Activation of Fgf4 Gene Expression in the Myotomes Is Regulated by Myogenic bHLH Factors and by Sonic Hedgehog

Abstract

The Fgf4 gene encodes an important signaling molecule which is expressed in specific developmental stages, including the inner cell mass of the blastocyst, the myotomes, and the limb bud apical ectodermal ridge (AER). Using a transgenic approach, we previously identified overlapping but distinct enhancer elements in the Fgf4 3' untranslated region necessary and sufficient for myotome and AER expression. Here we have investigated the hypothesis that Fgf4 is a target of myogenic bHLH factors. We show by mutational analysis that a conserved E box located in the Fgf4 myotome enhancer is required for Fgf4-lacZ expression in the myotomes. A DNA probe containing the E box binds MYF5, MYOD, and bHLH-like activities from nuclear extracts of differentiating C2-7 myoblast cells, and both MYF5 and MYOD can activate gene expression of reporter plasmids containing the E-box element. Analyses of Myf5 and MyoD knockout mice harboring Fgf4-lacZ transgenes show that Myf5 is required for Fgf4 expression in the myotomes, while MyoD is not, but MyoD can sustain Fgf4 expression in the ventral myotomes in the absence of Myf5. Sonic hedgehog (Shh) signaling has been shown to have an essential inductive function in the expression of Myf5 and MyoD in the epaxial myotomes, but not in the hypaxial myotomes. We show here that expression of an Fgf4-lacZ transgene in Shh-/- embryos is suppressed not only in the epaxial but also in the hypaxial myotomes, while it is maintained in the AER. This suggests that Shh mediates Fgf4 activation in the myotomes through mechanisms independent of its role in the activation of myogenic factors. Thus, a cascade of events, involving Shh and bHLH factors, is responsible for activating Fgf4 expression in the myotomes in a spatial- and temporal-specific manner.

Genetic analysis of interactions between the somitic muscle, cartilage and tendon cell lineages during mouse development

“…1C, blue and purple arrows). It has been previously shown that Myf5 and Myod1 are required for FGF expression in the mouse myotome (Fraidenraich et al, 2000;Grass et al, 1996), and that FGF signaling is essential for Scx induction in chick (Brent et al, 2003). Building on these findings, we decided to compare expression of Scx and the myotomal FGFs in mouse.…”

“…Interestingly, it appears that the events of specification of the somitic muscle progenitors and onset of myotomal FGF signaling are closely linked: in Myf5 -/-mutants, expression of the myotomal FGFs is delayed until induction of Myod1, and in Myf5 -/-; Myod1 -/-double mutants, expression is never initiated (Fraidenraich et al, 2000;Fraidenraich et al, 1998;Grass et al, 1996). Moreover, the dependency of FGF expression on induction of Myf5 and Myod1 appears to be direct.…”

“…Moreover, the dependency of FGF expression on induction of Myf5 and Myod1 appears to be direct. An Fgf4 myotomal enhancer element has been identified and found to contain E boxes binding Myf5 and Myod1, and it has been shown that these E boxes are required for Fgf4 expression in the myotome, and that an FGF4-lacZ transgene, driven by the myotome-specific enhancer, is not initiated in Myf5 -/-; Myod1 -/-mutants (Fraidenraich et al, 2000;Fraidenraich et al, 1998).…”

FGF acts directly on the somitic tendon progenitors through the Ets transcription factorsPea3andErmto regulate scleraxis expression

“…Interestingly, expression of Pea3, Erm, and Scx in the anterior and posterior sclerotome and dermomyotome appears to be a response to myotomal expression of Fgf8: while Fgf8 is expressed throughout somite development, Pea3, Erm and Scx only become restricted to their respective sclerotomal and dermomyotomal domains after Fgf8 has become localized in the myotome. In mouse, it has been shown that expression of FGFs in the myotome is directly controlled by a myotomespecific enhancer activated by the myogenic determinancy factors Myf5 and Myod; thus, it is only upon differentiation that the myofibers express FGFs (Fraidenraich et al, 2000;Grass et al, 1996). Additionally, there is evidence in both mouse and chick that sonic hedgehog signaling arising from the ventral midline is required for expression of the myotomal FGFs (Fraidenraich et al, 2000;Huang et al, 2003).…”

“…In mouse, it has been shown that expression of FGFs in the myotome is directly controlled by a myotomespecific enhancer activated by the myogenic determinancy factors Myf5 and Myod; thus, it is only upon differentiation that the myofibers express FGFs (Fraidenraich et al, 2000;Grass et al, 1996). Additionally, there is evidence in both mouse and chick that sonic hedgehog signaling arising from the ventral midline is required for expression of the myotomal FGFs (Fraidenraich et al, 2000;Huang et al, 2003).…”