Analysis of a gain-of-function FGFR2 Crouzon mutation provides evidence of loss of function activity in the etiology of cleft palate

Abstract

Cleft palate is a common birth defect in humans and is a common phenotype associated with syndromic mutations in fibroblast growth factor receptor 2 (Fgfr2). Cleft palate occurred in nearly all mice homozygous for the Crouzon syndrome mutation C342Y in the mesenchymal splice form of Fgfr2. Mutant embryos showed delayed palate elevation, stage-specific biphasic changes in palate mesenchymal proliferation, and reduced levels of mesenchymal glycosaminoglycans (GAGs). Reduced levels of feedback regulators of FGF signaling suggest that this gain-of-function mutation in FGFR2 ultimately resembles loss of FGF function in palate mesenchyme. Knowledge of how mesenchymal FGF signaling regulates palatal shelf development may ultimately lead to pharmacological approaches to reduce cleft palate incidence in genetically predisposed humans.Crouzon syndrome | fibroblast growth factor receptor 2 | cell proliferation | cell surface receptor | glycosaminoglycan C raniosynostosis syndromes, as well as syndromic and nonsyndromic cleft palate, have been associated with fibroblast growth factor receptor (FGFR) mutations. Of the four highly conserved FGFRs, FGFR2 is the most commonly mutated FGF receptor (1-4). The FGF-FGFR family is involved in multiple intracellular signaling mechanisms in embryonic development, cell growth, wound healing, and tumorigenesis. The FGFRs are receptor tyrosine kinases that signal via a ternary complex of FGFR, FGF, and glycosaminoglycans (GAGs) (5). The ternary complex formation leads to receptor dimerization, autophosphorylation, and activation of downstream signaling cascades (6).FGF-FGFR signaling is active during palatogenesis, and genetic FGF mutations may contribute to 5% of cases of nonsyndromic cleft lip and palate (7). In children with isolated cleft palate, mutations in Fgfr1, Fgfr2, Fgfr3, and Fgf8 have been identified. Additionally, single nucleotide polymorphisms in children with nonsyndromic cleft lip and palate are associated with Fgf3, Fgf7, Fgf10, Fgf18, and Fgfr1, providing confirmation of the importance of the FGF-FGFR system in palate development (7). Knockout mouse models for Fgf10 and Fgfr2b develop cleft palate (8, 9), establishing the necessity of epithelial FGF signaling in normal palatogenesis. Cleft palate in Fgf18 −/− mice (10) suggests that mesenchymal FGF signaling may also be important for palate development.Cleft palate has been associated with both gain-of-function and loss-of-function FGFR mutations (7,(11)(12)(13)(14)(15). Why gain-of-function or loss-of-function mutations result in the same palatal phenotype remains unclear, and understanding this phenomenon may provide additional insight into the pathogenesis of cleft palate. To address this question, we have focused on Crouzon syndrome (CS), a disorder resulting from a missense Fgfr2 mutation that displays an increased incidence of cleft palate in humans (14).CS occurs with an incidence of 12.5 per million births and results from genetic gain-of-function mutations in Fgfr2 (1). Craniofacial anomalies are its hallmark...

Golgb1 regulates protein glycosylation and is crucial for mammalian palate development

“…Brinkley and Vickerman showed that reducing HA in palatal mesenchyme through pharmacological enhancement of its degradation inhibited palatal shelf elevation in organ culture as well as in vivo (Brinkley and Vickerman, 1982). A palatal shelf elevation defect in mice homozygous for the Fgfr2 C342Y missense mutation was also associated with reduced HA accumulation in the palatal mesenchyme (Snyder-Warwick et al, 2010). We found that Golgb1 mutant embryos exhibit reduced HA accumulation and concomitantly increased cell density in the palatal mesenchyme by E13.5.…”

“…We found that mouse embryos deficient in Golgb1 consistently exhibit failure of palatal shelf elevation. Although failure of palatal shelf elevation has been described in several other mutant mouse strains (Jiang et al, 1998;Jin et al, 2010;Lan et al, 2004;Matsumura et al, 2011;Snyder-Warwick et al, 2010;Yu et al, 2010), the molecular and cellular mechanisms regulating palate shelf elevation are still not well understood (Bush and Jiang, 2012;Ferguson, 1988). It has been proposed that an intrinsic force is progressively generated to drive palatal shelf elevation and the chief component of this intrinsic force appears to be regionally specific accumulation of extracellular GAGs, predominantly HA (Ferguson, 1988).…”

Regulation of Tbx22 during facial and palatal development

“…We have previously shown that during early chicken and mouse facial development, FGF receptor-1 (FGFR1) and at lower levels also FGF receptor-2 (FGFR2) are widely expressed in the facial mesenchyme (Bachler and Neubü ser, 2001;Firnberg and Neubü ser, 2002). Mice deficient in Fgfr1 develop facial and palatal clefts with incomplete penetrance, and all embryos lacking Fgfr1 and Fgfr2 in the palatal mesenchyme develop a cleft secondary palate, suggesting that these two receptors might function partially redundantly in transducing FGF signals to the facial mesenchyme (Snyder-Warwick et al, 2010;Trokovic et al, 2003). Whether Tbx22 expression is affected in Fgfr1/Fgfr2 double mutants, however, has so far not been analyzed.…”

Type 1 Fibroblast Growth Factor Receptor in Cranial Neural Crest Cell-derived Mesenchyme Is Required for Palatogenesis

“…Embryos bearing the Fgfr2C342Y mutant show delayed palate elevation and reduced levels of mesenchymal GAGs. Reduced levels of feedback regulators of FGF signaling suggest that this gain-offunction mutation in FGFR2 ultimately resembles loss of FGF function in the palate mesenchyme (15). Future work to characterize GAG compositions in Fgfr1 cKO palate shelves is needed to assess the possibility that loss of Fgfr1 in CNC-derived mesenchymal cells changes GAG compositions and impairs palate shelf elevation required for palate shelf fusion.…”

“…In addition, the FGF signaling intensity during palatogenesis is delicately balanced. Expression of a constitutively active FGFR2 mutant in the epithelium increases palatal shelf mesenchyme proliferation and delays elevation of the shelves, resulting in cleft palate and other craniofacial disorders (15,16). Disruption of Sprouty 2 (Spry2), a negative feedback regulator of FGF signaling pathways, results in abnormal palate formation (17).…”