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Pownall ME, Isaacs HV. FGF Signalling in Vertebrate Development. San Rafael (CA): Morgan & Claypool Life Sciences; 2010.

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FGF Signalling in Vertebrate Development.

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FGF and Myogenesis

Skeletal muscle is derived from precursor cells called myoblasts that have been studied extensively in tissue culture over many years (Konigsberg, 1963). Myoblasts taken from embryos replicate clonally in culture and when the amount of growth factors in the media is reduced they differentiate. To do this, myoblasts (1) stop dividing, (2) express contractile protein genes (such as myosin, actin, and troponin, etc.), and (3) undergo cell–cell fusion to form multinucleated myofibres. The key growth factor repressing myogenic differentiation in these cultures was found to be FGF (Clegg et al., 1987). However, distinct clones of primary myoblasts were found to respond differently to FGF treatment: in some, FGF delayed differentiation, as predicted, while in other cases, FGF was found to be required for differentiation (Seed and Hauschka, 1988). In addition to these effects of FGF on cultured muscle cells, the expression of FGF ligands and receptors in the developing muscle of vertebrate embryos suggests that FGF signalling plays a role during skeletal myogenesis in vivo.

As already discussed, somites form sequentially from the posterior mesoderm adjacent to the neural tube and notochord. All skeletal muscles in the body originate from the somites and the earliest cells committed to the myogenic lineage are found in the medial part of the newly formed somite. These cells are discernible by their expression of one or more of the genes coding for the myogenic regulatory factors (MRFs). The MRFs, which include the genes myoD and myf5, are not only the earliest markers of skeletal muscle progenitors but are also essential regulators initiating the myogenic programme; they code for basic helix–loop–helix transcription factors that act as a developmental switch to drive a cell down the myogenic lineage (Weintraub, 1993). Some of these MRF expressing somite cells will differentiate in situ and form the deep muscles of the back; however, most skeletal muscle arise from myoblasts that have migrated away from the somites to populate, for instance, the abdomen and the limb. The migrating myoblasts do not express any MRF; however, they do express another transcription factor important for myogenesis called Pax3 (Buckingham and Relaix, 2007). Pax3, and the related gene Pax7, is expressed in migrating myoblasts but it is not until these cells populate the limb that they begin to express myf5 and myoD.

FGF signalling has been found to be an important regulator of myogenesis in the limb. The myogenic cells in the limb that express myoD and myf5 are also known to express FGF-R4 (Marcelle et al., 1995). Electroporation of a secreted, dominant negative form of FGF-R4 into chick limb buds interferes with muscle differentiation as shown by a down-regulation of myoD expression and myosin heavy chain protein (Marics et al., 2002). FGF-R4 also has been implicated as a myogenic regulator in somites. A screen for genes regulated by Pax3 in mouse embryos identified FGF-R4 as one transcriptional target that is dependent on Pax3 for its expression in the somites. Furthermore, this work found that the negative regulator of FGF signalling, Sprouty1, is also directly regulated by Pax3 (Lagha et al., 2008). Inhibition of FGF signalling by overexpressing Sprouty in limb muscles resulted in an increase in progenitors as compared to MRF expressing muscle cells, suggesting a role for FGF in regulating the balance between proliferation and differentiation. However, unlike the findings in tissue culture where FGF (for the most part) promotes the proliferation of myogenic precursors such that FGF must be inhibited to allow skeletal muscle differentiation, the findings of these in vivo studies suggest that FGF activity promotes myogenic differentiation.

In zebrafish, FGF8 is expressed in the anterior part of the early somites, while myoD is expressed in the posterior somites as well as in the medial, adaxial cells that line up adjacent to the midline structures (the neural tube and notochord). The adaxial cells are dependent on hedgehog signalling and will go on to form slow muscle fibres; some of these cells remain medial and express engrailed, while some will migrate from the midline to the most lateral part of the somite before differentiating. The rest of the cells in the lateral somite will form fast muscle fibres and this is dependent on FGF8 signalling (Groves et al., 2005). acerebellar (ace) mutant zebrafish, which carry a mutation in FGF8 or fish injected with an AMO directed against FGF8, or treated with the FGF inhibitor SU5402, all have reduced levels of myoD expression in the lateral somite. Interestingly, the levels of Pax3/7 expression were found to be higher in zebrafish lacking FGF8 signalling, suggesting that in the absence of FGF8 and myoD, these cells stay in a progenitor state (Hammond et al., 2007).

