Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regeneration

Abstract

BackgroundThe correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Current methods to visualize mineralized tissue in zebrafish rely on: 1) fixed specimens; 2) radiographic and μCT techniques, that are ultimately limited in resolution; or 3) vital stains with fluorochromes that are indistinguishable from the signal of green fluorescent protein (GFP)-labelled cells. Alizarin compounds, either in the form of alizarin red S (ARS) or alizarin complexone (ALC), have long been used to stain the mineralized skeleton in fixed specimens from all vertebrate groups. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to the water, can affect bone formation in juvenile and adult zebrafish, as ARS has been shown to inhibit skeletal growth and mineralization in mammals.ResultsHere we present a protocol for vital staining of mineralized structures in zebrafish with a low ARS concentration that does not affect bone mineralization, even after repetitive ARS staining events, as confirmed by careful imaging under fluorescent light. Early and late stages of bone development are equally unaffected by this vital staining protocol. From all tested concentrations, 0.01 % ARS yielded correct detection of bone calcium deposits without inducing additional stress to fish.ConclusionsThe proposed ARS vital staining protocol can be combined with GFP fluorescence associated with skeletal tissues and thus represents a powerful tool for in vivo monitoring of mineralized structures. We provide examples from wild type and transgenic GFP-expressing zebrafish, for endoskeletal development and dermal fin ray regeneration.

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

“…Nonetheless, potential effects of repetitive anaesthesia and staining procedures must not be ruled out. However, they did not impair regeneration and mineralization, as demonstrated by the normal regenerative outgrowth of the fin, according to a previous report37. Overall, this approach was shown to be a suitable alternative to the analysis of fixed samples, allowing a reduction in the number of used individuals.…”

“…Its successful detection may depend on the sensitivity of the imaging techniques (e.g. fluorescence versus bright-field microscopy) and on the use of demineralizing agents, like acid solutions, before or during staining procedures353637.…”

“…These imply monitoring at multiple time-points, the manipulation and sacrifice of more specimens and consequently the need for larger fish stocks and housing facilities. In this context, we explored the applicability of a recent methodology that allows the tracking of de novo bone formation in the same fish over time37, in a way that better attends the 3 R’s (replacement, reduction and refinement) guidelines to reduce animal experimentation39 and the Directive 2010/63/EU of the European Parliament. Mineralization of lepidotrichia was monitored in the course of fin regeneration in 11 individuals through vital staining with AR-S. Fluorescence signal was captured as for fixed samples although the exposure time had to be extended.…”

“…For live specimens, fish were immersed for 15 min in a 0.01% AR-S solution (pH 7.4)37, anaesthetized in MS222, photographed and returned to their containers. Staining was performed in groups of 5 (in 200 mL of staining solution) or individually (in 100 mL of staining solution), according to the need of tracking the same individuals over time.…”

Craniofacial skeleton of MEXICAN tetra (Astyanax mexicanus): As a bone disease model

“…These dyes can be traced during the development of the animal to track the pattern of normal and abnormal bone mineralization (Javidan and Schilling, ; Powers et al, ). These techniques have been used successfully to understand various mineralized tissue dynamics in the teleost skeleton including bone remodeling (Bruneel and Witten, ; Bensimon‐Brito et al, ), tooth regeneration (Bruneel et al, ; Ellis et al, ), and in different Mexican tetra morphs to trace the facial bone fragmentation (Powers et al, ).…”