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First published online 23 June 2005
doi: 10.1242/dev.01914

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Abnormalities in cartilage and bone development in the Apert syndrome FGFR2^+/S252W mouse

Yingli Wang^1,4,*, Ran Xiao^1,4,*, Fan Yang⁵, Baktiar O. Karim², Anthony J. Iacovelli^1,4, Juanliang Cai^1,4, Charles P. Lerner⁶, Joan T. Richtsmeier^4,7, Jen M. Leszl⁷, Cheryl A. Hill⁷, Kai Yu⁸, David M. Ornitz⁸, Jennifer Elisseeff⁵, David L. Huso² and Ethylin Wang Jabs^1,3,4,

¹ Institute of Genetic Medicine, Department of Pediatrics, The Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, MD 21205, USA
² Department of Comparative Medicine, The Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, MD 21205, USA
³ Department of Medicine and Plastic Surgery, The Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, MD 21205, USA
⁴ Center for Craniofacial Development and Disorders, The Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, MD 21205, USA
⁵ Department of Biomedical Engineering, The Johns Hopkins University, 3400 N. Charles Street, Clark 106, Baltimore, MD 21218, USA
⁶ Departments of Cell Biology/Microinjection and Microchemistry, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA
⁷ Department of Anthropology, The Pennsylvania State University, 409 Carpenter Building, University Park, PA 16802, USA
⁸ Department of Molecular Biology and Pharmacology, Washington University Medical School, 660 S. Euclid Avenue, St Louis, MO 63110, USA

View larger version (44K): [in a new window]   Fig. 1. Generation of the targeting construct and Fgfr2+/S252W mice. (A) Structure of a portion of the wild-type Fgfr2 gene with exons IIIa, IIIb, IIIc and 10; the targeting construct with the TK cassette and neo cassette with flanking loxP sequences; and the mutant allele produced by homologous recombination and with neo deletion mediated by Cre. The 755_756CA>GG, S252W mutation (*) was introduced into exon IIIa. Probes (–) and restriction enzymes (St, StyI; H, HindIII; S, SacI) used for Southern blot analysis, and PCR primers (F, Fn, R; arrowheads) used for genotyping are shown. (B) Identification of mutant and wild-type alleles in two independent ES cell clones using Southern blot analysis with 5' and 3' probes. (C) Genotype of a litter from +/S252Wfloxx+/Cre by PCR of tail DNA. Lanes 1, 3 and 5: heterozygote with +/S252Wflox, showing wild-type allele (521 bp) and hypomorphic allele with neo (1.1 kb). Lanes 2 and 4: heterozygote mutant +/S252W, showing mutant allele after neo deletion (581 bp). Lane 7: hypomorphic mutant +/S252Wflox/S252W. Lanes 6 and 8: wild type (+/+). (D) RT-PCR detection of mutant transcript in lung and skull tissues. Other tissues tested expressed the mutant allele (data not shown). The mutation introduces an additional SfiI restriction site. (E) Gross appearance of +/S252W and wild-type postnatal day (P)1 mice. Note the small body size of the mutant mouse. The difference of body weight between the mutant and control mice was significant.

View larger version (121K): [in a new window]   Fig. 2. Gross bony structures and histology of P1 Fgfr2+/S252W mice. (A,C,E,G,I,K,M,O,Q,S) Control littermates; (B,D,F,H,J,L,N,P,R,T) corresponding regions in mutant mice. (A-H) Alizarin Red S and Alcian Blue staining of (A-F) the skull and (G,H) the chest. (I-R) HE staining of the (I,J) brain, (K,L) palate, (M,N) trachea, (O,P) lung, and (Q,R) growth plates of long bone. (S,T) TRAP staining for growth plates. The mutant mice have: a wide interfrontal suture (B, arrows define the width of the suture); a shortened nasal snout (D, arrow); fusion of the joints between the zygomatic arch bones (F, arrows); a sternum with abnormal fusions and bifid xiphoid process (H, arrow); hydrocephalus with enlarged ventricles (J, arrows); a palate defect (L, arrow); a complete cartilage sleeve of the trachea with abnormal cartilage thickening (N); atelectasis of the lungs with alveolar proteinaceous fluid (P); chondrocyte hypertrophy and disorganization with irregular and thickened ossifying cartilaginous spicules in the growth plates of long bone (R); and decreased osteoclasts at the chondro-osseous junction (T; S, arrow shows normal osteoclasts stained red). LV, lateral ventricle; 3V, third ventricle; TC, tracheal cartilage; P, zone of chondrocyte proliferation; H, zone of hypertrophy; M, zone of mineralization; O, zone of ossification at the chondro-osseous junction. Scale bars: A-H, 1 mm; I-T, 50 µm.

