CHARACTERISATION OF A NOVEL MODEL OF BONE CRUSH REPAIR AND IN VIVO ANALYSIS OF SKELETAL REGENERATION IN ZEBRAFISH

In mammals, repair of bone fractures is believed to be achieved by local activation and differentiation of osteogenic progenitor cells. However, lineage-tracing studies after fin amputation have demonstrated that bone regenerates from mature osteoblasts in the zebrafish fin through a process of partial dedifferentiation, migration and proliferation. Thus, this raises the question whether osteoblast dedifferentiation is specific to appendage regeneration, or a process found more generally to repair bone in this animal. These studies used tail amputation which is a more severe form of damage than a bone fracture. Nevertheless, Sousa and colleagues (2012) proposed a novel crush assay for adult bones in zebrafish fin rays which showed promising results and was also developed for this project. The first objective of the thesis was to go beyond to find differences in zebrafish compared to what is already known in the mammalian fracture model during the different healing stages (inflammation, repair, remodeling). Interestingly, my analysis showed no remarkable differences at the cellular or molecular level in comparison to mammalian fracture repair. Notably, reactive oxygen species (ROS) production, which are one of the first signals to be induced after damage, depend on the lesion type in adult zebrafish. IL1β cytokine is induced early after bone damage and neutrophils are recruited at the fracture site after few hours as well. Both seem to induce directly or indirectly osteoclast recruitment. Osteoclasts participate early but also remained active after several days. Moreover, to determine whether osteoblasts dedifferentiation is restricted to appendage regeneration, efforts were done to establish the Cre-Lox system in adult zebrafish. As a first step, a set of six double transgenic lines were created to enable tracking of bone cells in vivo. Afterwards, different tamoxifen-induced Cre-recombination strategies were implemented but no successful results were obtained. Further research is needed to get positive results. The knowledge generated in this research can contribute to set the basis for the development of further studies of bone repair in zebrafish that can complement the ones performed in classical models.