There are numerous reasons to model a disease in a fish, including the rapid, five-day development of zebrafish and the ability to image whole organs in vivo and perform time-lapse analysis. Using in vivo time-lapse confocal microscopy, we show that zebrafish OPCs continuously extend and retract numerous filopodium-like processes as they migrate and settle into their final positions. The mechanism by which animal markings are formed is an intriguing problem that has remained unsolved for a long time. xڭ�]o�0F��+���"�? In vivo, high‐resolution, time‐lapse imaging characterized lens development in the zebrafish from 16 to 96 hr postfertilization (hpf). ... Washington University in St. Louis is now home to one of the largest zebrafish facilities in the world. Time-lapse imaging was performed using a Leica TCS SP8 upright microscope with a Leica HCX IRAPO L ×25/0.95 water-dipping objective and heating chamber, or on an upright 3i spinning-disc confocal using a Zeiss Plan-Apochromat 20×, 40× or 63×/1.0 NA water-dipping objective. By this time, the basic vertebrate body plan is recognizable and several organ systems are … We demonstrate the utility of vascular-specific transgenic zebrafish in conjunction with time-lapse multiphoton laser scanning microscopy by directly observing angiogenesis within … stream Time lapse imaging was conducted for a period of 14–17 h with the time interval between images set at 3 min. ( Send Message ) on 10-12-2007. Featured. Multiple embryo time-lapse imaging of zebrafish development. �i�����)�ڤ�ⴞ N�d뿯 �ҒL�Q��>>/����S�B�e)c*E�()��Z�� �c��WSfծ���3#)��%"��/���*�K�Ӌ��h��M��)��n̅+]m+�ֶ�dTH�5Ov�G�T*�q�d���'��y� �X#�[xǩE���9��x����[p�w�4�^)�4 Q[%Az�1a@zζ However, our time-lapse imaging indicates that, in zebrafish, microglial precursors appear to enter the optic tectum directly from the periphery . x����Z$�v��,���i�� �j��Yp�nh�� ���Rȳ�f?��P�F ���+X�!��u��N����*P������!+32"2��U ��c�^/�M����p���'߻7�Q��C�勑l�����:��O6��|I2]n�j直�=. Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Contrary to proposed models for vertebrate asymmetric divisions, no apico-basal cell divisions take place in the zebrafish retina during the generation of postmitotic neurons. endobj The most critical step for the mounting method is to identify the optimal concentration of agarose that will allow for unrestricted zebrafish embryo growth, and at the same time keep the embryos in a completely fixed position for confocal imaging. Delamination of the surface ectoderm resulted in the formation of the lens mass, which progressed to a solid sphere of cells separating from the developing cornea at approximately 24 hpf. 2 0 obj 2 Imaging Station - Formative Evaluation Zebrafish Development Time-Lapse Video - Holding Time Joyce Ma August 2002 PURPOSE To determine how long visitors stay to watch the time-lapse video of Zebrafish development To determine if visitors stay to watch the entire video To determine if there is any difference in holding time between the annotated and the non- This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. The mounting is performed in layers of agarose at different concentrations. This process have these following stepys. The video was captured for the first time by a new microscope imaging technique part-funded by the BHF. The acquired z stacks for both single time point and time lapse images were selected to encompass all regions of interest within the zebrafish larval brain. A variety of different transgenic zebrafish lines have been generated expressing green fluorescent protein (GFP) or other fluorescent proteins in different organs and tissues, permitting dynamic visualization of development of these organs and tissues in living animals via time-lapse … Time-lapse movie of zebrafish lens epithelial cells expressing GFP-histones and mCherry-zGem as they progress through the cell cycle. This process have these following stepys. The Roslin Institute. Intravital time-lapse imaging is a powerful technique for investigating continuous developmental processes without missing crucial events. 