<meta charset="utf8"></head><body>阐明斑马鱼视盖发育中的小胶质细胞行为

<ol><li>Wolf, S. A., Boddeke, H. W., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+in+physiology+and+disease%5BTitle%5D)+AND+%22Annu+Rev+Physiol%22%5BJournal%5D)">Microglia in physiology and disease.</a> Annu Rev Physiol. 79, 619-643 (2017).</li>
<li>Nimmerjahn, A., Kirchhoff, F., Helmchen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Resting+microglial+cells+are+highly+dynamic+surveillants+of+brain+parenchyma+in+vivo%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo.</a> Sci Rep. 308 (5726), 1314-1318 (2005).</li>
<li>Peri, F., Nusslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+imaging+of+neuronal+degradation+by+microglia+reveals+a+role+for+v0-ATPase+a1+in+phagosomal+fusion+in+vivo%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo.</a> Cell. 133 (5), 916-927 (2008).</li>
<li>Marın-Teva, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+promote+the+death+of+developing+Purkinje+cells%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Microglia promote the death of developing Purkinje cells.</a> Neuron. 41 (4), 535-547 (2004).</li>
<li>Paolicelli, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptic+pruning+by+microglia+is+necessary+for+normal+brain+development%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Synaptic pruning by microglia is necessary for normal brain development.</a> Science. 333 (6048), 1456-1458 (2011).</li>
<li>Hattori, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+microglia-blood+vessel+interactions+in+the+developing+brain%5BTitle%5D)+AND+%22Neurosci+Res%22%5BJournal%5D)">The microglia-blood vessel interactions in the developing brain.</a> Neurosci Res. 187, 58-66 (2023).</li>
<li>Colonna, M., Butovsky, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+function+in+the+central+nervous+system+during+health+and+neurodegeneration%5BTitle%5D)+AND+%22Annu+Rev+Immunol%22%5BJournal%5D)">Microglia function in the central nervous system during health and neurodegeneration.</a> Annu Rev Immunol. 35, 441-468 (2017).</li>
<li>Xu, H. -T., Pan, F., Yang, G., Gan, W. -B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Choice+of+cranial+window+type+for+in+vivo+imaging+affects+dendritic+spine+turnover+in+the+cortex%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Choice of cranial window type for in vivo imaging affects dendritic spine turnover in the cortex.</a> Nat Neurosci. 10 (5), 549-551 (2007).</li>
<li>Shin, J., Park, H. -C., Topczewska, J. M., Mawdsley, D. J., Appel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+cell+fate+analysis+in+zebrafish+using+olig2+BAC+transgenics%5BTitle%5D)+AND+%22Methods+Cell+Sci%22%5BJournal%5D)">Neural cell fate analysis in zebrafish using olig2 BAC transgenics.</a> Methods Cell Sci. 25 (1), 7-14 (2003).</li>
<li>Chi, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Foxn4+directly+regulates+tbx2b+expression+and+atrioventricular+canal+formation%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">Foxn4 directly regulates tbx2b expression and atrioventricular canal formation.</a> Genes Dev. 22 (6), 734-739 (2008).</li>
<li>Avdesh, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regular+care+and+maintenance+of+a+zebrafish+%28Danio+rerio%29+laboratory%3A+An+introduction%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: An introduction.</a> J Vis Exp. (69), e4196(2012).</li>
<li>Bohnsack, B. L., Gallina, D., Kahana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phenothiourea+sensitizes+zebrafish+cranial+neural+crest+and+extraocular+muscle+development+to+changes+in+retinoic+acid+and+IGF+signaling%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Phenothiourea sensitizes zebrafish cranial neural crest and extraocular muscle development to changes in retinoic acid and IGF signaling.</a> PLoS ONE. 6 (8), e22991(2011).</li>
<li>Ariga, J., Walker, S. L., Mumm, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multicolor+time-lapse+imaging+of+transgenic+zebrafish%3A+visualizing+retinal+stem+cells+activated+by+targeted+neuronal+cell+ablation%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Multicolor time-lapse imaging of transgenic zebrafish: visualizing retinal stem cells activated by targeted neuronal cell ablation.</a> J Vis Exp. (43), e2093(2010).</li>
<li>Ferrero, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryonic+microglia+derive+from+primitive+macrophages+and+are+replaced+by+cmyb-dependent+definitive+microglia+in+zebrafish%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Embryonic microglia derive from primitive macrophages and are replaced by cmyb-dependent definitive microglia in zebrafish.</a> Cell Rep. 24 (1), 130-141 (2018).</li>
<li>Xu, J., Wang, T., Wu, Y., Jin, W., Wen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+colonization+of+developing+zebrafish+midbrain+is+promoted+by+apoptotic+neuron+and+lysophosphatidylcholine%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Microglia colonization of developing zebrafish midbrain is promoted by apoptotic neuron and lysophosphatidylcholine.</a> Dev Cell. 38 (2), 214-222 (2016).</li>
<li>Svahn, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+ramified+microglia+from+early+macrophages+in+the+zebrafish+optic+tectum%5BTitle%5D)+AND+%22Dev+Neurobiol%22%5BJournal%5D)">Development of ramified microglia from early macrophages in the zebrafish optic tectum.</a> Dev Neurobiol. 73 (1), 60-71 (2013).</li>
<li>Thion, M. S., Garel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglial+ontogeny%2C+diversity+and+neurodevelopmental+functions%5BTitle%5D)+AND+%22Curr+Opin+Genet+Dev%22%5BJournal%5D)">Microglial ontogeny, diversity and neurodevelopmental functions.</a> Curr Opin Genet Dev. 65, 186-194 (2020).</li>
