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First published online 8 April 2004
doi: 10.1242/dev.01053

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VEGFA is necessary for chondrocyte survival during bone development

¹ Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA
² Department of Surgical Research, Children's Hospital and Harvard Medical School, Boston, MA 02115, USA
³ Department of Molecular Oncology, Genentech, South San Francisco, CA 94080, USA
⁴ Molecular Biology Section, Division of Biology, University of California, San Diego, CA 92093, USA
⁵ Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA

View larger version (48K): [in a new window]   Fig. 1. Reduced skeletal mineralization in Vegfa conditional knockout (CKO) mice. A comparison of the skeletons of unaffected (A,C,E,G) and Vegfa CKO (B,D,F,H) mice reveals reduced size of areas stained with Alizarin Red, suggesting reduced mineralization of mutant bones. Regions significantly affected (arrows) include the bones in hind limbs (A,B), ribs (C,D), sternum (E,F), and vertebral column (G,H).

View larger version (150K): [in a new window]   Fig. 2. Reduced angiogenesis in Vegfa conditional knockout (CKO) bones. Histology of unaffected and Vegfa CKO mice identifies marked differences in skeletal elements during development. In tibia and fibula at E15.5 in unaffected mice (A), blood vessel invasion into the hypertrophic cartilage and marrow cavity can be observed (arrowheads), while no invasion can be seen into hypertrophic cartilage in the Vegfa CKO mice (B). CD31 immunostaining of tibia at E16.0 in unaffected mice (C) shows vessels throughout the periosteum and in the marrow cavity. In the Vegfa CKO mice (D), there are vessels in the periosteum but no apparent invasion into the hypertrophic zone. In the radius and ulna at E16.5 in unaffected (E) mice, bone marrow and bone trabeculae are present below the hypertrophic zone (arrowhead). In Vegfa CKO mice the growth plate contains a much longer hypertrophic zone (F, arrowhead). At E18.5, unaffected ribs (G) contain a shorter hypertrophic zone. In contrast, the Vegfa CKO ribs contain a greatly expanded zone of hypertrophy (H, arrowhead).

View larger version (146K): [in a new window]   Fig. 3. Reduced removal of terminally differentiated chondrocytes in Vegfa conditional knockout (CKO) bones. Histological sections of E16.5 unaffected (A) and Vegfa CKO (B) humerus show the presence of an expanded hypertrophic zone in the Vegfa CKO growth plate (defined by brackets). Col2a1 expression is seen throughout the cartilage anlagen in both unaffected (C) and Vegfa CKO (D) mice. In unaffected (E) mice Col10a1 expression is seen in the hypertrophic zone (bracket) while in Vegfa CKO (F) mice, Col10a1 expression is seen only in part of the hypertrophic zone (bracket), suggesting that some of the hypertrophic chondrocytes are differentiated into terminal hypertrophic chondrocytes. Osp expression is seen in the last row of terminally differentiated hypertrophic chondrocytes and extensively in the osteoblasts under the growth plate in the unaffected mice (G), while in the Vegfa CKO growth plate, Osp expression is detected in several rows of terminally differentiated hypertrophic chondrocytes (H).

View larger version (100K): [in a new window]   Fig. 4. Cell death in Vegfa and Hif1a conditional knockout (CKO) bones. Histological sections of E16.5 Vegfa CKO humerus (B) show areas of cell death (arrow), unlike the unaffected bone (A). Compared with the femur and tibia of unaffected mice (C), the E18.5 Vegfa CKO bones (D) were misshapen with extensive regions of dead cells in the center of the bones, starting at the articular surface and continuing through the resting to the proliferating zones of chondrocytes and ending in a misshapen growth plate (arrows). Histological sections of unaffected (E) and Hif1a CKO (F) femur and tibia at E15.5 show misshapen bones with extensive regions of dead cells in the center of the bones (arrowheads).

View larger version (85K): [in a new window]   Fig. 5. Apoptosis in Vegfa conditional knockout (CKO) bones. Histological sections through the center of E18.5 unaffected (A,C) and Vegfa CKO (B,D) humerus. Areas within the squares in A and B are shown at high magnification in C and D. In the affected epiphysis (D), the presence of apoptotic cells with shrunken cytoplasm and condensed nuclei is observed. TUNEL assay of E18.5 unaffected (E) and Vegfa CKO (F) humerus and Vegfa CKO tibia and femur (G) shows strong TUNEL-positive signals in regions of apoptotic cells (arrowheads) in the Vegfa CKO bones (E and F are serial sections of A and B, while G is serial section of Fig. 4 D). Histological sections of E16.5 Prx1/Vegfa CKO scapula and humerus show misshapen bones with extensive regions of dead cells (H, arrowheads).

