We thank Monika Kilchenmann, Federica Bisignani and Agathe Duda for their excellent technical assistance.

所有实验坚持以声明的动物眼科及研究协会在视觉视觉研究与眼科（ARVO）使用。 1，动物<ol><li>维持公司（俄勒冈州）水标准条件下6-12个月之间以26.5℃的温度和14/10小时的光/暗周期15株野生型斑马鱼（ 斑马鱼 ）。 </li><li>按照参与机构的审批由州兽医局后，经动物实验动物照护指引。 </li></ol> 2，MNU处理<ol><li>制备含有氮甲基 -N- -nitrosourea（MNU）150毫克/升干燥物质的水。注意！ MNU是有毒的;可能会导致癌症，遗传性的损害或伤害胎儿。 </li><li>孵育斑马鱼中含有MNU，在室温下60分钟的水。 </li><li>迅速用清水冲洗斑马鱼淡水把鱼到一个新的鱼缸没有MNU。维持标准的条件，只要所需的实验下的鱼。 </li></ol> 3，视力测量<ol><li>启动optomotor系统，从菜单中选择"测试"，并设置选项，以"简单楼梯"，"视力（频率）"和"随机/独立"16。 </li><li>安装的输液瓶填充500ml水约1μm的optomotor系统之上。 </li><li>将一个斑马鱼在考试室，并把它连接到输液瓶。放置在optomotor系统的检查室。 </li><li>开始测量和观察计算机屏幕上实时眼球运动。因此，积极响应（"是"）被定义为连续扫视了正确的方向，而一个否定的答复（"无"）代表随机眼球运动相似，与那些观测站进制光栅。如果斑马鱼表现出的眼里三个或更多的后续视动反应（OKR），按"是";如果没有，请按"不"。 </li><li>提取的视力，其中已计算由软件通过确定视动刺激的空间频率的阈值时，根据菜单中的"结果"。 </li></ol> 4，组织学<ol><li>由乙基3 - 氨基苯甲酸酯（200-300毫克/升）的过量安乐死斑马鱼和去核的眼睛。 </li><li>在4℃下12小时固定整个眼睛用4％多聚甲醛（PFA）在磷酸盐缓冲盐水（PBS）中，然后脱水的样品在梯度醇系列。 </li><li>嵌入样品中石蜡，切5微米的部分通过视神经乳头（ONH）和它们安装在载玻片上。 </li><li>染色deparaffized石蜡切片用hemalum为4分钟，浸在幻灯片两次蒸馏水中后0.2％的盐酸ACID和0.8％的氨。染色用曙红的部分自来水中发展hemalum染色至少10分钟（H＆E）后3分钟。 </li><li>嵌入在安装介质中的脱水幻灯片，观察光镜下的幻灯片。 </li></ol> 5，免疫组化<ol><li>末端脱氧核苷酸转移酶缺口末端标记法（TUNEL） <ol><li>孵育20微克/毫升的蛋白酶K的去石蜡切片在室温下搅拌15分钟。用Tris洗部分三次，每次5分钟缓冲盐水（TBS）。 </li><li>孵化用50μlTUNEL反应混合物（10％标记溶液和90％的酶溶液）的部分，在湿润室中于37℃进行60分钟。用TBS，每次5分钟洗涤3次。 </li><li>安装带安装含有DAPI介质的幻灯片，并观察在荧光显微镜下的滑动。 </li></ol></li><li>增殖细胞核抗原（PCNA）染色<ol><li>沸腾在抗原修复缓冲液（Tris-EDTA + 0.05％吐温20，pH9.0）中的脱石蜡切片3分钟，洗三次，用TBS，每次5分钟。用100μl封闭溶液（TBS +10％山羊正常血清+1％牛血清白蛋白，pH7.6）中，在室温下搅拌1小时阻断。 </li><li>用在1抗PCNA的初级抗体染色1:200稀释，在湿润室中于4℃下过夜。用TBS +吐温20，每次5分钟洗涤3次。 </li><li>完成检测与在1相应的二级抗体1:500稀释，在室温下搅拌1小时。 </li><li>安装带安装含有DAPI介质的幻灯片，并观察在荧光显微镜下的滑动。 </li></ol></li></ol>

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The authors have nothing to disclose.

此前，本集团已转移光感受器变性的MNU模式从啮齿动物到斑马鱼系统9。随后的事件进行了描述，并随访了30天。在这段时间内完整视网膜形态退化和再生的初始治疗后发生。首先，组织学显示，从3天减少杆细胞计数与最小的相应8天，TUNEL染色MNU处理后第3天确定的视杆细胞的凋亡。再生开始的INL为3天，最长为5天外核层，增殖细胞可观察到的观察期，最大的结尾处5天，这是与此相反的INL在...

