Transcription factor scleraxis vitally contributes to progenitor lineage direction in wound healing of adult tendon in mice

Tendon is a dense connective tissue that transmits high mechanical forces from skeletal muscle to bone. The transcription factor scleraxis (Scx) is a highly specific marker of both precursor and mature tendon cells (tenocytes). Mice lacking scx exhibit a specific and virtually complete loss of tendons during development. However, the functional contribution of Scx to wound healing in adult tendon has not yet been fully characterized. Here, using ScxGFP-tracking and loss-of-function systems, we show in an adult mouse model of Achilles tendon injury that paratenon cells, representing a stem cell antigen-1 (Sca-1)–positive and Scx-negative progenitor subpopulation, display Scx induction, migrate to the wound site, and produce extracellular matrix (ECM) to bridge the defect, whereas resident tenocytes exhibit a delayed response. Scx induction in the progenitors is initiated by transforming growth factor β (TGF-β) signaling. scx-deficient mice had migration of Sca-1–positive progenitor cell to the lesion site but impaired ECM assembly to bridge the defect. Mechanistically, scx-null progenitors displayed higher chondrogenic potential with up-regulation of SRY-box 9 (Sox9) coactivator PPAR-γ coactivator-1α (PGC-1α) in vitro, and knock-in analysis revealed that forced expression of full-length scx significantly inhibited Sox9 expression. Accordingly, scx-null wounds formed cartilage-like tissues that developed ectopic ossification. Our findings indicate a critical role of Scx in a progenitor-cell lineage in wound healing of adult mouse tendon. These progenitor cells could represent targets in strategies to facilitate tendon repair. We propose that this lineage-regulatory mechanism in tissue progenitors could apply to a broader set of tissues or biological systems in the body

Local retinoic acid signaling directs emergence of the extraocular muscle functional unit

Coordinated development of muscles, tendons, and their attachment sites ensures emergence of functional musculoskeletal units that are adapted to diverse anatomical demands among different species. How these different tissues are patterned and functionally assembled during embryogenesis is poorly understood. Here, we investigated the morphogenesis of extraocular muscles (EOMs), an evolutionary conserved cranial muscle group that is crucial for the coordinated movement of the eyeballs and for visual acuity. By means of lineage analysis, we redefined the cellular origins of periocular connective tissues interacting with the EOMs, which do not arise exclusively from neural crest mesenchyme as previously thought. Using 3D imaging approaches, we established an integrative blueprint for the EOM functional unit. By doing so, we identified a developmental time window in which individual EOMs emerge from a unique muscle anlage and establish insertions in the sclera, which sets these muscles apart from classical muscle-to-bone type of insertions. Further, we demonstrate that the eyeballs are a source of diffusible all-trans retinoic acid (ATRA) that allow their targeting by the EOMs in a temporal and dose-dependent manner. Using genetically modified mice and inhibitor treatments, we find that endogenous local variations in the concentration of retinoids contribute to the establishment of tendon condensations and attachment sites that precede the initiation of muscle patterning. Collectively, our results highlight how global and site-specific programs are deployed for the assembly of muscle functional units with precise definition of muscle shapes and topographical wiring of their tendon attachments

Connecting muscles to tendons: tendons and musculoskeletal development in flies and vertebrates

The formation of the musculoskeletal system represents an intricate process of tissue assembly involving heterotypic inductive interactions between tendons, muscles and cartilage. An essential component of all musculoskeletal systems is the anchoring of the force-generating muscles to the solid support of the organism: the skeleton in vertebrates and the exoskeleton in invertebrates. Here, we discuss recent findings that illuminate musculoskeletal assembly in the vertebrate embryo, findings that emphasize the reciprocal interactions between the forming tendons, muscle and cartilage tissues. We also compare these events with those of the corresponding system in the Drosophila embryo, highlighting distinct and common pathways that promote efficient locomotion while preserving the form of the organism

MUSCULOSKELETAL INTEGRATION AT THE WRIST UNDERLIES MODULAR DEVELOPMENT OF LIMB TENDONS Development Accepted manuscript

