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 15 April 2009
doi: 10.1242/dev.030742

Development 136, 1697-1706 (2009)
Published by The Company of Biologists 2009
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
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 Cortes, M.
Right arrow Articles by Schwartz, N. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cortes, M.
Right arrow Articles by Schwartz, N. 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?

Sulfation of chondroitin sulfate proteoglycans is necessary for proper Indian hedgehog signaling in the developing growth plate

Mauricio Cortes1, Alexis T. Baria2 and Nancy B. Schwartz1,2,*

1 Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
2 Department of Pediatrics, The University of Chicago, Chicago, IL 60637, USA.


Figure 1
View larger version (71K):
[in this window]
[in a new window]

  		Fig. 1. Altered glycosaminoglycan sulfation in the bm growth plate. (A-D') Immunofluorescence of postnatal day 6 proximal tibia of wild-type (A-D) and bm (A'-D') mice with monoclonal antibodies (red) specific to distinct sulfated GAG epitopes ({alpha}-CS4, {alpha}-CS 6, {alpha}-CS0 and {alpha}-HS) counterstained with DAPI (blue). Note decreased staining of chondroitin-4-sulfate (A,A'), and chondroitin-6-sulfate (B,B') in the bm growth plate. Conversely, chondroitin-0-sulfate staining increased in the bm growth plate (C,C'), whereas there was no consistent difference in heparan sulfate antibody staining (D,D'). Zones are marked as follows: resting (R), proliferative (P) and hypertrophic (H). Use the zone references on left side for wild-type sections and on right side for bm sections.

 

Figure 2
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 2. FACE analysis of glycosaminoglycan content in the bm growth plate. Fluorophore-assisted carbohydrate electrophoresis (FACE) profile of wild-type and bm postnatal day 6 cartilage for chondroitin sulfate and heparan sulfate. (A) Relative percentage composition of the CS-4, CS-6 and CS-0 disaccharides generated upon chondroitinase digestion for wild-type and bm growth plates, demonstrating significant changes in sulfated CS content between bm and wild type. (B) HS-NS, HS-6S and HS-0S disaccharide composition generated upon heparatinase digestion for wild-type and bm cartilage, illustrating no significant differences in HS sulfation between wild-type and bm growth plates. For each experiment, cartilage samples were collected and pooled from the long bone epiphyses of at least six neonate pups. Three independent experiments were performed, each with triplicate samples for statistical purposes.

 

Figure 3
View larger version (104K):
[in this window]
[in a new window]

  		Fig. 3. Comparative analysis of wild-type and bm growth plate. (A-G) In situ hybridization of wild type and bm day 6 growth plates revealed comparable mRNA levels of Acan (B), Col10a1 (C) and Pthr1 (D), and varying degrees of decreased mRNA levels for Papss2 (A), Fgfr3 (E), Ihh (F) and Ptch1 (G). (H) Semi-quantitative RT-PCR for various growth plate markers showing a reduction in mRNA expression for Papss2, Fgfr3 and Ptch1 in the bm growth plate, and comparable expression of Acan, Col10a1, Pthr1 and Ihh.

 

Figure 4
View larger version (85K):
[in this window]
[in a new window]

  		Fig. 4. Abnormal Ihh signaling in the bm mouse growth plate. (A-B'') Representative immunostaining of wild-type (A-A'') and bm (B-B'') day 6 distal tibias for secreted Ihh (green), counterstained with DAPI (blue). The resting (R), proliferative (P) and hypertrophic (H) zones are indicated, respectively. Wild-type tissue shows graded distribution of Ihh throughout the ECM from the proliferative zone to the resting zone (A). By contrast, the bm growth plate displays abnormal Ihh distribution marked by aggregates in the proliferative zone (B). Higher magnification views (A',A'',B',B'') show the restricted diffusion of Ihh in the bm growth plate marked by the reduction in Ihh surrounding cells in the resting zone (A',B', arrowhead), and aggregation of Ihh in the proliferative zone (A'',B'', arrowheads). (C) β-Gal staining of proximal tibia growth plates from wild-type and bm mice heterozygous for the Ptch1lacZ mutant allele show that, in the bm mouse, there is a reduction in the range of β-gal-positive cells (black double-headed arrow), highlighted by an increase in the proportion of resting chondrocytes that are not β-gal positive (yellow double-headed arrow). (D) Semi-quantitative RT-PCR for the Ihh signaling activator (Gli1) and Ihh signaling repressor (Gli3), showing a reduction in Gli1 mRNA expression. Quantification of the shown RT-PCR, illustrating the reduction in both Gli1 mRNA and the ratio of Gli1/Gli3 in the bm growth plate.

