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- Central role of betaine-homocysteine S-methyltransferase 3 in chondral ossification and evidence for sub-functionalization in neoteleost fishPublication . Rosa, Joana; Tiago, Daniel; Marques, Cátia L.; Vijayakumar, Parameswaran; Fonseca, Luís; Cancela, Leonor; Laizé, VincentBackground: To better understand the complex mechanisms of bone formation it is fundamental that genes central to signaling/regulatory pathways and matrix formation are identified. Cell systems were used to analyze genes differentially expressed during extracellular matrix mineralization and bhmt3, coding for a betaine-homocysteine S-methyltransferase, was shown to be down-regulated in mineralizing gilthead seabream cells.Methods: Levels and sites of bhmt3 expression were determined by qPCR and in situ hybridization throughout seabream development and in adult tissues. Transcriptional regulation of bhmt3 was assessed from the activity of promoter constructs controlling luciferase gene expression. Molecular phylogeny of vertebrate BHMT was determined from maximum likelihood analysis of available sequences.Results: bhmt3 transcript is abundant in calcified tissues and localized in cartilaginous structures undergoing endo/perichondral ossification. Promoter activity is regulated by transcription factors involved in bone and cartilage development, further demonstrating the central role of Bhmt3 in chondrogenesis and/or osteogenesis. Molecular phylogeny revealed the explosive diversity of bhmt genes in neoteleost fish, while tissue distribution of bhmt genes in seabream suggested that neoteleostean Bhmt may have undergone several steps of sub-functionalization.Conclusions: Data on bhmt3 gene expression and promoter activity evidences a novel function for betaine-homocysteine S-methyltransferase in bone and cartilage development, while phylogenetic analysis provides new insights into the evolution of vertebrate BHMTs and suggests that multiple gene duplication events occurred in neoteleost fish lineage.General significance: High and specific expression of Bhmt3 in gilthead seabream calcified tissues suggests that bone-specific betaine-homocysteine S-methyltransferases could represent a suitable marker of chondral ossification.
- A new paradigm for Pharyngeal Tooth Formation?Publication . Oralova, V; Rosa, Joana; Larionova, D.; Witten, P. E.; Huysseune, A.As a corollary to the ‘modified outside in’ hypothesis about the evolutionary origin of vertebrate teeth (Huysseune et al., 2009, J. Anat. 214: 465-476), we previously hypothesized that the development of, allegedly endoderm-derived, pharyngeal teeth in teleost fish requires ectodermal contribution or signaling.
- Serum-specific stimulation of proliferation and mineralization of fish bone-derived cellsPublication . Rosa, Joana; Tiago, Daniel; Dias, J.; Cancela, Leonor; Laizé, VincentTeleost fish have recently been implemented as suitable model organisms to study vertebrate development, in particular skeletogenesis. In vitro cell systems derived from fish bone have been successfully established, although their development has been hampered by the limited availability of fish serum to supplement culture medium. Commercially available sera are mostly of mammalian origin and thus not necessarily adequate to fish cell growth. The main objective of this work was to compare proliferative and mineralogenic potential of bovine and fish sera using fish bone-derived cell lines VSa13 and VSa16. Fish serum was shown to (i) strongly stimulate cell proliferation in an apparent dose-dependent and cell type-specific manner, (ii) induce morphological changes, and (iii) enhance extracellular matrix mineralization of bone cells, although cytotoxic for fish osteoblast-like cells at the concentration tested. To better understand mechanisms underlying mineralogenic effect of fish serum in fish chondrocytes, expression of several mineralization-related genes was evaluated by qPCR. Regulation of matrix Gla protein (MGP) and bone morphogenetic protein 2 (BMP2) gene expression was modified upon culture with fish serum in a way compatible with an early onset and an increase in mineralization. In conclusion, fish serum was shown to be more adequate to proliferation and differentiation/mineralization of fish bone-derived cells.
- Periderm invasion contributes to the epithelial lining of the teleost pharynxPublication . Rosa, Joana; Oralova, V; Larionova, D.; Eisenhoffer, G. T.; Witten, P. E.; Huysseune, A.The gnathostome pharyngeal cavity functions in food transport and respiration. In amniotes the mouth and nares are the only channels allowing direct contact between internal and external epithelia. In teleost fsh, gill slits arise through opening of endodermal pouches and connect the pharynx to the exterior. Using transgenic zebrafsh lines, cell tracing, live imaging and diferent markers, we investigated if pharyngeal openings enable epithelial invasion and how this modifes the pharyngeal epithelium. We conclude that in zebrafsh the pharyngeal endoderm becomes overlain by cells with a peridermal phenotype. In a wave starting from pouch 2, peridermal cells from the outer skin layer invade the successive pouches until halfway their depth. Here the peridermal cells connect to a population of cells inside the pharyngeal cavity that express periderm markers, yet do not invade from outside. The latter population expands along the midline from anterior to posterior until the esophagusgut boundary. Together, our results show a novel role for the periderm as an internal epithelium becomes adapted to function as an external surface.
