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BackgroundZebrafish (Danio rerio) has a remarkable capacity to regenerate many organs and tissues. During larval stages the fin fold allows the possibility of performing long time-lapse imaging making this system very appealing to study the relationships between tissue movements, cell migration and proliferation necessary for the regeneration process.ResultsThrough the combined use of transgenic fluorescently-labeled animals and confocal microscopy imaging, we characterized in vivo the complete fin fold regeneration process. We show, for the first time, that there is an increase in the global rate of epidermal growth as a response to tissue loss. Also enhanced significantly is cell proliferation, which upon amputation happens in a broad area concerning the amputation level and not in a blastema-restricted way. This reveals a striking difference with regard to the adult fin regeneration system. Finally, an accumulation of migratory, shape-changing fibroblasts occurs proximally to the wound area, resembling a blastemal-like structure, which may act as a signaling center for the regeneration process to proceed.ConclusionsThese findings provide a novel in vivo description of fundamental mechanisms occurring during the fin fold regeneration process, thereby contributing to a better knowledge of this regenerative system and to reveal variations in the epimorphic regeneration field.

Over the last decade, zebrafish has proven to be a powerful model in cancer research. Zebrafish form tumors that histologically and genetically resemble human cancers. The live imaging and cost-effective compound screening possible with zebrafish especially complement classic mouse cancer models. Here, we report recent progress in the field, including genetically engineered zebrafish cancer models, xenotransplantation of human cancer cells into zebrafish, promising approaches toward live investigation of the tumor microenvironment, and identification of therapeutic strategies by performing compound screens on zebrafish cancer models. Given the recent advances in genome editing, personalized zebrafish cancer models are now a realistic possibility. In addition, ongoing automation will soon allow high-throughput compound screening using zebrafish cancer models to be part of preclinical precision medicine approaches.

Chimeric antigen receptor (CAR) T cells have proven to be a powerful cellular therapy for B cell malignancies. Massive efforts are now being undertaken to reproduce the high efficacy of CAR T cells in the treatment of other malignancies. Here, predictive preclinical model systems are important, and the current gold standard for preclinical evaluation of CAR T cells are mouse xenografts. However, mouse xenograft assays are expensive and slow. Therefore, an additional vertebrate in vivo assay would be beneficial to bridge the gap from in vitro to mouse xenografts. Here, we present a novel assay based on embryonic zebrafish xenografts to investigate CAR T cell-mediated killing of human cancer cells. Using a CD19-specific CAR and Nalm-6 leukemia cells, we show that live observation of killing of Nalm-6 cells by CAR T cells is possible in zebrafish embryos. Furthermore, we applied Fiji macros enabling automated quantification of Nalm-6 cells and CAR T cells over time. In conclusion, we provide a proof-of-principle study that embryonic zebrafish xenografts can be used to investigate CAR T cell-mediated killing of tumor cells. This assay is cost-effective, fast, and offers live imaging possibilities to directly investigate CAR T cell migration, engagement, and killing of effector cells.

Throughout the Animal Kingdom, the time of embryonic development is maintained and strictly controlled. Each step of the process is successful only when it occurs at the right time and place. This raises the question: how is time controlled during embryonic development? Time control is particularly crucial during embryo segmentation processes, where the number of generated segments, as well as the time of formation of each segment, is extraordinarily constant and specific for each species. Somitogenesis is the process through which the vertebrate presomitic mesoderm is segmented along its anterior-posterior axis into round-shaped masses of epithelial cells, named somites. In the chick embryo, a new pair of somites is formed every 90 min. The discovery that this clock-like precision is dictated by the somitogenesis molecular clock constituted a landmark in the Developmental Biology field. Several genes exhibit cyclic gene expression in the embryo presomitic mesoderm from which the somites arise, presenting a 90 min oscillation period, the time required to form a pair of somites. The combined levels of dynamic gene expression throughout the presomitic mesoderm enable cells to acquire positional information, thus giving them a notion of time. Anterior-posterior patterning of the vertebrate nervous system also involves partition into discrete territories. This is particularly evident in the hindbrain where overt segmentation occurs. Nevertheless, little is known about the segmentation genes and mechanisms that may be involved. This paper intends to describe the molecular clock associated with vertebrate somitogenesis, suggesting that it may be operating in many other patterning processes.

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BackgroundAccurate regulation of Notch signalling is central for developmental processes in a variety of tissues, but its function in pectoral fin development in zebrafish is still unknown.Methodology/Principal FindingsHere we show that core elements necessary for a functional Notch pathway are expressed in developing pectoral fins in or near prospective muscle territories. Blocking Notch signalling at different levels of the pathway consistently leads to the formation of thin, wavy, fragmented and mechanically weak muscles fibres and loss of stress fibres in endoskeletal disc cells in pectoral fins. Although the structural muscle genes encoding Desmin and Vinculin are normally transcribed in Notch-disrupted pectoral fins, their proteins levels are severely reduced, suggesting that weak mechanical forces produced by the muscle fibres are unable to stabilize/localize these proteins. Moreover, in Notch signalling disrupted pectoral fins there is a decrease in the number of Pax7-positive cells indicative of a defect in myogenesis.Conclusions/SignificanceWe propose that by controlling the differentiation of myogenic progenitor cells, Notch signalling might secure the formation of structurally stable muscle fibres in the zebrafish pectoral fin.

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