Transcriptomic and Genetic Analyses Identify the Krüppel-Like Factor Dar1 as a New Regulator of Tube-Shaped Long Tendon Development

To ensure locomotion and body stability, the active role of muscle contractions relies on a stereotyped muscle pattern set in place during development. This muscle patterning requires a precise assembly of the muscle fibers with the skeleton via a specialized connective tissue, the tendon. Like in vertebrate limbs, Drosophila leg muscles make connections with specific long tendons that extend through different segments. During the leg disc development, cell precursors of long tendons rearrange and collectively migrate to form a tube-shaped structure. A specific developmental program underlies this unique feature of tendon-like cells in the Drosophila model. We provide for the first time a transcriptomic profile of leg tendon precursors through fluorescence-based cell sorting. From promising candidates, we identified the Krüppel-like factor Dar1 as a critical actor of leg tendon development. Specifically expressed in the leg tendon precursors, loss of dar1 disrupts actin-rich filopodia formation and tendon elongation. Our findings show that Dar1 acts downstream of Stripe and is required to set up the correct number of tendon progenitors.

Muscle-tendon Crosstalk During Muscle Wasting

In organisms from flies to mammals, the initial formation of a functional tendon is completely dependent on chemical signals from muscle (myokines). However, how myokines affect the maturation, maintenance, and regeneration of tendons as a function of age is completely unstudied. Here we discuss the role of four myokines - fibroblast growth factors (FGF), myostatin, the secreted protein acidic and rich in cysteine (SPARC), and miR-29 - in tendon development and hypothesize a role for these factors in the progressive changes in tendon structure and function as a result of muscle wasting (disuse, aging and disease). Because of the close relationship between mechanical loading and muscle and tendon regulation, disentangling muscle-tendon crosstalk from simple mechanical loading is experimentally quite difficult. Therefore, we propose an experimental framework that hopefully will be useful in demonstrating muscle-tendon crosstalk in vivo. Though understudied, the promise of a better understanding of muscle-tendon crosstalk is the development of new interventions that will improve tendon development, regeneration, and function throughout the lifespan.

The Scleraxis Transcription Factor Directly Regulates Multiple Distinct Molecular and Cellular Processes During Early Tendon Cell Differentiation

Proper development of tendons is crucial for the integration and function of the musculoskeletal system. Currently little is known about the molecular mechanisms controlling tendon development and tendon cell differentiation. The transcription factor Scleraxis (Scx) is expressed throughout tendon development and plays essential roles in both embryonic tendon development and adult tendon healing, but few direct target genes of Scx in tendon development have been reported and genome-wide identification of Scx direct target genes in vivo has been lacking. In this study, we have generated a ScxFlag knockin mouse strain, which produces fully functional endogenous Scx proteins containing a 2xFLAG epitope tag at the carboxy terminus. We mapped the genome-wide Scx binding sites in the developing limb tendon tissues, identifying 12,097 high quality Scx regulatory cis-elements in-around 7,520 genes. Comparative analysis with previously reported embryonic tendon cell RNA-seq data identified 490 candidate Scx direct target genes in early tendon development. Furthermore, we characterized a new Scx gene-knockout mouse line and performed whole transcriptome RNA sequencing analysis of E15.5 forelimb tendon cells from Scx–/– embryos and control littermates, identifying 68 genes whose expression in the developing tendon tissues significantly depended on Scx function. Combined analysis of the ChIP-seq and RNA-seq data yielded 32 direct target genes that required Scx for activation and an additional 17 target genes whose expression was suppressed by Scx during early tendon development. We further analyzed and validated Scx-dependent tendon-specific expression patterns of a subset of the target genes, including Fmod, Kera, Htra3, Ssc5d, Tnmd, and Zfp185, by in situ hybridization and real-time quantitative polymerase chain reaction assays. These results provide novel insights into the molecular mechanisms mediating Scx function in tendon development and homeostasis. The ChIP-seq and RNA-seq data provide a rich resource for aiding design of further studies of the mechanisms regulating tendon cell differentiation and tendon tissue regeneration. The ScxFlag mice provide a valuable new tool for unraveling the molecular mechanisms involving Scx in the protein interaction and gene-regulatory networks underlying many developmental and disease processes.

