[85] Heparan Sulfate Is Required for Osteoblastic Linage Comitment.

Malgorzata I Wiweger, Pancras CW Hogendoorn. Leiden University Medical Center, Netherlands

Background: We have established a zebrafish (Danio rerio) model for multiple osteochondromas (MO), a syndrome caused by a mutation in the EXT1 or EXT2 genes leading to impaired biosynthesis of heparan sulfate. MO is mainly characterized by the development of benign cartilage-capped bone tumors at multiple sites of the skeleton. As shown in the zebrafish dackel (dak) mutant, heparan sulfate deficiency caused by a mutation in the ext2 gene, affects cartilage organization and bone development. Interestingly, dermal bones (intra-membranous bones in mammals) that do not require a cartilage template are also malformed and often lost in the homozygote mutant. It is known that mammalian mesenchymal precursors can differentiate towards: i) chondrocytes, ii) osteoblasts, iii) muscles or iii) apodiocytes. Previously, we have shown that differentiation of the chondrocytes is unaffected in the dak homozygote mutant. Here, we investigate if the impaired bone formation in the heparan sulfate-deficient fish might be explained by a switch in the commitment of osteoblast precursors towards fat and/or muscle cells.
Design: AB (wild type) and dak (ext2) homozygote mutant were raised under standard conditions. Fish were anesthetized in tricane, fixed in 4% formalin, dehydrated and stored in 100% methanol at -20 oC prior to use. Whole mount skeletons were stained with alizarin red and alcian blue. Fat deposition was visualized with Oil Red O. Expression patterns of osteoblast- and apodiocyte-related markers were studied by whole mount mRNA in situ hybridization. Anti-MF20 antibody was used for muscle analysis. Light microscopy observations were performed on at least ten fish.
Results: Even though the muscles in heparan-deficient fish were shorter and more spaced out, as they fitted the misshapen cartilaginous skeleton, the overall musculature of the dak homozygote mutant was similar to that of WT. However, homozygote mutant fish showed a clear phenotype related to an abnormally high accumulation of lipids. No cartilage-, fat-, bone- nor muscle-related phenotypes were observed in the heterozygote ext2 mutant. The molecular mechanisms underlying bone-to-fat switch are under investigation.
Conclusions: Heparan sulfate is involved in bone-to-fat switch during mesenchymal differentiation, without affecting chondrogenic- or muscle lineages. The presence of one functional copy of the EXT2 gene is sufficient for the maintenance of normal osteoblast and adipocyte differentiation. Hence, MO patients are expected to manifest mosaic bone-to-fat switch at locations where either loss of heterozygosity or haploinsuficiency occurred.
Category: Bone & Soft Tissue

Tuesday, March 1, 2011 9:30 AM

Poster Session III # 4, Tuesday Morning

 

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