Notch signalling acts in postmitotic avian myogenic cells to control MyoD activation

During Drosophila myogenesis, Notch signalling acts at multiple steps of the muscle differentiation process. In vertebrates, Notch activation has been shown to block MyoD activation and muscle differentiation in vitro, suggesting that this pathway may act to maintain the cells in an undifferentiated proliferative state. In this paper, we address the role of Notch signalling in vivo during chick myogenesis. We first demonstrate that the Notch1 receptor is expressed in postmitotic cells of the myotome and that the Notch ligands Delta1 and Serrate2 are detected in subsets of differentiating myogenic cells and are thus in position to signal to Notch1 during myogenic differentiation. We also reinvestigate the expression of MyoD and Myf5 during avian myogenesis, and observe that Myf5 is expressed earlier than MyoD, consistent with previous results in the mouse. We then show that forced expression of the Notch ligand, Delta1, during early myogenesis, using a retroviral system, has no effect on the expression of the early myogenic markers Pax3 and Myf5, but causes strong down-regulation of MyoD in infected somites. Although Delta1 overexpression results in the complete lack of differentiated muscles, detailed examination of the infected embryos shows that initial formation of a myotome is not prevented, indicating that exit from the cell cycle has not been blocked. These results suggest that Notch signalling acts in postmitotic myogenic cells to control a critical step of muscle differentiation.     

Ectodermal Wnt-6 promotes Myf5-dependent avian limb myogenesis

Limb muscles of vertebrates are derived from precursor cells that migrate from the lateral edge of the dermomyotome into the limb bud. Although several signaling molecules have been reported to be involved in the process of limb myogenesis, none of their activities has led to a consolidate idea about the limb myogenic pathway. Particularly, the role of ectodermal signals in limb myogenesis is still obscure. Here, we investigated the role of the ectoderm and ectodermal Wnt-6 during limb muscle development. We found that ectopic expression of Wnt-6 in the limb bud specifically extends the expression domains of Pax3, Paraxis, Myf5, Myogenin, Desmin and Myosin heavy chain (MyHC) but inhibits MyoD expression. Ectoderm removal results in a loss of expression of all of these myogenic markers. We show that Wnt-6 can compensate the absence of the ectoderm by rescuing the expression of Pax3, Paraxis, Myf5, Myogenin, Desmin and MyHC but not MyoD. These results show that, in chick, at least two signals from the limb ectoderm are necessary for muscle development. One of the signals is Wnt-6, which plays a unique role in promoting limb myogenesis via Pax3/Paraxis-Myf5, whereas the other putative signaling pathway involving MyoD expression is negatively regulated by Wnt-6 signaling.     

Divergent and conserved roles of Dll1 signaling in development of craniofacial and trunk muscle

Craniofacial and trunk skeletal muscles are evolutionarily distinct and derive from cranial and somitic mesoderm, respectively. Different regulatory hierarchies act upstream of myogenic regulatory factors in cranial and somitic mesoderm, but the same core regulatory network – MyoD, Myf5 and Mrf4 – executes the myogenic differentiation program. Notch signaling controls self-renewal of myogenic progenitors as well as satellite cell homing during formation of trunk muscle, but its role in craniofacial muscles has been little investigated. We show here that the pool of myogenic progenitor cells in craniofacial muscle of Dll1^LacZ/Ki mutant mice is depleted in early fetal development, which is accompanied by a major deficit in muscle growth. At the expense of progenitor cells, supernumerary differentiating myoblasts appear transiently and these express MyoD. The progenitor pool in craniofacial muscle of Dll1^LacZ/Ki mutants is largely rescued by an additional mutation of MyoD. We conclude from this that Notch exerts its decisive role in craniofacial myogenesis by repression of MyoD. This function is similar to the one previously observed in trunk myogenesis, and is thus conserved in cranial and trunk muscle. However, in cranial mesoderm-derived progenitors, Notch signaling is not required for Pax7 expression and impinges little on the homing of satellite cells. Thus, Dll1 functions in satellite cell homing and Pax7 expression diverge in cranial- and somite-derived muscle.     