 

Figure 5
View larger version (46K):
[in this window]
[in a new window]

  		Fig. 5. Proliferation defect in the bm growth plate. (A) Immunofluorescence of post-natal day 6 wild-type (+/+) and bm (–/–) growth plates with BrdU monoclonal antibody (red), and counterstained with DAPI nuclear stain (blue). The respective proliferative (P) zone for wild-type and bm sections is demarcated. (B) Quantification of BrdU incorporation in the proliferative region for wild-type and bm day 6 growth plates (*P<0.0001, n=9). The percentage of BrdU-positive cells was determined by dividing the number of BrdU-positive cells by the total number of DAPI-positive cells in the limb sections analyzed.

 

Figure 6
View larger version (18K):
[in this window]
[in a new window]

  		Fig. 6. Interaction of Ihh to sulfated glycosaminoglycans. (A) Alignment of the N-terminal domain of the three mammalian hedgehog family members: sonic (Shh), Indian (Ihh) and desert (Dhh). The basic stretch of amino acids is highlighted in blue. Sequence alignment was carried out using Accelerys DSGene software. (B) Binding curves of Ihh to various to GAGs. Ihh affinity to GAG chains was determined through binding of Ihh-alkaline phosphatase fusion protein (IhhAP) to distinct sulfated CS and HS chains. Similarly, the affinity of an IhhAP mutant (lacking the proteoglycan binding domain) was also determined. Relative fluorescent units (RFU) are plotted in relation to IhhAP concentration. Data are representative of three independent experiments. Binding of wild-type IhhAP fusion protein to various GAG chains revealed that Ihh binds to HS, CS4, CS6 and CS0 (closed symbols), with decreasing binding capacities and Ihh has the lowest binding capacity for non-sulfated CS. Binding assays with an IhhAP mutant harboring a mutation in the proteoglycan binding motif reveal complete loss of binding to all GAG chains tested (HS, CS4, CS6 and CSO, open symbols). (C) Curve fitting analysis for IhhAP binding affinity (Kd) and binding capacity (Bmax) for HS, CS4, CS6 and CS0, respectively. Curve fitting analysis was carried out using GraphPad Prism 4.

 

Figure 7
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 7. Co-immunoprecipitation of Ihh and aggrecan. Embryonic day 14 chick cartilage lysates incubated with IhhAP fusion protein were immunoprecipitated in the absence (–S103L) or presence (+S103L) of the aggrecan-specific monoclonal antibody S103L. Significantly more IhhAP activity was measured in immunoprecipitates in the presence of S103L antibody (*P<0.05, n=3). Treatment of cartilage lysates with chondroitinase ABC (+S103L/+ChABC) prior to immunoprecipitation resulted in a significant decrease in the amount of IhhAP protein bound to aggrecan (**P<0.05, n=3).

 

Figure 8
View larger version (52K):
[in this window]
[in a new window]

  		Fig. 8. Model for the function of sulfated CSPGs in modulating Ihh signaling in the developing growth plate. (A,B) The gradient CSPG expression is depicted in the wild-type (A) and bm (B) growth plates. Prehypertrophic chondrocytes (red) are the source of Ihh, which is secreted into the extracellular matrix and acts on the proliferative (blue) and resting (yellow) chondrocytes, inducing expression of Ptch1 and Pthrp. In the bm growth plate, undersulfation of CSPGs results in reduced and abnormal Ihh distribution, leading to reduced proliferation and diminished growth plate length. (C) In this model, a gradient of matrix-associated CSPGs (aggrecan) from the source of hedgehog expression to its target is established for the proper movement of hedgehog to its target cells (proliferative chondrocytes) by actively participating in the diffusion of hedgehog or by protecting hedgehog from degradation. Finally, cell-surface HSPGs (which have higher affinity for hedgehog) are required to be present on the target cells to bring hedgehog close to the plasma membrane for proper interaction with its receptor.

 

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 2009