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doi: 10.1242/10.1242/dev.00491

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Conditional inactivation of FGF receptor 2 reveals an essential role for FGF signaling in the regulation of osteoblast function and bone growth

¹ Department of Molecular Biology and Pharmacology, Washington University Medical School, Campus Box 8103, 660 S. Euclid Avenue, St. Louis, Missouri 63110, USA
² Department of Molecular Biology, UT Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA
³ Department of Internal Medicine, Washington University Medical School, Campus Box 8103, 660 S. Euclid Avenue, St. Louis, Missouri 63110, USA

View larger version (89K): [in a new window]   Fig. 1. Generation of Dermo1-cre mice and tissue-specific activity of the Dermo1-CRE recombinase. (A) A schematic representation of the Dermo1 genomic locus, the targeting vector and the mutant allele generated following homologous recombination. The arrow represents the cre gene and its transcriptional orientation. The length of diagnostic XhoI and EcoRV genomic restriction fragments are indicated by solid lines. Locations of the probes (5' and 3') used for DNA blot analysis and the primers (D1 and D2) used for PCR genotyping are indicated by filled boxes and arrowheads, respectively. FRT-neo contains a PGK-neo cassette (gray bar) flanked by flip recombination sites (black bar). (B) DNA blot analysis of XhoI-digested genomic DNA from embryonic stem cells hybridized with the 5' probe. (C) DNA blot analysis of EcoRV-digested tail DNA hybridized with the 3' probe. (D) Whole-mount detection of ß-gal activity in an E11.5 embryo. Note that neural tissue is not stained. (E) Frozen sagittal section of an E15.5 embryo stained for ß-gal activity. (F) Paraffin wax section of an E11.5 forelimb bud from the embryo shown in D. Note that the ectodermal epithelium is negative and the mesenchymal condensations (mc) are positive for ß-gal activity. (G) Frozen section of an E16.5 femur. The perichondrium and periosteum (arrows) are uniformly stained for ß-gal activity. (I,J,L) Frozen section of adult tibia, showing ß-gal activity in the growth plate (I), cortical bone (J) and osteoblasts lining bone trabecula (L). Bone marrow and muscle are not stained; F,G,I,J,L are counterstained with Nuclear Fast Red. Note that there are small clusters of unlabeled chondrocytes (*) in the growth plate (G,I). (H,K) In situ hybridization detection of Fgfr2 expression in developing bone using a probe that detects the tyrosine kinase domain. (H) E16.5 femur. (K) The diaphysis of a P7 femur. Note Fgfr2 expression in diaphyseal lacunae (arrowhead). Bs, BspEI; C, ClaI; E, EcoRI; H, HindIII; N, NotI; Rv, EcoRV; Xh, XhoI. bm, bone marrow; cb, cortical bone; h, hypertrophic zone; mc, mesenchymal condensation; ob, osteoblast; oc, osteoclast; p, proliferation zone; tb, trabecular bone.

View larger version (42K): [in a new window]   Fig. 2. Generation of a floxed Fgfr2 allele. (A) Schematic representation of the Fgfr2 genomic locus, the targeting vector and the Fgfr2flox allele following homologous recombination. The loxP sites are indicated by gray arrowheads. The length of diagnostic HindIII-EcoRV and EcoRV restriction fragments are indicated by solid lines. The probes (5' and 3') used for DNA bolt analysis and the primers (F1, F2 and F3) used for PCR genotyping are indicated by filled boxes and arrowheads, respectively. The allele, generated by CRE-mediated recombination to delete all sequences between the two loxP sites, is shown at the bottom. (B) Southern blot analysis of EcoRV-digested genomic DNA from embryonic stem cells hybridized with the 3' probe. (C) Southern blot analysis of HindIII-EcoRV-digested genomic DNA from embryonic stem cells hybridized with the 5' probe. (D) PCR analysis of tail DNA (genotype is shown on the left). Primer F1 and F2 distinguish the wild-type (142 bp) and Fgfr2flox (207 bp) alleles (bottom panel). Primer F1 and F3 produce a 471 bp fragment from the Fgfr2 allele (middle panel). The top panel shows PCR analysis of the Dermo1cre allele with primer D1 and D2 using the same DNA samples. A 370 bp fragment is produced from the Dermo1cre allele (lanes 1, 3 and 5). Since D2 is localized in the 5' end of the cre sequence, no PCR product is amplified from the wild-type allele (lanes 2 and 4). Note that the 471 bp Fgfr2 PCR fragment is amplified from Fgfr2+/flox; Dermo-1cre/+ tail DNA (lane 3). This is due to co-expression and deletion of the Fgfr2flox allele by Dermo1-CRE in tail tissue. B, BamHI; C, ClaI; E, EcoRI; H, HindIII; Rv, EcoRV; Xb, XbaI.