FGFR4 signaling is a necessary step in limb muscle differentiation

In chick embryos, most if not all, replicating myoblasts present within the skeletal muscle masses express high levels of the FGF receptor FREK/FGFR4, suggesting an important role for this molecule during myogenesis. We examined FGFR4 function during myogenesis, and we demonstrate that inhibition of FGFR4, but not FGFR1 signaling, leads to a dramatic loss of limb muscles. All muscle markers analyzed (such as Myf5, MyoD and the embryonic myosin heavy chain) are affected. We show that inhibition of FGFR4 signal results in an arrest of muscle progenitor differentiation, which can be rapidly reverted by the addition of exogenous FGF, rather than a modification in their proliferative capacities. Conversely, over-expression of FGF8 in somites promotes FGFR4 expression and muscle differentiation in this tissue. Together, these results demonstrate that in vivo, myogenic differentiation is positively controlled by FGF signaling, a notion that contrasts with the general view that FGF promotes myoblast proliferation and represses myogenic differentiation. Our data assign a novel role to FGF8 during chick myogenesis and demonstrate that FGFR4 signaling is a crucial step in the cascade of molecular events leading to terminal muscle differentiation.     

Notch signaling imposes two distinct blocks in the differentiation of C2C12 myoblasts

Notch signal transduction regulates expression of downstream genes through the activation of the DNA-binding protein Su(H)/CBF1. In Drosophila most of Notch signaling requires Su(H); however, some Notch-dependent processes occur in the absence of Su(H) suggesting that Notch signaling does not always involve activation of this factor. Using constitutively active forms of Notch lacking CBF1-interacting sequences we identified a Notch signaling pathway that inhibits myogenic differentiation of C2C12 myoblasts in the absence of CBF1 activation. Here we show that ligand-induced Notch signaling suppresses myogenesis in C2C12 myoblasts that express a dominant negative form of CBF1, providing additional evidence for CBF1-independent Notch signal transduction. Surprisingly mutant forms of Notch deficient in CBF1 activation are unable to antagonize MyoD activity, despite the fact that they inhibit myogenesis. Moreover, Notch-induced antagonism of MyoD requires CBF1 suggesting that the CBF1-dependent pathway mediates a cell-type-specific block in the myogenic program. However, Notch signaling in the absence of CBF1 activation blocks both myogenesis and osteogenesis, indicative of a general block in cellular differentiation. Taken together our data provide evidence for two distinct Notch signaling pathways that function to block differentiation at separate steps during the process of myogenesis in C2C12 myoblasts.     

Altered myogenesis in Six1-deficient mice

Six homeoproteins are expressed in several tissues, including muscle, during vertebrate embryogenesis, suggesting that they may be involved in diverse differentiation processes. To determine the functions of the Six1 gene during myogenesis, we constructed Six1-deficient mice by replacing its first exon with the lacZ gene. Mice lacking Six1 die at birth because of severe rib malformations and show extensive muscle hypoplasia affecting most of the body muscles in particular certain hypaxial muscles. Six1(-/-) embryos have impaired primary myogenesis, characterized, at E13.5, by a severe reduction and disorganisation of primary myofibers in most body muscles. While Myf5, MyoD and myogenin are correctly expressed in the somitic compartment in early Six1(-/-) embryos, by E11.5 MyoD and myogenin gene activation is reduced and delayed in limb buds. However, this is not the consequence of a reduced ability of myogenic precursor cells to migrate into the limb buds or of an abnormal apoptosis of myoblasts lacking Six1. It appears therefore that Six1 plays a specific role in hypaxial muscle differentiation, distinct from those of other hypaxial determinants such as Pax3, cMet, Lbx1 or Mox2.     

