这项工作是由卫生科学中西部大学基金研究促进格兰特授予英皇酒店与研究中西部大学办公室和赞助节目校内格兰特授予KJL支持。

该协议符合AVMA动物福利原则实验动物的照顾和使用，已被批准的机构动物护理和使用委员会（IACUC）在中西部的大学。 1.准备麻醉，设备，及媒体<ol><li>脱氯约1.5升使用一个商业脱氯剂（可在宠物商店），或者通过让水静置24小时自来水。获得3 1升的烧杯中，并填写每用500毫升水脱氯。标签之一任何操作之前，拿着鱼和一个用于恢复。到第三烧杯中，加入三卡因甲磺酸盐的100微克/毫升;这将成为其中的鱼类会被麻醉的烧杯中。 </li><li>填充浅盘用冰冷却，温热的培养基（RPMI 1640,10％FBS（胎牛血清），50微克/毫升庆大霉素，100μg/ ml的卡那霉素，25毫米的HEPES）至RT，并收集干净珠宝商的镊子。干净的镊子由音麦唱在70％的乙醇的前端，并穿过本生灯，允许乙醇以烧掉。 </li><li>决定是否将被放大，而不执行的程序，与商业，台式照明放大镜2 - 10X放大倍率，或解剖显微镜（完全依赖于个人偏好）。 </li></ol> 2.建立植文化<ol><li>捉在保持烧杯鱼和地点的所需数目。传输单个鱼麻醉烧杯中;最好是单独麻醉鱼。 </li><li>监测麻醉通过观察鱼的游泳和鳃运动的影响。随着麻醉的进展，刺运动减慢，鱼会游泳不正常，经常倒挂;考虑鱼只要鳃运动停止麻醉。不要让鱼更长时间的麻醉洗澡，因为这可能会导致鱼的死亡。 </li><li>从麻醉烧杯并转移除去鱼冰，水平放置鱼面临的手拿着镊子的尾巴。如果经历了该技术，如果多个鱼将被用于制造植体，考虑在此时将所述下一个鱼在麻醉烧杯中。 </li><li>要去除鳞屑，位置镊子平行与尖鱼的规模优势。踩下镊子的平侧面略成鱼的侧，以使刻度从鱼的身体提升。把握规模镊子，勇气，和地点（无反转）在组织培养皿。重复该过程，除去一个最大值，从一个大的成人（6从各侧）12秤，避免了鳃区域，侧线，和翅片。 </li><li>将鱼放入烧杯中恢复和监控，以确保它从麻醉的效果恢复。此时，鱼可以被转移回单个罐。让鱼恢复了至少21天，以便完整规模的再生。 </li><li>根据实验设计，将高达塑料35mm组织培养处理的培养皿20天平或4 - 每个玻璃底菜6尺度。让秤坚持的菜才轻轻添加媒体以免分离从盘刻度。 注意:使用的经验，前介质被添加到第一盘的多个鱼都可以加工。 </li><li>添加1.2毫升完整的媒体（见1.2以上）到每个35mm培养皿。 </li><li>在5％CO 2孵育优选外植体培养在28℃的时间有或没有额外的处理所需的时间。另外，孵育在加热的显微镜阶段或活细胞培养室视频显微镜内的文化。 </li></ol> 3，明场和视频显微镜<ol><li>含有细胞片上显微镜阶段，最初位置菜肴聚焦用4X物镜。 注:较高的放大倍数也可以使用，这取决于实验。标记的位置每个电池板和/或在舞台上的菜取向将有助于拍摄图像随着时间的推移与片材在大致相同的方向。 </li><li>去除碗碟从培养箱和照片在不同时间点，根据每个实验方案，并放置放回培养箱如果需要在实验时间帧仅静止图像。 </li><li>在执行视频显微镜，要注意以下几点: <ol><li>定位的视场，使得迁移细胞片仍然在视为视频的持续时间的字段中的尺度/新兴细胞片。 注意:这可能需要在观察细胞如何从规模下，迁移了一些经验。对于短视频，这是不到更长的录像的关注。 </li><li>对于较短的时间框架，在室温下孵育。对于较长的时间段（18+小时），使用加热阶段或孵化室要保持温度，酸碱度平衡，并尽量减少文化米蒸发edium。 </li></ol></li></ol> 4.固定的免疫荧光显微镜<ol><li>准备和预暖的所有解决方案，以28°C。为了查看角膜荧光下，上面用玻璃底的35mm组织培养皿中所描述的增长外植体文化。另外，使用玻璃玻片。 </li><li>在1.1倍PBS添加0.8毫升4％多聚甲醛的每一道菜。不先取出培养基。 在室温下孵育5分钟，然后吸出，除去该溶液中。 </li><li>在1.1倍PBS添加0.8毫升4％多聚甲醛的每一道菜。在室温下孵育5分钟，然后吸出，除去该溶液中。 </li><li>除非使用外部配体或外部的表位，通透细胞在1X PBS中加入0.5ml的0.2％的Triton X-100。在室温下孵育5分钟，然后吸出，除去该溶液中。 </li><li>加毫升1％的0.5 BSA的1×PBS中并孵育1小时（RT）或O / N（在4℃）。 注:根据实验条件，PHAlloidin可以在这个步骤中加入。 </li><li>孵育后，吸出物，除去溶液，并继续进行抗体染色。在4℃4周 - 2毫升1％的BSA在1×PBS中进行3 - 可替代地，通过将​​至少1存储培养皿。 </li></ol> 5.免疫荧光协议<ol><li>根据制造商的建议制备初级和次级抗体溶液。 注意:如果制造商的建议都无法使用，一个良好的开端稀释的初级抗体（以在1×PBS中的1％BSA）为1:500的多克隆和1:1000用于单克隆抗体;一个良好的开端稀释的二抗是1:1000。调节从制造商处获得更高的原始股票的这些稀释液，如果信号较弱，低级如果信号强和背景是一个问题。 </li><li>吸在1×PBS溶液中的1％BSA中。加入0.5毫升初级抗体溶液孵育1小时（RT）或O / N（在4℃）。 </li&gt; <li>除去第一抗体溶液，放入一个干净的和标记的1.5 ml离心管，并冷冻在-20℃。 注意:这允许第一抗体的再利用，如果需要的话。 </li><li>与1X PBS 5次，删除和每次冲洗后丢弃的PBS - 洗碟3。 </li><li>加入0.5毫升二抗溶液。如果需要的话，在该步骤中添加荧光标记的鬼笔环肽（以下任何制造商建议的浓度）。孵育1小时（RT）或O / N（在4℃）。 注:使用荧光标记的鬼笔允许肌动蛋白丝的可视化;我们已经发现，488标记的鬼笔环肽时，最好在我们的斑马鱼角膜，但其它的荧光团都可以使用。 </li><li>取下并丢弃二抗溶液。 </li><li>与1X PBS 5次，删除和每次冲洗后丢弃的PBS - 洗碟3。 注:该盘可以存储，涵盖了从光，在4℃，如果无法争夺瓦特立刻;请确保的1×PBS被加入到培养皿存储，以确保细胞不干燥而温暖至室温之前（20 - 30分钟），然后取出PBS中继续下一步骤之前，为了。 </li><li>当准备观察，加2 - 3滴的安装介质，有或没有DAPI，根据实验需要，放入盘中，并让坐在在室温10分钟。 注意:我们使用市售的，基于甘油的安装介质用DAPI，但也可以使用其它的安装介质。允许10分钟用于安装介质坐在盘便于容易除去玻璃盖玻片从培养皿的底部。 </li><li>孵化后，轻轻挤压在菜面松动并取出盖玻片。加安装媒体的附加压降到载玻片并放置盖玻片，将细胞下来，到滑动。 注意:考虑密封盖玻片使用明确指甲油滑动如果滑动需要保持时间超过1 - 2天。 </li><li>观察在荧光显微镜下的幻灯片。 注:我们的大多数的图像被下使用油浸一个40X或63X物镜拍摄，但具体的物镜使用将取决于实验方案。 </li></ol>

