Name: in vivo imaging of transgenic gene expression in individual retinal progenitors in chimeric zebrafish embryos to study cell nonautonomous influences
Uploaded: 2017-03-22T15:00:00.000+00:00
Duration: 10 min 36 s
Description: Watch this Scientific Journal Video about In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences at JoVE.c

这项工作是由ARC DECRA到PRJ（DE120101311）和德意志研究联合会（DFG）的研究经费以LP（PO 1440 / 1-1）的支持。澳大利亚再生医学研究所是由维多利亚州政府和澳大利亚联邦政府的资金支持。我们承认杰里米·伍枝记医生，谁进行公布在箕等人在这里描述的实验。 ，2016年我们是从Higashijima教授转基因鱼的规定，感激和感谢PROFS。特纳和鲁普为提供PCS2 +质粒和威尔金森博士生成H2B-RFP和H2A-GFP构造。我们感谢FishCore设施的工作人员（莫纳什大学）采取了动物的照顾。

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The authors have nothing to disclose.

理解的程度，邻近细胞影响的关键的细胞命运决定因子的表达的定时旨在有效地指示胚胎或诱导的多能干细胞分化成特定的有丝分裂后的细胞类型或甚至图案化的组织时是必不可少的。此外，检查在活的动物的显影细胞中的这些分子事件还提供相关的动态上与体内这些事件相关的特定蜂窝上下文（时间和空间）的信息。这种研究不能在所有脊椎动物模型可以轻松实现。 <p class="jove_content"...

<html>可视化在体内脊椎动物重要的发育过程的能力已经到使斑马鱼的关键模型为研究正常和疾病条件作出了贡献（在1综述，2）。具体地，神经视网膜是中枢神经系统的访问的一部分。视网膜适合于容易地进行神经发生的研究，由于其高度的组织，但结构较简单，并在脊椎动物物种3其高度保守的神经类型。细胞行为，如增殖，细胞周期出口，非对称的细胞分裂，命运规范，分化和神经电路形成的动力学可以遵循整个retinogenesis的整个过程中，这是在斑马鱼的中心视网膜由3天受精后完成（ dpf）的4，5，REF"&gt; 6，7。 另外，在每个上述的阶段的不同基因的功能要求，可伴随地评估在斑马鱼视网膜，用其他脊椎动物模型中，表型从基因敲除技术的应用导致提供的优点只能在验尸检查评估固定的组织。特别是，使用的转基因品系中，我们可以可视化和监控荧光蛋白如在视网膜报告基因的表达，使我们能够获得underlies一个特定的神经元细胞类型的起源的基因表达的时间分辨率。由于斑马鱼的迅速发展，这些事件可以在整个发育期间被可视化，从而提供更深入了解的基因表达相对于神经元细胞身份采集和细胞行为的时间的重要性。 最后，这些方法可有效地在通过移植的产生嵌合体的斑马鱼相结合，导致分析上市基因功能的两个关键方面。首先，检查从捐赠者的胚胎，其中一个特定基因被撞倒，而他们在未标记的野生型环境中开发移植的细胞，使我们能够获得有关基因功能的细胞自主的方式相关的信息。这导致了关于它们在此。正常表达是由祖细胞，可以不再从这些基因4,6产生功能性蛋白质的发育命运结果的检查例举的祖细胞内视网膜命运决定因素的功能是重要的见解， 8,9。利用这种方法，我们已经表明，很多命运的决定性因素（ 例如，VSX1，Atoh7，Ptf1a，Barhl2）细胞的作用-autonomously推动特定视网膜神经命运;缺乏的基因表达的主要导致命运开关，使得与基因拦截的细胞保持通过采用一个备用细胞命运4，6，7，10是可行的。其次，这种嵌合的实验可用于评估时不同遗传环境中开发类型的祖细胞如何野生表现。例如，通过比较野生型细胞的发展通常表达在一WT兴趣（和报告基因）与操纵宿主环境（ 例如，基因敲除/击倒）的基因，对基因表达和细胞命运所得效果可以评估。缺乏在主机环境中的某些神经类型的，例如，已被证明在细胞非自主的方式来影响野生型祖行为，以偏压它们朝向分化成的代表性不足Ò④短缺的神经类型4，7，11，12。鉴于视网膜神经元出生在通过特定的神经元的命运决定簇的基因的顺序定时表达式（命运的基因表达）（以13中综述）保守histogenic顺序，我们使用这些方法，以说明如何命运的基因表达在野生型祖细胞的定时当这种祖细胞在诱发异常细胞组合物视网膜主机环境发展会受到影响。这里，我们概述这些方法作为证据的相对标准和广泛使用的技术的组合如何能够命运的基因表达的时机在显影视网膜祖8,9的检查。 这个协议描述了一种实验方法组合延时成像用的放心的每形成在离体显影斑马鱼胚胎移植到跟随个别mosaically标记细胞整个发育retinogenesis的整个期间。通过执行功能基因操作或在宿主胚胎，供体胚胎，两者或两者都不是，人们可以评估基因功能的细胞自主性。这种方法可广泛适用于在任何其它系统的量这里概述的各个部件都是合适的类似研究问题。