View larger version (118K): [in a new window]   Fig. 6. Analysis of Vegfa expression in Hif1a conditional knockout (CKO) mice. Histological sections of E15.5 tibia of unaffected (A) and Hif1a CKO mice (B) reveal a delay in vessel invasion into the Hif1a CKO primary ossification center (arrowheads). Vegfa expression in the primary ossification center of unaffected tibia at E15.5 (G) shows expression in hypertrophic chondrocytes but no detectable expression in the epiphysis. At E18.5 (E), the expression of Vegfa in the hypertrophic zone is maintained and it is possible to detect a moderate level of Vegfa expression in the epiphysis (arrows). At E15.5 in the primary ossification center of tibia there is no apparent difference in Vegfa expression in the hypertrophic chondrocytes of unaffected (C) and Hif1a CKO mice (D), although the shape of the hypertrophic region is abnormal in the Hif1a CKO section (D). At E18.5 (F) the expression of Vegfa in the Hif1a CKO is dramatically decreased. At this stage, the extensive cell death in the Hif1a tissue makes it almost impossible to see any hypertrophic zone of chondrocytes and all remaining viable cells are proliferating Col2a1-positive cells (H).

View larger version (67K): [in a new window]   Fig. 7. Gene specific RT-PCR analyses of VEGFA receptors in epiphyseal chondrocytes. Total RNA was extracted from epiphyseal chondrocytes. RT-PCR reactions were performed in the presence of reverse transcriptase (RT) (+) or in its absence (–) (as a control for genomic DNA contamination). Cd31 was also amplified as a control for endothelial cell contamination. Nucleotide size markers are indicated at left. Amplified bands are only seen in the Vegfr3, Nrp1 and Nrp2 lanes, as indicated by asterisks.

View larger version (163K): [in a new window]   Fig. 8. Analysis of chondrocyte differentiation in Hif1a conditional knockout (CKO) mice. Col10a1, Ihh and Pthr1 expression domains are divided by cells that have lost the expression of these markers in the growth plates of unaffected (A,C,E) mice, while the Hif1a CKO (B,D,F) mice cells in the center of the hypertrophic domains express all three markers.

View larger version (148K): [in a new window]   Fig. 9. Vascularization in the vicinity of Vegfa conditional knockout (CKO) cartilaginous elements. CD31 immunostaining of unaffected (A) and Vegfa CKO (B,C,D) tibias at E16.0. Comparison of histologically comparable sections of unaffected (A) and Vegfa CKO (B) knee joint regions reveals no major difference in CD31 immunostaining (arrowheads). Extensive vascularization in the vicinity of the diaphyses and the epiphyses of Vegfa CKO knee joint region (C, arowheads). In D, an area indicated by a square in B shown at high magnification, CD31-positive blood vessels are seen in the vicinity of a region of initial cell death between the femur (f), tibia (t), and the anterior cruciate ligament (l).

View larger version (94K): [in a new window]   Fig. 10. Expression of Col2a1, Ihh, and Pthr1, in the E18.5 humerus. In Vegfa conditional knockout (CKO) mice (A), areas of increased proliferation (square) can be seen surrounding areas of cell death; square in A seen at higher magnification in B, arrowheads indicate proliferating cells. Col2a1 expression is seen throughout the cartilage in unaffected mice (C) while in Vegfa CKO (D) mice Col2a1 expression is lacking in the center of the cartilage (arrowhead). Ihh and Pthr1 expression in the unaffected humerus (E,G) is restricted to a narrow strip of prehypertrophic cells. In the Vegfa CKO growth plate the domain of expression of both Ihh and Pthr1 is expanded (F,H).

View larger version (29K): [in a new window]   Fig. 11. Two distinct roles of VEGFA during endochondral bone formation are regulated by different pathways. Diagram showing major components of the pathways that regulate expression of Vegfa in hypertrophic chondrocytes (right) and epiphyseal chondrocytes (left) during endochondral ossification. Angiogenesis into hypertrophic cartilage and establishment of the primary ossification center in developing long bones depend on a high level of Vegfa expression, controlled by the transcription factor Runx2, in hypertrophic chondrocytes (Zelzer et al., 2001). Since a reduction in the level of Vegfa was also observed in Ctgf null mice (Ivkovic et al., 2003), it is possible that Runx2 and Ctgf may be components of two independent or dependent regulatory pathways for control of Vegfa expression. In epiphyseal cartilage, cell survival depends on expression of Vegfa at a moderate level under the control of HIF1 and von Hippel-Lindau (VHL) protein. Whether Vegfa is the only critical target gene for HIF1 regulation in this chondrocyte survival pathway is not known, and it is possible that other genes may also play a role.

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