M. L. Cancela

Osteological development and abnormalities of the vertebral column and caudal skeleton in larval and juvenile stages of hatchery-reared Senegal sole (Solea senegalensis)

The Senegal sole is a species recently adapted to aquaculture for which little information on larval development is available. This study was designed to describe normal skeletal development and the occurrence of skeletal malformations in Senegal sole reared in captivity. Eggs were collected from natural spawning, incubated until hatching and larvae reared to the juvenile stage in a closed recirculating system. Samples were collected throughout development at regular intervals from hatching to fully formed juveniles. Specimens were stained with alcian blue and alizarin red and observed for skeletal development and detection of anomalies. A high number of malformations were detected, both in the caudal complex and the vertebral column. About 44% of the individuals observed showed at least one malformation and the highest occurrence of deformities was observed in the caudal region and in the vertebral column. Accordingly, 28% of the total deformities identified in this study were detected at those sites and in adjacent arches and spines. The causes were not identified in this study, but the high incidence of malformations may reflect culture problems due to rearing and/or feeding conditions that affect skeletal development.

Differentiated skeletal cells contribute to blastema formation during zebrafish fin regeneration

The origin of cells that generate the blastema following appendage amputation has been a long-standing question in epimorphic regeneration studies. The blastema is thought to originate from either stem (or progenitor) cells or differentiated cells of various tissues that undergo dedifferentiation. Here, we investigate the origin of cells that contribute to the regeneration of zebrafish caudal fin skeletal elements. We provide evidence that the process of lepidotrichia (bony rays) regeneration is initiated as early as 24 hours post-amputation and that differentiated scleroblasts acquire a proliferative state, detach from the lepidotrichia surface, migrate distally, integrate into the blastema and dedifferentiate. These findings provide novel insights into the origin of cells in epimorphic appendage regeneration in zebrafish and suggest conservation of regeneration mechanisms between fish and amphibians.

Distinct patterns of notochord mineralization in zebrafish coincide with the localization of Osteocalcin isoform 1 during early vertebral centra formation.

BackgroundIn chondrichthyans, basal osteichthyans and tetrapods, vertebral bodies have cartilaginous anlagen that subsequently mineralize (chondrichthyans) or ossify (osteichthyans). Chondrocytes that form the vertebral centra derive from somites. In teleost fish, vertebral centrum formation starts in the absence of cartilage, through direct mineralization of the notochord sheath. In a second step, the notochord is surrounded by somite-derived intramembranous bone. In several small teleost species, including zebrafish (Danio rerio), even haemal and neural arches form directly as intramembranous bone and only modified caudalmost arches remain cartilaginous. This study compares initial patterns of mineralization in different regions of the vertebral column in zebrafish. We ask if the absence or presence of cartilaginous arches influences the pattern of notochord sheath mineralization.ResultsTo reveal which cells are involved in mineralization of the notochord sheath we identify proliferating cells, we trace mineralization on the histological level and we analyze cell ultrastructure by TEM. Moreover, we localize proteins and genes that are typically expressed by skeletogenic cells such as Collagen type II, Alkaline phosphatase (ALP) and Osteocalcin (Oc). Mineralization of abdominal and caudal vertebrae starts with a complete ring within the notochord sheath and prior to the formation of the bony arches. In contrast, notochord mineralization of caudal fin centra starts with a broad ventral mineral deposition, associated with the bases of the modified cartilaginous arches. Similar, arch-related, patterns of mineralization occur in teleosts that maintain cartilaginous arches throughout the spine.Throughout the entire vertebral column, we were able to co-localize ALP-positive signal with chordacentrum mineralization sites, as well as Collagen II and Oc protein accumulation in the mineralizing notochord sheath. In the caudal fin region, ALP and Oc signals were clearly produced both by the notochord epithelium and cells outside the notochord, the cartilaginous arches. Based on immunostaining, real time PCR and oc2:gfp transgenic fish, we identify Oc in the mineralizing notochord sheath as osteocalcin isoform 1 (Oc1).ConclusionsIf notochord mineralization occurs prior to arch formation, mineralization of the notochord sheath is ring-shaped. If notochord mineralization occurs after cartilaginous arch formation, mineralization of the notochord sheath starts at the insertion point of the arches, with a basiventral origin. The presence of ALP and Oc1, not only in cells outside the notochord, but also in the notochord epithelium, suggests an active role of the notochord in the mineralization process. The same may apply to Col II-positive chondrocytes of the caudalmost haemal arches that show ALP activity and Oc1 accumulation, since these chondrocytes do not mineralize their own cartilage matrix. Even without cartilaginous preformed vertebral centra, the cartilaginous arches may have an inductive role in vertebral centrum formation, possibly contributing to the distinct mineralization patterns of zebrafish vertebral column and caudal fin vertebral fusion.

