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Differential regulation of endochondral bone growth and joint development by FGFR1 and FGFR3 tyrosine kinase domains

Department of Molecular Biology and Pharmacology, Washington University Medical School, Campus Box 8103, 660 S. Euclid Avenue, St Louis, MO 63110, USA

View larger version (52K): [in a new window]   Fig. 1. Generation of transgenic mice expressing the FGFR31ach transgene in chondrocytes. (A) Map of the ColII-FGFR31ach and ColII-FGFR3ach transgene. The transgene expression vector contains the type II collagen promoter (ColII-pr), the ß-globin splice donor and acceptor (black line), the FGFR cDNA including the G380R transmembrane domain mutation, the hGH gene including polyadenylation sequences, and the enhancer sequences of the rat type II collagen gene (ColII-en). FGFR3 sequences are in green. FGFR1 intracellular domain sequences are in red. (B) Comparison of transgene expression patterns and levels by in situ hybridization using an antisense hGH riboprobe on E18.5 tissues. The transgene expression domains are consistent between ColII-FGFR31ach and ColII-FGFR3ach mice, which include resting (r) and proliferating (p) chondrocytes. Expression is excluded from hypertrophic (h) chondrocytes. (C) Alizarin Red-stained skeletons of 1 month old transgenic mice and wild-type littermates. The ColII-FGFR31ach line 2 transgenic mice and ColII-FGFR3ach transgenic mice show a similar degree of dwarfism.

View larger version (138K): [in a new window]   Fig. 2. Histology and cell proliferation in the proximal tibial epiphyseal growth plate at postnatal day 10. (A-C) Hematoxylin and Eosin stained sections of wild-type mice (A), ColII-FGFR31ach transgenic mice (B) and ColII-FGFR31ach transgenic mice (C). (D-F) Immunohistochemical detection of BrdU-labeled growth plate chondrocytes of wild-type (D), ColII-FGFR31ach transgenic mice (E) and ColII-FGFR3ach transgenic mice (F). (G) Quantification of the percentage of BrdU-positive cells in the epiphyseal growth plate of the proximal tibia (n=4-5).

View larger version (147K): [in a new window]   Fig. 3. Synovial and non-synovial joint fusion in heterozygous ColII-FGFR31ach transgenic mice at postnatal day 30. (A,B) Section through the middle phalangeal joint of the front first digit of wild-type (A) and ColII-FGFR31ach transgenic mice (B). The boxed region is shown at a higher magnification in C,D. (E,F) Section through the sternebrae of wild-type mice (E) and ColII-FGFR31ach transgenic mice (F). The joints between S2, S3 and S4 in transgenic tissue are completely replaced by bone marrow.

View larger version (157K): [in a new window]   Fig. 4. Elbow and knee joint fusion in homozygous ColII-FGFR31ach transgenic mice (line 2) at E15.5. (A,B) Elbow joint of wild-type (A) and ColII-FGFR31ach transgenic line 2 (B). (C,D) Knee joints of wild-type (C) and ColII-FGFR31ach transgenic line 2 (D). f, femur; h, humerus; t, tibia; u, ulna.

View larger version (137K): [in a new window]   Fig. 5. Loss of phalangeal joints in homozygous ColII-FGFR31ach transgenic mice at E17.5. (A-C) Hematoxylin stained forelimb digits 2 and 3. (D-F) In situ hybridization showing transgene (hGH) expression. In both ColII-FGFR31ach transgenic line 1 (A,D) and line 2 (B,E), the joints in the second and third digits of the forefeet are completely lost and replaced with a single cartilaginous element. In homozygous ColII-FGFR3ach transgenic mice (C,F), the proximal phalangeal joints develop normally (indicated by an arrow), but the distal phalangeal joint is fused (indicated by an arrowhead).

View larger version (137K): [in a new window]   Fig. 6. Type II collagen expression and cell morphology in the knee joint of homozygous ColII-FGFR31ach mice at E13.5 and E14.5. (A) At E13.5, the presumptive joint position (indicated by an arrow) is well defined by a collagen II-negative zone in wild-type tissue. (B) The presumptive joint space in tissue from homozygous ColII-FGFR31ach transgenic mice has diffuse type II collagen expression, indicating the initiation of chondrification of the joint interzone. (C,D) Cell morphology in the developing joint. (C) Wild-type E13.5 joint tissue showing elongated mesenchymal cell shape. (D) Homozygous ColII-FGFR31ach line 2 transgenic tissue showing a rounded cell morphology. The presumptive joint space is located between the arrows. (E) The type II collagen-free region (arrow) in the developing knee joint in the wild-type animal is well established by E14.5. (F) The knee joint (arrow) is completely replaced by type II collagen-expressing cells in homozygous ColII-FGFR31ach line 2 tissue.

View larger version (148K): [in a new window]   Fig. 7. Pattern of cell proliferation in the knee joint at E13.5 in wild-type and homozygous ColII-FGFR31ach line 2 mice. (A) Wild-type and (B) homozygous transgenic tissue stained for BrdU incorporation. Box a and Box c outline the growth plates of the tibia and femur, respectively. Box b outlines the region of the presumptive joint. The number of proliferating cells in the growth plates of the tibia and femur is comparable between wild-type and homozygous transgenic tissue, whereas there is a significant decrease in the number of proliferating cells in the presumptive joint space in transgenic tissue compared with wild-type tissue.

View larger version (125K): [in a new window]   Fig. 8. Gdf5 expression in the joint space (arrows) of heterozygous (A,C) and homozygous (B,D) ColII-FGFR31ach line 2 mice at E13.5. (A,B) Hematoxylin and Eosin stained knee joint tissue. Inserts in (A,B) are the dark-field images showing transgene (hGH) expression. (C,D) Dark-field images showing Gdf5 expression. (C) In heterozygous tissue, Gdf5 is robustly expressed in the joint space and is complementary to the transgene expression domain. (D) In homozygous tissue, Gdf5 expression is dramatically reduced, even though ColII expression identifies the presumptive joint space. This indicates that loss of Gdf5 expression precedes complete joint fusion.

View larger version (21K): [in a new window]   Fig. 9. Model showing FGF regulation of joint development. The formation of the joint interzone is marked by the appearance of Wnt14 and Gdf5 expression, and decreased type II collagen expression. Joint interzone cells also have an elongated cell morphology. Adjacent cartilage elements express higher levels of type II collagen and express the mutant FGFR. At later stages in ColII-FGFR31ach and ColII-FGFR3ach mice, chondrification expands into the joint interzone owing to over activation of the FGFR signaling pathway in adjacent chondrocytes, resulting in a failure of joint formation.

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