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对于角膜移植培养的成功的最关键的步骤是使刻度附着到培养皿约2 - 除了培养基的前3分钟。大约鳞的75％将附着和生长片材（将需要建立每个实验培养物的数量，以进行相应的调整）。角膜片不形成围绕每个尺度从鱼取出，并放置在培养有几个原因。首先，外植体的DAPI染色揭示了一些鳞有少数细胞附着，因此，预计这些尺度不会有角膜的表。其次，外植体要坚持组织培养皿的能力是板材迁移的?...

<html>细胞运动性的​​研究已经传统上集中于单独迁移细胞。角膜从鱼类和两栖类植培养已证明是一个功能强大的模型系统使用实验和数学建模方法1-6来研究细胞运动的机制。上单独迁移细胞的实验工作是由细胞的快速，平稳的滑行动作的帮助下，同时保持一个统一的形状，速度和方向。然而， 在体内 ，细胞频繁移动的统称，同时保持细胞-细胞连接，尤其是在胚胎发育，伤口愈合，和转移。因此，集体细胞迁移是研究细胞迁移的领域内不断增长和日益突出的区域。这些细胞的迁移集体最近被描述7，它有很多独特的功能，这使它成为一个强大的系统来研究集体细胞迁移的伤口愈合模型。 ove_content"&gt;当秤从麻醉的成年斑马鱼弹拨，一些上皮组织保持附着到刻度的底面。当细胞培养基中加入，这些上皮细胞，或角膜，快速地迁移至自规模集体单元的外植体培养物可被看作是一种上皮伤口愈合模型中，其中细胞远离外植体，因为它们会在整个伤口床，以重新建立完整的上皮的临时基质迁移。最近的数据表明，这种体外培养模仿的许多特征在体内在成年斑马鱼伤口愈合。伤口闭合率8转换为类似于在体外系统7观察到的迁移速度，另外， 在体内伤口愈合模型中，与华法林治疗表明，止血对率没有影响愈合，而用氢化可的松治疗，以降低免疫反应不耽误表皮细胞再生8 </SUP&gt;。作为当秤从动物取出的斑马鱼不流血，止血是不是一个主要参与者。虽然我们已经发现免疫细胞外植体，我们还没有研究它们可能对集体细胞迁移的影响。 这里介绍的协议描述了如何建立斑马鱼角膜外植体培养，以确保在许多生物测定使用的一致性和可重复性。这些外植体培养在集体细胞迁移的体外模型中特别引人注目的几个原因。首先，斑马鱼角膜是建立植文化小时内使用原代细胞。因此，这些细胞未经历与主细胞9-13的通道相关联的形态和基因表达的变化。其次，该系统是一款型号为表皮细胞再生的响应研究伤人14，作为回应伤人，上皮间质潮流的一部分启动ansition（EMT）的过程就证明了基因表达的变化和细胞骨架重排14,15。因此，发生在未处理的细胞中的基因表达和运动的背景变化已被鉴定并提供了一​​个上下文来解释与治疗的变化。另外，鱼角膜和人类角质形成细胞在功能上是等效的;两者均为各自物种的初级上皮细胞和播放在上皮伤口愈合的关键作用。第三，成年斑马鱼正获得承认作为模型系统，适用于各种包括黑素瘤和其它癌症19-21，皮肤伤口愈合8,14和组织再生22-24人类疾病16-18中。 这个模型系统的技术优势也同样引人注目。可从非营利性的，集中的设施越来越多的突变和转基因株系。具体而言，斑马鱼突变项目（ZMP）在威康信托基金会桑格研究所的目标是创建在每个蛋白质编码基因的斑马鱼基因敲除等位基因。目前，他们已经突变11892个基因，基因组的大约45％，具有24088的等位基因特征。另外，ZMP接受建议，并会产生剔除装置收费。最近的基因敲除的方法在幼虫和成年斑马鱼25-28提供了灵活性，研究发育的重要基因，这些基因转基因方法是有问题的或不切实际的功能。 由于它们的非常迅速的〜145微米/小时运动速率建立培养物29后不久，测定可以迅速地完成（一般在24小时以下）。由于实验可以迅速完成，文化发展以及在室温，用几个涉及视频显微镜哺乳动物细胞迁移试验是避免相关的技术障碍。此外，在紧缩研究预算的年龄，斑马鱼肥胖是容易且廉价地维护。 外植体的结构和行为是复杂的。作为当比例尺被去除鱼不流血，这是不可能的比浅表皮层得多的规模被除去。角膜细胞似乎是主要的细胞类型的外植体时秤最初从鱼除去细胞的绝大部分出现染色用抗E-钙粘蛋白抗体14。此外，没有任何证据表明在初始培养的成纤维细胞作为判断不存在波形蛋白染色通过免疫荧光14。但是，随着反复除垢，似乎有在植众多嗜中性粒细胞（见视频1）。当暴露于LPS我们已经确定了这些细胞作为基于它们的大小，迅速地运动，并且其迁移出电池板的形态观察结果的中性粒细胞（数据未示出）。此外，这些细胞染色鲜艳的智慧h的抗体，以嗜中性粒细胞胞质因子，以及与各种次级IgG抗体，这表明它们具有在其表面上丰富FcR的（数据未示出）。多个细胞层是外植体中存在。共焦显微镜揭示邻近前缘的单个层的存在，以在靠近刻度角膜的两个以上的层，嗜中性粒细胞可见上方，下方，和之间的细胞角膜片。 在早期时间点，将细胞在多层植两极分化的边缘，引发角膜的集体的迁移，从外植体以约145微米/小时的初始速率。涵盖的外植体的面积急剧增加，从而导致细胞的片内扩散，张力，并前进前缘的下降速率。领导者和从动单元之间快速互变是在前缘的自发形成的孔的片材7内的形成和封闭过程中观察到的。由于与外植体开始的EMT过程继续，片材片段和角膜细胞失去其专业化，快速运动。于7天培养的基因表达，并与EMT一致的形态变化，伤口愈合，和炎症反应发生植正在考虑可行大约10天14。

