BMP2, BMP4 and BMP16 form a subfamily of bone morphogenetic proteins acting as pleiotropic growth factors during development and as a bone inducers during osteogenesis.BMP16 is the most recent member of this subfamily and basic data regarding protein structure and function, and spatio-temporal gene expression is still scarce. In this work, insights on BMP16 were provided through the comparative analysis of structural and functional data for zebrafish BMP2a, BMP2b, BMP4 and BMP16 genes and proteins, determined from threedimensional models, patterns of gene expression during development and in adult tissues, regulation by retinoic acid and capacity to activate BMP signaling pathway. Structures of Bmp2a, Bmp2b, Bmp4 and Bmp16 were found to be remarkably similar, with residues involved in receptor binding being highly conserved All proteins could activate the BMP signaling pathway, suggesting that they share a common function. On the contrary, stage-and tissue-specific expression of bmp2, bmp4 and bmp16 suggested the genes might be differentially regulated (e.g. different transcription factors, enhancers and/or regulatory modules) but also that they are involved in distinct physiological processes, although with the same function. Retinoic acid, a morphogen known to interact with BMP signaling during bone formation, was shown to down-regulate the expression of bmp2, bmp4 and bmp16, although to different extents. Taxonomic and phylogenetic analyses indicated that bmp16 diverged before bmp2 and bmp4, is not restricted to teleost fish lineage as previously reported, and that it probably arose from a whole genomic duplication event that occurred early in vertebrate evolution and disappeared in various tetrapod lineages through independent events.

Summary Polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are defining components of farmed fish feeds. They are known effectors of bone metabolism and may reduce the occurrence of skeleton deformities in farmed fish, given that dietary levels are optimized. Underlying mechanisms of bone‐related effects of PUFAs are however poorly understood. An in vitro cell system developed from gilthead seabream vertebra and capable of mineralization was used to assess the effect of AA, EPA and DHA on cell proliferation, extracellular matrix (ECM) mineralization and expression of a selection of marker genes. While all fatty acids promoted morphological changes and stimulated cell proliferation, AA and EPA inhibited ECM mineralization and DHA had the opposite effect. When fatty acids were combined, DHA apparently compensated for the inhibitory effect of AA and EPA. qPCR data indicated that, while expression of COL1A1, ON, MGP and RUNX2 genes remained unaltered, that of OP, BMP2, TNAP and COX2 genes was significantly regulated upon exposure to individual PUFAs. The concomitant increase in BMP2 and TNAP gene expression in DHA‐treated cells and the strong decrease in OP transcript in AA‐ and EPA‐treated cells may explain the pro‐ and anti‐mineralogenic effects of these fatty acids, respectively. An increase in COX2 gene expression was also observed in EPA‐ and DHA‐treated cells but the biological significance of this result remains ambiguous. This work also demonstrated the suitability of our in vitro cell systems to get insights into bone‐related effects of PUFAs and to investigate the intracellular pathways involved in their proliferative and mineralogenic effects.

Summary Bone morphogenetic proteins (BMPs) are involved in various physiological processes from early life stages throughout adulthood. Specific characteristics of BMPs have been used to define different subfamilies and BMP2/4 subfamily (composed of BMP2 and BMP4) has been linked to osteogenesis and skeleton development. BMP16 was recently identified as a new member of the BMP2/4 subfamily and reported as a teleost fish‐specific form. In this work, we collected a comprehensive set of ray‐finned fish BMP2, BMP4 and BMP16 sequences and demonstrated, through its presence in Holostei, that BMP16 is not restricted to teleost fish genome. Comparative analysis of BMP2, BMP4 and BMP16 primary structures revealed that most of the residues required for protein stabilization, dimer formation, glycosylation and receptor binding are substantially conserved between the three proteins, suggesting that BMP16, BMP2 and BMP4 may share similar mechanisms of action. In contrast, comparative analysis of gene expression profiles during Senegalese sole development revealed differences in onset and extent of gene expression, indicating that BMP16, BMP2 and BMP4 may contribute to different developmental and physiological processes. High levels of transcripts in adult calcified tissues and the up‐regulation of gene expression by retinoic acid, a known regulator of skeletal development, suggests that BMP16 shares with BMP2 and BMP4 a role in bone metabolism and skeletal development. This study provides new insights into the taxonomic distribution and the spatiotemporal expression of BMP16 gene, and suggests that it may share structural and functional similarities with other members of the BMP2/4 protein subfamily.

Among the many anthropogenic chemicals that end up in the aquatic ecosystem, heavy metals, in particular cadmium, are hazardous compounds that have been shown to affect developmental, reproductive, hepatic, hematological, and immunological functions in teleost fish. There is also evidence that cadmium disturbs bone formation and skeletal development, but data is scarce. In this work, zebrafish was used to further characterize the anti-osteogenic/osteotoxic effects of cadmium and gain insights into underlying mechanisms. Upon exposure to cadmium, a reduction of the opercular bone growth was observed in 6-days post-fertilization (dpf) larvae and an increase in the incidence of skeletal deformities was evidenced in 20-dpf post-larvae. The extent and stiffness of newly formed bone was also affected in adult zebrafish exposed to cadmium while regenerating their caudal fin. A pathway reporter assay revealed a possible role of the MTF-1 and cAMP/PKA signaling pathways in mechanisms of cadmium osteotoxicity, while the expression of genes involved in osteoblast differentiation and matrix production was strongly reduced in cadmium-exposed post-larvae. This work not only confirmed cadmium anti-osteogenic activity and identified targeted pathways and genes, but it also suggested that cadmium may affect biomechanical properties of bone.

Many chemicals produced by human activities end up in the aquatic ecosystem causing adverse developmental and reproductive effects in aquatic organisms. There is evidence that some anthropogenic chemicals disturb bone formation and skeletal development but the lack of suitable in vitro and in vivo systems for testing has hindered the identification of underlying mechanisms of osteotoxicity. Several fish systems - an in vitro cell system to study extracellular matrix mineralization and in vivo systems to evaluate bone formation and skeletogenesis - were combined to collect data on the osteotoxic activity of 3-methylcholanthrene (3-MC), a polycyclic aromatic hydrocarbon. Anti-mineralogenic effects, increased incidence of skeletal deformities and reduced bone formation and regeneration were observed in zebrafish upon exposure to 3-MC. Pathway reporter array revealed the role of the aryl hydrocarbon receptor 2 (Ahr2) in the mechanisms underlying 3-MC osteotoxicity in mineralogenic cell lines. Analysis of gene expression in zebrafish larvae confirmed the role of Ahr2 in the signaling of 3-MC toxicity. It also indicated a possible complementary action of the pregnane X receptor (Pxr) in the regulation of genes involved in bone cell activity and differentiation but also in xenobiotic metabolism. Data reported here demonstrated the osteotoxicity of 3-MC but also confirmed the suitability of fish systems to gain insights into the toxic mechanisms of compounds affecting skeletal and bone formation.