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Myofibres serve as the functional unit for locomotion, with the sarcomere as fundamental subunit. Running the entire length of this structure are hundreds of myonuclei, located at the periphery of the myofibre, juxtaposed to the plasma membrane. Myonuclear specialisation and clustering at the centre and ends of the fibre are known to be essential for muscle contraction, yet the molecular basis of this regionalisation has remained unclear. While the 'myonuclear domain hypothesis' helped explain how myonuclei can independently govern large cytoplasmic territories, novel technologies have provided granularity on the diverse transcriptional programs running simultaneously within the syncytia and added a new perspective on how myonuclei communicate. Building upon this, we explore the critical cellular and molecular sources of transcriptional and functional heterogeneity within myofibres, discussing the impact of intrinsic and extrinsic factors on myonuclear programs. This knowledge provides new insights for understanding muscle development, repair, and disease, but also opens avenues for the development of novel and precise therapeutic approaches. [ABSTRACT FROM AUTHOR]

Gene regulatory networks that act upstream of skeletal muscle fate determinants are distinct in different anatomical locations. Despite recent efforts, a clear understanding of the cascade of events underlying the emergence and maintenance of the stem cell pool in specific muscle groups remains unresolved and debated. Here, we invalidated Pitx2 with multiple Cre-driver mice prenatally, postnatally, and during lineage progression. We showed that this gene becomes progressively dispensable for specification and maintenance of the muscle stem (MuSC) cell pool in extraocular muscles (EOMs) despite being, together with Myf5, a major upstream regulator during early development. Moreover, constitutive inactivation of Pax7 postnatally led to a greater loss of MuSCs in the EOMs compared to the limb. Thus, we propose a relay between Pitx2, Myf5 and Pax7 for EOM stem cell maintenance. We demonstrate also that MuSCs in the EOMs adopt a quiescent state earlier that those in limb muscles and do not spontaneously proliferate in the adult, yet EOMs have a significantly higher content of Pax7+ MuSCs per area pre- and post-natally. Finally, while limb MuSCs proliferate in the mdx mouse model for Duchenne muscular dystrophy, significantly less MuSCs were present in the EOMs of the mdx mouse model compared to controls, and they were not proliferative. Overall, our study provides a comprehensive in vivo characterisation of MuSC heterogeneity along the body axis and brings further insights into the unusual sparing of EOMs during muscular dystrophy. Author summary: Skeletal myogenesis serves as a paradigm to study the mechanisms controlling stem cell commitment, proliferation, and differentiation. Understanding these mechanisms is crucial for addressing their dysregulation in various diseases and ageing. Although skeletal muscle is found throughout the body, differences in embryological origins, regenerative potential, and susceptibility to myopathies exist between head and trunk muscles. Notably, extraocular muscles (EOMs) demonstrate unique resilience in conditions like Duchenne muscular dystrophy. Most research on muscle stem cell (MuSC) biology has focused on limb muscles given their accessibility for experimentation. While the transcription factor Pitx2 plays a key role in EOM formation during embryonic development, the mechanisms governing the emergence and maintenance of MuSCs at this location remain unclear. Here, we demonstrate that MuSCs in the EOMs enter a quiescent state earlier than those in limb muscles and do not spontaneously proliferate in adulthood, challenging previous assumptions. Additionally, through genetic analyses, we elucidate the interplay between Pitx2, the MuSC marker Pax7, and the myogenic factor Myf5 in regulating extraocular MuSCs, thus refining our understanding of the genetic cascades that govern craniofacial myogenesis. [ABSTRACT FROM AUTHOR]

