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

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.

The zebrafish operculum: A powerful system to assess osteogenic bioactivities of molecules with pharmacological and toxicological relevance

“…using a panel of hundreds of compounds or small molecule libraries), the optimized operculum system described here, may provide a throughput higher than existing methodologies currently available to assess osteogenic compounds/activities. An approach using alizarin red S staining combined with morphometric analysis has already been demonstrated as an easy and accurate way to detect and quantify bone mineral deposition under fluorescence conditions (Bensimon-Brito et al, 2016;Cardeira et al, 2016). Other techniquese.g.…”

“…Other features, such as robustness, high fecundity and short generation time have reinforced the suitability of zebrafish as a laboratory animal. Various in vivo tools exist to study how drugs affect the skeleton (osteogenic or anti-osteogenic activities) (Laizé et al, 2014;Cardeira et al, 2016), including zebrafish mutant lines mimicking human skeletal disorders (Barrett et al, 2006;de Vrieze et al, 2014) and transgenic lines able to express fluorescent markers at sites of bone-related gene expression (Knopf et al, 2011a). These are valuable, both for the discovery of therapeutic molecules capable of rescuing skeletal pathologies and in studying their mechanisms of action.…”

Isolation, Culture, and Differentiation of Blastema Cells from the Regenerating Caudal Fin of Zebrafish

“…The effect of exogenous administration of RA is however controversial. It has been shown to either impair fin regeneration by blocking blastema formation [44,52,53] or to enhance RA signaling and positively influence fin regeneration [51]. RA is not only a potent and effective inducer of embryonic stem cell differentiation [54] but also of zebrafish blastema cell differentiation (this study).…”

New Insights into the Control of Cell Fate Choices and Differentiation by Retinoic Acid in Cranial, Axial and Caudal Structures

“…Osteoblasts may themselves exert a piloting function for other cell types, as the breakdown of ray-interray boundaries also affects other cell types, like fibroblasts and blood vessels [176]. An excess of RA results in a similar phenotype and induces an over-mineralized phenotype, by promoting bone matrix synthesis in osteoblasts [177]. Suppression of RA signaling by removing RA locally, as observed in the stump and in the proximal blastema, is a mechanism repeatedly utilized to guide osteoblast behavior towards the correct regenerative morphogenetic processes.…”

“…Reporter gene analyses showed that the fate of mature joint-forming osteoblast is not fixed, instead they differentiate to (regular) osteoblasts under RA, presumably by lifting an arrest in osteoblast differentiation or by transdifferentiation. If this effect contributes significantly to the over-ossification observed in RA-treated regenerates [176,177] has not been established yet. The findings underscore once more the requirement for precise spatio-temporal control of RA signaling during fin growth and regeneration.…”