Browsing by Author "Witten, Paul"
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- Determinism and causative factors for morphological anomalies in reared European fishesPublication . Boglione, C.; Fontagné, Stéphanie; Gavaia, Paulo J.; Gisbert, Enric; Kjorsvik, E.; Koumoundouros, Giorgos; Moren, M.; Moutou, K. A.; Witten, PaulThe presence of sublethal morphological deformities represents one of the main bottleneck of the industrial finfish hatchery production, resulting in major economic loss due to reduced growth and marketing ability of the final product, that has to be transformed (filets) or sold for fish flour. Furthermore, the elimination of deformed fishes from the productive cycle needs for periodic selections at present carried out by manual sorting. This represents an additional economic cost, and a stress for fishes.
- 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.
- 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.
- Skeletal anomalies in reared European fish larvae and juveniles. Part 1: normal and anomalous skeletogenic processesPublication . Boglione, C.; Gavaia, Paulo J.; Koumoundouros, Giorgos; Gisbert, Enric; Moren, M.; Fontagné, Stéphanie; Witten, PaulThis critical review summarises the knowledge about fish skeletal tissues and inherent normal and anomalous development. Particular emphasis is given to existing literature on reared European fishes. The aim was to identify the main gaps of knowledge that require to be fulfilled, in order to precociously identify anomalous developmental patterns that lead to skeletal anomalies in reared finfish larvae and juveniles. The review also aims at to extending our knowledge about the factors that are possibly be involved in the onset of skeletal anomalies. The long period goal is the optimization of the morphological quality of farmed juvenile fish.
- Skeletal anomalies in reared European fish larvae and juveniles. Part 2: main typologies, occurrences and causative factorsPublication . Boglione, C.; Gisbert, Enric; Gavaia, Paulo J.; Witten, Paul; Moren, M.; Fontagné, Stéphanie; Koumoundouros, GiorgosThe presence of skeletal anomalies in farmed teleost fish is currently a major problem in aquaculture, entailing economical, biological and ethical issues. The common occurrence of skeletal deformities in farmed fish and the absence of effective solutions for avoiding their onset or definitely culling out the affected individuals as early as possible from the productive cycle, highlight the need to improve our knowledge on the basic processes regulating fish skeletogenesis and skeletal tissues differentiation, modelling and remodelling. Severe skeletal anomalies may actually occur throughout the entire life cycle of fish, but their development often begins with slight aberrations of the internal elements. Comprehensive investigation efforts conducted on reared larvae and juveniles could provide a great contribute in fulfilling such gap of knowledge, as skeletogenesis and skeletal tissues differentiation occur during these early life stages. The aim of this review was to provide a synthetic but comprehensive picture of the actual knowledge on the ontogeny, typologies and occurrence of skeletal anomalies, and on the proposed causative factors for their onset in larvae and juveniles of European farmed fish. The state-of-art of knowledge on these issues is critically analysed intending to individualize the main gaps of knowledge that require to be filled up, in order to optimize the morphological quality of farmed juveniles.