M. Leonor Cancela

Development of two bone-derived cell lines from the marine teleost Sparus aurata; evidence for extracellular matrix mineralization and cell-type-specific expression of matrix Gla protein and osteocalcin

A growing interest in the understanding of the ontogeny and mineralization of fish skeleton has emerged from the recent implementation of fish as a vertebrate model, particularly for skeletal development. Whereas several in vivo studies dealing with the regulation of bone formation in fish have been published, in vitro studies have been hampered because of a complete lack of fish-bone-derived cell systems. We describe here the development and the characterization of two new cell lines, designated VSa13 and VSa16, derived from the vertebra of the gilthead sea bream. Both cell types exhibit a spindle-like phenotype and slow growth when cultured in Leibovitz’s L-15 medium and a polygonal phenotype and rapid growth in Dulbecco’s modified Eagle medium (D-MEM). Scanning electron microscopy and von Kossa staining have revealed that the VSa13 and VSa16 cells can only mineralize their extracellular matrix when cultured in D-MEM under mineralizing conditions, forming calcium-phosphate crystals similar to hydroxyapatite. We have also demonstrated the involvement of alkaline phosphatase, a marker of bone formation in vivo, and Gla proteins (osteocalcin and matrix Gla protein, MGP) in the process of mineralization. Finally, we have shown that VSa13 and VSa16 cell lines express osteocalcin and MGP in a mutually exclusive manner. Thus, both cell lines are capable of mineralizing in vitro and of expressing genes found in chondrocyte and osteoblast cell lineages, emphasizing the suitability of these new cell lines as valuable tools for analyzing the expression and regulation of cartilage- and bone-specific genes.

Phox2b function in the enteric nervous system is conserved in zebrafish and is sox10-dependent

Zebrafish lacking functional sox10 have defects in non-ectomesenchymal neural crest derivatives including the enteric nervous system (ENS) and as such provide an animal model for human Waardenburg Syndrome IV. Here, we characterize zebrafish phox2b as a functionally conserved marker of the developing ENS. We show that morpholino-mediated knockdown of Phox2b generates fish modeling Hirschsprung disease. Using markers, including phox2b, we investigate the ontogeny of the sox10 ENS phenotype. As previously shown for melanophore development, ENS progenitor fate specification fails in these mutant fish. However, in addition, we trace back the sox10 mutant ENS defect to an even earlier time point, finding that most neural crest cells fail to migrate ventrally to the gut primordium.

Gla-rich Protein (GRP), A New Vitamin K-dependent Protein Identified from Sturgeon Cartilage and Highly Conserved in Vertebrates

We report the isolation of a novel vitamin K-dependent protein from the calcified cartilage of Adriatic sturgeon (Acipenser nacarii). This 10.2-kDa secreted protein contains 16 γ-carboxyglutamic acid (Gla) residues in its 74-residue sequence, the highest Gla percent of any known protein, and we have therefore termed it Gla-rich protein (GRP). GRP has a high charge density (36 negative + 16 positive = 20 net negative) yet is insoluble at neutral pH. GRP has orthologs in all taxonomic groups of vertebrates, and a paralog (GRP2) in bony fish; no GRP homolog was found in invertebrates. There is no significant sequence homology between GRP and the Gla-containing region of any presently known vitamin K-dependent protein. Forty-seven GRP sequences were obtained by a combination of cDNA cloning and comparative genomics: all 47 have a propeptide that contains a γ-carboxylase recognition site and a mature protein with 14 highly conserved Glu residues, each of them being γ-carboxylated in sturgeon. The protein sequence of GRP is also highly conserved, with 78% identity between sturgeon and human GRP. Analysis of the corresponding gene structures suggests a highly constrained organization, particularly for exon 4, which encodes the core Gla domain. GRP mRNA is found in virtually all rat and sturgeon tissues examined, with the highest expression in cartilage. Cells expressing GRP include chondrocytes, chondroblasts, osteoblasts, and osteocytes. Because of its potential to bind calcium through Gla residues, we suggest that GRP may regulate calcium in the extracellular environment.

Detection of Mineralized Structures in Early Stages of Development of Marine Teleostei Using a Modified Alcian Blue-Alizarin Red Double Staining Technique for Bone and Cartilage

We have developed a procedure for staining cartilage and bone in fish larvae as small as 2 mm (notochord length), for which standard alcian blue/alizarin red procedures did not give positive and/or consistent results. Small calcified structures only 100–200 ixm in length can be clearly visualized. The method is suitable for both onto-genic studies during early stages of skeletal development in most marine fishes (e.g., Sporus aurata L., Solea senegalensis Kaup), whose larvae at hatching are often only a few millimeters long and for detecting skeletal abnormalities in small larvae. This procedure can also be used for specimens that have been preserved in 100% ethanol for up to two years.

Gla-Rich Protein Is a Novel Vitamin K-Dependent Protein Present in Serum That Accumulates at Sites of Pathological Calcifications

Mineralization of soft tissues is an abnormal process that occurs in any body tissue and can greatly increase morbidity and mortality. Vitamin K-dependent (VKD) proteins play a crucial role in these processes; matrix Gla protein is considered one of the most relevant physiological inhibitors of soft tissue calcification know to date. Several studies have suggested that other, still unknown, VKD proteins might also be involved in soft tissue calcification pathologies. We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), which contains the highest ratio between number of Gla residues and size of the mature protein so far identified. Although mainly expressed in cartilaginous tissues of sturgeon, in rat GRP is present in both cartilage and bone. We now show that GRP is a circulating protein that is also expressed and accumulated in soft tissues of rats and humans, including the skin and vascular system in which, when affected by pathological calcifications, GRP accumulates at high levels at sites of mineral deposition, indicating an association with calcification processes. The high number of Gla residues and consequent mineral binding affinity properties strongly suggest that GRP may directly influence mineral formation, thereby playing a role in processes involving connective tissue mineralization.

