spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 25 October 2006
doi: 10.1242/dev.02680

Development 133, 4667-4678 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Related articles in Development
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yoon, B. S.
Right arrow Articles by Lyons, K. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yoon, B. S.
Right arrow Articles by Lyons, K. M.

BMPs regulate multiple aspects of growth-plate chondrogenesis through opposing actions on FGF pathways

Byeong S. Yoon1, Robert Pogue2, Dmitri A. Ovchinnikov3, Isaac Yoshii1, Yuji Mishina4, Richard R. Behringer3 and Karen M. Lyons1,2,*

1 Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, CA 90095, USA.
2 Department of Orthopaedic Surgery, David Geffen School of Medicine at the University of California, Los Angeles, CA 90095, USA.
3 Department of Molecular Genetics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
4 Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.


Figure 1
View larger version (76K):

[in a new window]
  		Fig. 1. Localization of BMP receptors, and SMAD1, SMAD5 and SMAD8 activity in adjacent sections through an E16.5 proximal femur. (A) Alcian blue staining reveals the resting (res), columnar proliferative (prol), prehypertrophic (ph), and hypertrophic (hyp) zones. (B) Indirect immunofluorescence for BMPRIA reveals highest levels in the prehypertrophic and hypertrophic zones. (C) Staining for BMPRIB indicates expression throughout the growth plate, with higher levels in resting and prehypertrophic zones. (D) IHC for pSMAD1, pSMAD5 and pSMAD8 reveals highest levels of canonical BMP signaling in lower proliferative and prehypertrophic chondrocytes. (E) Percentages of pSMAD1-, pSMAD5- and pSMAD8-positive cells were determined in the consecutive segments delineated with black lines in D.

 

Figure 2
View larger version (38K):

[in a new window]
  		Fig. 2. Generalized chondrodysplasia in Bmpr1aCKO and Bmpr1aCKO;1b+/- neonates. (A,B) Chondrodysplasia in Bmpr1aCKO mice is exacerbated in Bmpr1aCKO;1b+/- mice. (C) Bmpr1aCKO and Bmpr1aCKO;1b+/- mice exhibit multiple axial defects. (D) The dorsal arches (da) of the vertebrae are short and fail to fuse in mutants. (E,F) Forelimb (E) and hindlimb (F) defects. All long bones are shortened in Bmpr1aCKO mice, and the scapula is hypoplastic. These defects are exacerbated in Bmpr1aCKO;1b+/- mice, in which the distal epiphyses of the humerus and femur are greatly reduced. f, femur; fi, fibula; h, humerus; r, radius; s, scapula; t, tibia; u, ulna; eo, exoccipital bone; so, supraoccipital bone; C1, first cervical vertebra.

 

Figure 3
View larger version (122K):

[in a new window]
  		Fig. 3. Growth-plate defects in Bmpr1aCKO and Bmpr1aCKO;1b+/- mice. (A,B) Alcian blue-stained sections through E14.5 (A) and E16.5 (B) distal femurs. Restriction in the width of the growth plate at the junction between the resting and proliferative zones is observed in Bmpr1aCKO and Bmpr1aCKO;1b+/- mice compared with wild-type. (C) H/E-stained sections through columnar zones of E16.5 wild-type, Bmpr1aCKO and Bmpr1aCKO;1b+/- littermates reveal a failure to adopt a flattened cell shape and intercalate in mutants. (D) H/E-stained sections through the hypertrophic zones of E16.5 wild-type, Bmpr1aCKO and Bmpr1aCKO;1b+/- littermates reveal the accumulation in mutants of cells with tightly packed nuclei (arrows), a characteristic feature of late hypertrophic chondrocytes. hyp, hypertrophic zone; ph, prehypertrophic zone; prol, proliferative zone; res, resting zone.

