Gly369Cys mutation in mouse FGFR3 causes achondroplasia by affecting both chondrogenesis and osteogenesis

Abstract

Missense mutations in fibroblast growth factor receptor 3 (FGFR3) result in several human skeletal dysplasias, including the most common form of dwarfism, achondroplasia. Here we show that a glycine-to-cysteine substitution at position 375 (Gly375Cys) in human FGFR3 causes ligand-independent dimerization and phosphorylation of FGFR3 and that the equivalent substitution at position 369 (Gly369Cys) in mouse FGFR3 causes dwarfism with features mimicking human achondroplasia. Accordingly, homozygous mice were more severely affected than heterozygotes. The resulting mutant mice exhibited macrocephaly and shortened limbs due to retarded endochondral bone growth and premature closure of cranial base synchondroses. Compared with their wild-type littermates, mutant mice growth plates shared an expanded resting zone and narrowed proliferating and hypertrophic zones, which is correlated with the activation of Stat proteins and upregulation of cell-cycle inhibitors. Reduced bone density is accompanied by increased activity of osteoclasts and upregulation of genes that are related to osteoblast differentiation, including osteopontin, osteonectin, and osteocalcin. These data reveal an essential role for FGF/FGFR3 signals in both chondrogenesis and osteogenesis during endochondral ossification.

Figure 1

Introduction of Gly369Cys mutation into the mouse Fgfr3 locus. (a) Targeting construct, pFgfr3-Gly369Cys, contained the Gly369Cys mutation in exon 10 and a pLoxpneo gene in intron 10 of the Fgfr3 gene. Of 120 G418^r/FIAU^r clones examined by Southern blot analysis using a 5′ flanking probe (probe 1), 5 clones showed an extra fragment of approximately 11 kb upon NotI + EcoRV digestion (c). The targeting events were confirmed by XbaI +EcoRV digestion using a 3′ internal probe (probe 2). Three of them had the point mutation co-transferred as indicated by sequencing (unpublished observations). (b) Removal of the pLoxpneo gene by breeding with EIIa-cre transgenic mice. The Cre-mediated deletion was genotyped by PCR as described in the Methods section.

Figure 2

Ligand-independent dimerization and activation of G375C mutant FGFR3. (a) Wild type, G380R, S371C, G375C and K650E mutants of FGFR3 were transiently expressed in 293T cells in the absence or presence of 50 ng/ml aFGF, lysed and immunoprecipitated with anti FGFR3 antibody. Blots were developed using anti-FGFR3 antiserum (upper panel) or anti-phosphotyrosine antibody (lower panel). (b) Receptor phosphotyrosine activity normalized for expression levels of FGFR3. In the absence of aFGF, FGFR3 mutants corresponding to the immunoblot above are phosphorylated. The K650E mutant has the highest basal activity, the G371C mutant is lower in basal activity but still higher than G375C and both are significantly higher than that of the wild type receptor. (c) Wild type and mutant FGFR3 transiently expressed in 293T cells were subjected to chemical cross-linking after being stimulated with 50 ng/ml aFGF. Ligand-independent receptor dimers were found for the S371C and G375C mutants, but not in the wild-type receptor.

Figure 3

The Gly369Cys mutation in mouse FGFR3 results in dwarf mice. (a) Morphology and (b) skeleton of 21 day old wild-type (WT), Fgfr3^369/+ (369/+), and Fgfr3^369/369 (369/369) mice. Notice dome-shaped heads in 369/+ and 369/369 mice (arrows). (c) Quantitative measurements of tail length of mice with 3 genotypes. Each point represents data from an average of 5 mice. Standard deviation is less than ± 5%. (d) X-ray images of 369/369 and WT mice. Notice the reduced bone density in 369/369 mice: F, femur; T, tibia.

Figure 4

Macrocephaly results from retarded growth of the cranial base. (a) Dorsal view of x-ray images of skulls of P21 day old 369/369, 369/+, and WT mice. (b) Cranial base of WT, and (c) 369/369 mice. Arrows point to synchondroses: basooccipital (oc), basosphenoid (ba) and presphenoid (pr). Notice the absence of synchondroses in 369/369 mice (arrows in c). (d) H&E image of a WT synchondrosis, which exhibits typical growth plate structures, including proliferating and maturing chondrocytes [positive for Fgfr3 (e)], and hypertrophic chondrocytes [positive for collagen type X (f)]. (g) H&E image of corresponding region of a 369/369 synchondrosis, which had completely turned into bone. (h–j) P6 synchondroses (arrows) of 369/369 (h), 369/+ (i) and WT (j) mice. The 369/369 synchondrosis was much thinner than the WT synchondrosis and was in a process of resolving, while the 369/+ synchondrosis (i) exhibited an intermediate phenotype between those of 369/369 and WT.

Figure 5

Histology and immunohistochemistry analyses of growth plates of P15 mice. Genotypes and markers were as indicated. (a) Chondrocytes in the WT growth plates are divided into 4 distinct zones, i.e. resting (Rc), proliferating (Pc), maturing (Mc) and hypertrophic (Hc) chondrocytes. (b and c) In the mutant growth plates, the demarcation of each zone is not clear. Arrows point to the secondary ossification center. (d–f) [³H]Thymidine incorporation. The labeled cells in WT mark the Pc zone. In mutant growth plates, the labeled cells are fewer in number and scattered, suggesting that the majority of the cells are in a quiescent state. (g–o) Immunohistochemical analysis of growth plates, using antibodies to FGFR3 (g–i), Stat5b (j–l, and m–o for higher magnification), and p19 (p–r). In all cases, the staining was the weakest in WT, intermediate in 369/+, and strongest in 369/369 growth plates.

Figure 6

The Gly369Cys mutation results in enhanced differentiation of osteoblast cells. Panel a–c, g–r are prepared from P15 mice and panel d–f from P1 mice. (a-c) Enhanced TRAP staining at the interface between mutant hypertrophic chondrocytes and primary spongiosa (arrows); the mutant also showed less primary spongiosa. (d–f) Alizarin Red S staining of undecalcified growth plates isolated from the knee joints of P1 mice. Arrows in (e) and (f) point to the advanced bone collars. (g–o) In situ hybridization using probes for osteopontin (Op, g–i), osteonectin (On, j–l) and osteocalcin (Oc, m–o). (p–r) Immunohistochemical staining of osteocalcin. Arrows in n and o point to ectopic expression of Oc in the maturing zone of chondrocytes. Both Op and On are also stronger in the 369/+ and 369/369 trabecular bones.