<table><tbody><tr><td>Agarose</td><td>Bioline</td><td>BIO-41025</td><td>Agarose coated dishes can be prepared a few days prior, sealed with parafilm to prevent drying and stored at 4 &amp;#176;C</td></tr><tr><td>Agarose low melt</td><td>Sigma</td><td>A9414-100G</td><td>Can be prepared in larger volume, microwaved to liquify and then kept molten in a 40 &amp;#176;C water-bath. 1 mL aliquots can be prepared (one for each group of embryos mounted).</td></tr><tr><td>borosillicate glass capillary tube w/o filament</td><td>SDR Scientfic</td><td>30-0035</td><td>alternatives with similar diameter can be used&amp;nbsp;</td></tr><tr><td>borosillicate glass capillary tube with filament</td><td>SDR Scientfic</td><td>30-0038</td><td>alternatives with similar diameter can be used&amp;nbsp;</td></tr><tr><td>glass petri dishes (60 mm diameter)</td><td>Science Supply</td><td>1070506</td><td>any glass alternative of any size</td></tr><tr><td>Injection tube (2 m)</td><td>Eppendorf</td><td>524616004</td><td>This connects the needle holder to the syringe during transplantation</td></tr><tr><td>Microinjection mold (plastic mold with wedge-shaped protrusions)</td><td>Adaptive Science Tools</td><td>PT-1</td><td>alternatives with similar shape can be use</td></tr><tr><td>microneedle holder</td><td>Narishige</td><td>M-152</td><td>any alternative that fits the outside diameter of the injection needles can be used</td></tr><tr><td>microinjector</td><td>Narishige or Eppendorf Femtojet</td><td></td><td>Pneumatic injector using gas pressure</td></tr><tr><td>micromanipulator</td><td>Coherent Scientific</td><td>M330IR</td><td>similar alternatives can be used</td></tr><tr><td>microloader tip</td><td>Eppendorf</td><td>5242956003</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma</td><td>M5904-500ML</td><td></td></tr><tr><td>needle puller</td><td>Sutter Instruments</td><td>Model P-2000</td><td>Settings used for this puller are: H 430, Fil 4, Vel 50, Del 225, Pul 75. Any needle puller can be used if it results in appropriate tip dimension</td></tr><tr><td>Parafilm</td><td>Interpath</td><td>PM996</td><td>any paraffin film</td></tr><tr><td>Pasteur pipette plastic</td><td>Samco Scientific</td><td>202</td><td></td></tr><tr><td>Pasteur pipette glass</td><td>Hirschmann Laborgeraete</td><td>9260101</td><td></td></tr><tr><td>Pronase</td><td>Sigma</td><td>P5942-25MG</td><td>&amp;nbsp;Protease Type XIV</td></tr><tr><td>N-phenyl thiourea</td><td>Sigma</td><td>P7629-25G</td><td>PTU is toxic and can be prepared as a stock solution to avoid frequent exposure to the powder, consult MSDS before purchase/use.</td></tr><tr><td>Qiaquick gel extraction</td><td>Qiagen</td><td>28704</td><td>any alternatives to purify DNA can be used</td></tr><tr><td>Rneasy Mini kit</td><td>Qiagen</td><td>74104</td><td>any alternatives to purify RNA can be used</td></tr><tr><td>SP6 mMessage kit</td><td>Qiagen</td><td>AM1340</td><td>any alternatives to transcribe DNA using SP6</td></tr></tbody></table>

