Early myotome specification regulates PDGFA expression and axial skeleton development

Reciprocal defects in signaling between the myotome and the sclerotome compartments of the somites in PDGFRalpha and Myf5 mutant embryos lead to alterations in the formation of the vertebrae and the ribs. To investigate the significance of these observations, we have examined the role of PDGF signaling in the developing somite. PDGFA ligand expression was not detected in the myotome of Myf5 null mutant embryos and PDGFA promoter activity was regulated by Myf5 in vitro. PDGFA stimulated chondrogenesis in somite micromass cultures as well as in embryos when PDGFA was knocked into the Myf5 locus, resulting in increased vertebral and rib development. PDGFA expression in the myotome was fully restored in embryos in which MyoD has been introduced at the Myf5 locus but to a lesser extent in similar myogenin knock-in embryos. These results underscore the importance of growth factor signaling within the developing somite and suggest an important role for myogenic determination factors in orchestrating normal development of the axial skeleton.     

The role of the notochord for epaxial myotome formation in the mouse.

The vertebrate somite is the source of all trunk skeletal muscles. Myogenesis in avian embryos is thought to depend on signals from notochord and neural tube for the epaxial muscles, and signals from lateral mesoderm and surface ectoderm for the hypaxial muscles. However, this hypothesis has to be tested because in mouse mutants lacking a notochord the presence of a fused myotome beneath the neural tube has been reported. We have compared the expression pattern of myogenic markers and markers for the hypaxial muscle precursors in the mutants Brachyury curtailed, truncate, Danforth's short tail and Pintail. In regions lacking notochord and sclerotome, we found small, ventrally located domains of Myf5 and MyoD expression, concomitant with ventrally expanded Pax3 signals and upregulated expression of the hypaxial marker Lbx1, suggesting that only the hypaxial program is active. We therefore hypothesise that in mammals, as in birds, the formation of the epaxial musculature depends on the presence of notochord derived signals.     

Different autonomous myogenic cell populations revealed by ablation of Myf5-expressing cells during mouse embryogenesis

The development of myogenic cells is mainly determined by expression of two myogenic factors, Myf5 and Myod1 (MyoD), which genetically compensate for each other during embryogenesis. Here, we demonstrate by conditional cell ablation in mice that Myf5 determines a distinct myogenic cell population, which also contains some Myod1-positive cells. Ablation of this lineage uncovers the presence of a second autonomous myogenic lineage, which superseded Myf5-dependent myogenic cells and expressed Myod1. By contrast, ablation of myogenin-expressing cells erased virtually all differentiated muscle cells, indicating that some aspects of the myogenic program are shared by most skeletal muscle cells. We conclude that Myf5 and Myod1 define different cell lineages with distinct contributions to muscle precursor cells and differentiated myotubes. Individual myogenic cell lineages seem to substitute for each other within the developing embryo.     

Sclerotome-derived Slit1 drives directional migration and differentiation of Robo2-expressing pioneer myoblasts

Pioneer myoblasts generate the first myotomal fibers and act as a scaffold to pattern further myotome development. From their origin in the medial epithelial somite, they dissociate and migrate towards the rostral edge of each somite, from which differentiation proceeds in both rostral-to-caudal and medial-to-lateral directions. The mechanisms underlying formation of this unique wave of pioneer myofibers remain unknown. We show that rostrocaudal or mediolateral somite inversions in avian embryos do not alter the original directions of pioneer myoblast migration and differentiation into fibers, demonstrating that regulation of pioneer patterning is somite-intrinsic. Furthermore, pioneer myoblasts express Robo2 downstream of MyoD and Myf5, whereas the dermomyotome and caudal sclerotome express Slit1. Loss of Robo2 or of sclerotome-derived Slit1 function perturbed both directional cell migration and fiber formation, and their effects were mediated through RhoA. Although myoblast specification was not affected, expression of the intermediate filament desmin was reduced. Hence, Slit1 and Robo2, via RhoA, act to pattern formation of the pioneer myotome through the regulation of cytoskeletal assembly.     

Developmental Plasticity of the Prospective Dermatome and the Prospective Sclerotome Region of an Avian Somite

The ventro-medial wall of a somite gives rise to the sclerotome and then to cartilaginous axial skeleton, while the dorso-lateral wall differentiates into the dermomyotome to form dermal mesenchyme and muscle. Although previous studies suggested pluri-potency of somite cell differentiation, apparent pluri-potency may be the result of migration of predetermined cells. To investigate whether the developmental fate of any region is determined, I isolated fragments of a region of a quail somite and transplanted them into chick embryos. When a fragment of the ventral wall of a quail somite, the prospective sclerotome, was transplanted into a chick embryo between the ectoderm and a newly formed somite, the transplanted quail cells were shown to form myotome and mesenchyme in 4-day chimera embryos and to form muscle and dermal tissue in 9-day chimeras. On the other hand, when a fragment of the dorsal wall of a quail somite, the prospective dermomyotome, was transplanted into a chick embryo between the neural tube and a newly formed somite, the graft gave rise to mesenchyme around the neural tube and notochord and then to vertebral cartilage. Thus the developmental fate of a region of a somite was shown not to be determined at the time of somite segmentation, confirming previous observations.     