SUMMARY The long tendons of the limb extend from muscles that reside in the zeugopod (arm/leg) to their skeletal insertions in the autopod (paw). How these connections are established along the length of the limb remains unknown. In this study, we show that limb tendons are formed in modular units that combine to form a functional contiguous structure; in muscle-less limbs, tendons develop in the autopod but do not extend into the zeugopod, and in the absence of limb cartilage, the zeugopod segments of tendons develop despite the absence of tendons in the autopod. Analyses of cell lineage and proliferation further indicate that distinct mechanisms govern growth of autopod and zeugopod tendon segments. To elucidate the integration of these autopod and zeugopod developmental programs, we reexamined early tendon development. At E12.5, muscles extend across the full length of a very short zeugopod and connect through short anlagen of tendon progenitors at the presumptive wrist to their respective autopod tendon segment, thereby initiating musculoskeletal integration. Zeugopod tendon segments are subsequently generated by proximal elongation of the wrist tendon anlagen, in parallel with skeletal growth, underscoring the dependence of zeugopod tendon development on muscles for tendon anchoring. Moreover, a subset of extensor tendons initially form as fused structures, due to initial attachment of their respective wrist tendon anlage to multiple muscles. Subsequent individuation of these tendons depends on muscle activity. Collectively, these results establish an integrated model for limb tendon development that we propose as a framework for future analyses of tendon and musculoskeletal phenotypes

Recruitment and maintenance of tendon progenitors by TGFβ signaling are essential for tendon formation

Tendons and ligaments mediate the attachment of muscle to bone and of bone to bone to provide connectivity and structural integrity in the musculoskeletal system. We show that TGFβ signaling plays a major role in the formation of these tissues. TGFβ signaling is a potent inducer of the tendon progenitor (TNP) marker scleraxis both in organ culture and in cultured cells, and disruption of TGFβ signaling in Tgfb2-/-;Tgfb3-/- double mutant embryos or through inactivation of the type II TGFβ receptor (TGFBR2; also known as TβRII) results in the loss of most tendons and ligaments in the limbs, trunk, tail and head. The induction of scleraxis-expressing TNPs is not affected in mutant embryos and the tendon phenotype is first manifested at E12.5, a developmental stage in which TNPs are positioned between the differentiating muscles and cartilage, and in which Tgfb2 or Tgfb3 is expressed both in TNPs and in the differentiating muscles and cartilage. TGFβ signaling is thus essential for maintenance of TNPs, and we propose that it also mediates the recruitment of new tendon cells by differentiating muscles and cartilage to establish the connections between tendon primordia and their respective musculoskeletal counterparts, leading to the formation of an interconnected and functionally integrated musculoskeletal system

The Atypical Homeodomain Transcription Factor Mohawk Controls Tendon Morphogenesis▿

The Mohawk homeobox (Mkx) gene encodes a new atypical homeodomain-containing protein with transcriptional repressor activity. Mkx mRNA exhibited dynamic expression patterns during development of the palate, somite, kidney, and testis, suggesting that it may be an important regulator of multiple developmental processes. To investigate the roles of Mkx in organogenesis, we generated mice carrying a null mutation in this gene. Mkx−/− mice survive postnatally and exhibit a unique wavy-tail phenotype. Close examination revealed that the mutant mice had smaller tendons than wild-type littermates and that the rapid postnatal growth of collagen fibrils in tendons was disrupted in Mkx−/− mice. Defects in tendon development were detected in the mutant mouse embryos as early as embryonic day 16.5 (E16.5). Although collagen fibril assembly initially appeared normal, the tendons of Mkx−/− embryos expressed significantly reduced amounts of collagen I, fibromodulin, and tenomodulin in comparison with control littermates. We found that Mkx mRNA was strongly expressed in differentiating tendon cells during embryogenesis and in the tendon sheath cells in postnatal stages. In addition to defects in tendon collagen fibrillogenesis, Mkx−/− mutant mice exhibited abnormal tendon sheaths. These results identify Mkx as an important regulator of tendon development

mTORC1 Signaling is a Critical Regulator of Postnatal Tendon Development

Abstract Tendons transmit contractile forces between musculoskeletal tissues. Whereas the biomechanical properties of tendons have been studied extensively, the molecular mechanisms regulating postnatal tendon development are not well understood. Here we examine the role of mTORC1 signaling in postnatal tendon development using mouse genetic approaches. Loss of mTORC1 signaling by removal of Raptor in tendons caused severe tendon defects postnatally, including decreased tendon thickness, indicating that mTORC1 is necessary for postnatal tendon development. By contrast, activation of mTORC1 signaling in tendons increased tendon cell numbers and proliferation. In addition, Tsc1 conditional knockout mice presented severely disorganized collagen fibers and neovascularization in the tendon midsubstance. Interestingly, collagen fibril diameter was significantly reduced in both Raptor and Tsc1 conditional knockout mice, albeit with variations in severity. We performed RNA-seq analysis using Achilles tendons to investigate the molecular changes underlying these tendon phenotypes. Raptor conditional knockout mice showed decreased extracellular matrix (ECM) structure-related gene expression, whereas Tsc1 conditional knockout mice exhibited changes in genes regulating TGF-β/BMP/FGF signaling, as well as in genes controlling ECM structure and disassembly. Collectively, our studies suggest that maintaining physiological levels of mTORC1 signaling is essential for postnatal tendon development and maturation