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  • ZFBONE: an ImageJ toolset for semi-automatic analysis of zebrafish bone structures
    Publication . Tarasco, Marco; Cordelieres, Fabrice P.; Cancela, M. Leonor; Laizé, Vincent
    The last decade has seen an increased interest in the discovery of compounds with bone anabolic activity to treat skeletal disorders such as osteoporosis and increase the well-being of patients. Due to the many technical advantages over classical rodent systems, zebrafish (Danio rerio) has been increasingly used in screening pipelines, in particular those aiming at identifying osteoactive compounds with pharmacological potential. Because compound osteoactivity is mostly determined in zebrafish through the morphometric analysis of bone structures, image analysis, rather than screening assay implementation, molecule availability and image acquisition, represents a bottleneck to the screening throughput. The absence of auto/semi-automatic tools for image analysis of fish bone structures is also a limitation to a broader usage of zebrafish screening pipelines. We present here ZFBONE (for ZebraFish BONE), an open-source, freely available, user-friendly, rapid and reliable toolset, aiming at accelerating image analysis by automating the morphometric assessment of zebrafish bone structures, but also at increasing data accuracy by reducing operator bias. Tools included in ZFBONE allow users to assess, from 2D images, morphometric parameters of several bone structures (e.g. operculum, caudal fin rays and scales) but also the extent and the intensity of bone-specific colorations. ZFBONE has been developed using the open-source ImageJ software, to make it available to the whole zebrafish research community, but also to have it easily modifiable according to user demands. ZFBONE can also be used toward the standardization of zebrafish screening protocols in academia and industry.
  • ZEB316: a small stand-alone housing system to study microplastics in small teleosts
    Publication . Tarasco, Marco; Martins, Gil; Gavaia, Paulo; Bebianno, Maria; Leonor Cancela, M.; Laizé, Vincent
    Many anthropogenic chemicals and plastic debris end up in the aquatic ecosystem worldwide, representing a major concern for the environment and human health. Small teleosts, such as zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes), offer significant advantages over classical animal models and are currently used as first-line organisms to assess environmental risks associated with many aquatic toxicants. Toxicological studies require the use of inert materials and controlled conditions. Yet, none of the available commercialized systems is adequate to assess the toxic effect of microplastics, because they contain components made of plastic polymers that may release micrometric plastic particles, leach manufacturing compounds, or adsorb chemicals. The ZEB316 stand-alone housing system presented in this study is meant to be a cost-effective and easy-to-built solution to perform state-of-the-art toxicological studies. It is built with inert and corrosion-resistant materials and provides good housing conditions through efficient recirculation and filtration systems. Assessment of water parameters and fish growth performance showed that the ZEB316 provides housing conditions comparable to those available from commercial housing systems.
  • The zebrafish operculum: a powerful system to assess osteogenic bioactivities of molecules with pharmacological and toxicological relevance
    Publication . Tarasco, Marco; Laizé, Vincent; Cardeira, João; Leonor Cancela, M.; Gavaia, Paulo
    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.
  • Effects of pristine or contaminated polyethylene microplastics on zebrafish development
    Publication . Tarasco, Marco; Gavaia, Paulo; Bensimon-Brito, Anabela; Cordelières, Fabrice P.; Santos, Tamara; Martins, Gil; De Castro, Daniela; Silva, Nadia; Cabrita, Elsa; Bebianno, Maria; Stainier, Didier Y.R.; Cancela, M. Leonor; Laizé, Vincent
    The presence of microplastics in the aquatic ecosystem represents a major issue for the environment and human health. The capacity of organic pollutants to adsorb onto microplastic particles raises additional concerns, as it creates a new route for toxic compounds to enter the food web. Current knowledge on the impact of pristine and/or contaminated microplastics on aquatic organisms remains insufficient, and we provide here new insights by evaluating their biological effects in zebrafish (Danio rerio). Zebrafish larvae were raised in ZEB316 stand-alone housing systems and chronically exposed throughout their development to polyethylene particles of 20-27 mu m, pristine (MP) or spiked with benzo[alpha]pyrene (MP-BaP), supplemented at 1% w/w in the fish diet. While they had no effect at 30 days post-fertilization (dpf), MP and MP-BaP affected growth parameters at 90 and 360 dpf. Relative fecundity, egg morphology, and yolk area were also impaired in zebrafish fed MP-BaP. Zebrafish exposed to experimental diets exhibited an increased incidence of skeletal deformities at 30 dpf as well as an impaired development of caudal fin/scales, and a decreased bone quality at 90 dpf. An intergenerational bone formation impairment was also observed in the offspring of parents exposed to MP or MP-BaP through a reduction of the opercular bone in 6 dpf larvae. Beside a clear effect on bone development, histological analysis of the gut revealed a reduced number of goblet cells in zebrafish fed MP-BaP diet, a sign of intestinal inflam-mation. Finally, exposure of larvae to MP-BaP up-regulated the expression of genes associated with the BaP response pathway, while negatively impacting the expression of genes involved in oxidative stress. Altogether, these data suggest that long-term exposure to pristine/contaminated microplastics not only jeopardizes fish growth, reproduction performance, and skeletal health, but also causes intergenerational effects.