Interactions between muscle fibers and segment boundaries in zebrafish

The most obvious segmental structures in the vertebrate embryo are somites: transient structures that give rise to vertebrae and much of the musculature. In zebrafish, most somitic cells give rise to long muscle fibers that are anchored to intersegmental boundaries. Therefore, this boundary is analogous to the mammalian tendon in that it transduces muscle-generated force to the skeletal system. We have investigated interactions between somite boundaries and muscle fibers. We define three stages of segment boundary formation. The first stage is the formation of the initial epithelial somite boundary. The second "transition" stage involves both the elongation of initially round muscle precursor cells and somite boundary maturation. The third stage is myotome boundary formation, where the boundary becomes rich in extracellular matrix and all muscle precursor cells have elongated to form long muscle fibers. It is known that formation of the initial epithelial somite boundary requires Notch signaling; vertebrate Notch pathway mutants show severe defects in somitogenesis. However, many zebrafish Notch pathway mutants are homozygous viable suggesting that segmentation of their larval and adult body plans at least partially recovers. We show that epithelial somite boundary formation and slow-twitch muscle morphogenesis are initially disrupted in after eight (aei) mutant embryos (which lack function of the Notch ligand, DeltaD); however, myotome boundaries form later ("recover") in a Hedgehog-dependent fashion. Inhibition of Hedgehog-induced slow muscle induction in aei/deltaD and deadly seven (des)/notch1a mutant embryos suggests that slow muscle is necessary for myotome boundary recovery in the absence of initial epithelial somite boundary formation. Because we have previously demonstrated that slow muscle migration triggers fast muscle cell elongation in zebrafish, we hypothesize that migrating slow muscle facilitates myotome boundary formation in aei/deltaD mutant embryos by patterning coordinated fast muscle cell elongation. In addition, we utilized genetic mosaic analysis to show that somite boundaries also function to limit the extent to which fast muscle cells can elongate. Combined, our results indicate that multiple interactions between somite boundaries and muscle fibers mediate zebrafish segmentation.     

Cooperative activity of noggin and gremlin 1 in axial skeleton development

Inductive signals from adjacent tissues initiate differentiation within the somite. In this study, we used mouse embryos mutant for the BMP antagonists noggin (Nog) and gremlin 1 (Grem1) to characterize the effects of BMP signaling on the specification of the sclerotome. We confirmed reduction of Pax1 and Pax9 expression in Nog mutants, but found that Nog;Grem1 double mutants completely fail to initiate sclerotome development. Furthermore, Nog mutants that also lack one allele of Grem1 exhibit a dramatic reduction in axial skeleton relative to animals mutant for Nog alone. By contrast, Pax3, Myf5 and Lbx1 expression indicates that dermomyotome induction occurs in Nog;Grem1 double mutants. Neither conditional Bmpr1a mutation nor treatment with the BMP type I receptor inhibitor dorsomorphin expands sclerotome marker expression, suggesting that BMP antagonists do not have an instructive function in sclerotome specification. Instead, we hypothesize that Nog- and Grem1-mediated inhibition of BMP is permissive for hedgehog (Hh) signal-mediated sclerotome specification. In support of this model, we found that culturing Nog;Grem1 double-mutant embryos with dorsomorphin restores sclerotome, whereas Pax1 expression in smoothened (Smo) mutants is not rescued, suggesting that inhibition of BMP is insufficient to induce sclerotome in the absence of Hh signaling. Confirming the dominant inhibitory effect of BMP signaling, Pax1 expression cannot be rescued in Nog;Grem1 double mutants by forced activation of Smo. We conclude that Nog and Grem1 cooperate to maintain a BMP signaling-free zone that is a crucial prerequisite for Hh-mediated sclerotome induction.     

A role for the myogenic determination gene Myf5 in adult regenerative myogenesis

The myogenic determination genes Myf5, Myod and Mrf4 direct skeletal muscle cell fate prenatally. In adult myogenesis, Myod has been shown to regulate myoblast differentiation, however, our understanding of satellite cell regulation is incomplete since the roles of Myf5 and Mrf4 had not been clearly defined. Here we examine the function of Myf5 and Mrf4 in the adult using recently generated alleles. Mrf4 is not expressed in normal or Myf5 null satellite cells and myoblasts, therefore excluding a role for this determination gene in adult muscle progenitors. Skeletal muscles of adult Myf5 null mice exhibit a subtle progressive myopathy. Crucially, adult Myf5 null mice exhibit perturbed muscle regeneration with a significant increase in muscle fibre hypertrophy, delayed differentiation, adipocyte accumulation, and fibrosis after freeze-injury. Satellite cell numbers are not significantly altered in Myf5 null animals and they show a modest impaired proliferation under some conditions in vitro. Mice double mutant for Myf5 and Dystrophin were more severely affected than single mutants, with enhanced necrosis and regeneration. Therefore, we show that Myf5 is a regulator of regenerative myogenesis and homeostasis, with functions distinct from those of Myod and Mrf4.