<li>Bohlen, C. J., Friedman, B. A., Dejanovic, B., Sheng, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+in+brain+development%2C+homeostasis%2C+and+neurodegeneration%5BTitle%5D)+AND+%22Annu+Rev+Genet%22%5BJournal%5D)">Microglia in brain development, homeostasis, and neurodegeneration.</a> Annu Rev Genet. 53, 263-288 (2019).</li>
<li>Squarzoni, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+modulate+wiring+of+the+embryonic+forebrain%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Microglia modulate wiring of the embryonic forebrain.</a> Cell Rep. 8 (5), 1271-1279 (2014).</li>
<li>Schafer, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+sculpt+postnatal+neural+circuits+in+an+activity+and+complement-dependent%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Microglia sculpt postnatal neural circuits in an activity and complement-dependent.</a> Neuron. 74 (4), 691-705 (2012).</li>
<li>Parkhurst, C. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+promote+learning-dependent+synapse+formation+through+brain-derived+neurotrophic+factor%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor.</a> Cell. 155 (7), 1596-1609 (2013).</li>
<li>Green, L. A., O'Dea, M. R., Hoover, C. A., DeSantis, D. F., Smith, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+embryonic+zebrafish+brain+is+seeded+by+a+lymphatic-dependent+population+of+mrc1%2B+microglia+precursors%5BTitle%5D)+AND+%22Nat.+Neurosci%22%5BJournal%5D)">The embryonic zebrafish brain is seeded by a lymphatic-dependent population of mrc1+ microglia precursors.</a> Nat. Neurosci. 25 (7), 849-864 (2022).</li>
<li>Hughes, A. N., Appel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+phagocytose+myelin+sheaths+to+modify+developmental+myelination%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Microglia phagocytose myelin sheaths to modify developmental myelination.</a> Nat Neurosci. 23 (9), 1055-1066 (2020).</li>
<li>Yu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((C1q+is+essential+for+myelination+in+the+central+nervous+system+%28CNS%29%5BTitle%5D)+AND+%22iScience%22%5BJournal%5D)">C1q is essential for myelination in the central nervous system (CNS).</a> iScience. 26 (12), 108518(2023).</li>
<li>Zengeler, K. E., Lukens, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Innate+immunity+at+the+crossroads+of+healthy+brain+maturation+and+neurodevelopmental+disorders%5BTitle%5D)+AND+%22Nat+Rev+Immunol%22%5BJournal%5D)">Innate immunity at the crossroads of healthy brain maturation and neurodevelopmental disorders.</a> Nat Rev Immunol. 21 (7), 454-468 (2021).</li>
<li>Paolicelli, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+states+and+nomenclature%3A+A+field+at+its+crossroads%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Microglia states and nomenclature: A field at its crossroads.</a> Neuron. 110 (21), 3458-3483 (2022).</li>
<li>Réu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+lifespan+and+turnover+of+microglia+in+the+human+brain%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">The lifespan and turnover of microglia in the human brain.</a> Cell Rep. 20 (4), 779-784 (2017).</li>
<li>Ajami, B., Bennett, J. L., Krieger, C., Tetzlaff, W., Rossi, F. M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Local+self-renewal+can+sustain+CNS+microglia+maintenance+and+function+throughout+adult+life%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.</a> Nat Neurosci. 10 (12), 1538-1543 (2007).</li>
<li>Mildner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+in+the+adult+brain+arise+from+Ly-6ChiCCR2%2B+monocytes+only+under+defined+host+conditions%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Microglia in the adult brain arise from Ly-6ChiCCR2+ monocytes only under defined host conditions.</a> Nat Neurosci. 10 (12), 1544-1553 (2007).</li>
<li>Cook, Z. T., Brockway, N. L., Weissman, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+the+developing+brain+in+living+zebrafish+using+Brainbow+and+time-lapse+confocal+imaging%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Visualizing the developing brain in living zebrafish using Brainbow and time-lapse confocal imaging.</a> J Vis Exp. (157), e60593(2020).</li>
<li>Tayanloo-Beik, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+modeling+of+autism+spectrum+disorders%2C+current+status+and+future+prospective%5BTitle%5D)+AND+%22Front+Psychiatry%22%5BJournal%5D)">Zebrafish modeling of autism spectrum disorders, current status and future prospective.</a> Front Psychiatry. 13, 911770(2022).</li>
<li>D'Amora, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+as+an+innovative+tool+for+epilepsy+modeling%3A+State+of+the+art+and+potential+future+directions%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Zebrafish as an innovative tool for epilepsy modeling: State of the art and potential future directions.</a> Int J Mol Sci. 24 (9), 7702(2023).</li>
<li>Langova, V., Vales, K., Horacek, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+zebrafish+and+laboratory+rodents+in+schizophrenia+research%5BTitle%5D)+AND+%22Front+Psychiatry%22%5BJournal%5D)">The role of zebrafish and laboratory rodents in schizophrenia research.</a> Front Psychiatry. 11, 549232(2020).</li>
<li>Chia, K., Klingseisen, A., Sieger, D., Priller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+as+a+model+organism+for+neurodegenerative+disease%5BTitle%5D)+AND+%22Front+Mol+Neurosci%22%5BJournal%5D)">Zebrafish as a model organism for neurodegenerative disease.</a> Front Mol Neurosci. 15, 940484(2022).</li>
<li>Astell, K. R., Sieger, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+in+vivo+models+of+cancer+and+metastasis%5BTitle%5D)+AND+%22Cold+Spring+Harb+Perspect+Med%22%5BJournal%5D)">Zebrafish in vivo models of cancer and metastasis.</a> Cold Spring Harb Perspect Med. 10 (8), 037077(2020).</li>
</ol>

