. Clinical Relevance: These findings provide clues regarding the clinical development of regenerative repair strategies for RC injury.

Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity. We previously conducted a genome-wide association study (GWAS) and detected two loci associated with AIS. To identify additional loci, we extended our GWAS by increasing the number of cohorts (2,109 affected subjects and 11,140 control subjects in total) and conducting a whole-genome imputation. Through the extended GWAS and replication studies using independent Japanese and Chinese populations, we identified a susceptibility locus on chromosome 9p22.2 (p = 2.46 × 10(-13); odds ratio = 1.21). The most significantly associated SNPs were in intron 3 of BNC2, which encodes a zinc finger transcription factor, basonuclin-2. Expression quantitative trait loci data suggested that the associated SNPs have the potential to regulate the BNC2 transcriptional activity and that the susceptibility alleles increase BNC2 expression. We identified a functional SNP, rs10738445 in BNC2, whose susceptibility allele showed both higher binding to a transcription factor, YY1 (yin and yang 1), and higher BNC2 enhancer activity than the non-susceptibility allele. BNC2 overexpression produced body curvature in developing zebrafish in a gene-dosage-dependent manner. Our results suggest that increased BNC2 expression is implicated in the etiology of AIS.

After tendon injuries, biomechanical properties of the injured tendon are not fully recovered in most cases. Modulation of signaling pathways, which are involved in tendon development and tendon repair, is one of attractive modalities to facilitate proper regeneration of the injured tendon. The roles of TGF-β signaling in tendon homeostasis and tendon development have been elucidated. In contrast, the roles of Wnt/β-catenin signaling in tendon remain mostly elusive. We found that the number of β-catenin-positive cells was increased at the injured site, suggesting involvement of Wnt/β-catenin signaling in tendon healing. Activation of Wnt/β-catenin signaling suppressed expressions of tenogenic genes of Scx, Mkx, and Tnmd in rat tendon-derived cells (TDCs) isolated from the Achilles tendons of 6-week old rats. Additionally, activation of Wnt/β-catenin reduced the amounts of Smad2 and Smad3, which are intracellular mediators for TGF-β signaling, and antagonized upregulation of Scx induced by TGF-β signaling in TDCs. We found that Wnt/β-catenin decreased Mkx and Tnmd expressions without suppressing Scx expression in Scx-programmed tendon progenitors. Our studies suggest that Wnt/β-catenin signaling is a repressor for tenogenic gene expressions.

Background: Transforming growth factor β1 (TGF-β1) positively regulates the tenogenic marker genes scleraxis ( Scx) and tenomodulin ( Tnmd) in mesenchymal progenitors in vitro. However, little is known about the effect of TGF-β1 on the expression of tenogenic markers during rotator cuff (RC) healing in rats. Hypothesis: TGF-β1 improves the biomechanical properties and histological maturity of reparative tissue in a rat RC repair model by stimulating the growth of tenogenic cells. Study Design: Controlled laboratory study. Methods: Adult male Sprague-Dawley rats (N = 180) underwent unilateral supraspinatus tendon-to-bone surgical repair and were randomly treated with a gelatin hydrogel presoaked in TGF-β1 (100 ng) or phosphate-buffered saline. The effects of TGF-β1 on RC healing were investigated at 2, 4, 6, 8, and 12 weeks postoperatively by immunostaining for proliferating cell nuclear antigen, by real-time reverse transcription polymerase chain reaction and in situ hybridization or immunostaining for enthesis-related markers (SRY-box containing gene 9 [ Sox9], Scx, and Tnmd), and by real-time reverse transcription polymerase chain reaction and immunostaining for type I and III collagen. At 6 and 12 weeks postoperatively, biomechanical testing, micro–computed tomography, and biochemical analysis were also performed. At 2 and 4 weeks postoperatively, mesenchymal stem cell–related markers, phospho-Smad2, and matrix metalloproteinase 9 (MMP-9) and MMP-13 were assessed by immunostaining. Results: The TGF-β1-treated group had significantly higher ultimate load to failure and tissue volume at 6 and 12 weeks postoperatively and a higher collagen content at 12 weeks compared with the saline group. Tendon-related gene expression, histological maturity, cell proliferation, and mesenchymal stem cell–related marker immunoreactivity were not affected by exogenously administrated TGF-β1 at all time points. In the TGF-β1-treated group, the percentage of phospho-Smad2-positive cells within the healing tissue increased, whereas the expression of MMP-9 and MMP-13 significantly decreased at 2 and 4 weeks postoperatively. Conclusion: TGF-β1 enhances formation of tough fibrous tissues at the healing site by inhibiting MMP-9 and MMP-13 expression to increase collagen accumulation but without the growth of tenogenic lineage cells. Clinical Relevance: These findings suggest that TGF-β1 could be used for enhancing biomechanical strength after RC surgical repair.

