A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis

Abstract

The b and c variants of fibroblast growth factor receptor 2 (FGFR2) differ in sequence, binding specificity, and localization. Fgfr2b, expressed in epithelia, is required for limb outgrowth and branching morphogenesis, whereas the mesenchymal Fgfr2c variant is required by the osteocyte lineage for normal skeletogenesis. Gain-of-function mutations in human FGFR2c are associated with craniosynostosis syndromes. To confirm and extend this evidence, we introduced a Cys342Tyr replacement into Fgfr2c to create a gain-of-function mutation equivalent to a mutation in human Crouzon and Pfeiffer syndromes. Fgfr2c(C342Y/)(+) heterozygote mice are viable and fertile with shortened face, protruding eyes, premature fusion of cranial sutures, and enhanced Spp1 expression in the calvaria. Homozygous mutants display multiple joint fusions, cleft palate, and trachea and lung defects, and die shortly after birth. They show enhanced Cbfa1/Runx2 expression without significant change in chondrocyte-specific Ihh, PTHrP, Sox9, Col2a, or Col10a gene expression. Histomorphometric analysis and bone marrow stromal cell culture showed a significant increase of osteoblast progenitors with no change in osteoclastogenic cells. Chondrocyte proliferation was decreased in the skull base at embryonic day 14.5 but not later. These results suggest that long-term aspects of the mutant phenotype, including craniosynostosis, are related to the Fgfr2c regulation of the osteoblast lineage. The effect on early chondrocyte proliferation but not gene expression suggests cooperation of Fgfr2c with Fgfr3 in the formation of the cartilage model for endochondral bone.

Fig. 1.

Targeted activation of the Fgfr2c transcriptional alternative. (A) Genomic structure and targeting events; exons are shaded, with the exon number above and the protein domain name underneath. X and Y, 3′ and internal probe, respectively. (B) DNA sequence of the region used for site-directed mutagenesis, showing the C342Y mutation and the newly formed RsaI site. (C) Southern blot analysis of the homologous recombination in embryonic stem cells, probed with the 3′ external probe after EcoRI digestion. (D) Head of wild-type (left) and viable Fgfr2c^C342Y/+ heterozygote (right). Shortened face and slightly bulging, widely spaced eyes are clearly visible. (E) Wild-type (left) and perinatal lethal homozygous Fgfr2c^C342Y/C342Y (right) littermate pups: mutants show smaller size, greatly shortened face, cyanosis, lack of milk spot, and open eyelids. B, BamHI; H, HindIII; RI, EcoRI; S, SacI; TM, transmembrane exon; #, site of point mutation.

Fig. 2.

Skull phenotype of Fgfr2^C342Y/+ heterozygotes. (A and B) Littermates at postnatal day 29 (P29). (A) Arrows point to the obliterated coronal and fused lambdoid sutures, with multiple bone inserts. (B) Arrowheads indicate the normal basioccipital–basisphenoid–exoccipital synchondroses. (C) Histological sections of the coronal suture showing increased overlap of frontal (f) and parietal (p) bones at E16.5 and obliterated suture at P29 (arrows). (Scale bars, 200 μm.)

Fig. 3.

Shortening of the nasomaxillary area and cleft palate in the P1 homozygous mutant skull. (A–D) Lateral and ventral views of alizarin-stained preparations. Note the shortened nasomaxillary and sphenoid regions. (E and F) Fixed heads and palatal views showing overt cleft palate in the mutant. wt, wild type.

Fig. 5.

Ossification-related gene expression in wild type (A, C, E, G, I, K, M, and O) and heterozygote (B) or Fgfr2^C342Y/C342Y homozygote (D, F, H, J, L, N, and P). (A and B) Spp1 expression is enhanced in the heterozygote skull vault at E18.5. (C–L) Cbfa1/Runx2 expression in sagittal sections of the P1 skull base (C and D), with higher-power views of the basioccipital–basisphenoid synchondrosis (E–H) and in longitudinal sections of the E13.5 humerus (I–L), showing enhanced signal in the perichondrium and periosteum of the mutant. (M–P) There is a slight increase in Ihh expression in the E13.5 humerus. pit, pituitary.

Fig. 4.

Synarthroses and trachea/lung defects in the homozygous mutant neonate. (A and B) Alizarin red staining. (C–E) Alizarin red and alcian blue staining. (A and B) Knee-joint synarthrosis; arrowhead shows mineralized bridge between the femur and tibia of the mutant. (C) Fused sternebrae and asymmetric rib cage in the mutant (arrows). (D) Fusion of cervical vertebral arches (arrowheads) but lack of ossification of vertebral bodies. (E) Tracheal rings replaced by a cartilaginous sheath. (F) Reduced lungs in the homozygote (right) but not heterozygote (center), compared with wild type (left).