View larger version (100K): [in a new window]   Fig. 3. Generation of Fgfr2 conditional knockout (Fgfr2cko) mice. (A) Mating scheme used to produce Fgfr2cko mice (Fgf2flox/; Dermo1cre/+). (B-G) In situ hybridization detection of Fgfr1 and Fgfr2 in femurs of E16.5 embryos (dark-field images). (B,C), Fgfr2 expression detected with the transmembrane domain probe, or (D,E) with the tyrosine kinase domain probe. (F,G), Fgfr1 expression. (B,D,F) Sections from normal control embryos. (C,E,G) Sections from Fgfr2cko embryos. (H) RT-PCR analysis of Fgfr2 expression from wild-type and targeted alleles. Total RNA was prepared from E10.5 whole embryos (lanes 1 and 2) and from leg bones of E16.5 embryos (lane 3-5). Genotype of the embryo in each lane: 1, Fgfr2+/+; 2, Fgfr2/; 3, Fgfr2/flox; 4, Fgfr2+/flox and 5, Fgfr2/flox; Dermo1cre/+. A 820 bp fragment is generated from Fgfr2 transcripts of both wild-type and Fgfr2flox alleles (lanes 1, 3 and 4) while a 473 bp fragment is generated from the Fgfr2 allele (lanes 2, 3 and 5). Note that both Fgfr2 alleles are transcribed in Fgfr2/flox mice (lane 3) and full-length Fgfr2 transcripts are undetectable in skeletal tissues of Fgfr2cko mice (lane 5). (I) The appearance of Fgfr2cko (right) and control mice (left) at 2 weeks of age. (J) Growth curves of control (triangle) and Fgfr2cko (circle) mice.

View larger version (89K): [in a new window]   Fig. 4. Skeletal abnormalities and decreased bone density in Fgfr2cko mice. (A,B) Alizarin Red stained skulls from P30 control (A) and Fgfr2cko (B) mice, showing a dome-shaped skull in the Fgfr2cko mouse. (C,D) Dorsal view of the Alizarin Red-stained axial skeleton from a P30 control (C) and Fgfr2cko (D) mouse, showing a non-ossified gap in the midline of the vertebrae and the absence of the spinous process (arrows) in the Fgfr2cko mouse. (E,F) Alizarin Red- and Alcian Blue-stained tarsal bones from a P7 control (E) and Fgfr2cko (F) mouse. All of 13 mice examined showed tarsal bone fusion (arrows). (G,H) Alizarin Red stained tarsal bones from a P60 control (G) and Fgfr2cko (H) mouse. The cuneiforme 3 bone is fused with naviculare and cuboideum (arrows) in the Fgfr2cko mice. Note that in the adult, the cuneiforme 2 and 3 bones are also fused in the Fgfr2cko mice (arrows). (I,J) Radiological analysis of bones from Fgfr2cko mice and normal littermates showing decreased bone length and increased radiolucency in Fgfr2cko mice. (I) Femurs, P22; (J) femurs, P43; (K) quantitative analysis of bone mineral density by DEXA of femur (black) and lumbar vertebra (red) of mice age 1-58 weeks. Solid bars, control mice; open bars, Fgfr2cko mice. *P<0.05.

View larger version (164K): [in a new window]   Fig. 5. In situ hybridization detection of osteoblast marker expression in femurs at E16.5 and P7. (A,D) Bright-field images, von Kossa stain. (B,C,E,F,G-L) Dark-field images. (B,E) Cbfa1 expression at E16.5. (C,F) osteopontin expression at E16.5. (G,H) collagen type I expression at P7. (I,J) osteopontin expression at P7. (K,L) osteocalcin expression at P7. (A,B,C,G,I,K) Sections from a control littermate. (D,E,F,H,J,L) Sections from a Fgfr2cko mouse.

View larger version (152K): [in a new window]   Fig. 6. Defective osteogenesis in Fgfr2cko mice. (A,B) H&E stained sections of the distal tibia of P7 mice, showing the epiphyseal growth plate. The length of the metaphysis is indicated by the double-headed arrows. (C,D) Enlarged views showing the morphology of metaphyseal osteoblasts in the proximal tibia. Note that osteoblasts are plump cuboidal cells in the wild-type mouse and become atrophic and irregular in the Fgfr2cko mouse (arrows). (E,F) Enlarged view of the boxed regions in A and B showing the morphology of the perichondrium. The thickness of the perichondrium is indicated by double-headed arrows. (G,H) H&E stained sections showing the femoral diaphysis of P7 mice. The thickness of the cortical bone plus the periosteum is indicated by double-headed arrows. Diaphyseal lacunae occupied by active osteoblasts are indicated by arrows. (A,C,E,G) Sections from control littermates. (B,D,F,H) Sections from the Fgfr2cko mice. Objective magnification, (A,B) 10x; (C-F) 40x; (G,H) 20x. bm, bone marrow; cb, cortical bone; fc, fibroblast-like cell layer; h, hypertrophic zone; mu, muscle; op, osteoprogenitor layer; p; proliferation zone, po, periosteum; tb, trabecular bone.

View larger version (128K): [in a new window]   Fig. 7. Mineral apposition rate (MAR), osteoblast proliferation and osteoclast density in Fgfr2cko mice. (A,B) MAR was assessed by calcein double labeling over a 9-day interval beginning at P8. (C,D) BrdU immunohistochemistry on sections of the femur from P15 mice showing the distal growth plate. The dashed lines parallel to the chondro-osseous junction demarcate the 100 µm region of the metaphysis that was counted (see Materials and Methods). The labeling index of the growth plate proliferating zone was similar for Fgfr2cko mice and controls. In contrast, the osteoblast proliferation index in the primary spongiosa was reduced in Fgfr2cko mice compared to controls (Table 3). (E,F) BrdU immunohistochemistry of the femoral diaphysis at P15. Note the proliferating osteoprogenitor cells located in the perichondrium (arrows). (G,H) TRAP staining of sections of the proximal femur at P4. Note that osteoclasts (stained in red) at the chondro-osseous junction appear larger and more numerous in Fgfr2cko mice than in control littermates. Osteoclast densities were 1.29±0.29/0.01 mm2 in control mice (n=6) and 1.82±0.37/0.01 mm2 in Fgfr2cko mice (n=6, P<0.001). (A,C,E,G) control mice. (B,D,F,H) Fgfr2cko mice.