Cloning of the bone Gla protein gene from the teleost fish Sparus aurata. Evidence for overall conservation in gene organization and bone-specific expression from fish to man.

Bone Gla protein (BGP, Osteocalcin) is a bone-specific vitamin K-dependent protein which has been intensively studied in mammals. Although BGP is the most abundant non-collagenous protein of bone, its mode of action at the molecular level remains unclear. From an evolutionary point of view, the appearance of BGP seems to parallel the appearance of hydroxyapatite-containing bone structures since it has never been found in elasmobranchs, whose skeleton is composed of calcified cartilage. Accordingly, recent work indicates that, in mammalian bone, BGP is required for adequate maturation of the hydroxyapatite crystal. Taken together, these data suggest that teleost fishes, presumably the first vertebrates to develop a BGP-containing skeleton, may be a useful model to further investigate BGP function. In addition, fish offer several advantages over mammalian models, due to a large progeny, external embryonic development and transparency of larvae. In the present work, the BGP cDNA and gene were cloned from a teleost fish, Sparus aurata, and its tissue distribution, pattern of developmental expression and evolutionary pathways analyzed. The molecular organization of the Sparus BGP (spBGP) gene is similar to mammalian BGP genes, and its expression throughout development follows the onset of calcification. The spBGP gene encodes a pre-propeptide of 97 amino acid residues, expressed only in bone and showing extensive homology to its mammalian homologs. Phylogenetic analysis of the available BGP sequences supports the hypothesis that all BGPs have a single origin and share a common ancestor with a related vitamin K-dependent protein (Matrix Gla protein).

Oligopeptide transporter PepT1 in Atlantic cod (Gadus morhua L.): cloning, tissue expression and comparative aspects.

SUMMARY A novel full-length cDNA that encodes for the Atlantic cod (Gadus morhua L.) PepT1-type oligopeptide transporter has been cloned. This cDNA (named codPepT1) was 2838 bp long, with an open reading frame of 2190 bp encoding a putative protein of 729 amino acids. Comparison of the predicted Atlantic cod PepT1 protein with zebrafish, bird and mammalian orthologs allowed detection of many structural features that are highly conserved among all the vertebrate proteins analysed, including (1) a larger than expected area of hydrophobic amino acids in close proximity to the N terminus; (2) a single highly conserved cAMP/cGMP-dependent protein kinase phosphorylation motif; (3) a large N-glycosylation-rich region within the large extracellular loop; and (4) a conserved and previously undescribed stretch of 8–12 amino acid residues within the large extracellular loop. Expression analysis at the mRNA level indicated that Atlantic cod PepT1 is mainly expressed at intestinal level, but that it is also present in kidney and spleen. Analysis of its regional distribution along the intestinal tract of the fish revealed that PepT1 is ubiquitously expressed in all segments beyond the stomach, including the pyloric caeca, and through the whole midgut. Only in the last segment, which included the hindgut, was there a lower expression. Atlantic cod PepT1, the second teleost fish PepT1-type transporter documented to date, will contribute to the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful tool for the study of gut functional regionalization, as well as a marker for the analysis of temporal and spatial expression during ontogeny.

