RESUMO
OBJECTIVE: Increasing evidence points to a strong genetic component to osteoarthritis (OA) and that certain changes that occur in osteoarthritic cartilage recapitulate the developmental process of endochondral ossification. As zebrafish are a well validated model for genetic studies and developmental biology, our objective was to establish the spatiotemporal expression pattern of a number of OA susceptibility genes in the larval zebrafish providing a platform for functional studies into the role of these genes in OA. DESIGN: We identified the zebrafish homologues for Mcf2l, Gdf5, PthrP/Pthlh, Col9a2, and Col10a1 from the Ensembl genome browser. Labelled probes were generated for these genes and in situ hybridisations were performed on wild type zebrafish larvae. In addition, we generated transgenic reporter lines by modification of bacterial artificial chromosomes (BACs) containing full length promoters for col2a1 and col10a1. RESULTS: For the first time, we show the spatiotemporal expression pattern of Mcf2l. Furthermore, we show that all six putative OA genes are dynamically expressed during zebrafish larval development, and that all are expressed in the developing skeletal system. Furthermore, we demonstrate that the transgenic reporters we have generated for col2a1 and col10a1 can be used to visualise chondrocyte hypertrophy in vivo. CONCLUSION: In this study we describe the expression pattern of six OA susceptibility genes in zebrafish larvae and the generation of two new transgenic lines marking chondrocytes at different stages of maturation. Moreover, the tools used demonstrate the utility of the zebrafish model for functional studies on genes identified as playing a role in OA.
Assuntos
Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Predisposição Genética para Doença/genética , Osteoartrite/genética , Osteoartrite/fisiopatologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Condrócitos/patologia , Cromossomos Artificiais Bacterianos/genética , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo II/fisiologia , Colágeno Tipo IX/genética , Colágeno Tipo IX/metabolismo , Colágeno Tipo IX/fisiologia , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Colágeno Tipo X/fisiologia , Fator 5 de Diferenciação de Crescimento/genética , Fator 5 de Diferenciação de Crescimento/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Hipertrofia/genética , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/fisiologia , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/fisiologiaRESUMO
The aim of this study was to test whether the nitric oxide (NO) donor sodium nitroprusside (SNP) has an effect on mineralization in ATDC5 cells. Mineralization in ATDC5 cell culture was induced by addition of beta-glycerophosphate or inorganic phosphate, visualized by staining precipitated calcium with an alizarin red stain, and quantified using atomic absorption spectrometry. SNP was shown to inhibit the mineralization of ADTC5 cells. This inhibition was not affected by inhibitors of guanylyl cyclase nor mimicked by a cyclic guanosine monophosphate (cGMP) analog. Furthermore, SNP did not inhibit phosphate uptake or inhibit apoptosis in ATDC5 cells. These findings indicate that SNP can specifically inhibit matrix mineralization via a cGMP-independent pathway and that the effect is not mediated by inhibition of phosphate transport or apoptosis. These results suggest a preventive role of NO in premature or pathological mineralization.
