The contribution of connective tissues to muscle morphogenesis : an unexpected role for CXCL12 and CXCL14 chemokines

Skeletal muscle development mostly relies on intrinsic capacities of muscle progenitors to proliferate and differentiate. However, extrinsic signals arising from non-myogenic cells also contribute to the establishment of functional skeletal muscles. The aim of this PhD project was to investigate the role of connective-tissue (CT) on the development of skeletal muscle, using the chick embryonic limb as a model. We particularly investigated the influence of the two chemokines CXCL12 and CXCL14, which have been previously shown as expressed in limb mesenchyme giving rise to the different types of CTs during development. The involvement of CXCL12 and CXCL14 in limb CT differentiation was studied, as well as the role of these chemokines in skeletal muscle development mediated by CT. We first showed that CXCL12 and CXCL14 display distinct restricted expression patterns in limb CT of chick embryos and demonstrated that CXCL12 promotes the expression of OSR1, OSR2 and COL3A1 genes, three markers of irregular CT, while CXCL14 enhances the expression of a regular CT gene, SCX. In addition, the expression of CXCL12, CXCL14 and their putative CT target genes were all negatively regulated by the anti-fibrotic BMP signalling, but also in the absence of musculoskeletal mechanical forces. These results show for the first time the involvement of CXCL12 and CXCL14 chemokines in the differentiation of CTs. The putative role of both chemokines on CT-mediated myogenesis was then analysed. We observed that CXCR7, one CXCL12 receptor, was expressed both in muscle progenitors and differentiated muscle cells in embryonic chick limbs. Using gain- and loss-of-function approaches in primary cultures of chick limb myoblasts, we revealed that CXCR7 promoted myogenesis by regulating muscle cell fusion, while CXCL12 did not influence muscle differentiation. CXCL14 dramatically inhibits in vitro myogenesis. Functional assays performed in chick embryonic forelimbs in vivo demonstrate that overexpression of CXCL12, CXCR7 or a dominant-negative form of CXCR7 all resulted in abnormal and mispatterned muscles in chick limbs. Similarly, CXCL14 overexpression in chick limb in vivo led to profound anomalies in muscle differentiation. All together, our results demonstrate an essential contribution of CXCL12 and CXCL14 chemokines in CT differentiation and in CTmediated muscle development in embryonic limb.