Purification of Matrix Gla Protein From a Marine Teleost Fish, Argyrosomus regius: Calcified Cartilage and Not Bone as the Primary Site of MGP Accumulation in Fish†

Matrix Gla protein (MGP) belongs to the family of vitamin K‐dependent, Gla‐containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas the protein was found to accumulate mainly in bone. However, at that time, it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells because both coexist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. To analyze MGP structure and function in bony fish, MGP was acid‐extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius, The A. regius MGP and BGP cDNA sequences were submitted to the GenBank database with accession numbers AF334473 and AF459030, respectively. separated from the mineral phase by dialysis, and purified by Sephacryl S‐100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N‐terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription‐polymerase chain reaction (RT‐PCR) and 5′rapid amplification of cDNA (RACE)‐PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was analyzed by Northern blot, in situ hybridization, and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in cartilage from branchial arches, with no expression detected in the different types of bone analyzed, whereas BGP mRNA was located in bone tissue as expected. Accordingly, the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. In soft tissues, MGP mRNA was mainly expressed in heart but in situ hybridization, indicated that cells expressing the MGP gene were located in the bulbus arteriosus and aortic wall, rich in smooth muscle and endothelial cells, whereas no expression was detected in the striated muscle myocardial fibers of the ventricle. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in cartilage, heart, and kidney tissues, but in contrast with results obtained in Xenopus and higher vertebrates, the protein does not accumulate in vertebra of non‐osteocytic teleost fish, but only in calcified cartilage. In addition, our results also indicate that the presence of MGP mRNA in heart tissue is due, at least in fish, to the expression of the MGP gene in only two specific cell types, smooth muscle and endothelial cells, whereas no expression was found in the striated muscle fibers of the ventricle. In light of these results and recent information on expression of MGP gene in these same cell types in mammalian aorta, it is likely that the levels of MGP mRNA previously detected in Xenopus, birds, and mammalian heart tissue may be restricted toregions rich in smooth muscle and endothelial cells. Our results also emphasize the need to re‐evaluate which cell types are involved in MGP gene expression in other soft tissues and bring further evidence that fish are a valuable model system to study MGP gene expression and regulation.

Matrix Gla protein gene expression and protein accumulation colocalize with cartilage distribution during development of the teleost fish Sparus aurata

Matrix Gla protein (MGP) is a member of the family of extracellular mineral-binding Gla proteins, expressed in several tissues with high accumulation in bone and cartilage. Although the precise molecular mechanism of action of this protein remains unknown, all available evidence indicates that MGP plays a role as an inhibitor of mineralization. We investigated the sites of gene expression and protein accumulation of MGP throughout development of the bony fish Sparus aurata, by in situ hybridization, Northern and RT-PCR Southern hybridization, and immunohistochemistry. The results obtained were compared with the patterns of developmental appearance of cartilaginous and mineralized structures in this species, identified by histological techniques and by detection of mRNA presence and protein accumulation of osteocalcin (Bone Gla protein), a marker for osteoblasts known to accumulate in bone mineralized extracellular matrix. The expression of MGP mRNA was first detected at 2 days posthatching (dph) by Northern analysis, RT-PCR amplification, and in situ hybridization, and thereafter continuously detected at various levels of intensity, until 130 dph. In situ hybridization analysis performed in parallel with immunohistochemistry indicated that until ca. 45 dph, the MGP gene was highly expressed in a number of different tissues including skull, jaw, neural and hemal arches, and heart and the protein accumulated in cartilaginous tissues. At 85 dph, a stage when most skeletal structures are mineralized, MGP gene expression and protein accumulation were restricted to the remaining cartilaginous structures, whereas osteocalcin gene expression and protein accumulation were localized in most mineralized structures. MGP gene expression was also detected in heart and kidney, although in situ hybridization only detected MGP mRNA in heart, located in the arterial bulbus and not in the cardiac muscle. Our results are in agreement with those recently described for MGP localization in adult tissues of another teleost fish, as well as available data from higher vertebrates, strengthening the hypothesis of a conserved function for MGP from teleost fish to human, a period of more than 200 million years of evolution. In addition, Sparus aurata, a marine teleost fish routinely grown in captivity, appears to be a good model to further analyze MGP gene expression and regulation.

