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The IIIc alternative of Fgfr2 is a positive regulator of bone formation

¹ Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot Israel
² Institute for Animal Science, The Volcani Center, Beit Dagan, Israel
³ Department of Human Anatomy and Genetics, University of Oxford, Oxford, UK

View larger version (31K): [in a new window]   Fig. 1. Targeted disruption of the Fgfr2IIIc transcriptional alternative. (A) Genomic structure and targeting events; exons are shaded, with the exon number above, and the protein domain name underneath. and , 5' and 3' probes respectively; B, BamHI; H, HindIII; RI, EcoRI; S, SacI; TM, transmembrane exon; #, site of point mutation. (B) DNA sequence of the region used for site-directed mutagenesis, showing the newly formed HindIII site and the translational stop codon (*). (C) Southern blot analysis of the homologous recombinant ES cells, probed with the 5' and 3' fragments designated in A.

View larger version (110K): [in a new window]   Fig. 2. Fgfr2IIIb expression is normal in the homozygous Fgfr2IIIc mutant. In situ hybridization analysis with a probe specific for exon 8 of Fgfr2IIIb. (A,C,E,G) bright-field; (B,D,F,H) dark-field views. (A-D) E12.5 limb bud, showing Fgfr2IIIb expression in surface ectoderm. (E-H) Parasagittal sections of the mid-trunk region of E14.5 embryos, showing Fgfr2IIIb expression in the bronchial epithelium of the lungs and the perichondrium of prevertebrae. pv, prevertebrae; lg, lung.

View larger version (180K): [in a new window]   Fig. 3. Localization of Fgfr2IIIc transcripts in skeletogenic tissues. (A,B) In the E12.5 limb bud, Fgfr2IIIc transcripts are in skeletogenic mesenchymal condensations. (C,D) In the E14.5 tibia, transcripts are localized to the perichondrium (periosteal collar) and nascent epiphyseal plate. (E,F) In the E18.5 skull base, transcripts are in the perichondrium and ossification zone. ep, position of future epiphyseal plate; hc, hypertrophic chondrocyte zone; pc, proliferating chondrocyte zone; pit, pituitary, po, periosteal collar; rc, resting chondrocyte zone; tr, trabecular bone.

View larger version (36K): [in a new window]   Fig. 4. Dwarfism and abnormal skull shape in homozygous Fgfr2IIIc mutants. (A) Live P14 wt and mutant mice. (B) Alizarin Red skeletal staining of P14 wild-type and mutant mice, showing overall small size, rounded skull, lower incisor overgrowth and shorter facial area in the mutant. (C) Growth curves demonstrating 40-50% growth retardation in the mutant.

View larger version (62K): [in a new window]   Fig. 5. Delayed onset of ossification in the Fgfr2IIIc–/– mutant: Alizarin Red S staining of skeletal preparations. (A,B) E14.5 embryos, showing delayed mineralization of the whole skeleton. (C-F) First and second cervical vertebrae at P14, showing incomplete vertebral arches in the mutant.

View larger version (72K): [in a new window]   Fig. 6. Mineralization of the skull base. After the slight delay of ossification observed in the mutant at E16.5, sutures between exoccipital and basioccipital bones begin to close as early as E18.5. White arrows point to the basioccipital-exoccipital junction, where in the mutant precocious fusion takes place. Black arrows on the P14 mutant specimen indicate complete fusion of the basioccipital-exoccipital suture and contact at the basioccipital-basisphenoid suture; the vertical arrow indicates the abnormal position of the basisphenoid-presphenoid suture beneath the palate, due to shortening of the sphenoid area in the mutant. bo, basioccipital bone; pal, palate; bs, basisphenoid bone; eo, exoccipital bone; ps, pre-sphenoid so, supraoccipital bone.

View larger version (96K): [in a new window]   Fig. 7. Differentiation and mineralization of the skull vault. (A,B) Spp1 expression at E16.5 shows delayed formation of the metopic suture and is absent in the nasal bones in the mutant (arrow). (C-F) Alizarin-stained P14 skeletal preparations, showing premature fusion of the medial part of the coronal suture in the Fgfr2IIIc mutant. cs, coronal suture; ls, lambdoid suture; ss, sagittal suture.

View larger version (126K): [in a new window]   Fig. 8. Decreased cell proliferation in the coronal suture of Fgfr2IIIc–/– fetuses during skull vault growth. (A,B) Levels of BrdU incorporation are normal at E14.5. (C,D) Numbers of BrdU-labelled cells are reduced at E16.5. (E,F) At P1, only a few BrdU-positive cells are observed in the mutant. (G,H) Parallel Mallory-stained P1 sections showing the ossification fronts. al, alisphenoid cartilage; f, frontal bone; p, parietal bone. Scale bar: 100 µm.

View larger version (134K): [in a new window]   Fig. 9. Decreased Spp1 expression in the skull and in the tibial growth plate of the Fgfr2IIIc mutant: bright-field views of radioactive in situ hybridization. (A-D) Parasagittal sections of the E18.5 head; Cand D are higher magnification of A and B, respectively. (E,F) P14 tibia. bo, basioccipital; bs, basisphenoid; pit, pituitary; ps, presphenoid bone. Scale bar: 1 mm.

View larger version (112K): [in a new window]   Fig. 10. Histological structure of the epiphyseal plate. (A,B) Haematoxylin and Eosin-stained histological section of P7 tibia, showing reduced length of proliferative and hypertrophic chondrocyte columns in the Fgfr2IIIc mutant. (C) Morphometric analysis: cell number in the chondrocyte columns. bc, bone collar; ep-oc, epiphysial ossification center; hc, hypetrophic chondrocyte zone; pc, proliferating chondrocyte zone; rc, resting chondrocyte zone; tr, trabecular bone; PZ, zone of proliferating chondrocytes; HZ, zone of hypertrophic chondrocytes.

View larger version (108K): [in a new window]   Fig. 11. Diminished expression of chondrocyte and osteocyte markers in the Fgfr2IIIc mutant. (A-L) Skull base at P1. (M-P) Tibia at P7. Cbfa1 and PTHrP expression are greatly decreased in the mutant; the Ihh expression domain is reduced, reflecting the reduced size of proliferating chondrocyte zones in both the skull base (I-L) and the tibial growth plate (M-P). hc, hypertrophic chondrocyte zone; pit, pituitary; pc, proliferating chondrocyte zone; rc, resting chondrocyte zone; tr, trabecular bone.