视觉是最重要的意义的人及其损害具有较高的社会经济影响。在发达国家，视网膜变性疾病是视力减退和失明的老年人中1的主要原因。最退行性视网膜疾病的原因只是部分了解和治疗方案，以夺回失去的视力是非常有限的。视网膜色素变性是视网膜变性疾病的原发性感光损失2-3。n的典型例子甲基 -N -nitrosourea（MNU）诱导的视网膜变性，因此被广泛应用在啮齿类动物模型与主光感受器细胞死亡4疾病。它是一种烷化剂，并导致良性和恶性肿瘤，通常会出现曝光5-7后几个月。此外，它会导致短期的观察期间内特定光感受器细胞死亡。由此，视网膜层structu的损失重和显著视网膜变薄了以浓度依赖性方式观察到。视网膜神经胶质细胞被激活，但在视网膜色素上皮细胞（RPE）没有变化被发现。内质网（ER）应激相关的细胞凋亡似乎是MNU作用的主要途径在视网膜8。 我们最近推出的MNU作为一种化学模型诱导光感受器变性斑马鱼9。除了 ​​其他原因，斑马鱼（ 斑马鱼 ）已成为因为它的视觉系统对其他脊椎动物10的相似之处在视觉研究很重要。外层视网膜包含感光体，它可被分成四个不同的锥面类型具有峰值灵敏度的紫外线，短，中等，和可见光谱的长波长和一个视杆型。在内核层（INL）的双极性，水平和无长突的interneurons细胞体被发现，如WELl与穆勒胶质细胞的胞体。在所述外丛状层（OPL）的光感受器和视网膜内层之间的突触联系形成的，而最接近透镜的细胞层是神经节细胞层（GC），该组件形成长轴突包括视神经和视束。神经节细胞和细胞中的内核层之间的突触联系形成于内网层（IPL）11。视网膜色素上皮所在的视网膜神经上皮层外，并围绕光感受器外段长微绒毛顶端12。此外，斑马鱼是高度再生，并能够再生毁损脑，视网膜，脊髓，心脏和其他组织13。当视网膜损伤时，细胞中的INL，这被认为是Müller细胞，被激活并具有分化成多种视网膜细胞类型的潜力。此外，它们还产生棒祖细胞，它们分别位于外核层。另一个让urce供给成年斑马鱼视网膜有新生细胞的纤毛边缘区。这个源是需要实现的视杆细胞的恒定密度的不断增长的斑马鱼眼14。 在MNU模型可以用作视网膜组织的简单和可再现的变性/再生方法。由于在斑马鱼中的生物过程的某些相似性和在人体中，这可能打开门，以确定参与细胞死亡途径，筛选潜在的神经保护药物。根据我们组先前的研究，我们现在说明这MNU诱导斑马鱼视网膜脱离和随之而来的再生包括根据实验室影片9功能变化模型的方法。

<table><tbody><tr><td>Acetic acid</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>A6283</td><td></td></tr><tr><td>Ammonia</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>294993</td><td>0.80%</td></tr><tr><td>Bovine serum albumine (BSA)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>05470</td><td></td></tr><tr><td>Dako Pen</td><td>Dako, Glostrup, Danmark</td><td>S2002</td><td></td></tr><tr><td>DAPI mounting medium</td><td>Vector Labs, Burlingame, CA, USA</td><td>H-1200</td><td></td></tr><tr><td>Eosin</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>45260</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>2860</td><td>100%, 96%, 70%</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>ED</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate salt</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>E10521</td><td>Tricaine</td></tr><tr><td>Eukitt</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>3989</td><td></td></tr><tr><td>Goat anti-rabbit Alexa 594</td><td>Life Technologies, Zug, Switzerland&amp;nbsp;</td><td>A11012</td><td></td></tr><tr><td>Goat normal serum</td><td>Dako, Glostrup, Danmark</td><td>X0907</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>320331</td><td>0.20%</td></tr><tr><td>In situ Cell Death Detection Kit</td><td>Roche Applied Sciences, Rotkreuz, Switzerland</td><td>11684795910</td><td>TUNEL Kit</td></tr><tr><td>Mayer&#039;s hemalum solution</td><td>Merck, Darmstadt, Germany</td><td>109249</td><td></td></tr><tr><td>Methylnitrosourea (MNU)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>N4766</td><td>Toxic</td></tr><tr><td>OptoMotry</td><td>CerebralMechanics, Lethbridge, AB, Canada</td><td>n.a.</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>P6148</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>P5368</td><td></td></tr><tr><td>Proteinase K</td><td>Dako, Glostrup, Danmark</td><td>S3004</td><td></td></tr><tr><td>Rabbit anti-PCNA&amp;nbsp;</td><td>Santa Cruz Biotechnology, Santa Cruz, USA</td><td>sc-33756</td><td></td></tr><tr><td>Superfrost Plus glass slides</td><td>Gehard Menzel GmbH, Braunschweig, Germany</td><td>10149870</td><td></td></tr><tr><td>Tris buffered saline (TBS)</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>P5912</td><td></td></tr><tr><td>Trizma base</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>T1503</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>P1379</td><td></td></tr><tr><td>Xylene</td><td>Sigma-Aldrich, Buchs, Switzerland</td><td>534056</td><td></td></tr><tr><td>Zebrafish (Danio rerio) AB (Oregon) strain</td><td>University of Fribourg, Dept. of Biology</td><td>n.a.</td><td>Own fish facility</td></tr></tbody></table>

methylnitrosourea (mnu)-induced retinal degeneration and regeneration in the zebrafish: histological and functional characteristics