Reticulocalbin 3 is involved in postnatal tendon development by regulating collagen fibrillogenesis and cellular maturation

Tendon plays a critical role in the joint movement by transmitting force from muscle to bone. This transmission of force is facilitated by its specialized structure, which consists of highly aligned extracellular matrix consisting predominantly of type I collagen. Tenocytes, fibroblast-like tendon cells residing between the parallel collagen fibers, regulate this specialized tendon matrix. Despite the importance of collagen structure and tenocyte function, the biological mechanisms regulating fibrillogenesis and tenocyte maturation are not well understood. Here we examine the function of Reticulocalbin 3 (Rcn3) in collagen fibrillogenesis and tenocyte maturation during postnatal tendon development using a genetic mouse model. Loss of Rcn3 in tendon caused decreased tendon thickness, abnormal tendon cell maturation, and decreased mechanical properties. Interestingly, Rcn3 deficient mice exhibited a smaller collagen fibril distribution and over-hydroxylation in C-telopeptide cross-linking lysine from α1(1) chain. Additionally, the proline 3-hydroxylation sites in type I collagen were also over-hydroxylated in Rcn3 deficient mice. Our data collectively suggest that Rcn3 is a pivotal regulator of collagen fibrillogenesis and tenocyte maturation during postnatal tendon development.

Transcriptomic and genetic analyses identify the Krüppel-like factor dar1 as a master regulator of tube-shaped long tendon development

To ensure locomotion and body stability, the active role of muscle contractions relies on a stereotyped muscle pattern set in place during development. This muscle patterning requires a precise assembly of the muscle fibers with the skeleton via a specialized connective tissue, the tendon. Despite evident disparities, little is known about the molecular basis of tendon diversity. Like in vertebrate limbs, Drosophila leg muscles make connections with specific long tendons that extend through different segments. During leg disc development, cell precursors of long tendons rearrange and collectively migrate to form a tube-shaped structure. A specific developmental program underlies this unique feature of tendon-like cells in the Drosophila model. We provide for the first time a transcriptomic profile of leg tendon precursors through fluorescence-based cell sorting. From promising candidates, we identified the Krüppel-like factor dar1 as a critical actor of leg tendon development. Specifically expressed in leg tendon precursors, loss of dar1 disrupts actin-rich filopodia formation and tendon elongation. Our findings show that dar1 acts downstream of stripe as a critical regulator of cytoskeleton remodeling and mediates the recruitment of new stripe-positive tendon progenitors in a cell non-autonomous manner.

Rcn3 is Involved in Postnatal Tendon Development by Regulating Collagen Modification and Fibrillogenesis

Tendon plays a critical role in the joint movement by transmitting force from muscle to bone. This transmission of force is facilitated by its structure, which consists of an aligned and organized type I collagen. Despite the importance of collagen structure, the biological mechanisms regulating fibrillogenesis are not well understood. Here we examine the function of Rcn3 in postnatal tendon development and collagen fibrillogenesis using a genetic mouse model. Loss of Rcn3 in tendon caused decreased tendon thickness, abnormal tendon cellular maturation, and decreased mechanical properties. Interestingly, Rcn3 deficient mice exhibited a relatively smaller distribution of collagen fibril and over-hydroxylation in C-telopeptide cross-linking lysine from α1(1) chain. Besides, the proline 3-hydroxylation sites in type I collagen were also over-hydroxylated in Rcn3 deficient mice. Our data collectively suggest that Rcn3 is required for proper postnatal tendon development via regulation of collagen modification and fibrillogenesis.

Models of tendon development and injury

Tendons link muscle to bone and transfer forces necessary for normal movement. Tendon injuries can be debilitating and their intrinsic healing potential is limited. These challenges have motivated the development of model systems to study the factors that regulate tendon formation and tendon injury. Recent advances in understanding of embryonic and postnatal tendon formation have inspired approaches that aimed to mimic key aspects of tendon development. Model systems have also been developed to explore factors that regulate tendon injury and healing. We highlight current model systems that explore developmentally inspired cellular, mechanical, and biochemical factors in tendon formation and tenogenic stem cell differentiation. Next, we discuss in vivo, in vitro, ex vivo, and computational models of tendon injury that examine how mechanical loading and biochemical factors contribute to tendon pathologies and healing. These tendon development and injury models show promise for identifying the factors guiding tendon formation and tendon pathologies, and will ultimately improve regenerative tissue engineering strategies and clinical outcomes.