Age-related degenerative changes within the vertebral column are a significant cause of morbidity with considerable socio-economic impact worldwide. An improved understanding of these changes through the development of experimental models may lead to improvements in existing clinical treatment options. The zebrafish is a well-established model for the study of skeletogenesis with significant potential in gerontological research. With advancing age, zebrafish frequently develop gross deformities of their vertebral column, previously ascribed to reduced trunk muscle tone. In this study, we assess degenerative changes specifically within the bone and cartilage of the vertebral column of zebrafish at 1, 2 and 3-years of age. We show increased frequency and severity of spinal deformities/curvatures with age. Underlying the most severe phenotypes are partial or complete vertebral dislocations and focal thickening of the vertebral bone at the joint margins. MicroCT examination demonstrates small defects, fractures and morphological evidence suggestive of bone erosion and remodeling (i.e. osteophytes) within the vertebrae during aging, but no significant change in bone density. Light and electron microscopic examination reveal striking age-related changes in cell morphology, suggestive of chondroptosis, and tissue remodelling of the vertebral cartilage, particularly within the pericellular micro-environment. Glycosaminoglycan analysis of the vertebral column by HPLC demonstrates a consistent, age-related increase in the yield of total chondroitin sulfate disaccharide, but no change in sulfation pattern, supported by immunohistochemical analysis. Immunohistochemistry strongly identifies all three chondroitin/dermatan sulphate isoforms (C-0-S, C-4-S/DS and C-6-S) within the vertebral cartilage, particularly within the pericellular micro-environment. In contrast, keratan sulfate immunolocalises specifically with the notochordal tissue of the intervertebral disc, and its labelling diminishes with age. In summary, these observations raise the prospect that zebrafish, in addition to modelling skeletal development, may have utility in modelling age-related degenerative changes that affect the skeleton during senescence.
Background: Chondroitin sulfate (CS) and heparan sulfate (HS) are important for cartilage formation. Results: Analyses of zebrafish mutants demonstrate interplay between HS and CS biosynthesis in vivo.Conclusion: HS biosynthesis is prioritized over CS biosynthesis. The balance between extl3 and csgalnact1/csgalnact2 function determines the HS/CS ratio. Significance: Disease progression caused by HS deficiency may be affected by altered CS biosynthesis.
Background: Chondroitin/dermatan sulfate (CS/DS) proteoglycans present in the extracellular matrix have important structural and regulatory functions. Results: Six human genes have previously been shown to catalyze CS/DS polymerization. Here we show that one of these genes, chpf, is represented by two copies in the zebrafish genome, chpfa and chpfb, while the other five human CS/DS glycosyltransferases csgalnact1, csgalnact2, chpf2, chsy1, and chsy3 all have single zebrafish orthologues. The putative zebrafish CS/DS glycosyltransferases are spatially and temporally expressed. Interestingly, overlapping expression of multiple glycosyltransferases coincides with high CS/DS deposition. Finally, whereas the relative levels of the related polysaccharide HS reach steady-state at around 2 days post fertilization, there is a continued relative increase of the CS amounts per larvae during the first 6 days of development, matching the increased cartilage formation. Conclusions: There are 7 CS/DS glycosyltransferases in zebrafish, which, based on homology, can be divided into the CSGALNACT, CHSY, and CHPF families. The overlap between intense CS/DS production and the expression of multiple CS/DS glycosyltransferases suggests that efficient CS/DS biosynthesis requires a combination of several glycosyltransferases. Developmental Dynamics 242:964-975, 2013. V C 2013 Wiley Periodicals, Inc.Key words: chondroitin sulfate; polymerase; CSGALNACT; CHSY; CHPF; zebrafish Key Findings:The zebrafish genes csgalnact1, csgalnact2, chsy1, chsy3, chpf2, chpfa, and chpfb are orthologues to the human genes that mediate CS/DS polymerization. Chordate CS/DS glycosyltransferases may be divided into the CSGALNACT, CHSY, and CHPF families. Sites of high CS/DS production in zebrafish embryos are characterized by the overlapping expression of multiple CS/DS producing glycosyltransferases. The content of CS/DS per fish is massively increased during zebrafish larval development whereas the heparan sulfate content gets stabilized around 48 hpf.
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