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- 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.
- Warfarin effects in the skeletal development of zebrafish (Danio rerio)Publication . Peres dos Santos, R.; Bensimon-Brito, A.; Gavaia, Paulo J.; Cancela, LeonorVitamin K-dependent gamma carboxylation (VKGC) is crucial for posttranslational modification of glutamate residues to form α-carboxy glutamic acid (Gla) in the presence of reduced vitamin K, molecular oxygen, and carbon dioxide (1). This modification has important implications, mainly physiological, like homeostasis, signal transduction and bone calcification. This mechanism ensures complete carboxylation of coagulation factors, and proteins like bone Gla protein (BGP) and matrix Gla protein (MGP), being essential for their biological activity. Warfarin is a known anticoagulant that inhibits the action of VKGC (2), inhibiting consequently the activity of the referred proteins.
- 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.
- Matrix Gla protein expression: a complex process involving the use of alternative promoters, multiple splicing events and microRNAsPublication . Cancela, Leonor; Laizé, Vincent; Conceição, N.; Tiago, Daniel; Maia, Ana-Teresa; Bensimon-Brito, A.; Gavaia, Paulo J.Matrix Gla protein (MGP) is a secreted vitamin K-dependent protein (VKD) located in the extracellular matrix and capable of binding calcium through its -carboxyglutamate residues. Although identified in 1983, transcriptional and post-transcriptional mechanisms regulating its expression remain unclear.
- 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.
- Effect of warfarin in zebrafish (Danio rerio) bone formation during caudal fin regenerationPublication . Dionísio, Gisela; Bensimon-Brito, A.; Gavaia, Paulo J.; Cancela, LeonorIn the last decade, fish has emerged as an important organism for studies on skeletal development in vertebrates, and evidence has been accumulated showing that zebrafish is a suitable system to perform phenotype-based drug screens. The ability to regenerate epidermal injuries is a general feature of most organisms yet only a few can fully regenerate severed appendages comprising several different tissues. Zebrafish is one of the most used models for regeneration studies, creating a powerful tool to study de novo bone formation without affecting vital development processes.
- Differentiated skeletal cells contribute to blastema formation during zebrafish fin regenerationPublication . Sousa, Sara; Afonso, Nuno; Bensimon-Brito, Anabela; Fonseca, Mariana; Simões, Mariana; Leon, Joaquin; Roehl, Henry; Cancela, M. Leonor; Jacinto, AntónioThe 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.
- Four-and-a-half LIM domains protein 2 (FHL2) is associated with the development of craniofacial musculature in the teleost fish Sparus aurataPublication . Rafael, Marta S.; Laizé, Vincent; Bensimon-Brito, A.; Leite, Ricardo; Schüle, R.; Cancela, LeonorFour-and-a-half LIM domains protein 2 (FHL2) is involved in major cellular mechanisms such as regulation of gene transcription and cytoskeleton modulation, participating in physiological control of cardiogenesis and osteogenesis. Knowledge on underlying mechanisms is, however, limited. We present here new data on FHL2 protein and its role during vertebrate development using a marine teleost fish, the gilthead seabream (Sparus aurata L.). In silico comparison of vertebrate protein sequences and prediction of LIM domain three-dimensional structure revealed a high degree of conservation, suggesting a conserved function throughout evolution. Determination of sites and levels of FHL2 gene expression in seabream indicated a central role for FHL2 in the development of heart and craniofacial musculature, and a potential role in tissue calcification. Our data confirmed the key role of FHL2 protein during vertebrate development and gave new insights into its particular involvement in craniofacial muscle development and specificity for slow fibers.
- Revisiting in vivo staining with alizarin red S - a valuable approach to analyse zebrafish skeletal mineralization during development and regenerationPublication . Bensimon-Brito, A.; Cardeira Da Silva, João; Dionísio, Gisela; Huysseune, Ann; Cancela, Leonor; Witten, PaulBackground The correct evaluation of mineralization is fundamental for the study of skeletal development, maintenance, and regeneration. Current methods to visualize mineralized tissue in zebrafish rely on: 1) fixed specimens; 2) radiographic and μCT techniques, that are ultimately limited in resolution; or 3) vital stains with fluorochromes that are indistinguishable from the signal of green fluorescent protein (GFP)-labelled cells. Alizarin compounds, either in the form of alizarin red S (ARS) or alizarin complexone (ALC), have long been used to stain the mineralized skeleton in fixed specimens from all vertebrate groups. Recent works have used ARS vital staining in zebrafish and medaka, yet not based on consistent protocols. There is a fundamental concern on whether ARS vital staining, achieved by adding ARS to the water, can affect bone formation in juvenile and adult zebrafish, as ARS has been shown to inhibit skeletal growth and mineralization in mammals. Results Here we present a protocol for vital staining of mineralized structures in zebrafish with a low ARS concentration that does not affect bone mineralization, even after repetitive ARS staining events, as confirmed by careful imaging under fluorescent light. Early and late stages of bone development are equally unaffected by this vital staining protocol. From all tested concentrations, 0.01 % ARS yielded correct detection of bone calcium deposits without inducing additional stress to fish. Conclusions The proposed ARS vital staining protocol can be combined with GFP fluorescence associated with skeletal tissues and thus represents a powerful tool for in vivo monitoring of mineralized structures. We provide examples from wild type and transgenic GFP-expressing zebrafish, for endoskeletal development and dermal fin ray regeneration.