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 ScxCre 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 ScxCre/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 ScxCre/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., 筋骨格系を繋ぐ組織の成熟には転写因子Scleraxisが重要な役割を果たすことを解明. 京都大学プレスリリース. 2017-03-27.

Tendons transmit the mechanical force of skeletal muscle contraction to the bones, whereas ligaments connect the two bones together to stabilize the joint. During embryonic development, each component in the musculoskeletal system, initially develops as an individual primordium of tendon, ligament, skeletal muscle, and cartilage. Later, mutual interaction between these tissues plays an important role for the integration of the musculoskeletal components. Accumulating evidence suggests that myotendinous and osteotendinous/osteoligamentous junctions are important structures to maintain homeostasis of the integrated musculoskeletal components. In this review, we will focus on the establishment and maintenance of these junctions.

SRY-box containing gene 9 (Sox9) and scleraxis (Scx) regulate cartilage and tendon formation, respectively. Here we report that murine Scx(+)/Sox9(+) progenitors differentiate into chondrocytes and tenocytes/ligamentocytes to form the junction between cartilage and tendon/ligament. Sox9 lineage tracing in the Scx(+) domain revealed that Scx(+) progenitors can be subdivided into two distinct populations with regard to their Sox9 expression history: Scx(+)/Sox9(+) and Scx(+)/Sox9(-) progenitors. Tenocytes are derived from Scx(+)/Sox9(+) and Scx(+)/Sox9(-) progenitors. The closer the tendon is to the cartilaginous primordium, the more tenocytes arise from Scx(+)/Sox9(+) progenitors. Ligamentocytes as well as the annulus fibrosus cells of the intervertebral discs are descendants of Scx(+)/Sox9(+) progenitors. Conditional inactivation of Sox9 in Scx(+)/Sox9(+) cells causes defective formation in the attachment sites of tendons/ligaments into the cartilage, and in the annulus fibrosus of the intervertebral discs. Thus, the Scx(+)/Sox9(+) progenitor pool is a unique multipotent cell population that gives rise to tenocytes, ligamentocytes and chondrocytes for the establishment of the chondro-tendinous/ligamentous junction.

Adolescent idiopathic scoliosis (AIS) is the most common pediatric skeletal disease. We previously reported a locus on chromosome 10q24.31 associated with AIS susceptibility in Japanese using a genome-wide association study (GWAS) consisting of 1,033 cases and 1,473 controls. To identify additional AIS-associated loci, we expanded the study by adding X-chromosome SNPs in the GWAS and increasing the size of the replication cohorts. Through a stepwise association study including 1,819 cases and 25,939 controls, we identified a new susceptibility locus on chromosome 6q24.1 in Japanese (P = 2.25 × 10(-10); odds ratio (OR) = 1.28). The most significantly associated SNP, rs6570507, was in GPR126 (encoding G protein-coupled receptor 126). Its association was replicated in Han Chinese and European-ancestry populations (combined P = 1.27 × 10(-14); OR = 1.27). GPR126 was highly expressed in cartilage, and the knockdown of gpr126 in zebrafish caused delayed ossification of the developing spine. Our results should provide insights into the etiology and pathogenesis of AIS.