Myf5 and MyoD activation define independent myogenic compartments during embryonic development

Gene targeting has indicated that Myf5 and MyoD are required for myogenic determination because skeletal myoblasts and myofibers are missing in mouse embryos lacking both Myf5 and MyoD. To investigate the fate of Myf5:MyoD-deficient myogenic precursor cells during embryogenesis, we examined the sites of epaxial, hypaxial, and cephalic myogenesis at different developmental stages. In newborn mice, excessive amounts of adipose tissue were found in the place of muscles whose progenitor cells have undergone long-range migrations as mesenchymal cells. Analysis of the expression pattern of Myogenin-lacZ transgene and muscle proteins revealed that myogenic precursor cells were not able to acquire a myogenic fate in the trunk (myotome) nor at sites of MyoD induction in the limb buds. Importantly, the Myf5-dependent precursors, as defined by Myf5(nlacZ)-expression, deficient for both Myf5 and MyoD, were observed early in development to assume nonmuscle fates (e.g., cartilage) and, later in development, to extensively proliferate without cell death. Their fate appeared to significantly differ from the fate of MyoD-dependent precursors, as defined by 258/-2.5lacZ-expression (-20 kb enhancer of MyoD), of which a significant proportion failed to proliferate and underwent apoptosis. Taken together, these data strongly suggest that Myf5 and MyoD regulatory elements respond differentially in different compartments.     

Wnt signalling regulates myogenic differentiation in the developing avian wing

The limb musculature arises by delamination of premyogenic cells from the lateral dermomyotome. Initially the cells express Pax3 but, upon entering the limb bud, they switch on the expression of MyoD and Myf5 and undergo terminal differentiation into slow or fast fibres, which have distinct contractile properties that determine how a muscle will function. In the chick, the premyogenic cells express the Wnt antagonist Sfrp2, which is downregulated as the cells differentiate, suggesting that Wnts might regulate myogenic differentiation. Here, we have investigated the role of Wnt signalling during myogenic differentiation in the developing chick wing bud by gain- and loss-of-function studies in vitro and in vivo. We show that Wnt signalling changes the number of fast and/or slow fibres. For example, in vivo, Wnt11 decreases and increases the number of slow and fast fibres, respectively, whereas overexpression of Wnt5a or a dominant-negative Wnt11 protein have the opposite effect. The latter shows that endogenous Wnt11 signalling determines the number of fast and slow myocytes. The distinct effects of Wnt5a and Wnt11 are consistent with their different expression patterns, which correlate with the ultimate distribution of slow and fast fibres in the wing. Overexpression of activated calmodulin kinase II mimics the effect of Wnt5a, suggesting that it uses this pathway. Finally, we show that overexpression of the Wnt antagonist Sfrp2 and DeltaLef1 reduces the number of myocytes. In Sfrp2-infected limbs, the number of Pax3 expressing cells was increased, suggesting that Sfrp2 blocks myogenic differentiation. Therefore, Wnt signalling modulates both the number of terminally differentiated myogenic cells and the intricate slow/fast patterning of the limb musculature.     

In vitro indeterminate teleost myogenesis appears to be dependent on Pax3

The zebrafish (Danio rerio) has been used extensively as a model system for developmental studies but, unlike most teleost fish, it grows in a determinate-like manner. A close relative, the giant danio (Devario cf. aequipinnatus), grows indeterminately, displaying both hyperplasia and hypertrophy of skeletal myofibers as an adult. To better understand adult muscle hyperplasia, a postlarval/postnatal process that closely resembles secondary myogenesis during development, we characterized the expression of Pax3/7, c-Met, syndecan-4, Myf5, MyoD1, myogenin, and myostatin during in vitro myogenesis, a technique that allows for the complete progression of myogenic precursor cells to myotubes. Pax7 appears to be expressed only in newly activated MPCs while Pax3 is expressed through most of the myogenic program, as are c-Met and syndecan-4. MyoD1 appears important in all stages of myogenesis, while Myf5 is likely expressed at low to background levels, and myogenin expression is enriched in myotubes. Myostatin, like MyoD1, appears to be ubiquitous at all stages. This is the first comprehensive report of key myogenic factor expression patterns in an indeterminate teleost, one that strongly suggests that Pax3 and/or Myf5 may be involved in the regulation of this paradigm. Further, it validates this species as a model organism for studying adult myogenesis in vitro, especially mechanisms underlying nascent myofiber recruitment.