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- Development of chondroid tissues in lordotic-kyphotic Senegalese sole (Solea senegalensis)Publication . Cardeira Da Silva, João; Mendes, A. C.; Pousão-Ferreira, P.; Cancela, Leonor; Gavaia, Paulo J.Vertebral deformities are frequent and constitute a major drawback for many aquaculture produced species. Lordosis and kyphosis, which are severe curvatures of the vertebral column, are particularly problematic, compromising fish marketing and profitability. Senegalese sole is a species with increasing interest in this industry and, like many others, presents high frequencies of vertebral deformities under rearing conditions.
- The use of enhanced green fluorescent protein to track matrix Gla protein expression during zebrafish (Danio rerio) developmentPublication . Cardeira Da Silva, João; Gavaia, Paulo J.; Brito, A. B.; Cancela, LeonorMatrix Gla protein (mgp), a vitamin K-dependent protein, is an important element of skeleton development as it regulates mineralization of both cartilaginous and bony structures, among others. Although mgp is found in a wide range of tissues, it is known to be expressed mostly by chondrocytes and vascular smooth muscle cells. Accordingly, inhibition of Mgp function results in over-mineralization of cartilages and growth plates and in ectopic mineralizations, particularly in the vascular system. Besides being a well characterized protein and the existence of developmental studies in zebrafish, there is still no description of monitoring mgp expression in live specimens.
- Lordotic-kyphotic vertebrae develop ectopic cartilage-like tissue in Senegalese sole (Solea senegalensis)Publication . Cardeira Da Silva, João; Bensimon-Brito, A.; Pousão-Ferreira, P.; Cancela, Leonor; Gavaia, Paulo J.Lordosis and kyphosis are curvatures of the vertebral column that commonly affect Senegalese sole (Solea senegalensis) under intensive rearing conditions, constituting a problem for the aquaculture industry by increasing labor and operation costs and consequently reducing profits. However, no studies have yet addressed microanatomical changes in vertebral column curvature in this species. We have conducted histological analyses in order to determine the alterations at tissue and cellular levels in these deformities. Development of an ectopic cartilage-like tissue within bone matrix at the growth regions (contact area between two vertebrae) of affected vertebrae was observed in lordotic-kyphotic fish. As previously suggested, the increase in strain supported by deformed vertebrae may trigger the onset of metaplastic tissue formation through an osteochondroprogenitor precursor from the condensation of mesenchymal cells or by trans-differentiation of (pre-)osteoblastic cells to a chondrocytic lineage. The possibility of a notochord contribution, as well as the molecular events related to bone stress and bone types in terms of cellularity, are also discussed.
- Early axial and appendicular skeletal development of the meagre (Argyrosomus regius). A comparison of two rearing systemsPublication . Valles, E.; Cardeira Da Silva, João; Dionísio, Gisela; Estévez, A.; Pousão-Ferreira, P.; Cancela, Leonor; Gavaia, Paulo J.The meagre (Argyrosomus regius) is a new species for aquaculture with high economical value, presenting growth rates of 1 kg/year. Skeletal deformities generally appear in cultured larvae at early stages, reducing the value of the fish. The present study describes the osteological development of the vertebral column and fins in meagre larvae. Meagre larvae were obtained from wild broodstocks, kept at IRTA and IPIMAR, and induced to spawn using GnRHa injection.
- Skeletal development and performance of zebrafish Danio rerio (Hamilton, 1822) larvae and juvenile fish fed with different dietsPublication . Cardeira Da Silva, João; Dionísio, Gisela; Bensimon-Brito, A.; Cancela, Leonor; Gavaia, Paulo J.The appearance of skeletal deformities is a major problem associated to intensive, large-scale culture of fish species, affecting growth, development and ultimately, survival of produced fish. Although the mechanisms working behind it are still unclear, it is known that environmental, genetic and nutritional factors play fundamental roles. Given its importance as a vertebrate model to study skeleton-related diseases, the selection of an appropriate dietary protocol is a critical step in order to diminish the nutritional effect on bone and cartilage development.