Author(s): Meryem B. Baghdadi [sup.1] [sup.2] [sup.3] , David Castel [sup.4] [sup.5] , Léo Machado [sup.6] , So-ichiro Fukada [sup.7] , David E. Birk [sup.8] , Frederic Relaix [sup.6] , [...], The cell microenvironment, which is critical for stem cell maintenance, contains both cellular and non-cellular components, including secreted growth factors and the extracellular matrix.sup.1-3. Although Notch and other signalling pathways have previously been reported to regulate quiescence of stem cells.sup.4-9, the composition and source of molecules that maintain the stem cell niche remain largely unknown. Here we show that adult muscle satellite (stem) cells in mice produce extracellular matrix collagens to maintain quiescence in a cell-autonomous manner. Using chromatin immunoprecipitation followed by sequencing, we identified NOTCH1/RBPJ-bound regulatory elements adjacent to specific collagen genes, the expression of which is deregulated in Notch-mutant mice. Moreover, we show that Collagen V (COLV) produced by satellite cells is a critical component of the quiescent niche, as depletion of COLV by conditional deletion of the Col5a1 gene leads to anomalous cell cycle entry and gradual diminution of the stem cell pool. Notably, the interaction of COLV with satellite cells is mediated by the Calcitonin receptor, for which COLV acts as a surrogate local ligand. Systemic administration of a calcitonin derivative is sufficient to rescue the quiescence and self-renewal defects found in COLV-null satellite cells. This study reveals a Notch-COLV-Calcitonin receptor signalling cascade that maintains satellite cells in a quiescent state in a cell-autonomous fashion, and raises the possibility that similar reciprocal mechanisms act in diverse stem cell populations.Muscle stem cell quiescence in mice is maintained by a Notch-Collagen V-CALCR signalling pathway that is activated and sustained in a cell-autonomous fashion.

Skeletal muscle stem cells (MuSCs) are recognised as functionally heterogeneous. Cranial MuSCs are reported to have greater proliferative and regenerative capacity when compared with those in the limb. A comprehensive understanding of the mechanisms underlying this functional heterogeneity is lacking. Here, we have used clonal analysis, live imaging and single cell transcriptomic analysis to identify crucial features that distinguish extraocular muscle (EOM) from limb muscle stem cell populations. A MyogeninntdTom reporter showed that the increased proliferation capacity of EOM MuSCs correlates with deferred differentiation and lower expression of the myogenic commitment gene Myod. Unexpectedly, EOM MuSCs activated in vitro expressed a large array of extracellular matrix components typical of mesenchymal non-muscle cells. Computational analysis underscored a distinct co-regulatory module, which is absent in limb MuSCs, as driver of these features. The EOM transcription factor network, with Foxc1 as key player, appears to be hardwired to EOM identity as it persists during growth, disease and in vitro after several passages. Our findings shed light on how high-performing MuSCs regulate myogenic commitment by remodelling their local environment and adopting properties not generally associated with myogenic cells. [ABSTRACT FROM AUTHOR]

Four SIX homeoproteins display a combinatorial expression throughout embryonic developmental myogenesis and they modulate the expression of the myogenic regulatory factors. Here, we provide a deep characterization of their role in distinct mouse developmental territories. We showed, at the hypaxial level, that the Six1:Six4 double knockout (dKO) somitic precursor cells adopt a smooth muscle fate and lose their myogenic identity. At the epaxial level, we demonstrated by the analysis of Six quadruple KO (qKO) embryos, that SIX are required for fetal myogenesis, and for the maintenance of PAX7+ progenitor cells, which differentiated prematurely and are lost by the end of fetal development in qKO embryos. Finally, we showed that Six1 and Six2 are required to establish craniofacial myogenesis by controlling the expression of Myf5. We have thus described an unknown role for SIX proteins in the control of myogenesis at different embryonic levels and refined their involvement in the genetic cascades operating at the head level and in the genesis of myogenic stem cells. Author summary: We demonstrate with double, triple and quadruple Six KO mouse embryos that specific Six combinations are required for proper myogenesis depending on the level of the mouse embryonic body axis. We show that the Six1 and Six2 genes are required for craniofacial myogenesis by controlling the engagement of unsegmented cranial paraxial mesodermal cells in the myogenic pathway. We also show that hypaxial somitic dermomyotomal cells from embryos mutant for the Six1 and Six4 genes are unable to engage in the skeletal muscle lineage. Last, we show that embryos mutant for the four Six genes expressed in the myogenic lineage exhibit a defect in self-renewal of PAX7+ stem cells present in their residual muscle masses, and that SIX proteins interact directly with several enhancer elements at the Pax7 locus to control its expression. We have thus characterized new functions of SIX proteins in the control of myogenesis at different embryonic levels and refined their involvement in the genetic cascades that govern the genesis of myogenic stem cells. [ABSTRACT FROM AUTHOR]