The analysis of growth factors that activate myogenesis in Xenopus was accelerated by mesoderm induction studies (see section on Mesoderm Induction); indeed, the first molecular marker used in these assays was skeletal muscle actin (Gurdon et al., 1989). In the amphibian embryo, mesoderm is induced at the equator of the blastula embryo in response to nodal signals emanating from the vegetal hemisphere. Subsequent to induction, mesodermal cells move inside the embryo during gastrulation and eventually give rise to a wide spectrum of tissue types including skeletal muscle. Several FGF ligands are expressed very early in the nascent mesoderm and in the somites (Isaacs et al., 1995; Isaacs et al., 1992; Lea et al., 2009).

Nodal activity nodal is mimicked by the related TGF-β family protein activin. The expression of FGF4 (eFGF) is an immediate early response to activin (Fisher, 2002) and is essential for the activation of myoD expression in the mesoderm (Figure 13). Moreover, it has been demonstrated that a functional FGF signalling pathway is required for activin to induce mesoderm (Cornell and Kimelman, 1994). Explants expressing a dominant negative FGFR cannot form mesoderm in response to activin, despite there being no defect in the activin signalling pathway. Activin can induce myoD expression in animal caps, but this requires protein synthesis (Hopwood et al., 1989); very likely this reflects the requirement for the production of FGF ligands to activate many mesodermal genes. Explants with impaired FGF signalling do not activate myoD expression in response to activin signals (Cornell and Kimelman, 1994). However, FGF4 induces myoD expression in animal caps in the absence of protein synthesis and is required for myoD expression in vivo (Fisher, 2002). These data put FGF4 at the head of the myogenic programme in Xenopus. There is some evidence that the molecular mechanism underlying the induction of myoD expression by FGF could be through MAPK-dependent inhibition of a transcriptional repressor such as Hes6 or Groucho (Burks et al., 2009; Murai et al., 2007).

FIGURE 13. Early mesodermal expression of MyoD requires FGF4.

FIGURE 13

Early mesodermal expression of MyoD requires FGF4. The embryo on the left is a control Xenopus gastrula (NF stage 10.5) showing the expression of MyoD in the mesoderm around the blastopore. The embryos on the right have been injected into one cell at (more...)

In Xenopus, myoD mRNA and protein are present at high levels in the myogenic lineage before differentiation (Hopwood et al., 1989; Hopwood et al., 1992). In addition, a single myogenic precursor cell taken from a late gastrula embryo behaves as a determined myoblast: when transplanted to a ventral region, it differentiates as skeletal muscle (Kato and Gurdon, 1993). Therefore, these cells in the early mesoderm of the Xenopus gastrula represent a population skeletal muscle precursor cells. In amniotes, a similar pool of muscle progenitors is present in the medial edge of the dermomyotome as determined by lineage labeling (Denetclaw et al., 1997; Pownall et al., 2002). To maintain the stable expression of myoD and myf5, these precursor cells need to remain in contact with each other; this interaction is known as a “community effect” and is thought to help groups of muscle precursor cells coordinate their differentiation (Gurdon, 1988). When mesoderm cells are explanted from Xenopus gastrulae and cultured as single cells, they extinguish the expression of myoD and myf5, while intact explants or reaggregated cells express normal levels of the MRF genes. Adding FGF4 to the dispersed cell cultures rescues the expression of myoD and myf5 (Standley et al., 2001); this result, together with the coexpression of MRF genes and several FGF ligands, suggests that the community effect is mediated by FGF signalling.

Copyright © 2010 by Morgan & Claypool Life Sciences.
Bookshelf ID: NBK53152

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