Development of an In Vitro Clonal Culture and Characterization of the rRNA Gene Cluster of Perkinsus atlanticus, a Protistan Parasite of the Clam Tapes decussatus

Abstract Perkinsus atlanticus cultures were established either with trophozoites isolated from fresh gills, with hypnospores isolated from tissues incubated in fluid thioglycollate medium, or directly from infected hemocytes of carpet shell clams Tapes decussatus from Algarve (Southern Portugal), using a culture medium and conditions optimized for Perkinsus marinus. Perkinsus atlanticus isolates were cloned by limiting dilution, and their identity unequivocally established by PCR-based species-specific diagnostic assays, and by sequencing the complete rRNA gene cluster. The rRNA gene cluster is 7.5-kb in length including 5S, IGS, SSU, ITS1, 5.8S, ITS2, LSU, and an inter–cluster spacer. rDNA sequences of the P. atlanticus clone were between 98.3–100% identical to P. atlanticus sequences previously obtained from clam tissue (non-clonal) isolates. Based on the IGS sequences available from Perkinsus species, a set of primers was designed to amplify P. atlanticus and the two clonally cultured Perkinsus species (P. marinus and P. andrewsi) currently available from a recognized repository. This Perkinsus “genus-specific” PCR-based assay complements the species-specific assays developed earlier and strengthen the detection of Perkinsus species for which specific detection assays are not yet available.

Alternatively spliced transcripts of Sparus aurata insulin-like growth factor 1 are differentially expressed in adult tissues and during early development

Spliced 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.

Suppression subtractive hybridization for studying gene expression during aerial exposure and desiccation in fucoid algae

Gene expression in Fucus vesiculosus L. from the Ria Formosa, Faro, Portugal was investigated by screening a cDNA library generated by suppression subtractive hybridization (SSH) for algae undergoing mild desiccation stress (60–70%tissue water content). The subtractive library was constructed from small amounts (1 µg) of total RNA using PCR-based techniques. Screening by reverse Northern analysis (cDNA Southerns) revealed that between 60% and 70% of clones randomly selected from the library were differentially regulated in desiccated and hydrated algae. Most genes could not be directly identified based on sequence homology with known gene sequences. However, several cDNAs for chloroplast encoded transcripts were identified and shown to be up-regulated or differentially regulated in desiccated algae relative to hydrated controls. These included partial sequences for ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco; rbcL/rbcS), chloroplast coupling factor ATPase (atpH/atpI) and a photosystem I P700 chlorophyll a binding protein (psaA). The usefulness of reverse Northern analysis was confirmed with conventional Northern analysis of transcript abundance for Rubisco, which varied rapidly in response to light and the hydration status of the algae. These results show that SSH is a useful technique for analysing the responses of gene expression to environmental stress, and as a starting point for the subsequent identification of stress-responsive genes in macroalgae.

Vanadate proliferative and anti-mineralogenic effects are mediated by MAPK and PI-3K/Ras/Erk pathways in a fish chondrocyte cell line.

We recently reported proliferative and anti‐mineralogenic effects of vanadate on fish chondrocytes and here we investigate the signalling pathways associated with these effects. Our data show that vanadate stimulates chondrocyte proliferation through the MAPK pathway, using signalling mechanisms similar to those used by IGF‐1, while it inhibits chondrocyte differentiation/mineralization through a putative PI‐3K/Ras/Erk signalling, a pathway shared with insulin. Our data also suggest that vanadate impairs ECM mineralization not only by interfering with regulatory pathways but also by inhibiting enzymatic activity of ALP. Finally, this work provides additional evidence for the conservation, throughout evolution, of mechanisms regulating chondrocyte proliferation and differentiation.

Identification of an osteopontin‐like protein in fish associated with mineral formation

Fish has been recently recognized as a suitable vertebrate model and represents a promising alternative to mammals for studying mechanisms of tissue mineralization and unravelling specific questions related to vertebrate bone formation. The recently developed Sparus aurata (gilthead seabream) osteoblast‐like cell line VSa16 was used to construct a cDNA subtractive library aimed at the identification of genes associated with fish tissue mineralization. Suppression subtractive hybridization, combined with mirror orientation selection, identified 194 cDNA clones representing 20 different genes up‐regulated during the mineralization of the VSa16 extracellular matrix. One of these genes accounted for 69% of the total number of clones obtained and was later identified as theS. aurata osteopontin‐like gene. The 2138‐bp full‐length S. aurata osteopontin‐like cDNA was shown to encode a 374 amino‐acid protein containing domains and motifs characteristic of osteopontins, such as an integrin receptor‐binding RGD motif, a negatively charged domain and numerous post‐translational modifications (e.g. phosphorylations and glycosylations). The common origin of mammalian osteopontin and fish osteopontin‐like proteins was indicated through an in silico analysis of available sequences showing similar gene and protein structures and was further demonstrated by their specific expression in mineralized tissues and cell cultures. Accordingly, and given its proven association with mineral formation and its characteristic protein domains, we propose that the fish osteopontin‐like protein may play a role in hard tissue mineralization, in a manner similar to osteopontin in higher vertebrates.