Vestiges, rudiments and fusion events: the zebrafish caudal fin endoskeleton in an evo-devo perspective

The vertebral column results from a controlled segmentation process associated with two main structures, the notochord and the somites. Pathological fusion of vertebral bodies can result from impaired segmentation during embryonic development or occur postnatally. Here, we explore the process of formation and subsequent fusion of the caudalmost vertebral bodies in zebrafish, where fusion is a normal process, mechanically required to support the caudal fin. To reveal whether the product of fusion is on an evolutionary or a developmental scale, we analyze the mode of formation of vertebral bodies, identify transitory rudiments, and characterize vestiges that indicate previous fusion events. Based on a series of closely spaced ontogenetic stages of cleared and stained zebrafish, parasagittal sections, and detection methods for elastin and mineral, we conclude that the formation of the urostyle involves four fusion events. Although fusion of preural 1 (PU1+) with ural 1 (U1) and fusion within ural 2 (U2+) are no longer traceable during centrum formation (phylogenetic fusion), fusion between the compound centrum [PU1++U1] and U2+ (ontogenetic fusion) occurs after individualization of the centra. This slow process is the last fusion and perhaps the latest fusion during the evolution of the zebrafish caudal fin endoskeleton. Newly described characters, such as a mineralized subdivision within U2+, together with the reinterpretation of known features in an evolutionary–developmental context, strongly suggest that the zebrafish caudal fin endoskeleton is made from more fused vertebral bodies than previously assumed. In addition, these fusion events occur at different developmental levels depending on their evolutionary status, allowing the dissection of fusion processes that have taken place over different evolutionary times.

Multiple paternity in Norway lobster (Nephrops norvegicus L.) assessed with microsatellite markers.

We investigated genetic diversity and the mating system of the Norway lobster (Nephrops norvegicus) in a wild population off the Portuguese coast. Approximately 100 individuals were screened for 2 microsatellite loci. For 11 ovigerous lobsters both the female and a sample of her offspring (24 eggs) were genotyped. High genetic diversity was observed for the 2 markers in the population. Paternity within broods was analyzed by comparing multilocus genotypes of each egg with the corresponding mother, and the male parent contribution was then deduced. Multiple paternity was observed in 6 of the 11 broods studied. In those cases, 2 to 3 male parents were likely to have contributed to the fertilization of the eggs. When multiple paternity was involved, the comparative reproductive success of the male parents was quite even. This is the first report of multiple paternity in the Norway lobster. Comparisons with other taxa are presented, and consequences of multiple paternity are discussed.

MiR-29a is an enhancer of mineral deposition in bone-derived systems.

MicroRNAs (miRNAs) provide a mechanism for fine-tuning of intricate cellular processes through post-transcriptional regulation. Emerging evidences indicate that miRNAs play key roles in regulation of osteogenesis. The miR-29 family was previously implicated in mammalian osteoblast differentiation by targeting extracellular matrix molecules and modulating Wnt signaling. Nevertheless, the function of miR-29 in bone formation and homeostasis is not completely understood. Here, we provide novel insights into the biological effect of miR-29a overexpression in a mineralogenic cell system (ABSa15). MiR-29a gain-of-function resulted in significant increase of extracellular matrix mineralization, probably due to accelerated differentiation. We also demonstrated for the first time that miR-29a induced β-catenin protein levels, implying a stimulation of canonical Wnt signaling. Our data also suggests that SPARC is a conserved target of miR-29a, and may contribute to the phenotype observed in ABSa15 cells. Finally, we provide evidences for miR-29a conservation throughout evolution based on sequence homology, synteny analysis and expression patterns. Concluding, miR-29a is a key player in osteogenic differentiation, leading to increased mineralization in vitro, and this function seems to be conserved throughout vertebrate evolution by interaction with canonical Wnt signaling and conservation of targets.