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-/-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.

SUMMARY During the assembly of the musculoskeletal system, bone ridges provide a stable anchoring point and stress dissipation for the attachment of muscles via tendons to the skeleton. In this study, we investigate the development of the deltoid tuberosity as a model for bone ridge formation. We show that the deltoid tuberosity develops through endochondral ossification in a two-phase process: Initiation is regulated by a signal from the tendons, whereas the subsequent growth phase is muscle-dependent. We then show that the transcription factor scleraxis (SCX) regulates Bmp4 in tendon cells at their insertion site. The inhibition of deltoid tuberosity formation and several other bone ridges in embryos in which Bmp4 expression was blocked specifically in Scx-expressing cells implicates BMP4 as a key mediator of tendon effects on bone ridge formation. This study establishes a mechanistic basis for tendon-skeleton regulatory interactions during musculoskeletal assembly and bone secondary patterning.

Defects in tendon patterning and differentiation are seldom assessed in mouse mutants due to the difficulty in visualizing connective tissue structures. To facilitate tendon analysis, we have generated mouse lines harboring two different transgene reporters, alkaline phosphatase (AP) and green fluorescent protein (GFP), each expressed using regulatory elements derived from the endogenous Scleraxis (Scx) locus. Scx encodes a transcription factor expressed in all developing tendons and ligaments as well as in their progenitors. Both the ScxGFP and ScxAP transgenes are expressed in patterns recapitulating almost entirely the endogenous developmental expression of Scx including very robust expression in the tendons and ligaments. These reporter lines will facilitate isolation of tendon cells and phenotypic analysis of these tissues in a variety of genetic backgrounds.

Myoblast fusion is essential for the formation and regeneration of skeletal muscle. In a genetic screen for regulators of muscle development in Drosophila, we discovered a gene encoding a guanine nucleotide exchange factor, called loner, which is required for myoblast fusion. Loner localizes to subcellular sites of fusion and acts downstream of cell surface fusion receptors by recruiting the small GTPase ARF6 and stimulating guanine nucleotide exchange. Accordingly, a dominant-negative ARF6 disrupts myoblast fusion in Drosophila embryos and in mammalian myoblasts in culture, mimicking the fusion defects caused by loss of Loner. Loner and ARF6, which also control the proper membrane localization of another small GTPase, Rac, are key components of a cellular apparatus required for myoblast fusion and muscle development. In muscle cells, this fusigenic mechanism is coupled to fusion receptors; in other fusion-competent cell types it may be triggered by different upstream signals.

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. Here, we show that mouse 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 indicate that distinct mechanisms govern the growth of autopod and zeugopod tendon segments. To elucidate the integration of these autopod and zeugopod developmental programs, we re-examined 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. These results establish an integrated model for limb tendon development that provides a framework for future analyses of tendon and musculoskeletal phenotypes.

Studies of cell fate focus on specification, but little is known about maintenance of the differentiated state. In this study, we find that the mouse tendon cell fate requires continuous maintenance in vivo and identify an essential role for TGFβ signaling in maintenance of the tendon cell fate. To examine the role of TGFβ signaling in tenocyte function the TGFβ type II receptor (Tgfbr2) was targeted in the Scleraxis-expressing cell lineage using the ScxCre deletor. Tendon development was not disrupted in mutant embryos, but shortly after birth tenocytes lost differentiation markers and reverted to a more stem/progenitor state. Viral reintroduction of Tgfbr2 to mutants prevented and even rescued tenocyte dedifferentiation suggesting a continuous and cell autonomous role for TGFβ signaling in cell fate maintenance. These results uncover the critical importance of molecular pathways that maintain the differentiated cell fate and a key role for TGFβ signaling in these processes.

Tendon-bone attachment sites, called entheses, are essential for musculoskeletal function. They are formed embryonically by Sox9+ progenitors and continue to develop postnatally, utilizing Gli1 lineage cells. Despite their importance, we lack information on the transition from embryonic to mature enthesis and on the relation between Sox9+ progenitors and the Gli1 lineage. Here, by performing a series of lineage tracing experiments in mice, we identify the onset of Gli1 lineage contribution to different entheses. We show that Gli1 expression is regulated embryonically by SHH signaling, whereas postnatally it is maintained by IHH signaling. During bone elongation, some entheses migrate along the bone shaft, whereas others remain stationary. Interestingly, in stationary entheses Sox9 + cells differentiate into the Gli1 lineage, but in migrating entheses this lineage is replaced by Gli1 lineage. These Gli1 + progenitors are defined embryonically to occupy the different domains of the mature enthesis. Overall, these findings demonstrate a developmental strategy whereby one progenitor population establishes a simple embryonic tissue, whereas another population contributes to its maturation. Moreover, they suggest that different cell populations may be considered for cell-based therapy of enthesis injuries.

The range and precision of limb movements are dependent on the specific patterns of muscles and tendons. To facilitate analyses of tendon and muscle phenotypes we compiled a description of these tissues in the forelimb of developing mouse embryos. Individual tendons, muscles, and ligaments were annotated in a series of transverse sections through the forelimb of an embryo at day 18.5 of embryonic development (E18.5). Transverse sections present a distinctive and highly reproducible pattern of the muscles and tendons at different limb levels that can be used as a simple reference in analyses of mutant phenotypes. A comparable set of sections from an embryo at E14.5 was included to highlight structural features that change during the maturation of the musculoskeletal system. The ability to define the precise position of transverse sections along the proximal-distal axis of the limb may also be useful in studies of other features in developing limbs. Developmental Dynamics 238:693-700, 2009.