<table><tbody><tr><td>1 L Breeding tanks</td><td>Tecniplast</td><td>ZB10BTE</td><td></td></tr><tr><td>1-phenyl-2-thiourea (PTU)</td><td>Sigma-Aldrich</td><td>P7629</td><td>Diluted to 0.2 mM in E3 to prevent embryo pigmentation</td></tr><tr><td>Bottom glass imaging dish</td><td>FluoroDish</td><td>FD3510-100</td><td></td></tr><tr><td>Disposable Graduated transfer pipette</td><td>avantor</td><td>16001-188</td><td></td></tr><tr><td>Dry block heater</td><td>Novolab</td><td>Grant QBD4</td><td>To keep low melting agarose at 37 &amp;deg;C</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td><td>Sigma-Aldrich</td><td>E10521-50G</td><td></td></tr><tr><td>Imaris 10.0</td><td>Oxford Instruments</td><td></td><td>analysis software</td></tr><tr><td>Imaris File Converter</td><td>Oxford Instruments</td><td></td><td>https://imaris.oxinst.com/big-data</td></tr><tr><td>Laser-scanning confocal microscope</td><td>Nikon</td><td>Eclipse Ti2-E</td><td></td></tr><tr><td>Methylene blue</td><td>Sigma-Aldrich</td><td>M9140-25G</td><td></td></tr><tr><td>microloader tips</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>NuSieve GTG Agarose</td><td>Lonza</td><td>&amp;nbsp;50081</td><td></td></tr><tr><td>Petri dishes (90 mm)</td><td>avantor</td><td>391-0559</td><td></td></tr><tr><td>Pronase</td><td>Sigma-Aldrich</td><td>11459643001</td><td></td></tr><tr><td>&amp;nbsp;Stainless Steel Forceps Dumont No. 5</td><td>FineScienceTools</td><td>11254-20</td><td></td></tr><tr><td>Stereo microscope</td><td>Leica</td><td>Leica M80</td><td>To mount the embryos</td></tr><tr><td>teasing needle</td><td>avantor</td><td>76549-024</td><td></td></tr></tbody></table>

