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First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00772

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Defective endochondral ossification in mice with strongly compromised expression of JunB

Jochen Hess*, Bettina Hartenstein*, Sibylle Teurich, Dirk Schmidt{ddagger}, Marina Schorpp-Kistner and Peter Angel§

Deutsches Krebsforschungszentrum Heidelberg (DKFZ), Division of Signal Transduction and Growth Control (A100), Im Neuenheimer Feld 280, D-69120 Heidelberg



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  		Fig. 1. Compromised JunB expression in mutant mice leads to growth retardation and shortening of long bones. (A) Comparison of an 8-week-old control (311K; bottom) and a junB–/– Ubi-junB (311R; top) mouse, (B) reduced length of tibiae and femurs of age matched female junB–/– Ubi-junB (311R; open circles) compared to control mice (filled circles). Black bars indicate the mean values of control (n=10, wt and 311K) and mutant animals (n=7), (C) RT-PCR analysis of junB mRNA with total RNA derived from long bone epiphysis of 8-week-old junB–/– Ubi-junB mice (311R) and control (311K) littermates. RT-PCR analysis for ß-Tubulin served as a control for quality and quantity of the cDNA samples. (D) Expression of JunB protein was detected by immunohistochemistry in proliferative (PC) and prehypertrophic chondrocytes, but not in hypertrophic chondrocytes (HC), on 6 µm growth plate sections from 4-week-old control (311K) mice. Additionally, expression of JunB protein was demonstrated in control osteoblasts and osteocytes in the trabecular bone area (arrows). In junB–/– Ubi-junB bone sections only very few chondrocytes, but no osteoblasts, were positive for JunB protein (arrows). Control sections were incubated only with the second antibody (left panel). Sections were counterstained with Haematoxylin; magnification was 20x.

 


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  		Fig. 2. Hypochondrodysplasia-like phenotype in growth plates of junB–/– Ubi-junB mice. (A) Growth plate morphology of proximal tibiae from 4-, 8- and 16-week-old control (311K) and junB–/– Ubi-junB (311R) littermates. Black lines indicate differences in the size of control and mutant growth plates. Sections were stained with Haematoxylin and Eosin. PC, proliferative chondrocytes; HC, hypertrophic chondrocytes. Magnification was 20x (4 weeks, 8 weeks) and 40x (16 weeks). (B) In situ detection of type-II (col-II), type-X collagen (col-X), alkaline phosphatase (alp) and vascular endothelial growth factor (vegf) mRNAs in growth plates of femur sections prepared from 4-week-old control (middle panel) and junB–/– Ubi-junB (right panel) littermates. Hybridisation of control sections with sense probes served as control for specificity (left panel). Dark-field images were used to demonstrate vegf expression in hypertrophic chondrocytes. Sections were counterstained with Haematoxylin and Eosin; magnification was 20x.

 


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  		Fig. 4. Expression of osteoblast-specific marker genes in long bones. In situ detection of marker genes expressed in osteoblasts of proximal tibiae sections derived from 4-week-old control (middle panel) and junB–/– Ubi-junB littermates (right panel): type-I collagen (col-I, A-C), matrix metalloproteinase 13 (mmp13, D-F), osteopontin (opn, G-I) and osteocalcin (oc, J-L). The arrow indicates accumulation of osteocalcin-positive osteoblasts at the chondrocyte/osteoblast junction in mutant sections. Hybridisation of control sections with sense probes (left panel) served as controls for specificity. Sections were counterstained with Haematoxylin and Eosin. Magnification was 10x.

 


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  		Fig. 3. Reduced trabecular and cortical bone in junB–/– Ubi-junB animals. (A) Bone sections of 4-week-old (top), 8-week-old (middle) and 16-week-old (bottom) control and mutant littermates were stained with Haematoxylin and Eosin, magnification was 10x (top and middle) or 5x (bottom). (B) TRAP staining of osteoclasts in distal tibiae sections of 4-week-old control and junB–/– Ubi-junB littermates. (C) In situ detection of MMP-9-expressing osteoclasts and chondroclasts in proximal tibia sections of 4-week-old control (middle) and mutant mice (right). Hybridisation of control section with sense probe served as control for specificity (left). Sections were counterstained with Haematoxylin and Eosin; magnification was 20x (B,C).

 


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  		Fig. 5. In vitro differentiation of bone marrow-derived stromal cells. (A) In vitro differentiated control (left panel) and junB–/– Ubi-junB cultures (right panel) derived from marrow stromal cells were stained for ALP activity to measure osteoblast differentiation, or with alizarin Red for osteoblast function. Stromal cells from transgenic mutant lines 311R (right panel) and 288R (data not shown) revealed similar defects in the process of in vitro differentiation. (B) Colony forming unit assay with bone marrow cells from control and junB–/– Ubi-junB littermates. Colonies were stained for ALP activity and counterstained with Haematoxylin. Representative images are shown on the right. Relative number of colonies (each containing a minimum of 20 cells) was calculated after the total number of control colonies was set to 100%. Bars represent mean ± s.e.m. calculated from two independent experiments done in triplicates.

 


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  		Fig. 6. Impaired proliferation and deregulated expression of cell cycle regulators in junB–/– Ubi-junB cultures. (A) Growth rate of bone marrow-derived control (filled symbols) and junB–/– Ubi-junB (open symbols) stromal cells. 1x104 cells were plated in 96-well plates and total number of cells was measured at indicated time points. Values are means ± s.e.m. of three independent experiments done in duplicates. (B) Reduced number of BrdU-positive cells in junB–/– Ubi-junB stromal cell cultures. Cells were cultured in the presence of 60 µM BrdU for 2 hours and the BrdU-positive cells were analysed by immunohistochemistry with an anti-BrdU antibody. Bars represent mean ± s.e.m. of two independent experiments done in triplicates. (C) Expression of cell cycle regulators (Cyclin A, Cyclin D1, p16INK4 and p21) was measured by RT-PCR analysis with cDNA prepared from asynchronously growing control (lane 1) and mutant (lane 2) stromal cells. RT-PCR analysis for JunB demonstrates lack of JunB expression in bone marrow-derived stromal cell cultures and ß-Tubulin served as control for quantity and quality of used cDNA samples.

 


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  		Fig. 7. Deregulated expression of Cyclin A and p16INK4a in junB–/– Ubi-junB chondrocytes and osteoblasts. Reduced proliferation of growth plate chondrocytes and osteoblasts in junB–/– Ubi-junB long bones was demonstrated by immunohistochemistry with a monoclonal anti-PCNA antibody (A-E). Cyclin A (F-J) and p16INK4 (K-O) protein levels were determined by immunohistochemistry using polyclonal anti-Cyclin A or anti-p16INK4a antibodies, respectively. Arrowheads indicate Cyclin A-positive chondrocytes and osteoblasts in control sections (G,I) and chondrocytes and osteoblasts with increased expression of p16INK4 in mutant sections (M,O). PCNA-positive (E) and Cyclin A positive (J) haematopoietic cells were still present in junB–/– Ubi-junB bone marrow (arrows). Control sections were incubated only with second antibody (A,F,K). Sections were counterstained with Haematoxylin; 40x magnification.

 

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© The Company of Biologists Ltd 2003