Browsing by Author "Cancela, Leonor"
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- Alternatively spliced transcripts of Sparus aurata insulin-like growth factor 1 are differentially expressed in adult tissues and during early developmentPublication . Tiago, Daniel; Laizé, Vincent; Cancela, LeonorSpliced variants of insulin-like growth factor 1 (IGF-1), a small peptide with a critical role in metabolism and growth, have been identified in various vertebrate species. However, despite recent functional data in mammalian systems suggesting specific roles (e.g. in muscle formation) for their pro-peptides and/or E domains, their function remains unclear. In this study, three alternatively spliced variants of Sparus aurata proIGF-1 (1a, 1b, and 1c) were identified and their expression analyzed. In adult fish, IGF-1 gene expression was observed in various soft tissues (highest levels in liver) and calcified tissues, with IGF-1c being always the most expressed isoform. In developing larvae, each isoform presented a specific pattern of expression, characterized by different onset and extent and consistent with a possible role of IGF-1a and 1b during early post-hatching events (e.g. bone or muscle formation), while IGF-1c would be rather involved in early larvae formation but probably acts in concerted action with other isoforms at later stages. We also propose that, in adults, IGF-1a and 1b isoforms may have a local action, while isoform 1c would assume a systemic action, as its mammalian counterpart. This hypothesis was further supported by in silico analysis of isoform distribution, revealing that only IGF-1c/Ea isoform has been conserved throughout evolution and that other fish isoforms (i.e. 1a and 1b) may be associated with mechanisms of osmoregulation. We finally propose that IGF-1 variants may exhibit different modes of action (systemic or local) and may be involved in different developmental and adaptive mechanisms.
- 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.
- Characterization of osteocalcin (BGP) and matrix gla protein (MGP) fish specific antibodies: validation for immunodetection studies in lower vertebratesPublication . Simes, D; Williamson, M. K.; Schaff, Brian J.; Gavaia, Paulo J.; Ingleton, P. M.; Price, P. A.; Cancela, LeonorIn fish species the basic mechanisms of bone development and bone remodeling are not fully understood. The classification of bone tissue in teleosts as cellular or acellular and the presence of transitional states between bone and cartilage and the finding of different types of cartilage in teleosts not previously recognized in higher vertebrates emphasizes the need for a study on the accumulation of the Gla-containing proteins MGP and BGP at the cellular level. In the present study, polyclonal antibodies developed against BGP and MGP from A. regius (a local marine teleost fish) and against MGP from G. galeus (a Pacific Ocean shark), were tested by Western blot for their specificity against BGP and MGP from several other species of teleost fish and shark. For this purpose we extracted and purified both proteins from various marine and freshwater teleosts, identified them by N-terminal amino acid sequence analysis and confirmed the presence of gamma- carboxylation in the proteins with the use of a stain specific for Gla residues. Each antibody recognized either BGP or MGP with no cross-reaction between proteins detected. All purified fish BGPs and MGPs tested were shown to be specifically recognized, thus validating the use of these antibodies for further studies.
- Characterization of Sparus aurata osteonectin cDNA and in silico analysis of protein conserved features: Evidence for more than one osteonectin in SalmonidaePublication . Laizé, Vincent; Pombinho, A. R.; Cancela, LeonorOsteonectin is a matricellular protein involved in various cellular mechanisms but its exact function remains unclear despite numerous studies. We present here the cloning of Sparus aurata partial osteonectin cDNA and the reconstruction of 15 other sequences from both vertebrates and invertebrates, almost doubling the set of available sequences (a total of 35 sequences is now available). Taking advantage of the resulting large amount of data, we have created multiple sequence alignments and identified osteonectin putative conserved features (intra- and inter-disulfide bonds, collagen- and calcium-binding domains and phosphorylation sites) likely to be important for protein structure and function. This work also provides the first evidence for the presence of more than one osteonectin in some species. Finally, S. aurata osteonectin gene expression has been shown to initiate during larval development shortly after gastrulation, and to be high in bone-derived cell lines while down-regulated during extracellular matrix mineralization, further emphasizing the important role of osteonectin in skeletal development and bone formation.