View larger version (90K): [in a new window]   Fig. 3. Skeletal features of hypomorphic Fgfr2+/S252Wflox/S252W mice. (A) The gross appearance shows the smaller body size of the mutant mouse at 8 weeks of age. (B) X-ray of the whole body at 8 weeks of age shows short nasal bones and maxillary bones with overriding lower incisors. (C) Micro-CT of the skull of a 10-month-old Fgfr2+/S252Wflox/S252W mouse (right) shows extreme brachycephaly, diminutive and curved nasal bones, extreme reduction anteroposteriorly of the frontal bones, obliterated fronto-nasal, fronto-premaxillary and maxillary-premaxillary sutures, and a defect of the interfrontal suture in the skull (arrow).

View larger version (61K): [in a new window]   Fig. 4. Histological analysis of the calvarial sutures in Fgfr2+/S252W mice. (A,C,E,G,I,K) Control littermates; (B,D,F,H,J,L) corresponding regions in mutant mice. (A-F) HE staining shows the development of the sagittal suture from E16.5 to P1. (A,B) No obvious difference of the sagittal sutures between the mutant and control at E16.5. (C,D) More proximate osteogenic fronts are evident at the mutant sagittal suture at E18.5. (E,F) Osteoid deposition is seen across the mutant sagittal suture at P1. (G-L) HE staining shows: a wide gap between the osteogenic fronts at the mutant interfrontal suture at P1 (G,H); presynostosis/synostosis with osteoid deposition at the mutant coronal suture at P1 (I,J); and presynostosis/synostosis at the mutant lambdoid suture at P1 (K,L). Arrows, osteogenic fronts; arrowheads, presynostosis/synostosis with osteoid formation; s, skin; p, parietal bone; b, brain; f, frontal bone; ip, interparietal bone. Scale bars: A-H,K,L, 50 µm; I,J, 25 µm.

View larger version (73K): [in a new window]   Fig. 5. Proliferation, differentiation and apoptosis at the midline sagittal suture in Fgfr2+/S252W mice at E18.5. (A,B) Immunohistochemical staining of Ki67 shows increased numbers and abnormal distribution of positive cells indicating abnormal proliferation in mutants (arrows). (C-H) Expression of osteogenic markers shows increased or abnormal differentiation in mutants. (C,D) Osteonectin in situ hybridization (arrows). (E,F) ALP staining (arrows). (G,H) Osteopontin in situ hybridization (arrows). (I,J) TUNEL staining does not show an obvious difference in apoptosis between mutants and controls. (A,C,E,G,I) Control littermates; (B,D,F,H,J) corresponding regions in mutant mice. s, skin; p, parietal bone; b, brain. Scale bars: A-J, 50 µm.

View larger version (109K): [in a new window]   Fig. 6. Proliferation and differentiation at the midline interfrontal suture in Fgfr2+/S252W mice at E18.5. (A,B) HE staining shows a gap in mutants. (C,D) Ki67 staining shows abnormal distribution of positive cells in the mid-sutural mesenchyme in mutants. (E-H) Expression of osteogenic markers shows displaced, decreased or delayed differentiation in mutants. (E,F) Osteonectin in situ hybridization; (G,H) osteopontin in situ hybridization. (A,C,E,G) Control littermates; (B,D,F,H) corresponding regions in mutant mice. Arrows, osteogenic fronts; s, skin; f, frontal bone; b, brain. Scale bars: A-H, 50 µm.

View larger version (91K): [in a new window]   Fig. 7. Abnormal cartilage at the midline sagittal suture in Fgfr2+/S252W mice from E16.5 to E18.5. (A-D) HE staining, (E-H) Sox9 in situ hybridization, and (I-L) collagen type II immunohistochemical staining, showing the abnormal cartilage in mutants from E16.5 (left panels) to E18.5 (right panels). (A,E,I,C,G,K) Control littermates; (B,F,J,D,H,L) corresponding regions in mutant mice. Arrows, abnormal cartilage; s, skin; p, parietal bone; b, brain. Scale bars: A-L, 50 µm.

View larger version (135K): [in a new window]   Fig. 8. Chondrocytes in cultured cells from the limb of P1 Fgfr2+/S252W mice. (A,B) Von Kossa staining at the pericellular regions and (C,D) collagen type I immunohistochemical staining are similar for both mutants and controls. (E,F) Collagen type II staining is positive for the mutants, but is absent in controls. (A,C,E) Control littermates; (B,D,F) mutants. Scale bars: A-F, 100 µm.

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