视网膜退行性疾病， 如视网膜色素变性，用得到的，为广大视力下降，在工业领域感光伤害帐户。动物模型是至关重要的意义，研究这种疾病。在这方面，在感光体特异性毒素N-甲基-N- -nitrosourea（MNU）已被广泛用于在啮齿动物药理学诱导的视网膜变性。此前，我们已经建立了MNU诱导的视网膜变性模型中的斑马鱼，在视觉研究的另一种流行的模型系统。 一个迷人的区别是哺乳动物在成年斑马鱼视网膜损伤后的再生持续神经。为了量化这一观察，我们在成年斑马鱼使用视力的测量。由此，视动反射被用于跟随在非麻醉的鱼的功能变化。这补充了组织学以及免疫staini吴凋亡（TUNEL）和增殖（PCNA）关联的发展形态变化。 综上所述，光感受器细胞凋亡MNU治疗，随后是细胞中的外核层（ONL）的显着降低三天之后出现。此后，将细胞在内核层（INL）和ONL的增殖被观察。在此，我们表明，不仅是一个完整的组织，而且功能性再生发生在30天的时间过程。现在我们说明的方法来量化和跟进的视频格式使用的MNU斑马鱼视网膜脱离和再生。

视力: 该实验装置[空间频率:0.042圆/度（C / D）;对比度:100％;漂移速度:20度/秒（D / SRC）;背光亮度:本研究的152 cd /米2]启用成年斑马鱼OKR评估。 VA测量的平均持续时间大约为5 - 10分钟，每一个斑马鱼，其耐受性的方法好。 MNU曝光前视力为0.577±0.014周/度（C / D）， 图1示出了视觉灵敏度当然150毫克/升的MNU后应用。从第1天开始，将测量结果显示在视敏度有显?...

Watch this Scientific Journal Video about Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics at JoVE.com

Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

在这里我们展示视网膜脱离和再生，对以N甲基 -N- -nitrosourea在成年斑马鱼的视觉功能的影响进行量化。其次是在内核层扩散视力丧失和降低光感受器号。发生完整的形态和功能恢复的初步治疗后30天。

Methylnitrosourea（MNU）诱导的视网膜变性和再生的斑马鱼:组织学和功能特征

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial ...

methylnitrosourea-mnu-induced-retinal-degeneration-regeneration

Research

JoVE Journal

Biology

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

11.4K 次观看.  University of Bern. 在这里我们展示视网膜脱离和再生，对以N甲基 -N- -nitrosourea在成年斑马鱼的视觉功能的影响进行量化。其次是在内核层扩散视力丧失和降低光感受器号。发生完整的形态和功能恢复的初步治疗后30天。 

视频: Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

科研

发育生物学

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 ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

在斑马鱼和镜头移植在眼突变分析中的应用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

生物学

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

神经科学

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire retina.

成年斑马鱼视网膜再生研究中使用的一种新型的光损伤范式

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

斑马鱼作为一种模型来评估亚硝酸盐的潜在致畸

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

免疫与感染

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 ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, a&#160;vertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform RpEGEN for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

This protocol describes the methodology to genetically ablate the retinal pigment epithelium (RPE) using a transgenic zebrafish model. Adapting the protocol to incorporate signaling pathway modulation using pharmacological compounds is extensively detailed. A MATLAB platform for quantifying RPE regeneration based on pigmentation was developed and is presented and discussed.

硝基还原酶/甲硝唑介导的消融和MATLAB平台（RpEGEN）研究斑马鱼视网膜色素上皮的再生

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

Methylnitrosourea（MNU）诱导的视网膜变性和再生的斑马鱼:组织学和功能特征

摘要

探索更多视频

此视频中的章节

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的肝细胞的再生

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

在斑马鱼和镜头移植在眼突变分析中的应用

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

成年斑马鱼视网膜再生研究中使用的一种新型的光损伤范式

斑马鱼作为一种模型来评估亚硝酸盐的潜在致畸

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

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

在体内电穿孔Morpholinos到成年斑马鱼视网膜

硝基还原酶/甲硝唑介导的消融和MATLAB平台（RpEGEN）研究斑马鱼视网膜色素上皮的再生

Methylnitrosourea（MNU）诱导的视网膜变性和再生的斑马鱼:组织学和功能特征

摘要

探索更多视频

此视频中的章节

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

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

在斑马鱼和镜头移植在眼突变分析中的应用

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

成年斑马鱼视网膜再生研究中使用的一种新型的光损伤范式

斑马鱼作为一种模型来评估亚硝酸盐的潜在致畸

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

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

在体内电穿孔Morpholinos到成年斑马鱼视网膜

硝基还原酶/甲硝唑介导的消融和MATLAB平台（RpEGEN）研究斑马鱼视网膜色素上皮的再生

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的肝细胞的再生