live imaging and characterization of microglia dynamics in the zebrafish embryo

Read this Scientific Article Protocol about Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo at JoVE.com

<meta charset="utf8"></head><body>作者聚焦:使用 Zebrafish 探索大脑发育过程中的小胶质细胞迁移

斑马鱼胚胎中小胶质细胞动力学的实时成像和表征

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and ...

live-imaging-characterization-microglia-dynamics-zebrafish

Research

JoVE Journal

Immunology and Infection

Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo

1.2K 次观看.  Université Libre de Bruxelles (ULB). 我的研究项目侧重于表征斑马鱼胚胎发育第一周的小胶质细胞动力学、形态和功能。小胶质细胞是高度动态的细胞，我们相信，对其行为模式和形态变化的更深入描述可以让我们对它们在发育中的大脑中的成熟过程和活动有一个有趣的见解。此类成像的主要挑战在于以肉眼质量和出色的临时分辨率捕获多个通道。然而，共聚焦技术的进步现在使...

视频: 作者聚焦:使用 Zebrafish 探索大脑发育过程中的小胶质细胞迁移

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

在斑马鱼后脑细胞标记和成像

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

科研

神经科学

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

发育生物学

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

多光子时间推移成像实时可视化发展: 迁移神经嵴细胞在斑马鱼胚胎中的可视化

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

斑马鱼幼虫显微注射和成像的微装置

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

生物学

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

多色成像定时转基因斑马鱼:可视化由针对性的神经细胞消融激活视网膜干细胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Long-term Live Imaging Device for Improved Experimental Manipulation of Zebrafish Larvae

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for live high-resolution microscopic imaging of dynamic biological phenomena in space and time with cellular resolution. However, the traditional method of agarose encapsulation for live imaging can impede larval development and tissue regrowth. Therefore, this manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which was designed and fabricated as a functionally compartmentalized device to orient larvae for high-resolution microscopy while permitting caudal fin transection within the device and subsequent unrestrained tail development and re-growth. This device allows for wounding and long-term imaging while maintaining viability. Given that the zWEDGI mold is 3D printed, the customizability of its geometries make it easily modified for diverse zebrafish imaging applications. Furthermore, the zWEDGI offers numerous benefits, such as access to the larva during experimentation for wounding or for the application of reagents, paralleled orientation of multiple larvae for streamlined imaging, and reusability of the device.

This manuscript describes the zWEDGI (zebrafish Wounding and Entrapment Device for Growth and Imaging), which is a compartmentalized device designed to orient and restrain zebrafish larvae. The design permits tail transection and long-term collection of high-resolution fluorescent microscopy images of wound healing and regeneration.

改善斑马鱼幼虫实验操作的长期活体成像装置

The zebrafish larva is an important model organism for both developmental biology and wound healing. Further, the zebrafish larva is a valuable system for ...

生物工程

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

斑马鱼光学杯形态形成的四维成像

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

在体内成像完全活跃的脑组织在清醒斑马鱼幼虫和青少年由骷髅和皮肤去除

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...