Formation and role of functional domains within syncytial myofiber during muscle growth – MyoDom

The objective of MyoDom is to dissect the cellular mechanisms and molecular networks that govern the establishment and maintenance of discrete functional domains within the syncytial myofiber. Skeletal muscle is composed of multinucleated myofibers and muscle stem cells (MuSCs) in addition to non-myogenic cells such as connective tissue fibroblasts. The muscle developmental program involves the step of proliferation, differentiation and fusion of MuSC-derived myoblasts. During foetal and postnatal skeletal development, muscle growth relies on the fusion of millions of MuSC-derived myoblasts to form multinucleated myofibers, the functional unit responsible for muscle contraction. Along the myofiber, myonuclei are spatially specialized to define functional domains. Myonuclei in the center of myofibers are juxtaposed to the motor neurons to form neuromuscular junctions (NMJ), which initiate muscle contraction. Myonuclei at the tips of fibers interface with tendon cells to establish myotendinous junctions (MTJ) that buffer mechanical forces applied to the myofibers. The mechanisms underlying the establishment of functional domains of myonuclei are established during the fusion-mediated muscle growth and maintained during homeostasis remains largely unknown. We propose to dissect the cellular mechanisms and molecular networks that govern the establishment and maintenance of discrete functional domains within the myofibers.

As preliminary data, the partners identify a heterogeneity of myonuclei and MuSCs spatially associated with the MTJ and NMJ muscle domains. This heterogeneity is visible at a cellular level with a preferential location of myoblast fusion at the NMJ domain and with the unexpected recruitment of fibroblast nuclei at the MTJ domain. The cellular heterogeneity is associated with a regionalisation of gene expression in the MTJ (tip) and NMJ (central) muscle domains from development to adult. The 3 partners have generated (published and unpublished) single-nucleus RNA-sequencing (snRNAseq) datasets from limb tissues at foetal stages in chicken and from mouse muscles at postnatal and adult stages. The combination of all these snRNAseq datasets covering all stages constitutes a powerful source of information.
We will define the functional domains of a syncytium by combining mouse genetics, chicken electroporation, snRNAseq approaches, fluorescent in situ hybridization and RNAscope technologies. In WP1, we propose to analyse the cellular mechanisms underlying the emergence and maintenance of myonucleus domains. We will the contribution of different as a mean to generate and maintain distinct domains. We will look at the regionalization of myoblast fusion along the myofiber and cellular heterogeneity at the MTJ during muscle growth. We will also address the nuclear flux underlying the maintenance of muscle domains in homeostasis. In WP2, we will address the function of the regionalized dialogues between myofibers and adjacent cells. From snRNAseq datasets, we already identified candidate that were validated as being regionalized within MTJ or NMJ myonuclei. We will also examine new candidate genes linked to the establishment of the MTJ domain.

Skeletal muscle has been studied for many years with the assumption that myogenic cells are all alike. The originality of the project focuses on the cellular and molecular regionalization during muscle growth and homeostasis. We believe that the function of nucleus heterogeneity is medically relevant since major transcriptional modifications are observed in MTJ and NMJ myonuclei in pathological situations.