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 18 January 2006
doi: 10.1242/dev.02258

Development 133, 651-662 (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 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 Provot, S.
Right arrow Articles by Lassar, A. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Provot, S.
Right arrow Articles by Lassar, A. B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Nkx3.2/Bapx1 acts as a negative regulator of chondrocyte maturation

Sylvain Provot1,2,*, Hervé Kempf1,*, L. Charles Murtaugh1, Ung-il Chung2, Dae-Won Kim1, Jay Chyung1, Henry M. Kronenberg2 and Andrew B. Lassar1,{dagger}

1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
2 Endocrine Unit, Massachusetts General Hospital-Harvard Medical School, 50 Blossom Street, Boston, MA 02114, USA.


Figure 1
View larger version (63K):

[in a new window]
  		Fig. 1. Expression of chick Nkx3.2 is restricted to immature proliferative chondrocytes during endochondral bone development. Chick embryos, stages as indicated, were pulse labeled with BrdU before harvesting and processing for section in situ hybridization (ISH; A-J) or stained with an anti-BrdU antibody (K,L). Arrowheads indicate approximate boundary of BrdU-positive zone in parallel sections. Arrows (A-F) indicate distal-most boundary of cartilage, marked by col IX and Sox9 expression.

 

Figure 2
View larger version (72K):

[in a new window]
  		Fig. 2. Misexpression of Nkx3.2 inhibits chondrocyte maturation. The right wing buds of late E3 chick embryos (HH stage 16-18) were infected with RCAS(A)-Nkx3.2. The embryos were harvested on E9, and processed for Alcian Blue/Alizarin Red staining (A), or pulsed for three hours with BrdU prior to harvesting on E10 for ISH and BrdU incorporation (B,C). Red arrowhead (A) indicates the nascent bone collar. Double-headed arrows (B) compare the peripheral-most extent of Ihh and col X expression in the RCAS-Nkx3.2-infected and contralateral control wings. Arrowhead (C) indicates the approximate boundary between proliferative and non-dividing cells in the cartilage of the control wing; arrows indicate proliferative cells in central regions of the RCAS-Nkx3.2-infected wing cartilage. hu, humerus; ra, radius; ul, ulna.

 

Figure 3
View larger version (89K):

[in a new window]
  		Fig. 3. DNA binding and transcriptional repression by Nkx3.2 are required to block chondrocyte maturation. The right forelimb buds of day 3 chick embryos were infected with RCAS viruses encoding mutant forms of Nkx3.2, as indicated. The embryos were harvested at E10 for skeletal preparation (A-F), or section ISH (G-L) with the indicated probes.

 

Figure 4
View larger version (44K):

[in a new window]
  		Fig. 4. Nkx3.2-{Delta}C-VP16 acts as a reverse function form of Nkx3.2 and accelerates chondrocyte maturation. The right wing buds of day 3 chick embryos were infected with RCAS(A)-Nkx3.2-{Delta}C-VP16 virus. Embryos were harvested at either E10 (A,C) or E8.5 (B), and processed for Alcian Blue/Alizarin Red staining (A) or section ISH (B,C). The extent of mineralized bone (red bar in A) is increased in limbs infected with RCAS-Nkx3.2-{Delta}C-VP16 relative to the contralateral control limb. Double-headed arrows (B,C) compare either the peripheral-most extent of col X expression or the peripheral-most extent lacking col II expression.

 

Figure 5
View larger version (79K):

[in a new window]
  		Fig. 5. PTHrP signals are necessary to drive Nkx3.2 expression in the growth plate. (A) Infection of limbs with RCAS-Nkx3.2 results in the loss of PTHrP and endogenous Nkx3.2 expression. Arrowheads (part a) indicate strong PTHrP expression in the periarticular region of the control wing; arrowheads (part c) indicate normal expression of endogenous Nkx3.2 in control wing, whereas arrows (d) indicate the absence of endogenous Nkx3.2 expression in RCAS(A)Nkx3.2-infected cartilage. (B) Ectopic PTHrP delays chondrocyte maturation and maintains uniform Nkx3.2 expression throughout the cartilage. Wing buds of E3 chick embryos were infected with RCAS(A)-PTHrP virus. Embryos were harvested at E10, and RCAS(A)-PTHrP-infected wings and contralateral control wings were analyzed by Alcian Blue/Alizarin Red staining (AB/AR, parts a,b) or section ISH (c-l). Arrowheads (c,e,g) indicate the central region of the ulna in which expression of Sox9, Nkx3.2 and col II is downregulated in the control wing. Arrows (d,f,h) indicate the central region of the ulna in which expression of these markers is maintained in the RCAS-PTHrP infected wing. (C) Nkx3.2/Bapx1 expression is lost in PTHrP and PTHrP-receptor null animals. Bright field (a,d,f) or ISH (b,c,e,g) of E18.5 mouse tibiae with the indicated probes, taken from mice of indicated genotypes. (D) Loss of p57 expression rescues the absence of immature chondrocytes but not Nkx3.2/Bapx1 expression in the absence of PTHrP signals. ISH of E17.5 mouse ulnae, taken from mice of indicated genotypes, with indicated probes. (E) PTHrP signals induce the expression of Nkx3.2 in cultured chondrocytes. Upper sternal chondrocytes (USC) were isolated from the cephalic portion of 15-day-old chick embryo sternae and cultured for 3 days in either the absence (lane 1) or presence (lane 2) of 100nM PTHrP. Gene expression was assayed by RT-PCR.

