Tyrosine phosphorylation of FRS2 is prevented by mutations in the juxtamembrane domain of FGFR2. L6 myoblasts devoid of endogenous FGFR expressing WT Fgfr2c, Fgfr2c-LR, -C342Y, or -CLR mutants were used in these experiments (A–D). A FLAG tag was inserted to the C-terminal end of all of the receptors. Serum-starved L6 myoblasts were stimulated with FGF1 for 10 min at 37°C. The cell lysates were subjected to immunoprecipitation with anti-FGFR2 antibodies (A), anti-FRS2 antibodies (B), anti-Shc antibodies (C), or antiphospholipase-Cγ antibodies (D) followed by immunoblotting with anti-pTyr antibodies. Tyrosine phosphorylation of FRS2α (B), Shc (C), and phospholipase-Cγ (D) in lysates from L6 cells expressing WT Fgfr2c, Fgfr2c-LR, -C342Y, or -CLR mutants are shown. As a control, blots were also probed with anti-FLAG (A), anti-FRS2 (B), anti-Shc (C), or anti-PLCγ (D) antibodies. It was previously demonstrated that the different electrophoretic mobility of the C342Y mutant is caused by altered glycosylation pattern (9). Also, note that FGF1 stimulation does not enhance the activity of Fgfr2c-C342Y and -CLR mutants (A–D).

Frs2α-dependent and independent biological responses in Crouzon-like syndrome. (A–C) Coronal sections through the head showing intact hard palate of E18.5 embryos of WT mice (A) and cleft palates in Fgfr2c^C342Y/C342Y (B) and Fgfr2c^CLR/CLR mice (C). (D–F) Trachea showing the cartilaginous rings in WT mice (D), whereas in Fgfr2c^C342Y/C342Y (E) and Fgfr2c^CLR/CLR (F) mice, the cricoid is not separated from the tracheal cartilage. In the mutants, there is no separation of the tracheal cartilage into rings, and the trachea appears like a smooth cartilaginous tube. (G–I) Knee joints of E18.5 embryos of WT mice (G) are similar to the knee joints of Fgfr2c^CLR/CLR mutants (I), exhibiting normal development of the joint space. The joint spaces did not form in the Fgfr2c^C342Y/C342Y mice (H). (J–L) Normal elbow joint development in WT (J) and Fgfr2c^CLR/CLR mice (L). Fgfr2c^C342Y/C342Y mice (K) exhibit joint agenesis between the humerus and radius and secondary agenesis of the coronoid process and trochelar fossa in the ulna. C, coronoid process; Cr, cricoid cartilage; F, femur; Fi, fibula; Hu, humerus; HP, hard palate; N, nasal septum; O, olecranon process; P, patella; R, radius; T, tongue; Tc, tracheal cartilage rings; Ti, tibia; Tr, trochlear notch; Ty, thyroid cartilage; and U, ulna. Staining, Mason's trichrome (A–C and G–L); Safranin O (D–F). (Scale bars: 200 μm.)

Generation of targeting vector for Crouzon syndrome-like Fgfr2c mutant deficient in recruitment of FRS2α. (A) Schematic diagram showing the genomic structure of Fgfr2. The targeting vector contains 8.6 kb of the Fgfr2 genomic fragment, interrupted by the loxp-flanked mc1-neo gene inserted at the HindIII site of intron 9. A PGK-TK gene was inserted at the BamHI site of intron 10. Cys-342 was replaced by a Tyr residue in exon 9 (marked #). L424 and R426 were mutated to Alanine in exon 10 (marked ∗). The genomic portions used as probes for Southern blot analyses were indicated as X (3′ external probe) and Y (5′ internal probe). RI, EcoRI; H, HindIII; B, BamHI; S, SalI. (B) Sequences from exons 9 and 10 showing the site of mutations and the newly created restriction sites. (C) PCR analysis of the genotypes of newborn pups from the Fgfr2c^CLR/+ intercross. Arrows show the location of PCR primers. WT allele gives ≈225-bp product, and the recombinant allele gives ≈315-bp PCR product (M, CLR mutant).

Craniosynostosis is prevented by uncoupling Frs2α from Crouzon-like Fgfr2c mutant in Fgfr2c^CLR/+ mice. Photographs of the heads of 6-week-old live mice (A–C) and Alizarin-stained skulls (D–F). Note the open sutures in the WT and Fgfr2c^CLR/+ skulls (arrows); microCT scans showing 3D images of calvaria (G–I). Note completely fused coronal suture in Fgfr2c^C342Y/+ mutant mice (arrows) and open sutures in WT and Fgfr2c^CLR/+ mice. (J–L) 2D images of crosssection of coronal suture showing open suture of WT mice, fused suture of Fgfr2c^C342Y/+ mice (arrows) and open suture of Fgfr2c^CLR/+ mice. (M–O) Histological section of coronal sutures of 1-week-old mice stained with von Kossa and methyl green. Note the presence of bone trabeculae in the sutural mesenchyme in the Fgfr2c^C342Y/+ mutant. (Left) WT, (Center) Fgfr2c^C342Y/+, and (Right) Fgfr2c^CLR/+. C, coronal suture; S, sagittal suture; L, lambdoid suture; F, frontal suture; fb, frontal bone; pb, parietal bone; of, ossification front; me, mesenchyme; and Tb, bone trabecula. [Scale bars: 2 mm (D–I); 100 μm (M–O).]

Suture fusion in calvaria tissue explants is prevented by treatment with FGFR inhibitor. Calvaria harvested from E18.5-day-old embryos of WT (a–h) and Fgfr2c^C342Y/+ mutant (i–p) mice were cultured either with vehicle (0.2% DMSO) alone (a–d and i–l) or with 1 μM PLX052 (e–h and m–p) for 2 weeks. Histological sections of cultured calvaria were made perpendicular to the coronal suture, which passes through the frontal bone (fb) and the parietal bone (pb). Sections were stained with toluidine blue (a, c, e, g, i, k, m, and o), and adjacent sections were stained by von Kossa followed by methyl green counter staining (b, d, f, h, j, l, n, and p). Lower [c, d, g, h, k, l, o, and p (Scale bars: 100 μm)] depicts the higher magnification of the coronal sutures shown in the boxed regions of Upper [a, b, e, f, i, j, m, and n (Scale bars: 500 μm)], respectively. Open (arrow) and fused coronal sutures (arrowhead) are indicated. Fused sutures are observed in untreated calvaria from mutant mice (i–l), and open sutures are observed in calvaria from mutant mice treated with PLX052 (m–p).