1 cell, 2 cell, 4 cell, 8 cell, early blastula, mid-blastula, late blastula, gastrula stage, 24 hour, one day, two day, three day embryo, newly hatched three-day fish and adult formation. << /ProcSet [ /PDF /Text /ImageB /ImageC /ImageI ] /ColorSpace << /Cs1 Or press "Snap Photo" again to take another image! [0 0 612 792] >> One of the most important questions is whether the positional information for the pattern formation is derived from a covert prepattern or an autonomous mechanism. Research methodologies include the following: gene expression studies, gain or loss of gene function studies, cryopreservation of zebrafish lines, importation of new lines, and characterization of phenotypes by time-lapse photography. 3 0 obj Assessment of heart function via time‐lapse video microscopy in Zebrafish. Time-lapse of a 2 day old zebrafish embryo with green erythrocytes and red blood vessels. Time-lapse images of a zebrafish spinal neuron growth cone in culture loaded with a fluorescent calcium sensor. Here we reveal stem/precursor cell diversity during wound repair in larval zebrafish somitic body muscle using time-lapse 3D confocal microscopy on reporter lines. In this study, using the zebrafish as the model system, we attempted to answer this classic question. The ventricular or apical surface is adjacent to the retinal pigment epithelium (pe), and the basal surface is adjacent to the lens (le). • 85% of dissociated cells expressed serotonin, indicating neuronal subtypes present. Swinburne IA, Mosaliganti KR, Green AA, Megason SG. 4x. The zebrafish system affords several significant advantages to these studies, including high-resolution in vivo time-lapse imaging and large-scale genetic and chemical screening. Methods Mol Biol. National Institutes of Health. Annotated zebrafish development timelapse. A mounting method for extended time-lapse confocal microscopy of whole zebrafish embryos is described here. • * Interoperability. Improved long-term imaging of embryos with genetically encoded α … Others. 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. In Vivo Time-Lapse Imaging of Zebrafish Embryonic Development -- Distel and Köster 2007 (16): pdb.prot4816 -- Cold Spring Harbor Protocols Reagent Amount to add 2.9 M NaCl 60 mL You need to login to download this video. 7 0 R >> /Font << /F1.0 8 0 R /F2.0 11 0 R >> /XObject << /Im1 5 0 R Check out this time-lapse 3D video of a zebrafish heart growing over a day! The transparency and rapid embryogenesis of zebrafish, together with fluorescent reporters, allow for the study of developmental processes with single-cell resolution using in vivo time-lapse imaging (Beis and Stainier, 2006). Unit or Division: Developmental Neurobiology Unit (Ichiro Masai) Free for anyone to re-use, but must be credited to OIST. This specimen, stained with Bodipy-FL-C 5-Ceramide, shows the outline of all cells in the retinal neuroepithelium (ne). Tubing carrying water is … For time-lapse imaging Tg(sox10:mRFP; olig2:GFP) larvae were used. At 56–57 hpf, larvae were anaesthetised in 600 μM tricaine and manually mounted in 1.5% low melting point agarose in embryo medium containing either SKP2-C25 (1% DMSO) or 1% DMSO and 600 μM tricaine. 4 0 obj Annotated zebrafish development timelapse is shown in this video. endobj An efficient method for isolating embryonic zebrafish neurons from neural tissue. In Vivo Time-Lapse Imaging of Zebrafish Embryonic Development -- Distel and Köster 2007 (16): pdb.prot4816 -- Cold Spring Harbor Protocols Reagent Amount to add 2.9 M NaCl 60 mL 1 cell, 2 cell, 4 cell, 8 cell, early blastula, mid-blastula, late blastula, gastrula stage, 24 hour, one day, two day, three day embryo, newly hatched three-day fish and adult formation. %PDF-1.3 Cryopreservation of transgenic and mutant zebrafish lines; Importation of existing zebrafish lines; Characterization of phenotypes by time-lapse photography Get started. Time-lapse imaging of zebrafish embryos is also described by Köster and Fraser (2004) and Distel et al. adult formation. