Muscle and tendon interactions during development: Growth factors, transcription factors and collagens – Muscolten

This project has for objective to understand the muscle and tendon development and the interactions between both tissues. With the aim of identifying the genes involved in these processes, we undertook a global approach to list all the genes involved in tendon formation, during development. Furthermore, we also targeted certain number of relevant candidate genes for our study. The function of these genes is studied by using various animal models, used classically in Developmental Biology.
We use the chick model, which presents the advantage to allow the experiments in the embryo. We intend to modify the expression of genes (selected in our global list) in chick limbs and to study the consequences for tendon and muscle formation.
We use zebrafish model for the numerous advantages that it presents: very small-sized, abundance of embryos, fast development (3 days) and transparent embryos facilitating the observations on the living animal, easy experiments) of loss of expression, numerous transgenic lineages and available mutants.
We also use the mouse model, common to 3 partners, which allows us to study the consequences of the invalidation of a gene for tendon and muscle formation

With our transcriptomic analysis of mouse tendon cells, we listed the genes involved in tendon formations during development. The bioinformatic analysis of this list of genes allowed us to identify pathways important for tendon differentiation.

We also studied the implication of a transcription factor (identified from our list) in the tendon formation using chick and mouse models. We showed that this gene is important for the production of the collagen in developing tendons.

During development, muscle becomes attached to tendon through a structure called the myotendinous junction. Defects in the formation of this junction lead irreparably to a failure of the attachment of the muscle on the tendon and to a dysfunction of the musculo-skeletal system. The formation of this region is little informed. Using the model of the zebrafish, we showed that the extracellular protein, collagen XXII, which is exclusively expressed by the muscle during development, deposited at the myotendinous junction, plays a crucial role in the stabilization of the myotendinous junction and thus in the attachment of muscle to tendon. This collagen, which represents the first extracellular marker of the myotendinous junction will allow us to undertake mecanistic studies of the formation of this junction.

The identification of new molecular actors (transcription factors or secreted molecules) involved in tendon biology during the development is an important step for the understanding of mechanisms involved in tendon repair.

The collagen XXII represents the first real extracellular marker of the myotendinous junction, what allows us to approach mecanistic studies of the formation of this junction, but also to analyse the signals emanating from the muscle and/or from the tendon in its formation. The absence of collagen XXII during the development leads to the development of a severe muscular dystrophy. This result can have an impact in the field of the health because it positions the collagen XXII as a candidate gene in the cases of muscular dystrophies not solved.

We have identified the zinc finger transcription factor, Egr1, as being involved in tendon differentiation by regulating the transcription of tendon-associated collagens during chick and mouse development (Lejard and al., 2011). We have characterized one zebrafish orthologue of the gene col5a1 genes (Bretaud and al., 2011) and shown the implication of the Collagen V in the interface dermis-skin (Bonod-Bidaud and al., 2011). We are also interested in the formation of the myotendinous junction in zebrafish (Charvet and al., 2011, 2012).
We have established a technique allowing stable gene misexpression in chick embryos (Wang and al., 2011).

The proper development of the musculoskeletal system requires the coupled development of three tissues: muscle, tendon and cartilage/bone. In contrast to muscle and cartilage/bone tissues, tendons did not attract much attention from developmental biologists. Moreover, no treatment currently exists to restore injured tendon or ligament to its normal condition. Damaged tendons are observed following injuries but also in genetic diseases affecting extracellular matrix components such as Ehlers-Danlos syndromes, or in muscle dystrophies as a consequence of a muscle defect.
The major constituents of tendon are collagens, which provides structural support and strength of tendons. The molecules driving the synthesis of tendon collagens are not known. We shall establish the transcriptome of embryonic tendon cells, in order to identify the molecular network regulating collagen expression and organisation in tendons. We expect to identify new genes involved in tendon specification and differentiation.
During development, tendon differentiation relies on the presence of muscles, indicating the existence of muscle-tendon interactions. We have already identified candidate molecules involved in muscle and tendon formation that we are currently focusing on: the signalling pathways, Fgf (Fibroblast growth factor) and Bmp (Bone morphogenetic protein), the transcription factors, Egrs (Early growth response) and the collagens V, XII, XV and XXII.

This project aims to decipher the molecular aspects of muscle and tendon interactions and how growth factors, transcription factors and collagens (produced by muscles and/or tendons) integrate to form mature and functional tendons. We will follow in vivo strategies using the chick, zebrafish and mouse models to characterise tendon development and its interaction with muscle. Adult stem cells will be used in order to determine which molecules have the capacity to trigger collagen synthesis. One biochemical part will be to analyse the interactions between growth factors and collagens. The project aims at understanding the biomechanical and physiological properties of tendon and muscle and tendon interface from native tissues deficient for developmental genes (identified in the project). We expect that a better understanding of the molecular mechanisms underlying tendon development will open new perspectives for treatment of tendon injuries and genetic disorders affecting tendons directly or indirectly.