 

Figure 6
View larger version (71K):

[in a new window]
  		Fig. 6. Nkx3.2 represses Runx2 expression. (A) Nkx3.2 and Runx2 are expressed in a reciprocal pattern in the developing cartilage elements of the limb. ISH for the indicated probes is displayed for E8 chick limbs. In the cartilage element, Runx2 expression is most intense where Nkx3.2 expression is diminished (marked by arrowheads in a,b). (B) Forced expression of Nkx3.2 represses Runx2 expression in vivo. The right wing buds of E3 chick embryos were infected with either RCAS(A)-Nkx3.2 (b,d) or RCAS(A)-Nkx3.2-{Delta}C-VP16 (f,h) viruses. Embryos were harvested at either E8.5 (a-d) or E8 (e-h), and sections of the infected or contralateral control wings were hybridized with the indicated probes. (C) The intensity of the Runx2 ISH signal displayed in B and in two other experiments was quantified, in both the peripheral (blue rectangles in B) and central regions (orange rectangles in B) of the cartilage in both control and infected wings, as indicated. (D) Forced expression of Nkx3.2 represses Runx2 expression in presomitic mesoderm explants in vitro. Explants of presomitic mesoderm were either cultured for 2 days in either Shh (lanes 1 and 3) or infected with RCAS-Nkx3.2 (lanes 2 and 4). The explants were cultured in Bmp4 for either an additional 6 (lanes 1 and 2) or 12 (lanes 3 and 4) days. Expression of indicated markers was assayed by RT-PCR.

 

Figure 7
View larger version (73K):

[in a new window]
  		Fig. 7. Forced Runx2 expression rescues an Nkx3.2-induced but not a PTHrP-induced blockade of chondrocyte maturation. (A) Forced Runx2 expression rescues an Nkx3.2-induced blockade of chondrocyte maturation in vivo. The right wing buds of E3 chick embryos were co-infected with RCAS(A)-Nkx3.2 plus RCAS(B)-Alkaline Phosphatase (AP) viruses (b,e,h,k,n), or RCAS(A)-Nkx3.2 plus RCAS(B)-Runx2 viruses (c,f,i,l,o), and reincubated until E10. The control contralateral uninfected wing (a,d,g,j,m) and the co-infected wings were analyzed either by Alcian Blue/Alizarin Red staining (AB/AR, a-c), or by section ISH with the indicated probes (d-o). (B) Nkx3.2 does not block chondrocyte hypertrophy induced by ectopic Runx2 in vitro. Explants of presomitic mesoderm were co-infected with the indicated viruses and cultured sequentially in Shh for 2 days and Bmp4 for 3 days. Gene expression was assayed by RT-PCR. (C) PTHrP and forskolin can block chondrocyte hypertrophy induced by ectopic Runx2 in vitro. Explants of presomitic mesoderm were infected with RCAS(A)-Runx2 and cultured as described in part B, in either the absence (lanes 1 and 5) or in the presence of increasing amounts of either PTHrP (lanes 2-4) or forskolin (lanes 6 and 7), as indicated. Gene expression was assayed by RT-PCR.

 

Figure 8
View larger version (45K):

[in a new window]
  		Fig. 8. PTHrP signals block chondrocyte hypertrophy by both Nkx3.2/Bapx1-dependent and -independent pathways. Our findings indicate that PTHrP signals are necessary and sufficient to maintain the expression of Nkx3.2/Bapx1 in the proliferative region of the growth plate, and suggest that Nkx3.2/Bapx1 blocks chondrocyte maturation in this region of the growth plate by repressing the expression of Runx2 mRNA and possibly other co-factors necessary for chondrocyte hypertrophy. In addition, PTHrP signals can signal via a cAMP-dependent pathway to block the ability of Runx2 to induce chondrocyte maturation in a post-transcriptional manner.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006