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First published online May 5, 2004
doi: 10.1242/10.1242/dev.01138

Development 131, 2497-2508 (2004)
Published by The Company of Biologists 2004
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Deletion of Vhlh in chondrocytes reduces cell proliferation and increases matrix deposition during growth plate development

David Pfander1,2,3, Tatsuya Kobayashi1, Melissa C. Knight1, Elazar Zelzer4, Denise A. Chan5, Bjorn R. Olsen4, Amato J. Giaccia5, Randall S. Johnson3, Volker H. Haase6 and Ernestina Schipani1,*

1 Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA
2 Division of Orthopedic Rheumatology, Department of Orthopedic Surgery, University of Erlangen-Nuremberg, 91054, Germany
3 Molecular Biology Section, Division of Biology, University of San Diego, San Diego, CA 92093, USA
4 Department of Cell Biology, Harvard Medical School, Boston, MA 02215, USA
5 Program in Cancer Biology, Department of Radiation Oncology Stanford University, Stanford, CA 94305, USA
6 Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA



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  		Fig. 1. Alizarin Red S staining of forelimbs and hindlimbs of E18.5 control and Vhlh null (Cre transgenic line b) mice (A,C), and of newborn control and Vhlh null (Cre transgenic line a) mice (B,D). (E) Four-week-old control and Vhlh null mice.

 


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  		Fig. 2. Analysis of growth plates from control and Vhlh null mice with Cre transgenic lines a and b. Hematoxylin and Eosin staining of histological sections of control (A,D,G) and Vhlh null (B,C,E,F,H,I) tibia proximal growth plates at birth. (D-I) Magnification of the resting zone (D-F) and proliferative zone (G-I) of newborn control (D,G) and Vhlh null (E,F,H,I) growth plates. Arrowheads (B,C) indicate areas occupied with atypical enlarged chondrocytes; arrows indicate areas of increased matrix deposition within the Vhlh growth plate.

 


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  		Fig. 3. In situ hybridization analysis of histological sections of proximal tibia growth plates from control (A-D) and Vhlh null (E-H) newborn mice, with type II collagen (A,B,E,F), type X collagen (C,G) and PTH/PTHrP receptor (D,H) cRNA; darkfield and brightfield images are shown.

 


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  		Fig. 7. (A) Evidence for stabilization of HIF1{alpha} in Vhlh null primary chondrocytes. Western blot analysis of whole-cell lysate from primary chondrocytes isolated from Vhlh+f/+f newborn mice and infected with ß-galactosidase (WT) or Cre recombinase (Vhlh null) adenoviral constructs. Levels of Pro564-hydroxylated HIF1{alpha} and {alpha}-tubulin were analyzed. (B) Evidence for increased HIF1{alpha} activity in Vhlh null primary chondrocytes. Elisa measurements of soluble VEGF in the supernatant from primary chondrocytes isolated from Vhlh+f/+f newborn mice and infected with ß-galactosidase (WT) or Cre recombinase (Vhlh null) adenoviral constructs. (C) VEGF, PGK and type II collagen mRNA expression measured by real-time PCR of total RNA isolated from primary chondrocytes isolated from Vhlh+f/+f newborn mice and infected with ß-galactosidase (WT) or Cre recombinase (Vhlh null) adenoviral constructs.

 


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  		Fig. 4. Delay of the secondary ossification center in the Vhlh null growth plate. (A-D) Hematoxylin and Eosin staining of histological sections of control (A,C) and null (B,D) proximal tibia growth plates from 5-day-(A,B), and 1-month-old (C,D) mice. (E-J) In situ hybridization analysis of histological sections of tibia proximal growth plates from control (E,G,I) and Vhlh null (F,H,J) 5-day-old mice, with type X collagen (E,F), PTH/PTHrP receptor (G,H) and Indian Hedgehog (I,J) cRNA; darkfield images are shown. (K,L) Articular cartilage of control and Vhlh null animals, one month after birth, at higher magnification.

 


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  		Fig. 5. Evidence for a decreased cell proliferation rate within the Vhlh null growth plate. (A,B) Detection of BrdU-labeled chondrocytes in histological sections of E16.5 control (A) and Vhlh null (B) proximal tibia growth plates. (C,D) In situ hybridization analysis with p57kip2 cRNA on histological sections of newborn control (C) and Vhlh null (D) growth plates; darkfield images are shown. (E) Percentage of BrdU-labeled cells; bars represent mean percentages (±s.d.) of BrdU-labeled chondrocytes in Vhlh null and wild-type (WT) growth plates at E14.5, E15.5 and E16.5, separated into proliferating zone (PZ) and resting zone (RZ). Statistical differences in each zone were identified using the unpaired t-test. **P<0.01.

 


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  		Fig. 6. (A,B) Evidence for stabilization of HIF1{alpha} in the Vhlh null growth plate. Immunohistochemical detection of HIF1{alpha} in cryosections of newborn control (A) and Vhlh null (B) proximal tibia growth plates. (C-F) Evidence for an increased HIF1{alpha} activity in Vhlh null growth plates. In situ hybridization analyses of histological sections of proximal tibia growth plates from control (C,E) and Vhlh null (D,F) newborns, with PGK (C,D) and VEGF (E,F) cRNA; darkfield images are shown.

 


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  		Fig. 8. Analysis of growth plates from control, Hif1a null and Hif1a/Vhlh null mice. (A-C,H-J) Hematoxylin and Eosin staining of histological sections of control (A,H), Hif1a null (B,I) and Hif1a/Vhlh null mice (C,J), at E14.5 (A-C) and birth (H-J). (E-G) Evidence for increased cell proliferation rates in Hif1a null and Hif1a/Vhlhnull chondrocytes. Detection of BrdU-labeled chondrocytes in histological sections of E14.5 control (E), Hif1a null (F) and Hif1a/Vhlh null (G) proximal tibia growth plates. (D) Percentage of BrdU-labeled cells; bars represent mean percentages (±s.d.) of BrdU-labeled chondrocytes in Hif1a null, Hif1a/Vhlh null and wild-type (WT) growth plates at E14.5, separated into proliferating zone (PZ) and resting zone (RZ). Statistical differences in each zone were identified using the unpaired t-test. **P<0.01.

 

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© The Company of Biologists Ltd 2004