Abstract
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.
Highlights
Caudal fin) that secrete and maintain the bone matrix[12]
No detailed chronology of regeneration events is yet available at this temperature, a general delay is expected in the orchestration of the different events
We proposed a method for investigating regeneration and de novo bone matrix mineralization in the zebrafish caudal fin
Summary
Caudal fin) that secrete and maintain the bone matrix[12]. Given its remarkable regenerative potential, the zebrafish caudal fin system has been increasingly used for drug screening and discovery of factors regulating regeneration and de novo osteogenesis, mineralogenesis[13,14,15,16]. Three main events can be distinguished: wound healing, blastema formation and regenerative outgrowth[2,5,6] This specific type of regeneration (i.e. epimorphosis) relies on the dedifferentiation of cells resting below the amputation plane to form a blastema, an intermediate structure composed of undifferentiated and highly proliferative cells[17,18,19]. In this work we provide a detailed description of the regenerative and de novo mineralogenic profiles and of the main events occurring during fin regeneration and lepidotrichia mineralization. Such data is used for the development of a methodology to rapidly and accurately assess the overall regenerative and mineralogenic performances in this system. The suitability of the present methodology for the study and identification of compounds with regenerative and/or mineralogenic activities is addressed through proof-of-concept experiments using retinoic acid and warfarin, two molecules with described effects on bone formation[26,27]
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