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

Abstract

Bone disorders affect millions of people worldwide and available therapeutics have a limited efficacy, often presenting undesirable side effects. As such, there is a need for novel molecules with bone anabolic properties. The aim of this work was to establish a rapid, reliable and reproducible method to screen for molecules with osteogenic activities, using the zebrafish operculum to assess bone formation. Exposure parameters were optimized through morphological analysis of the developing operculum of larvae exposed to calcitriol, a molecule with known pro-osteogenic properties. An exposure of 3 days initiated at 3 days post-fertilization was sufficient to stimulate operculum formation, while not affecting survival or development of the larvae. Dose-dependent pro-and anti-osteogenic effects of calcitriol and cobalt chloride, respectively, demonstrated the sensitivity of the method and the suitability of the operculum system. A double transgenic reporter line expressing fluorescent markers for early and mature osteoblasts was used to gain insights into the effects of calcitriol and cobalt at the cellular level, with osteoblast maturation shown to be stimulated and inhibited, respectively, in the operculum of exposed fish. The zebrafish operculum represents a consistent, robust and rapid screening system for the discovery of novel molecules with osteogenic, anti-osteoporotic or osteotoxic activity.

Generation and Validation of a Complete Knockout Model of abcc6a in Zebrafish

“…The craniofacial bone elements appeared to be normally developed and mineralized. Specifically, the operculum and cleithrum-reported as a marker for osteogenesis-did not reveal any structural anomalies or advanced mineralization ( Figure 3b) (Tarasco et al, 2017). Over time, fish developed problems with swimming and feeding, requiring euthanasia.…”

Anti-Inflammatory Activity of Exopolysaccharides from Phormidium sp. ETS05, the Most Abundant Cyanobacterium of the Therapeutic Euganean Thermal Muds, Using the Zebrafish Model

“…Operculum area was determined in 5 dpf zebrafish larvae after treatment with CuSO 4 •5H 2 O followed by EPS recovery of inflammation, following the indication of Tarasco and co-worker (2017) [46]. Briefly, five days post fertilization larvae were euthanized, stained for 15 min at room temperature with 0.01% alizarin red S (AR-S, A5533, Sigma), and then, washed twice with distilled water for 5 min.…”

Anti-Osteogenic Activity of Cadmium in Zebrafish

“…Data presented in this article-i.e., the reduced growth of the opercular bone, the increased incidence of skeletal deformities, the impaired stiffness of regenerated bone, and the differential expression of bone marker genes-provide clear evidence of an anti-osteogenic or osteotoxic effect of cadmium at doses that can be found in the aquatic environment [31,32]. A decreased operculum growth rate was recently evidenced in zebrafish larvae exposed to cobalt chloride [33] and 3-methylcholanthrene [30] through mechanisms still poorly understood but possibly related to an impairment of osteoblast (bone forming cells) maturation and function. In this regard, cadmium was found to inhibit extracellular matrix mineralization in osteoblast MC3T3-E1 cell cultures [34] but also to decrease the expression marker genes central to osteoblast maturation and function (this study).…”

“…Brightness and contrast were optimized to enhance the visibility of the cranial bones. Operculum growth was assessed through morphometric analysis of fluorescence images according to [33].…”