 

Figure 4
View larger version (66K):

[in a new window]
  		Fig. 4. Proliferative defects in Bmpr1aCKO;1b+/- mice. All sections are E16.5 femurs. (A) Immunostaining for PCNA. (B) TUNEL assay reveals abundant apoptotic cells in the resting and columnar zones of mutants. (C) Quantification of the rates of proliferation (PCNA) and apoptosis (TUNEL) in wild-type and mutant littermates. Values are expressed as percent labeled cells. (D) Immunostaining for CKIs reveals increased expression of p16, p21 and p27 in mutant proliferative zones. *P<0.001.

 

Figure 5
View larger version (111K):

[in a new window]
  		Fig. 5. Defects in hypertrophic differentiation in Bmpr1aCKO mice. (A) E14.5 and (B) E16.5 distal femoral growth plates immunostained for markers of hypertrophic differentiation. In B, the length of the hypertrophic zone is demarcated by a black bar. The region of marker expression is demarcated by blue double-headed arrows. There is an increase in the overall length of the hypertrophic zone and a disproportionately enlarged zone of late hypertrophic chondrocytes in mutants at E16.5.

 

Figure 6
View larger version (147K):

[in a new window]
  		Fig. 6. Impaired growth-plate angiogenesis in Bmpr1aCKO mice. (A) H/E-stained sections from E16.5 distal femurs. (B) Adjacent sections immunostained for PECAM. In wild-type mice, blood vessels are restricted to the chondro-osseous junction (boxed area in B), where apoptotic chondrocytes are located. In Bmpr1aCKO mutants, blood vessels can be seen invading the region of the growth plate occupied by late hypertrophic chondrocytes.

 

Figure 7
View larger version (47K):

[in a new window]
  		Fig. 7. Impaired IHH signaling in Bmpr1aCKO mice. (A) Expression of Ihh, and Ptc1 by in situ hybridization. Expression of Ihh and its target, Ptc, is reduced in mutants. (B) No differences in expression levels of the PTHrP receptor Pthr1 are observed in mutants. (C) Ihh may be a direct target of BMP pathways in chondrocytes. p994 luc consists of a 994 bp fragment of the mouse Ihh promoter (-889/+105) (Yoshida et al., 2004Go). 2HC8 consists of a 429 bp fragment of the mouse Ihh promoter (-423/+1) (Seki and Hata, 2004Go). These were transiently transfected into RCS chondrocytes in the presence or absence of BMP2 (60 ng/ml) and a SMAD1 expression construct. *P<0.05; **P<0.005.

 

Figure 8
View larger version (63K):

[in a new window]
  		Fig. 8. Elevated pSTAT and pERK1/2 in Bmpr1aCKO;1b+/- growth plates. All sections are E16.5 distal femurs. (A) p-STAT1 immunostaining. (B) Quantification of increased p-STAT1 in mutants, expressed as percent positive cells. (C) p-ERK1/2 immunostaining in the hypertrophic (upper) and periarticular (lower) region. P-ERK1/2 immunostaining can be detected in hypertrophic chondrocytes, but there is no detectable difference in the number of positive cells in this region. An increased percentage of p-ERK1/2-staining cells can be observed in the outlined periarticular (resting) zone in mutants. *P<0.01; **P<0.001.

 

Figure 9
View larger version (69K):

[in a new window]
  		Fig. 9. Expanded domain of FGFR1 expression in Bmpra1CKO and Bmpr1aCKO;1b+/- growth plates. Immunofluorescence staining for FGFR1 in (A) wild-type, (B) Bmpr1aCKO and (C) Bmpr1aCKO;1b+/- distal femurs at E16.5. Intense staining is restricted to the hypertrophic zone (arrow) in wild-type mice. In Bmpr1aCKO mice, intense staining for FGFR1 persists in the expanded hypertrophic zone in (arrow), but is also seen in epiphyseal and columnar chondrocytes. A similar expansion of the domain of FGFR1 expression is observed in Bmpr1aCKO;1b+/- growth plates.

 




© The Company of Biologists Ltd 2006