Scleraxis (Scx) is a basic helix-loop-helix transcription factor that is a marker for the tendon/ligament cell lineage. The ∼11 kb genomic region from the mouse Scx gene locus faithfully recapitulates the endogenous Scx expression pattern in ScxGFP transgenic (Tg) mice. We have established two Tg mouse lines expressing Cre-recombinase (Cre) using this regulatory region (ScxCre-L and ScxCre-H). The specificity and efficiency of Cre recombination in these Tg lines are evaluated by crossing with Rosa-CAG-LSL-tdTomato (Ai14) or ROSA26R (R26R) reporter mice. The recombination in ScxCre-H;Ai14 mice is efficiently achieved in the endogenous Scx expression domains including the branchial arches, the syndetome, and the lateral plate mesoderm. Further analysis of ScxCre-H;Ai14;ScxGFP embryos reveal that expression of the ScxGFP transgene largely overlaps with Cre activity detected by tdTomato at embryonic day 12.5 (E12.5). In ScxCre-L;R26R or ScxCre-H;R26R neonates, Cre activity is detected in tendons, ligaments, intervertebral discs, joints, and cartilage around the chondro-tendinous/ligamentous junction, the prospective enthesis. The present results suggest that ScxCre Tg lines are useful for targeting the gene specifically in the Scx-expressing domains.

Tenomodulin (TNMD, Tnmd) is a gene highly expressed in tendon known to be important for tendon maturation with key implications for the residing tendon stem/progenitor cells as well as for the regulation of endothelial cell migration in chordae tendineae cordis in the heart and in experimental tumour models. This review aims at providing an encompassing overview of this gene and its protein. In addition, its known expression pattern as well as putative signalling pathways will be described. A chronological overview of the discovered functions of this gene in tendon and other tissues and cells is provided as well as its use as a tendon and ligament lineage marker is assessed in detail and discussed. Last, information about the possible connections between TNMD genomic mutations and mRNA expression to various diseases is delivered. Taken together this review offers a solid synopsis on the up-to-date information available about TNMD and aids at directing and focusing the future research to fully uncover the roles and implications of this interesting gene.

Previously, we identified an adolescent idiopathic scoliosis susceptibility locus near human ladybird homeobox 1 (LBX1) and FLJ41350 by a genome-wide association study. Here, we characterized the associated non-coding variant and investigated the function of these genes. A chromosome conformation capture assay revealed that the genome region with the most significantly associated single nucleotide polymorphism (rs11190870) physically interacted with the promoter region of LBX1-FLJ41350. The promoter in the direction of LBX1, combined with a 590-bp region including rs11190870, had higher transcriptional activity with the risk allele than that with the non-risk allele in HEK 293T cells. The ubiquitous overexpression of human LBX1 or either of the zebrafish lbx genes (lbx1a, lbx1b, and lbx2), but not FLJ41350, in zebrafish embryos caused body curvature followed by death prior to vertebral column formation. Such body axis deformation was not observed in transcription activator-like effector nucleases mediated knockout zebrafish of lbx1b or lbx2. Mosaic expression of lbx1b driven by the GATA2 minimal promoter and the lbx1b enhancer in zebrafish significantly alleviated the embryonic lethal phenotype to allow observation of the later onset of the spinal curvature with or without vertebral malformation. Deformation of the embryonic body axis by lbx1b overexpression was associated with defects in convergent extension, which is a component of the main axis-elongation machinery in gastrulating embryos. In embryos overexpressing lbx1b, wnt5b, a ligand of the non-canonical Wnt/planar cell polarity (PCP) pathway, was significantly downregulated. Injection of mRNA for wnt5b or RhoA, a key downstream effector of Wnt/PCP signaling, rescued the defective convergent extension phenotype and attenuated the lbx1b-induced curvature of the body axis. Thus, our study presents a novel pathological feature of LBX1 and its zebrafish homologs in body axis deformation at various stages of embryonic and subsequent growth in zebrafish., 思春期特発性側弯症(AIS)の原因遺伝子LBX1が側弯を引き起こす仕組みを解明 --AIS治療法の確立へ期待--. 京都大学プレスリリース. 2016-02-01.