- Analysis of sperm quality in a type I diabetes zebrafish modelPublication . Diogo, Patricia; Eufrásio, Ana; Martins, Gil; Cardeira, João; Cancela, M. Leonor; Cabrita, Elsa; Gavaia, PauloDiabetes is a fast growing disease in human populaon and the study of its impact on mammalian reproducve traits has been con-troversial. Some authors showed a negave eect on sperm mol-ity and DNA fragmentaon in some species, while others failed to detect any eects. In the present study zebrash was used as a model to study the eect of diabetes in sperm traits such as mol-ity, viability and DNA fragmentaon
- Quantitative assessment of the regenerative and mineralogenic performances of the zebrafish caudal finPublication . Cardeira Da Silva, João; Gavaia, Paulo J.; Fernandez, Ignacio; Cengiz, Ibrahim Fatih; Moreira-Silva, Joana; Oliveira, Joaquim Miguel; Reis, Rui L.; Cancela, Leonor; Laizé, VincentThe ability of zebrafish to fully regenerate its caudal fin has been explored to better understand the mechanisms underlying de novo bone formation and to develop screening methods towards the discovery of compounds with therapeutic potential. Quantifying caudal fin regeneration largely depends on successfully measuring new tissue formation through methods that require optimization and standardization. Here, we present an improved methodology to characterize and analyse overall caudal fin and bone regeneration in adult zebrafish. First, regenerated and mineralized areas are evaluated through broad, rapid and specific chronological and morphometric analysis in alizarin red stained fins. Then, following a more refined strategy, the intensity of the staining within a 2D longitudinal plane is determined through pixel intensity analysis, as an indicator of density or thickness/volume. The applicability of this methodology on live specimens, to reduce animal experimentation and provide a tool for in vivo tracking of the regenerative process, was successfully demonstrated. Finally, the methodology was validated on retinoic acid-and warfarin-treated specimens, and further confirmed by micro-computed tomography. Because it is easily implementable, accurate and does not require sophisticated equipment, the present methodology will certainly provide valuable technical standardization for research in tissue engineering, regenerative medicine and skeletal biology.
- The zebrafish operculum: a powerful system to assess osteogenic bioactivities of molecules with pharmacological and toxicological relevancePublication . Tarasco, Marco; Laizé, Vincent; Cardeira, João; Leonor Cancela, M.; Gavaia, PauloBone disorders affect millions of people worldwide and available therapeutics have a limited efficacy, often presenting undesirable side effects. As such, there is a need for novel molecules with bone anabolic properties. The aim of this work was to establish a rapid, reliable and reproducible method to screen for molecules with osteogenic activities, using the zebrafish operculum to assess bone formation. Exposure parameters were optimized through morphological analysis of the developing operculum of larvae exposed to calcitriol, a molecule with known pro-osteogenic properties. An exposure of 3 days initiated at 3 days post-fertilization was sufficient to stimulate operculum formation, while not affecting survival or development of the larvae. Dose dependent pro- and anti-osteogenic effects of calcitriol and cobalt chloride, respectively, demonstrated the sensitivity of the method and the suitability of the operculum system. A double transgenic reporter line expressing fluorescent markers for early and mature osteoblasts was used to gain insights into the effects of calcitriol and cobalt at the cellular level, with osteoblast maturation shown to be stimulated and inhibited, respectively, in the operculum of exposed fish. The zebrafish operculum represents a consistent, robust and rapid screening system for the discovery of novel molecules with osteogenic, anti-osteoporotic or osteotoxic activity.
- Development of an in vitro cell system from zebrafish suitable to study bone cell differentiation and extracellular matrix mineralizationPublication . Vijayakumar, Parameswaran; Laizé, Vincent; Cardeira Da Silva, João; Trindade, Marlene; Cancela, M. LeonorMechanisms of bone formation and skeletal development have been successfully investigated in zebrafish using a variety of in vivo approaches, but in vitro studies have been hindered due to a lack of homologous cell lines capable of producing an extracellular matrix (ECM) suitable for mineral deposition. Here we describe the development and characterization of a new cell line termed ZFB1, derived from zebrafish calcified tissues. ZFB1 cells have an epithelium-like phenotype, grow at 28 degrees C in a regular L-15 medium supplemented with 15% of fetal bovine serum, and are maintained and manipulated using standard methods (e.g., trypsinization, cryopreservation, and transfection). They can therefore be propagated and maintained easily in most cell culture facilities. ZFB1 cells show aneuploidy with 2n=78 chromosomes, indicative of cell transformation. Furthermore, because DNA can be efficiently delivered into their intracellular space by nucleofection, ZFB1 cells are suitable for gene targeting approaches and for assessing gene promoter activity. ZFB1 cells can also differentiate toward osteoblast or chondroblast lineages, as demonstrated by expression of osteoblast- and chondrocyte-specific markers, they exhibit an alkaline phosphatase activity, a marker of bone formation in vivo, and they can mineralize their ECM. Therefore, they represent a valuable zebrafish-derived in vitro system for investigating bone cell differentiation and extracellular matrix mineralization.
- Distinct patterns of notochord mineralization in zebrafish coincide with the localization of Osteocalcin isoform 1 during early vertebral centra formationPublication . Bensimon-Brito, A.; Cardeira Da Silva, João; Cancela, Leonor; Huysseune, Ann; Witten, PaulIn 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. Results - To 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). Conclusions - If 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.