Browsing by Author "Witten, Paul Eckhard"
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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.
- Skeletal anomalies in reared European fish larvae and juveniles. Part 1: normal and anomalous skeletogenic processesPublication . Boglione, Clara; Gavaia, Paulo; Koumoundouros, Giorgos; Gisbert, Enric; Moren, Mari; Fontagne, Stephanie; Witten, Paul EckhardThis critical review summarizes 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 filled, in order to precociously identify anomalous developmental patterns that lead to skeletal anomalies in reared finfish larvae and juveniles. The review also aims to extend our knowledge about the factors that are possibly involved in the onset of skeletal anomalies. The final goal is the optimization of the morphological quality of farmed juvenile fish.