A crystal-clear zebrafish for in vivo imaging.
Ontology highlight
ABSTRACT:
SUBMITTER: Antinucci P
PROVIDER: S-EPMC4933947 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Zebrafish are excellent at regenerating their heart by reinitiating proliferation in pre-existing cardiomyocytes. Studying how zebrafish achieve this holds great potential in developing new strategies to boost mammalian heart regeneration. Nevertheless, the lack of appropriate live-imaging tools for the adult zebrafish heart has limited detailed studies into the dynamics underlying cardiomyocyte proliferation. Here, we address this by developing a system in which cardiac slices of the injured zebrafish heart are cultured ex vivo for several days while retaining key regenerative characteristics, including cardiomyocyte proliferation. In addition, we show that the cardiac slice culture system is compatible with live timelapse imaging and allows manipulation of regenerating cardiomyocytes with drugs that normally would have toxic effects that prevent their use. Finally, we use the cardiac slices to demonstrate that adult cardiomyocytes with fully assembled sarcomeres can partially disassemble their sarcomeres in a calpain- and proteasome-dependent manner to progress through nuclear division and cytokinesis. In conclusion, we have developed a cardiac slice culture system, which allows imaging of native cardiomyocyte dynamics in real time to discover cellular mechanisms during heart regeneration.
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Project description:Type I collagen plays a pivotal role in shaping bone morphology and determining its physical properties by serving as a template for ossification. Nevertheless, the mechanisms underlying bone collagen formation, particularly the principles governing its orientation, remain unknown owing to the lack of a method that enables continuous in vivo observations. To address this challenge, we constructed a method to visualize bone collagen by tagging with green fluorescent protein (GFP) in zebrafish and observed the interactions between osteoblasts and collagen fibers during bone formation in vivo. When collagen type I alpha 2 chain (Col1a2)-GFP was expressed under the control of the osteoblast-specific promoters osx or osc in zebrafish, bone collagen was observed clearly enough to identify its localization, whereas collagen from other organs was not. Therefore, we determined that this method was of sufficient quality for the detailed in vivo observation of bone collagen. Next, bone collagen in the scales, fin rays, and opercular bones of zebrafish was observed in detail, when bone formation is more active. High-magnification imaging showed that Col1a2-GFP can visualize collagen sufficiently to analyze the collagen fiber orientation and microstructure of bones. Furthermore, by simultaneously observation of bone collagen and osteoblasts, we successfully observed dynamic changes in the morphology and position of osteoblasts from the early stages of bone formation. It was also found that the localization pattern and orientation of bone collagen significantly differed depending on the choice of the expression promoter. Both promoters (osx and osc) used in this study are osteoblast-specific, but their Col1a2-GFP localizing regions within the bone were exclusive, with osx region localizing mainly to the outer edge of the bone and osc region localizing to the central area of the bone. This suggests the existence of distinct osteoblast subpopulations with different gene expression profiles, each of which may play a unique role in osteogenesis. These findings would contribute to a better understanding of the mechanisms governing bone collagen formation by osteoblasts.
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Project description:Whether mature oligodendrocytes (mOLs) participate in remyelination has been disputed for several decades. Recently, some studies have shown that mOLs participate in remyelination by producing new sheaths. However, whether mOLs can produce new oligodendrocytes by asymmetric division has not been proven. Zebrafish is a perfect model to research remyelination compared to other species. In this study, optic nerve crushing did not induce local mOLs death. After optic nerve transplantation from olig2:eGFP fish to AB/WT fish, olig2+ cells from the donor settled and rewrapped axons in the recipient. After identifying these rewrapping olig2+ cells as mOLs at 3 months posttransplantation, in vivo imaging showed that olig2+ cells proliferated. Additionally, in vivo imaging of new olig2+ cell division from mOLs was also captured within the retina. Finally, fine visual function was renewed after the remyelination program was completed. In conclusion, our in vivo imaging results showed that new olig2+ cells were born from mOLs by asymmetric division in adult zebrafish, which highlights the role of mOLs in the progression of remyelination in the mammalian CNS.
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Project description:Zebrafish embryos provide a unique opportunity to visualize complex biological processes, yet conventional imaging modalities are unable to access intricate biomolecular information without compromising the integrity of the embryos. Here, we report the use of confocal Raman spectroscopic imaging for the visualization and multivariate analysis of biomolecular information extracted from unlabeled zebrafish embryos. We outline broad applications of this method in: (i) visualizing the biomolecular distribution of whole embryos in three dimensions, (ii) resolving anatomical features at subcellular spatial resolution, (iii) biomolecular profiling and discrimination of wild type and ?RD1 mutant Mycobacterium marinum strains in a zebrafish embryo model of tuberculosis and (iv) in vivo temporal monitoring of the wound response in living zebrafish embryos. Overall, this study demonstrates the application of confocal Raman spectroscopic imaging for the comparative bimolecular analysis of fully intact and living zebrafish embryos.
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Project description:BackgroundThe zebrafish visual system is a good research model because the zebrafish retina is very similar to that of humans in terms of the morphologies and functions. Studies of the retina have been facilitated by improvements in imaging techniques. In vitro techniques such as immunohistochemistry and in vivo imaging using transgenic zebrafish have been proven useful for visualizing specific subtypes of retinal cells. In contrast, in vivo imaging using organic fluorescent molecules such as fluorescent sphingolipids allows non-invasive staining and visualization of retinal cells en masse. However, these fluorescent molecules also localize to the interstitial fluid and stain whole larvae.ResultsWe screened fluorescent coumarin derivatives that might preferentially stain neuronal cells including retinal cells. We identified four coumarin derivatives that could be used for in vivo imaging of zebrafish retinal cells. The retinas of living zebrafish could be stained by simply immersing larvae in water containing 1?g/ml of a coumarin derivative for 30 min. By using confocal laser scanning microscopy, the lamination of the zebrafish retina was clearly visualized. Using these coumarin derivatives, we were able to assess the development of the zebrafish retina and the morphological abnormalities induced by genetic or chemical interventions. The coumarin derivatives were also suitable for counter-staining of transgenic zebrafish expressing fluorescent proteins in specific subtypes of retinal cells.ConclusionsThe coumarin derivatives identified in this study can stain zebrafish retinal cells in a relatively short time and at low concentrations, making them suitable for in vivo imaging of the zebrafish retina. Therefore, they will be useful tools in genetic and chemical screenings using zebrafish to identify genes and chemicals that may have crucial functions in the retina.
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