Fin ray branching is defined by TRAP+ osteolytic tubules in zebrafish.

The shaping of bone structures relies on various cell types and signaling pathways. Here, we use the zebrafish bifurcating fin rays during regeneration to investigate bone patterning. We found that the regenerating fin rays form via two mineralization fronts that undergo an osteoblast-dependent fusion/stitching until the branchpoint, and that bifurcation is not simply the splitting of one unit into two. We identified tartrate-resistant acid phosphatase-positive osteolytic tubular structures at the branchpoints, hereafter named osteolytic tubules (OLTs). Chemical inhibition of their bone-resorbing activity strongly impairs ray bifurcation, indicating that OLTs counteract the stitching process. Furthermore, by testing different osteoactive compounds, we show that the position of the branchpoint depends on the balance between bone mineralization and resorption activities. Overall, these findings provide a unique perspective on fin ray formation and bifurcation, and reveal a key role for OLTs in defining the proximo-distal position of the branchpoint.

The osteogenic and mineralogenic potential of the microalgae Skeletonema costatum and Tetraselmis striata CTP4 in fish models.

Skeletal disorders are problematic aspects for the aquaculture industry as skeletal deformities, which affect most species of farmed fish, increase production costs and affect fish welfare. Following recent findings that show the presence of osteoactive compounds in marine organisms, we evaluated the osteogenic and mineralogenic potential of commercially available microalgae strains Skeletonema costatum and Tetraselmis striata CTP4 in several fish systems. Ethanolic extracts increased extracellular matrix mineralization in gilthead seabream (Sparus aurata) bone-derived cell cultures and promoted osteoblastic differentiation in zebrafish (Danio rerio) larvae. Long-term dietary exposure to both extracts increased bone mineralization in zebrafish and upregulated the expression of genes involved in bone formation (sp7, col1a1a, oc1, and oc2), bone remodeling (acp5a), and antioxidant defenses (cat, sod1). Extracts also improved the skeletal status of zebrafish juveniles by reducing the incidence of skeletal anomalies. Our results indicate that both strains of microalgae contain osteogenic and mineralogenic compounds, and that ethanolic extracts have the potential for an application in the aquaculture sector as dietary supplements to support fish bone health. Future studies should also identify osteoactive compounds and establish whether they can be used in human health to broaden the therapeutic options for bone erosive disorders such as osteoporosis.

Screening of Mineralogenic and Osteogenic Compounds in Zebrafish-Tools to Improve Assay Throughput and Data Accuracy.

Bone disorders affect millions of people worldwide and treatments currently available often produce undesirable secondary effects or have limited efficacy. It is therefore of the utmost interest for patients to develop more efficient drugs with reduced off-target activities. In the long process of drug development, screening and preclinical validation have recently gained momentum with the increased use of zebrafish as a model organism to study pathological processes related to human bone disorders, and the development of zebrafish high-throughput screening assays to identify bone anabolic compounds. In this review, we provided a comprehensive overview of the literature on zebrafish bone-related assays and evaluated their performance towards an integration into screening pipelines for the discovery of mineralogenic/osteogenic compounds. Tools available to standardize fish housing and feeding procedures, synchronize embryo production, and automatize specimen sorting and image acquisition/analysis toward faster and more accurate screening outputs were also presented.

Cells at the Edge: The Dentin-Bone Interface in Zebrafish Teeth.

Bone-producing osteoblasts and dentin-producing odontoblasts are closely related cell types, a result from their shared evolutionary history in the ancient dermal skeleton. In mammals, the two cell types can be distinguished based on histological characters and the cells' position in the pulp cavity or in the tripartite periodontal complex. Different from mammals, teleost fish feature a broad diversity in tooth attachment modes, ranging from fibrous attachment to firm ankylosis to the underlying bone. The connection between dentin and jaw bone is often mediated by a collar of mineralized tissue, a part of the dental unit that has been termed "bone of attachment". Its nature (bone, dentin, or an intermediate tissue type) is still debated. Likewise, there is a debate about the nature of the cells secreting this tissue: osteoblasts, odontoblasts, or yet another (intermediate) type of scleroblast. Here, we use expression of the P/Q rich secretory calcium-binding phosphoprotein 5 (scpp5) to characterize the cells lining the so-called bone of attachment in the zebrafish dentition. scpp5 is expressed in late cytodifferentiation stage odontoblasts but not in the cells depositing the "bone of attachment". nor in bona fide osteoblasts lining the supporting pharyngeal jaw bone. Together with the presence of the osteoblast marker Zns-5, and the absence of covering epithelium, this links the cells depositing the "bone of attachment" to osteoblasts rather than to odontoblasts. The presence of dentinal tubule-like cell extensions and the near absence of osteocytes, nevertheless distinguishes the "bone of attachment" from true bone. These results suggest that the "bone of attachment" in zebrafish has characters intermediate between bone and dentin, and, as a tissue, is better termed "dentinous bone". In other teleosts, the tissue may adopt different properties. The data furthermore support the view that these two tissues are part of a continuum of mineralized tissues. Expression of scpp5 can be a valuable tool to investigate how differentiation pathways diverge between osteoblasts and odontoblasts in teleost models and help resolving the evolutionary history of tooth attachment structures in actinopterygians.

Fish Models of Induced Osteoporosis.

Osteopenia and osteoporosis are bone disorders characterized by reduced bone mineral density (BMD), altered bone microarchitecture and increased bone fragility. Because of global aging, their incidence is rapidly increasing worldwide and novel treatments that would be more efficient at preventing disease progression and at reducing the risk of bone fractures are needed. Preclinical studies are today a major bottleneck to the collection of new data and the discovery of new drugs, since they are commonly based on rodent in vivo systems that are time consuming and expensive, or in vitro systems that do not exactly recapitulate the complexity of low BMD disorders. In this regard, teleost fish, in particular zebrafish and medaka, have recently emerged as suitable alternatives to study bone formation and mineralization and to model human bone disorders. In addition to the many technical advantages that allow faster and larger studies, the availability of several fish models that efficiently mimic human osteopenia and osteoporosis phenotypes has stimulated the interest of the academia and industry toward a better understanding of the mechanisms of pathogenesis but also toward the discovery of new bone anabolic or antiresorptive compounds. This mini review recapitulates the in vivo teleost fish systems available to study low BMD disorders and highlights their applications and the recent advances in the field.

Beyond the whole-mount phenotype: high-resolution imaging in fluorescence-based applications on zebrafish.

Zebrafish is now widely used in biomedical research as a model for human diseases, but the relevance of the model depends on a rigorous analysis of the phenotypes obtained. Many zebrafish disease models, experimental techniques and manipulations take advantage of fluorescent reporter molecules. However, phenotypic analysis often does not go beyond establishing overall distribution patterns of the fluorophore in whole-mount embryos or using vibratome or paraffin sections with poor preservation of tissue architecture and limited resolution. Obtaining high-resolution data of fluorescent signals at the cellular level from internal structures mostly depends on the availability of expensive imaging technology. Here, we propose a new and easily applicable protocol for embedding and sectioning of zebrafish embryos using in-house prepared glycol methacrylate (GMA) plastic that is suited for preservation of fluorescent signals (including photoactivatable fluorophores) without the need for antibodies. Four main approaches are described, all involving imaging fluorescent signals on semithin (3 µm or less) sections. These include sectioning transgenic animals, whole-mount immunostained embryos, cell tracking, as well as on-section enzyme histochemistry.