Fibroblast growth factors (FGFs) comprise a large family of multifunctional, heparin-binding polypeptides that show diverse patterns of interaction with a family of receptors (FGFR1 to -4) that are subject to alternative splicing. FGFR binding specificity is an essential mechanism in the regulation of FGF signaling and is achieved through primary sequence differences among FGFs and FGFRs and through usage of two alternative exons, IIIc and IIIb, for the second half of immunoglobulin-like domain 3 (D3) in FGFRs. While FGF4 binds and activates the IIIc splice forms of FGFR1 to -3 at comparable levels, it shows little activity towards the IIIb splice forms of FGFR1 to -3 as well as towards FGFR4. To begin to explore the structural determinants for this differential affinity, we determined the crystal structure of FGF4 at a 1.8-Å resolution. FGF4 adopts a ␤-trefoil fold similar to other FGFs. To identify potential receptor and heparin binding sites in FGF4, a ternary FGF4-FGFR1-heparin model was constructed by superimposing the FGF4 structure onto FGF2 in the FGF2-FGFR1-heparin structure. Mutation of several key residues in FGF4, observed to interact with FGFR1 or with heparin in the model, produced ligands with reduced receptor binding and concomitant low mitogenic potential. Based on the modeling and mutational data, we propose that FGF4, like FGF2, but unlike FGF1, engages the ␤C-␤E loop in D3 and thus can differentiate between the IIIc and IIIb splice isoforms of FGFRs for binding. Moreover, we show that FGF4 needs to interact with both the 2-O-and 6-O-sulfates in heparin to exert its optimal biological activity.The fibroblast growth factor (FGF) family consists of 22 polypeptides (FGF1 to-22) with diverse biological activities (16,21,41). FGFs modulate proliferation and differentiation of a variety of cells of mesenchymal and neuroectodermal origin (1). FGFs play critical roles during embryonic processes such as mesoderm induction, postimplantation blastocyst development, and limb and lung development (7,40). Increased FGF signaling leads to a variety of human skeletal disorders, including dwarfism and craniosynostosis syndromes (15,19,39). In adult organisms, FGFs are thought to be involved in physiological angiogenesis and wound healing as well as in pathological angiogenesis, such as in tumor neovascularization and diabetic retinopathy (1).The diverse effects of FGFs are mediated by four receptor tyrosine kinases, FGFR1 to -4, which are composed of an extracellular ligand binding portion consisting of three immunoglobulin (Ig)-like domains (D1 to -3), a single transmembrane helix, and a cytoplasmic portion with protein tyrosine kinase activity. Ligand binding and specificity reside in D2, D3, and the short D2-D3 linker (29,30,34).Receptor dimerization is a prerequisite for FGF signaling and requires heparin or heparan sulfate proteoglycans (HSPGs) (22, 31). The recent crystal structure of a ternary FGF2-FGFR1-heparin complex has provided a mechanistic view of the process by which heparin aids FGFs to induce ...

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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.

FGF4 is the earliest member of the fibroblast growth factor (FGF) family expressed during embryogenesis where it plays essential roles in post-implantation development and limb growth and patterning. The expression of the Fgf4 gene in specific developmental stages, including the ICM of the blastocyst, the myotomes, and the limb bud AER, is regulated by distinct enhancer elements (Hom) in the 3' UTR. We previously identified the Hom3a region as the major DNA element responsible for Fgf4 expression in the myotomes and AER, and showed that a conserved E-box is a target for the myogenic bHLH transcription factors MYF5 and MYOD. To further define the cis- and trans-acting elements that determine Hom3a activity, we conducted a mutational analysis of the ability of the Hom3a region to drive lacZ expression in the myotomes of transgenic mice. We identified a minimal enhancer of 226nt that contains four elements, including the E-box, necessary to drive gene expression in the myotomes. One of these elements is a binding site for the GATA family of transcription factors, and we show here that GATA 1-4 and 6 can synergize with MYF5 or MYOD to activate transcription of a reporter plasmid driven by a portion of the Hom3a enhancer including the GATA site and the E-box. In line with this finding, we could show a direct interaction between MYF5/MYOD and GATA-3 or GATA-4, mediated by the N-terminal and bHLH domains of MYF5/MYOD and the C-terminal zing finger domain of GATA-3/4. To further study the role of the Hom3a enhancer in directing Fgf4 expression and the function of FGF4 in limb and muscle development, we generated mutant mice in which the Fgf4 Hom3a region had been deleted (Delta3a). In situ hybridization analysis of sections from Delta3a/ Delta3a embryos at E11.5 showed a drastically reduced expression of Fgf4 mRNA in the myotomes and AER. However, these mice developed normally and show no limb or muscle defects, and the same was true of heterozygous mice in which one Fgf4 allele carried the Hom3a deletion and the other was a null allele (Delta3a/Fgf4(-)). Together, these results show that Hom3a is the major DNA enhancer element directing Fgf4 expression in myotomes and limb bud AER, and that its activity in the myotomes results at least in part from the synergistic action of GATA and bHLH myogenic factors that bind to evolutionary conserved sequences in the Hom3a enhancer. However, expression of Fgf4 in the myotomes or AER of murine embryos does not appear to be essential for muscle or limb development.

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