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. Although methods have been devised for shortto medium-term time-lapse imaging of transgenic zebrafish, these methods are not suitable for longer term imaging because of poor control over temperature, evaporation, and anoxia. Extended time-lapse imaging of vascular, neuronal and muscle development After optimizing the mounting method described above, time lapse confocal microscopy images were captured over a span of 55 h. We imaged live transgenic zebrafish expressing GFP or RFP in different tissues. Uploaded by: We describe a new imaging chamber that provides continuously circulating flow of warm, oxygenated aqueous media. Neuron 37 , 597 - 609 . Annotated zebrafish development timelapse is shown in this video. 1 0 obj Observing the development of a zebrafish as a model organism could help students develop an understanding of the importance of cell division and specialization during the development of a multicellular organism. However, a surprising shift in the orientation of cell division from central-peripheral to … Single time point images were obtained at 0–4 days posttransplantation (dpt). Adaptive prospective optical gating enables day-long 3D time-lapse imaging of the beating embryonic zebrafish heart, … Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. Right click on the image below and then select "Save image as" option to save the image for your powerpoint presentations. Time-lapse images of a zebrafish spinal neuron growth cone in culture loaded with fluorescent Ca 2+ sensor Fluo-8H (pseudocolored according to scale in Figure 6A). We describe a new imaging chamber that provides continuously circulating flow of warm, oxygenated aqueous media. Zebrafish Core in the NICHD Annual Report Time-lapse imaging shows that Vsx1 is upregulated only in RPCs that downregulate Vsx2, consistent with the data in mice showing that Vsx2 represses vsx1 . Schematic diagram of the apparatus used for long-term time-lapse imaging of developing zebrafish. 2009;546:243–54. Zebrafish larvae were treated at 48 hpf with SKP2-C25 (1% DMSO). << /Type /Page /Parent 14 0 R /Resources 3 0 R /Contents 2 0 R /MediaBox In vivo time-lapse imaging of cell divisions during neurogenesis in the developing zebrafish retina. For serial time-lapse experiments (see below) we typically mount six fish at a time and try to limit imaging sessions to 1 hour per group. Zebrafish Embryogenesis Brightfield imaging Time-lapse Morphogenetic movements Population statistics Quantification Temperature control This is a … Somitogenesis in the zebrafish embryo is shown in Movie 1 . For Core Members This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. doi: 10.1016/S0896-6273(03)00066-7 OpenUrl CrossRef PubMed Web … In vivo time-lapse imaging of cell divisions during neurogenesis in the developing zebrafish retina. More information: Jonathan M. Taylor et al. Here, we report the … Between 15 and 20 min, the embryos were treated with SU5402 (0.2 μg/ml) or PD184352 (3.0 μM). Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function 1,2.To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. gastrula stage, 24 hour, one day, two day, three day embryo, newly hatched three-day fish and The timelapse movie shows a developing zebrafish embryo Tg(kdrl:GFP) from 24-96 hpf imaged using selective plane illumination microscopy (SPIM). Early in the time-lapse video, cells can be seen dividing with little change in their shape or appearance. Apparatus for long-term time-lapse imaging. Field of Research: Developmental Neuroscience. In vivo, high‐resolution, time‐lapse imaging characterized lens development in the zebrafish from 16 to 96 hr postfertilization (hpf). This protocol describes a method to visualize clones of progenitor cells and neurons in the developing zebrafish hindbrain and follow them in vivo using Brainbow and time-lapse confocal microscopy 11.The major advantage of this protocol in comparison to in vitro or ex vivo studies is the ability to directly observe the proliferative zone of the vertebrate brain in its natural milieu over time. To determine whether the Erk biosensor could be used to monitor Erk activity in zebrafish embryos, 8SS embryos carrying the Erk biosensor were observed for 60 or 75 min at 5 min intervals at 28.5 °C. *To avoid redundant efforts, please check whether a suitable zebrafish line is already available through existing databases. Although traditionally used for developmental biology, zebrafish has recently been used to investigate metabolic diseases. Because of the rapid embryogenesis, external development, and transparency of zebrafish embryos, their developmental processes can be visualized in time-lapse studies in the context of the living organism. Researchers from the universities of Glasgow and Edinburgh were able to record individual heart cells growing and dividing in … Materials and methods Time-lapse image recording reveals that nephrogenesis in control morpholino injected embryos is unaltered in comparison to uninjected ones (results not shown) and follows the described steps of early zebrafish kidney development 3. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. Collectively, these data demonstrate that the endothelium of the ventral wall of CA in the PBI is indeed capable of giving rise to a transient wave of T lymphopoiesis independent of HSCs via EHT. Here, we report the use of confocal Raman spectroscopic imaging for … While mounting other fish check back to make sure previous subjects maintain desired orientation until agarose solidifies (~3 min). Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. You need to login to download this video. 3, K–P; and Video 1). Time‐lapse imaging is often the only way to appreciate fully the many dynamic cell movements critical to neural development. Rapid embryonic development A major benefit of using the zebrafish as a model is its rapid early development. �AH�8V��?̬���ť٘ҭ*�l�W��h.t�&v���)���C�������1�vH���\�ݩ�s�09^�g�',�R$J�޻rn�7#�?���hىυ�,`��;���i������_���QV>�|`����^߬��/��Pqȉ.5dBeg)���(�j��}����8�BP�"�rX�O�������\����(��4�5��4 ��[;����e�5�9�f��7~�[b~�W��Oa� Uhr� High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). The upper part shows… This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. The Zebrafish Virtual Atlas and ... not time), an atlas of reference images indexed by anatomical structure and developmental age, annotated 3-D reconstructions and time lapse movies. (2006). A time-lapse 3D video of a zebrafish heart growing over a day has been captured for the first time by a new microscope imaging technique. The zebrafish is a favorite model organism to study tissue morphogenesis during development at a subcellular level. Enterprise . Get your team aligned with all the tools you need on one secure, reliable video platform. Annotated zebrafish development timelapse. Dr. Nils Lindstrom. In zebrafish, the lens placode appeared in the head ectoderm, similar to mammals. endobj A: Brightfield image of a 3‐dpf transparent Zebrafish embryo. Time-lapse FRET imaging. scitan In zebrafish, the lens placode appeared in the head ectoderm, similar to mammals. Neuron 37 , 597 - 609 . Indeed, time-lapse imaging showed that EHT occur in the ventral wall of CA from about 32 hpf until 72 hpf (Fig. In zebrafish, our high-resolution time-lapse imaging allowed us to definitively determine that endoderm-derived cells of Seessel’s pouch detach … Summary. To support this observation, we carried out time-lapse imaging analysis with Tg(kdrl:eGFP;coro1a:DsRedx) embryos in which the brain vessels and microglia are marked by GFP and DsRedx, respectively. Edinburgh, Scotland . Two-photon excitation microscopy was used to reconstruct cell divisions in living zebrafish embryonic retinas. endstream Boxes designate the ventricle and dorsal aorta regions. 490 Annotated zebrafish development timelapse. Time-Lapse Video of Zebrafish “Inner Ear” Development Wins Small World in Motion Competition Posted On May 5, 2015 Nikon Instruments Inc. recently announced the winners of the fourth annual Nikon Small World in Motion Photomicrography Competition. B: M‐mode imaging through short axis of the ventricle. Zebrafish Embryogenesis Brightfield imaging Time-lapse Morphogenetic movements Population statistics Quantification Temperature control This is a … Compound transgenic fish … • Primary cells can survive 9 days in vitro, expressing markers for neurons and glia. << /Length 4 0 R /Filter /FlateDecode >> For example, the time lapse movie to the right depicts the first 18 hours of development. Confocal. Zebrafish possess many advantages that make them the best vertebrate model organism for live imaging of dynamic development events. The optical transparency of its embryos permits time lapse live imaging (Hall et al., 2009; Herrgen et al., 2009; Feierstein et al., 2015). Zebrafish Development Time-Lapse Video - Holding Time Joyce Ma August 2002 PURPOSE To determine how long visitors stay to watch the time-lapse video of Zebrafish development To determine if visitors stay to watch the entire video To determine if there is any difference in holding time between the annotated and the non- 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. This process have these following stepys. Denne video handler om Timelapse zebrafish. Process remodeling and migration paths are highly variable and seem to be influenced by contact with neighboring OPCs. Bethesda, Maryland, USA. Dr. Jeremy Logue. Figure 2c shows three timepoints of a single time-lapse recording with 34 positions, 2 stitched FOVs, 2 channels (transgenes cldnb:GFP and cxcr4b:H2B-RFP), imaged with a 10 min. Annotated zebrafish development timelapse is shown in this video. Developing mouse embryonic kidneys . 5 0 obj We analysed time-lapse movies of chevron formation during zebrafish development at 28°C, which were acquired in our previous studies (Herrgen et al., … Article Google Scholar 23. %��������� Although methods have been devised for shortto medium-term time-lapse imaging of transgenic zebrafish, these methods are not suitable for longer term imaging because of poor control over temperature, evaporation, and anoxia. doi: 10.1016/S0896-6273(03)00066-7 OpenUrl CrossRef PubMed Web of Science At time = 0∶00 min, BDNF is applied in the bath and initiates a Ca 2+ signal in the growth cone by 5 min. 1 cell, 2 cell, 4 cell, 8 cell, early blastula, mid-blastula, late blastula, Time-Lapse Analysis of Cell Division in the Zebrafish Embryonic Retina. 100 /ColorSpace 7 0 R /BitsPerComponent 8 /Filter /FlateDecode >> This largely results from the fact that zebrafish embryos are transparent and thus accessible to various imaging techniques, such as confocal and two-photon excitation (2PE) microscopy. • Cell migration was studied using time-lapse microscopy of glial cells and neurons. Time-lapse movie of zebrafish lens epithelial cells expressing GFP-histones and mCherry-zGem as they progress through the cell cycle. Fluorescence. /Im2 9 0 R /Im3 12 0 R >> >> Skeletal muscle with incision wounds rapidly regenerates both slow and fast muscle fibre types. Still image of the spinal cord from time lapse imaging of a 46 hours post-fertilization embryo harboring a transgenic reporter marking sox10+ cells. Using in vivo lineage tracing by time-lapse confocal microscopy combined with morpholino oligonucleotide-mediated gene inactivation, we report the function of FGF3 during zebrafish POMC cell ontogeny, defining a genetic and developmental boundary between two distinct pituitary POMC lineages in a gene-dose-dependent fashion. endobj login or signup, Channels: << /Length 6 0 R /Type /XObject /Subtype /Image /Width 1800 /Height stream interval over a period of 13 h (Additional file 1: Movie S1). ゼブラフィッシュの上皮細胞が、細胞周期の進行に伴いGFP-histoneとmCherry-zGemを発現する様子を示したタイムラプス動画。 The Aquatic Zebrafish Core provides services using the small vertebrate zebrafish as a model for human disease and to study gene function. Heterogeneity of stem cells or their niches is likely to influence tissue regeneration. Hr postfertilization ( hpf ) all cells in the developing zebrafish retina from about 32 until... Characterization of phenotypes by time-lapse photography Get started 32 hpf until 72 hpf ( Fig technique by! 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The largest zebrafish facilities in the developing zebrafish retina promoter-driven colors that restrict the motility the. Developmental Neurobiology unit ( Ichiro Masai ) Free for anyone to re-use, but must credited. • cell migration was studied using time-lapse 3D video of a zebrafish spinal neuron growth cone culture! S1 ) embryonic zebrafish neurons from neural tissue rapidly regenerates both slow and fast muscle fibre types living. Formed is an intriguing problem that has remained unsolved for a period of 14–17 h the! Example, the time lapse Movie to the right depicts the first 18 hours of development,! Muscle fibre types 0–4 days posttransplantation ( dpt ) best vertebrate model organism study!