<table><tbody><tr><td>35 mm Glass Bottom Culture Dishes (Poly-D Lysine Coated)</td><td>MatTek</td><td>P35GC-1.5-14-C</td><td>1.5 mm thickness is best for coverslip removal. 14 mm diameter provides more culture area.</td></tr><tr><td>Swiss Jewelers Style Forceps, Integra, Miltex 17-303</td><td>VWR</td><td>21909-460</td><td>We find that narrow, fine forceps work the best for scale removal.</td></tr></tbody></table>

zebrafish keratocyte explants to study collective cell migration and reepithelialization in cutaneous wound healing

由于其独特的游动性，鱼角膜分离从外植体培养物早已被用于研究单个细胞迁移的机制。然而，当外植体建立，这些细胞还集体移动，保持了许多这使得个体角膜一个有吸引力的模型来研究移民的特点:蠕动较快速度，广泛的肌动蛋白丰富的片层具有垂直肌动线，相对恒定的速度和迁移的方向。在早期的外植体，细胞与那些迁移统称单独迁移的快速互允许的细胞 - 细胞粘连在确定迁移模式中的作用的研究，并强调迁移的两种模式之间的分子联系。在后来的外植体的细胞失去其迅速地和集体迁移能力，上皮至间质转变发生和伤口愈合和炎症相关的基因的差异表达。因此，角膜外植体可以作为一种体外模型为皮肤创伤愈合过程中发生，并且可以代表一个独特的系统来研究集体细胞迁移的机制中所定义的基因表达的变化方案的情况下的表皮细胞再生。多种突变体和转基因斑马鱼线是可用的，其允许外植体可以从鱼具有不同遗传背景的确立。这允许不同的蛋白质，这些过程中的作用，以唯一地寻址。这里所概述的协议描述一种简单而有效的方法，用于在各种相关的集体细胞迁移测定的建立这些外植体培养中使用。

如示于图1中 ，角膜的片可以观察到从规模下建立的外植体培养的小时内迁移出去。有时，单独移植角膜已经从集中迁移片剖开可以观察到。另外，作为外植体是从已被预先弹拨鱼成立，有丰富的嗜中性粒细胞通过片材迁移。在较长的文化时期，角膜板碎片为细胞进行EMT 14。 片前进的初始速率是极其迅速，但这个速率很快变慢作为细胞扩散（面积和核间?...

Watch this Scientific Journal Video about Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing at JoVE.com

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

斑马鱼角膜基质细胞的表外植迁移，并提供了一个体外模型集体细胞迁移的上皮伤口愈合的情况下机制的研究。这些协议的细节，以建立初级外植体培养在集体细胞迁移测定法使用的有效方式。

斑马鱼角膜植研究集体细胞迁移和再上皮化在皮肤伤口愈合

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

zebrafish-keratocyte-explants-to-study-collective-cell-migration

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.9K 次观看.  Midwestern University. 斑马鱼角膜基质细胞的表外植迁移，并提供了一个体外模型集体细胞迁移的上皮伤口愈合的情况下机制的研究。这些协议的细节，以建立初级外植体培养在集体细胞迁移测定法使用的有效方式。 

视频: Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

科研

发育生物学

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

在斑马鱼胚胎的机械血管损伤

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

使用提顿体系的研究斑马鱼再生的分子机制

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

建立临床相关<em&gt;离体</em&gt;调查上皮创伤修复的微环境本地人模拟白内障手术模型

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

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

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

发展中国家斑马鱼的表皮细胞挤压实时成像

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

生物学

<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 ...

斑马鱼角膜植研究集体细胞迁移和再上皮化在皮肤伤口愈合

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此视频中的章节

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

在斑马鱼胚胎的机械血管损伤

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

使用提顿体系的研究斑马鱼再生的分子机制

建立临床相关

改善斑马鱼幼虫实验操作的长期活体成像装置

发展中国家斑马鱼的表皮细胞挤压实时成像

胚胎和幼体斑马鱼的高分辨率细胞移植

胚胎和幼体斑马鱼的高分辨率细胞移植

胚胎和幼体斑马鱼的高分辨率细胞移植