in vivo imaging of transgenic gene expression in individual retinal progenitors in chimeric zebrafish embryos to study cell nonautonomous influences

遗传和技术优势作出了斑马鱼脊椎动物一个关键的模式生物，其中基因操作的后果可在体内在整个快速发育期间进行跟踪。多个进程可以研究包括细胞增殖，基因表达，细胞迁移和形态发生。重要的是，嵌合体通过移植的产生可以容易地执行，允许主机环境的影响下镶嵌标签和单个细胞的跟踪。例如，通过结合宿主胚的个体移植供体细胞中有功能的基因操作（ 例如，通过显微注射吗啉）和实时成像，外在的影响，细胞非自治信号（由经遗传修饰的环境提供）进行评估。在这里，我们证明这种方法是如何使用荧光的转基因表达的开始比较作为细胞命运determin的定时的代理在通货膨胀不同的遗传主机环境。 在本文中，我们提供的协议为显微注射斑马鱼胚胎标记供体细胞，并引起在宿主胚胎的基因敲除，用于产生嵌合胚胎移植技术的说明，以及用于制备和体内的时间推移共焦运行协议多个胚胎成像。特别是，在执行多位置成像比较事件定时例如基因表达的开始时是至关重要的。这需要从多个控制和同时处理的胚胎实验数据收集。这种方法可以很容易地扩展为在任何器官或访问的实时成像选择的组织外在影响的研究，其前提是移植可根据已建立的胚胎命运地图容易定位。

这项工作提出了一个实验性协议时，野生型视网膜祖细胞morphant胚胎主机内发展，以评估基因表达变化的时序。实验宿主胚胎Ptf1a morphants，缺乏视网膜7，26的中间生的水平和无长突的interneurons。这些已相比，控制宿主胚胎，其中注射了一个标准的控制啉。 因为在整个中枢神经系统的?...

Watch this Scientific Journal Video about In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences at JoVE.c

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

在不同的遗传背景的个体WT视网膜祖细胞的现场跟踪允许细胞非自主性的信令神经过程中贡献进行评估。这里，通过胚胎移植和体内的时间推移共焦成像的基因敲除，嵌合体的产生的组合物用于此目的。

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

in-vivo-imaging-transgenic-gene-expression-individual-retinal

Research

JoVE Journal

Developmental Biology

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

7.6K 次观看.  The University of Melbourne. 在不同的遗传背景的个体WT视网膜祖细胞的现场跟踪允许细胞非自主性的信令神经过程中贡献进行评估。这里，通过胚胎移植和体内的时间推移共焦成像的基因敲除，嵌合体的产生的组合物用于此目的。 

视频: In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

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 ...

科研

发育生物学

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. 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. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

在发展中国家斑马鱼的前脑的神经祖细胞克隆相关的定时实时成像

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

神经科学

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

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 ...

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

移植绿色荧光蛋白表达对视网膜和大脑发育的实时成像嵌合斑马鱼胚胎的卵裂球

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

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 ...

<meta charset="utf8"></head><body>用于再生和发育研究的稳健斑马鱼细胞移植

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>作者聚焦:绘制发育和再生过程中斑马鱼神经系统中的细胞连接图

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

胚胎和幼体斑马鱼的高分辨率细胞移植

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...