Browsing by Author "Huysseune, Ann"
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- Beyond the whole-mount phenotype: high-resolution imaging in fluorescence-based applications on zebrafishPublication . Oralová, Veronika; Rosa, Joana; Soenens, Mieke; Bek, Jan Willem; Willaert, Andy; Witten, Paul Eckhard; Huysseune, AnnZebrafish is now widely used in biomedical research as a model for human diseases, but the relevance of the model depends on a rigorous analysis of the phenotypes obtained. Many zebrafish disease models, experimental techniques and manipulations take advantage of fluorescent reporter molecules. However, phenotypic analysis often does not go beyond establishing overall distribution patterns of the fluorophore in whole-mount embryos or using vibratome or paraffin sections with poor preservation of tissue architecture and limited resolution. Obtaining high-resolution data of fluorescent signals at the cellular level from internal structures mostly depends on the availability of expensive imaging technology. Here, we propose a new and easily applicable protocol for embedding and sectioning of zebrafish embryos using in-house prepared glycol methacrylate (GMA) plastic that is suited for preservation of fluorescent signals (including photoactivatable fluorophores) without the need for antibodies. Four main approaches are described, all involving imaging fluorescent signals on semithin (3 µm or less) sections. These include sectioning transgenic animals, whole-mount immunostained embryos, cell tracking, as well as on-section enzyme histochemistry.
- Cells at the edge: the dentin–bone interface in Zebrafish teethPublication . Rosa, Joana; Witten, Paul Eckhard; Huysseune, AnnBone-producing osteoblasts and dentin-producing odontoblasts are closely related cell types, a result from their shared evolutionary history in the ancient dermal skeleton. In mammals, the two cell types can be distinguished based on histological characters and the cells’ position in the pulp cavity or in the tripartite periodontal complex. Different from mammals, teleost fish feature a broad diversity in tooth attachment modes, ranging from fibrous attachment to firm ankylosis to the underlying bone. The connection between dentin and jaw bone is often mediated by a collar of mineralized tissue, a part of the dental unit that has been termed “bone of attachment”. Its nature (bone, dentin, or an intermediate tissue type) is still debated. Likewise, there is a debate about the nature of the cells secreting this tissue: osteoblasts, odontoblasts, or yet another (intermediate) type of scleroblast. Here, we use expression of the P/Q rich secretory calcium-binding phosphoprotein 5 (scpp5) to characterize the cells lining the so-called bone of attachment in the zebrafish dentition. scpp5 is expressed in late cytodifferentiation stage odontoblasts but not in the cells depositing the “bone of attachment”. nor in bona fide osteoblasts lining the supporting pharyngeal jaw bone. Together with the presence of the osteoblast marker Zns-5, and the absence of covering epithelium, this links the cells depositing the “bone of attachment” to osteoblasts rather than to odontoblasts. The presence of dentinal tubule-like cell extensions and the near absence of osteocytes, nevertheless distinguishes the “bone of attachment” from true bone. These results suggest that the “bone of attachment” in zebrafish has characters intermediate between bone and dentin, and, as a tissue, is better termed “dentinous bone”. In other teleosts, the tissue may adopt different properties. The data furthermore support the view that these two tissues are part of a continuum of mineralized tissues. Expression of scpp5 can be a valuable tool to investigate how differentiation pathways diverge between osteoblasts and odontoblasts in teleost models and help resolving the evolutionary history of tooth attachment structures in actinopterygians.
- 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.
- Periderm invasion contributes to epithelial formation in the teleost pharynxPublication . Rosa, Joana; Oralová, Veronika; Larionova, Daria; Eisenhoffer, G. T.; Eckhard Witten, P.; Huysseune, AnnThe gnathostome pharyngeal cavity functions in food transport and respiration. In amniotes the mouth and nares are the only channels allowing direct contact between internal and external epithelia. In teleost fish, gill slits arise through opening of endodermal pouches and connect the pharynx to the exterior. Using transgenic zebrafish lines, cell tracing, live imaging and different markers, we investigated if pharyngeal openings enable epithelial invasion and how this modifies the pharyngeal epithelium. We conclude that in zebrafish the pharyngeal endoderm becomes overlain by cells with a peridermal phenotype. In a wave starting from pouch 2, peridermal cells from the outer skin layer invade the successive pouches until halfway their depth. Here the peridermal cells connect to a population of cells inside the pharyngeal cavity that express periderm markers, yet do not invade from outside. The latter population expands along the midline from anterior to posterior until the esophagus-gut boundary. Together, our results show a novel role for the periderm as an internal epithelium becomes adapted to function as an external surface.
- 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.