Quantitative assessment of the regenerative and mineralogenic performances of the zebrafish caudal fin

The ability of zebrafish to fully regenerate its caudal fin has been explored to better understand the mechanisms underlying de novo bone formation and to develop screening methods towards the discovery of compounds with therapeutic potential. Quantifying caudal fin regeneration largely depends on successfully measuring new tissue formation through methods that require optimization and standardization. Here, we present an improved methodology to characterize and analyse overall caudal fin and bone regeneration in adult zebrafish. First, regenerated and mineralized areas are evaluated through broad, rapid and specific chronological and morphometric analysis in alizarin red stained fins. Then, following a more refined strategy, the intensity of the staining within a 2D longitudinal plane is determined through pixel intensity analysis, as an indicator of density or thickness/ volume. The applicability of this methodology on live specimens, to reduce animal experimentation and provide a tool for in vivo tracking of the regenerative process, was successfully demonstrated. Finally, the methodology was validated on retinoic acid- and warfarin-treated specimens, and further confirmed by micro-computed tomography. Because it is easily implementable, accurate and does not require sophisticated equipment, the present methodology will certainly provide valuable technical standardization for research in tissue engineering, regenerative medicine and skeletal biology.J.C., I.F. and I.F.C. acknowledge the financial support from the Portuguese Foundation for Science and Technology (FCT) through the doctoral grant PD/BD/52425/2013 within the ProRegeM PhD Programme (PD/00117/2012), the post-doctoral grant SFRH/BDP/82049/2011, and the doctoral grant SFRH/BD/99555/2014, respectively. J.M.O. also thanks FCT for the funds provided under the program Investigador FCT 2012 (IF/00423/2012) and through the post-doctoral grant SFRH/BPD/70230/2010. This work was financed by the FCT through research project PTDC/MAR/112992/2009 (AQUATOX) and project UID/Multi/04326/2013, and co-financed by the FCT and the European Commission (ERDF-COMPETE) through PEst-C/MAR/LA0015/2011 project. The help of Gil Martins, Luís Granadeiro and Pedro Catela in fish maintenance is gratefully acknowledged