Uncoupling fibroblast growth factor receptor 2 ligand binding specificity leads to Apert syndrome-like phenotypes.

D missense mutations in the genes encoding fibroblast growth factor receptors (FGFRs) 1–3 are the etiology of many craniosynostosis (premature fusion of the cranial sutures) and chondrodysplasia (dwarfism) syndromes (1–3). Mutations in Fgfr2 cause craniosynostosis syndromes including Crouzon syndrome, Pfeiffer syndrome, and Apert syndrome (AS). The article in this issue of PNAS by Hajihosseini et al. (4) presents the first animal model for a mutation in Fgfr2 that is associated with AS, one of the most severe of the human craniosynostosis syndromes. FGFRs are transmembrane receptor tyrosine kinase proteins that are activated by many of the 22 members of the FGF family (5, 6). The extracellular region of the FGFR contains two or three Ig-like domains and mediates ligand binding (7). In the presence of the cofactor, heparin, or heparan sulfate, ligand binding induces receptor dimerization and subsequent activation (5, 8–10). Importantly, the affinity and specificity of FGFRs are regulated by tissue-specific alternative splicing. The paper by Hajihosseini et al. (4), and two additional studies (11, 12), show that mutations that cause AS circumvent the biochemical and developmental regulatory mechanisms that are normally imposed by tissue-specific alternative splicing of Fgfr2 and result in ectopic ligand-dependent receptor activation. Alternative splicing in the region encoding the carboxyl-terminal half of Ig domain III creates receptor isoforms with distinct ligand binding specificity by incorporating either a b or a c exon (Fig. 1A). For FGFR2, this splicing event is very tissue-specific with b exon usage in epithelial tissue and c exon usage in mesenchymal tissue (13). Directional epithelialmesenchymal signaling is maintained because mesenchymally expressed ligands, such as FGF7 and FGF10, can activate only epithelially spliced FGFR2b. Similarly, ligands such as FGFs 2, 4, 6, 8, 9 and 17, which tend to be expressed in epithelial tissues activate mesenchymally spliced FGFR2c (14–16). The reciprocal nature of this signaling mechanism is most eloquently illustrated in the developing limb where FGF10 is a required mesenchymal signal that induces formation of the apical ectodermal ridge and FGF8 (and possibly FGFs 4, 9, and 17) is an epithelial factor that signals to distal mesenchyme (Fig. 1B) (17–21). The importance of FGFR2 signaling in organogenesis is illustrated by gene targeting studies. Mouse embryos lacking Fgfr2 die at stages before the limbs or lungs develop (22, 23). Embryos in which only the b isoform of Fgfr2 has been deleted survive until birth but also fail to develop limb buds, lung, and other organs (24). These phenotypes are remarkably similar to those seen in mice lacking Fgf10 (25–27) and demonstrate the importance of the mesenchymal to epithelial FGF signaling pathway. The effects of loss of FGFR2 signaling in mesenchymal tissues are obscured by the early embryonic lethality and the agenesis of the limbs and other structures in the null mutant. Biochemical studies show that many of these mutations result in ligand independent FGFR activation, often involving the formation of an intermolecular disulfide bond. AS, although allelic with other craniosynostosis syndromes, is much more severe and is characterized by premature fusion of the coronal sutures, severe syndactyly in the hands and feet, brain malformations, and mental retardation. The severe syndactyly and neurological disorders are not associated with other craniosynostosis syndromes. These phenotypic differences suggest that the signals transduced by receptors harboring AS mutations differ from those in other craniosynostosis syndrome mutations. Differences could lie in the intensity of the signal or in the spatial and temporal patterns of receptor activation. The vast majority of AS patients harbor one of two missense mutations (S252W or P253R) in the highly conserved region linking Ig domain II and III of FGFR2 (11, 28). Biochemical analysis revealed that these genetic alterations render the mesenchymally expressed mutant FGFR2c abnormally susceptible to activation by mesenchymally expressed ligands such as FGF7 and FGF10 and the epithelially expressed mutant FGFR2b abnormally susceptible to activation by epithelially expressed ligands such as FGF2, 6, and 9, thus circumventing the normal epithelialmesenchymal signaling restrictions (Fig. 1C) (12). The mechanism by which these mutations affect receptor signaling is fundamentally different from that of other mutations that activate FGFR2, which generally cause ligand independent receptor dimerization, stabilized by intermolecular disulfide bonds (1, 3). Recently Oldridge et al. (11) have identified two cases of AS (of 260) that do not have missense mutations in Fgfr2. Interestingly, these patients were found to have de novo Alu-insertions upstream or within the c exon (exon 9) of Fgfr2. Molecular analysis showed that these Alu insertions affect alternative splicing of Fgfr2, resulting in the ectopic expression of FGFR2b in tissues that normally would express FGFR2c (Fig. 1D). Thus mesenchymal tissue from these patients coexpresses both FGFR2c and FGFR2b. Interestingly, those authors also have identified patients with Pfeiffer syndrome with a mutation in FGFR2 that affects the 39 splice donor site of the c exon. The phenotype of these patients is more severe than that of Pfeiffer syndrome patients with different mutations in FGFR2 and suggests that alternative splicing may be affected and could contribute to the more severe phenotype. Hajihosseini et al. (4) have modeled these types of mutations in the mouse by specifically deleting the c exon of FGFR2