- Characterization of specific antibodies for fish osteocalcin and its usefulness to investigate osteocalcin tissue distribution in lower vertebrates.Publication . Simes, D; Gavaia, Paulo J.; Ortiz-Delgado, J. B.; Pinto, Jorge; Cancela, LeonorOsteocalcin (BGP or Bone Gla protein) is a small acidic protein with 46-50 residues (pI»4.0) that belongs to the family of the vitamin K dependent, Gla containing proteins. This protein is the most abundant non-collagenous bone protein in mammals and has been isolated only from bone and dentine suggesting that it may be expressed only in hydroxyapatite-containing bone tissue. Previous studies suggest that in mammals BGP is an ossification regulator, but its mode of action at the molecular level, in particular in non-mammalian organisms, remains unclear
- Cloning and ontogenetic expression of the oligopeptide transporter PepT1 (Solute carrier family 15, member 1), in fed and starved larval Atlantic cod, Gadus morhua LPublication . Rønnestad, I.; Gavaia, Paulo J.; S B Viegas, Carla; Cancela, LeonorFunctional characteristics of marine fish larvae digestive system at the onset of exogenous feeding have long been a subject for discussion. Absorption of digested proteins in vertebrates has been shown to include transporter systems for single amino acids and small peptides.
- Comparative analysis of gene expression profiles among distantly related fish species. Insight into skeletal and immunological systemsPublication . Mariani, Valentina; Viegas, C. A.; Gavaia, Paulo J.; Cancela, Leonor; Giuffra, ElisabettaThe aim of this project is to identify and characterize the polymorphism of genes relevant for the physiology of skeletal and immunological tissues, and highly conserved across fish species. To achieve this goal, the expression profiles of an ancient species relevant for caviar and fillet production(A. transmontanus) are compared to available and in progress information (Marine Genomics network of Excellence, UE) of modern Teleosts.
- Comparative analysis of zebrafish bone morphogenetic proteins 2, 4 and 16: molecular and evolutionary perspectivesPublication . Marques, Cátia L.; Fernandez, Ignacio; Viegas, Michael; Cox, C. J.; Martel, Paulo; Rosa, Joana; Cancela, Leonor; Laizé, VincentBMP2, BMP4 and BMP16 form a subfamily of bone morphogenetic proteins acting as pleiotropic growth factors during development and as 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 three-dimensional 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 downregulate 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.
- Comparative promoter analysis and its application to the identification of candidate regulatory factors of cartilage-expressed genesPublication . Conceição, N.; Cox, C. J.; Simões, B.; Viegas, M.; Cancela, LeonorChondrocyte gene regulation is important for the generation and maintenance of cartilage tissues. Analysis of the transcriptional regulation of cartilage-specific genes, encoding both collagenous and noncollagenous proteins, provides a useful strategy to identify transcription factors (TFs) that control chondrocyte specification and differentiation. Our work aims at the identification of candidate TFs important for cartilage maintenance and development through an in silico approach. In order to better define the transcriptional regulatory networks that affect chondrogenesis in zebrafish we propose a combination of comparative promoter analysis and transcription factor binding site analysis using a TRANSFAC position weight matrix search to identify cis-regulatory transcription factor binding motifs in a set of cartilage characteristic genes. With this methodology we have successfully identified several transcription factors known to be important for chondrogenesis thus validating our in silico approach.
- Comparing skeletal development of wild and hatchery-reared Senegalese sole (Solea senegalensis, Kaup 1858): Evaluation in larval and postlarval stagesPublication . Gavaia, Paulo J.; Domingues, S.; Engrola, S.; Drake, P.; Sarasquete, C.; Dinis, Maria Teresa; Cancela, LeonorThe Senegalese sole is a marine pleuronectiform that naturally occurs in Southern Europe and Mediterranean region where it is being produced in aquaculture, in particular in Portugal and Spain. The aim of this study was to assess the quality of hatchery-reared larvae in comparison with those reared in the wild, and determine to which extension wild growing larvae are also affected by skeletal deformities. The main structures affected included those forming the axial skeleton, the caudal fin complex and both anal and dorsal fins, with the most prevalent anomalies affecting caudal vertebrae and arches. Hatchery-reared fish presented a higher incidence of deformities (79%) compared with the 19% observed in wild specimens. In wild postlarvae collected in Autumn no deformities were observed. This work clearly shows that wild Senegalese sole present less skeletal deformities than those hatchery-reared during larval stages, indicating a selective mortality of wild deformed fish and/or an effect of aquaculture-related rearing conditions in the development of skeletal deformities in sole.