- Identification of a new cartilage-specific S100-like protein up-regulated during endo/perichondral mineralization in gilthead seabreamPublication . Fonseca, V. G.; Rosa, Joana; Laizé, Vincent; Gavaia, Paulo J.; Cancela, LeonorCalcium ions and calcium-binding proteins play a major role in many cellular processes, in particular skeletogenesis and bone formation. We report here the discovery of a novel S100 protein in fish and the analysis of its gene expression patterns. A 648-bp full-length cDNA encoding an 86-amino acid S100-like calcium-binding protein was identified through the subtractive hybridization of a gilthead seabream (Sparus aurata) cDNA library constructed to identify genes associated with in vitro mineralization. Deduced protein lacks an identifiable signal peptide and exhibits two EF-hand motifs characteristic of S100 proteins. Phylogenetic and bioinformatic analyses of S100 sequences suggested that gilthead seabream protein represents a novel and fish-specific member of the S100 protein family. Expression of S100-like gene was up-regulated during the in vitro mineralization of bone-derived cell lines and during seabream development, from larvae throughout adulthood, reflecting skeletogenesis. Restriction of S100-like gene expression to chondrocytes of cartilaginous tissues undergoing endo/perichondral mineralization in juvenile fish further confirmed the mineralogenic role of the protein in fish and emphasized the potential of S100-like as a marker of mineralizing cartilage in developing fish.
- The osteogenic and mineralogenic potential of the microalgae Skeletonema costatum and Tetraselmis striata CTP4 in fish modelsPublication . Carletti, Alessio; Rosa, Joana; Pes, Katia; Borges, Inês; Santos, Tamara; Barreira, Luísa; Varela, João; Pereira, Hugo; Cancela, M. Leonor; J. Gavaia, Paulo; Laizé, VincentSkeletal 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.
- Beyond the whole-mount phenotype: high-resolution imaging in fluorescence-based applications on zebrafishPublication . Oralová, Veronika; Rosa, Joana; Soenens, Mieke; Bek, Jan Willem; Willaert, Andy; Witten, Paul Eckhard; Huysseune, AnnZebrafish 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.
- Cells at the edge: the dentin–bone interface in Zebrafish teethPublication . Rosa, Joana; Witten, Paul Eckhard; Huysseune, AnnBone-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.
- Periderm invasion contributes to epithelial formation in the teleost pharynxPublication . Rosa, Joana; Oralová, Veronika; Larionova, Daria; Eisenhoffer, G. T.; Eckhard Witten, P.; Huysseune, AnnThe gnathostome pharyngeal cavity functions in food transport and respiration. In amniotes the mouth and nares are the only channels allowing direct contact between internal and external epithelia. In teleost fish, gill slits arise through opening of endodermal pouches and connect the pharynx to the exterior. Using transgenic zebrafish lines, cell tracing, live imaging and different markers, we investigated if pharyngeal openings enable epithelial invasion and how this modifies the pharyngeal epithelium. We conclude that in zebrafish the pharyngeal endoderm becomes overlain by cells with a peridermal phenotype. In a wave starting from pouch 2, peridermal cells from the outer skin layer invade the successive pouches until halfway their depth. Here the peridermal cells connect to a population of cells inside the pharyngeal cavity that express periderm markers, yet do not invade from outside. The latter population expands along the midline from anterior to posterior until the esophagus-gut boundary. Together, our results show a novel role for the periderm as an internal epithelium becomes adapted to function as an external surface.
- Dietary lipid quality regulates bone composition and metabolism in gilthead seabream (Sparus aurata) juvenilesPublication . Dias, J.; Rodrigues, V.; Colen, Rita; Rosa, Joana; Viegas, Michael; Cardeira Da Silva, João; Cancela, Leonor; Gavaia, Paulo J.; Laizé, VincentReplacement of significant amounts of marine fish oils by vegetable oils is a major trend in the aquaculture feed industry. However, knowledge on the mechanisms underlying the nutritional regulation of bone metabolism is extremely scarce in fish. We speculate that changes in the dietary ratio of fatty acids may modulate tissue eicosanoids production and affect bone formation in fastgrowing gilthead seabream, an important fish species for aquaculture in the Mediterranean region.