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.

Fig. 1.

Fgfr2 expression in the wild-type murine palate. (A–D) H&E stained coronal sections through the soft palate, posterior, middle and anterior regions of the pre-elevation E14.5 WT mouse palate. (A′–D′) Fgfr2 in situ hybridization (red) on adjacent sections. The greatest Fgfr2 expression is seen in the superomedial portion of the posterior palate (arrow). p, palate; t, tongue. (Scale bar: 200 μM.)

Fig. 2.

Palate development in WT, Fgfr2^C342Y/+, and Fgfr2^C342Y/C342Y mice. (A, D, and G) E13.5, (B, E, and H) E14.5, and (C, F, and I) E15.5 H&E stained coronal sections of developing mouse palates. Normal palate development consists of palate shelf outgrowth (A), elevation (B), and fusion (C), whereas development of the Fgfr2^C342Y/C342Y palate (G–I) is notable for narrower shelves, delayed elevation, and a cleft palate. Fgfr2^C342Y/+ palate development (D–F) includes a spectrum of phenotypes, such as delayed elevation and normal fusion, shown here. n, nasal cavity; o, oral cavity; p, palate; t, tongue. (Scale bar: 200 μM.)

Fig. 3.

Cellular proliferation in developing WT, Fgfr2^C342Y/+, and Fgfr2^C342Y/C342Y palates. (A) Palatal shelf division into four regions to quantify BrdU staining. (B–D) At E13.5, Fgfr2^C342Y/C342Y palates (C) have increased proliferation in regions III and IV compared with WT (B) palates (D; a, P < 0.004; b, P < 0.02, respectively; n = 4 or more for all genotypes). (E) At E14.5, Fgfr2^C342Y/C342Y show decreased BrdU incorporation compared to WT palates throughout the palate mesenchyme (a, P < 0.02 region I; b, P < 0.002 region II; c, P < 0.0007 region III; d, P < 0.02 region IV; n = 4 for all genotypes). Black bars, WT; gray bars, Fgfr2^C342Y/+; white bars, Fgfr2^C342Y/C342Y.

Fig. 4.

Glycosaminoglycan accumulation, composition, and synthesis in developing WT, Fgfr2^C342Y/+, and Fgfr2^C342Y/C342Y palates. (A) GAG accumulation during palate development (a, P < 0.001; b, P < 0.02; c, P < 0.001). (B) Compared with WT, Fgfr2^C342Y/C342Y palates have less total GAG accumulation in the anterior (a, P < 0.05) and posterior (b, P < 0.02) regions at E14.25. (C–E) WT (C) and Fgfr2^C342Y/+ (D) palates show increased Alcian blue staining compared with Fgfr2^C342Y/C342Y (E) in the posterior region at E14.5 (representative of three embryos for each genotype). (F) Decreased GalN composition is seen in both regions of the Fgfr2^C342Y/C342Y palate at E14.25 compared with WT (a, P < 0.04; b, P < 0.02). (G) GalN/GlcN ratio in whole palatal shelves. All genotypes have a decreased ratio of GalN/GlcN in the whole palate just before elevation (E14.25; a, P < 0.001), and the Fgfr2^C342Y/C342Y GalN/GlcN ratio is further decreased compared with WT at E14.25 (b, P < 0.01). At E14.5, all genotypes show an increased GalN/GlcN ratio (c, P < 0.001), and the Fgfr2^C342Y/C342Y GalN/GlcN ratio is further increased compared with WT (d, P < 0.02). (H) Has2 expression was decreased in the posterior region of the Fgfr2^C342Y/C342Y palate compared with WT at E14.25 (a, P < 0.05). Black bars, WT; gray bars, Fgfr2^C342Y/+; white bars, Fgfr2^C342Y/C342Y.

Fig. 5.

FGF signaling and palate development: In vitro FGF signaling manipulation in WT palate and qPCR studies in WT and Fgfr2^C342Y/C342Y posterior palates. (A) WT palate explant cultures treated with FGF2 (1 μg/mL) or PD173074 (2 μM) exhibited cleft palate (arrows) compared with the fused palates of untreated and DMSO-treated control palates. (B) Compared with WT, the posterior palates of Fgfr2^C342Y/C342Y mice exhibit a 2-fold increase in relative expression of Fgfr2c at E15.5 (a, P < 0.02; n = 4 or more), but no differences at earlier time points. (C) Barx1 expression in the Fgfr2^C342Y/C342Y posterior palate increases greater than 2.5-fold (a, P < 0.05) at E14.25 (n = 7 or more). (D) Expression of Spry2 is decreased in Fgfr2^C342Y/C342Y mouse compared with WT at E15.5 (a, P < 0.04, n = 4 or more). (E) Expression of Spry4 is decreased throughout palate development (a, P < 0.01; b, P < 0.01; c, P < 0.03; n = 4 or more). Black bars, WT; white bars, Fgfr2^C342Y/C342Y.