Dominant osteogenesis imperfecta (OI) is a bone disease mainly caused by collagen type I mutations and characterized by bone fragility and growth delay. Nowadays no definitive cure is available. A zebrafish OI model (Chihuahua) carrying an heterozygous G574D substitution in the α1 chain of collagen type I was generated by ENU mutagenesis and is available in our laboratory. Control (WT) and mutant (Chi+/−) fish growth was followed up from day 1 post fertilization to 5 month old. Skeleton was analyzed by X-ray, μCT and specific bone staining. Collagen I was extracted from skin, scales and bone and analyzed by SDS-PAGE. Collagen melting stability was evaluated by Differential Scanning Calorimetry (DSC). Electron microscopy of osteoblasts and confocal microscopy of fibroblasts were also performed. At morphological level, Chi+/− embryos show a typical fin fold bending, whereas at adult age a smaller size compared to WT was significant starting from 3 months. X-ray and μCT revealed reduced mineralization in adult Chi+/− vs WT. Skeletal staining highlighted multiple bone fractures and deformities, such as alterations in neural spine inclination. A delayed mineralization was also observed in mutant larvae. Collagen type I presents a delay in migration and a broadening of the α bands in SDS-PAGE, revealing the presence of overmodification, while DSC showed ~2 °C reduction in the Tm of mutant collagen compared to WT. The presence of an endoplasmic reticulum (ER) enlargement in osteoblasts and fibroblasts of mutant fish was evident by microscopy analysis. The collagen overmodification and ER retention and the skeletal phenotype found in Chi+/− fish reproduce the main features reported in OI patients, validating this model for the study of the disease and for the evaluation of novel possible therapy.
All animal experiments were in agreement with EU Directive 2010/63/EU.
14 May 2016 - 17 May 2016