Name: assessment of zebrafish lens nucleus localization and sutural integrity
Uploaded: 2019-05-06T14:00:00
Description: Watch this Scientific Journal Video about Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity at JoVE.com

我们要感谢我们的资金来源: NIH R01 Y05661 至 j. e. h, ins Gehring 协助产生aqp0a\ b 双突变体和斑马鱼饲养, Daniel Dranow 博士进行讨论, 导致产生转基因, 布鲁斯博士布隆伯格和肯·乔博士的实验室使用他们的解剖显微镜, 梅根·史密斯博士寻求统计分析方面的帮助。

本研究中使用的动物规程符合 ARVO 关于在眼科和视力研究中使用动物的声明, 并已获得加州大学欧文分校动物护理和使用机构委员会 (IACUC) 的批准。

1. 斑马鱼畜牧业和安乐死
<ol><li>在标准实验室条件下饲养和维护斑马鱼 (AB 菌株)12。在胚胎培养基 (EM) 中培养胚胎 (EM)12。在受精后 20-24小时 (原发5期之前) 向 EM 添加0.003% 的 PTU, 以防止胚?...

<ol>
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斑马鱼晶状体形态分析是了解突变体表型或旨在研究晶状体生物学的药理干预作用的第一步。我们概述了分析晶状体缝合线, 皮质纤维细胞形态和镜头核的各个方面的方法。这些方法是体外和体内的结合 (与表1 相比)。体外方法允许更详细的外皮质细胞形态, 以及进入成人镜片的后缝合线。
使用转基因标记可以进行体内分析。我们在这里...

斑马鱼是研究晶状体发育和生理的一个很好的模型, 因为它与哺乳动物的镜片有解剖上的相似性, 遗传和药理操作相对容易, 胚胎眼睛发育的速度快, 体积小, 透明度高在早期阶段允许体内成像。本文提出了分析斑马鱼晶状体在胚胎和成虫阶段形态的方法, 重点是结构完整性、体外和体内皮质膜形态以及晶状体核位置沿胚胎和成人阶段的位置。前、后轴. 这些方法作为研究晶状体发育和生理功能的起点, 以及斑马鱼晶状体表型的反向基因屏幕。
斑马鱼晶状体形态的影像学
Aquaporin 0 (AQP0) 是最丰富的晶状体膜蛋白, 由于哺乳动物的多种基本功能, 对晶状体的发育和清晰度都至关重要。斑马鱼有两个 A斯洛0 (Aqp0a 和 Aqp0a), 我们已经开发出方法来分析他们的功能在胚胎和成人镜片中的丧失。我们的研究表明, 由于前缝合不稳定, a qp0a/突变体出现前极性不透明度,而 aqp0a/双突变体发展为核不透明度1。Aqp0 已被证明在水运2、粘附3、4、细胞骨架锚固5和折射率梯度 6的生成中发挥着重要作用, 但这些研究主要是在体外。斑马鱼提供了一个独特的机会, 研究如何失去功能, 或不安的功能, Aqp0a 或 Aqp0b 将如何影响形态和功能在一个活的镜头。为了评估晶状体细胞的形态和发育过程中的完整性, 我们对现有的体外免疫组化方法7进行了改进, 用于胚胎和成人晶状体, 并生成了转基因技术来监测这一过程在体内的情况.
对整个固定胚胎和成人镜片进行了质膜结构和结构完整性的免疫组化分析。与哺乳动物相比, 斑马鱼镜片非常小 (胚胎镜片直径约为 100μm, 成人镜片直径高达1毫米), 并有点缝合 8, 这在冷冻中很少被捕获。因此, 整个镜头对于分析结构完整性是必不可少的。对于前缝合形成的体内分析, 以及精确晶状体细胞结构的成像, 我们生成了表达苹果特异性标记晶状体膜的转基因。
晶状体膜转基因实时成像的优点包括: 1) 避免固定伪影, 2) 研究延时电影的动态形态变化, 3) 使早期事件与后期事件相关的纵向研究表。虹膜的色素沉着通常会阻止晶状体外围的清晰成像。在原始数-5 (prim-5) 阶段9之前添加1苯基 2-硫脲 (PTU) 可防止黑色素生成和眼睛色素沉着, 时间约为受精 (dpf)。然而, 4 dpf 后, 晶状体周围在体内被遮挡, 特别是在较老的阶段。此外, 镜头本身的密度掩盖了它的后极的成像。因此, 要研究晶状体周围或后部缝合的形态, 需要切除和固定 4 dpf 后的晶状体。
转基因斑马鱼系已被用于体内胚胎晶状体膜结构的分析10.Q01转基因表达了在ef1 α启动子和 df4 pax6 增强剂元素的 Hexamer 驱动下, 融合到膜靶向序列 gap 的青色荧光蛋白, 在晶状体纤维细胞 11中普遍存在。Q01确实有超镜表达, 包括视网膜中的阿玛可拉细胞, 如果主要兴趣是晶状体, 这将增加背景信号。我们开发了一种新的转基因线, 它在镜头中特别表达膜系带的 mApple, 目的是避免任何超透镜信号。
镜头核定位
我们发现, 晶状体核从幼虫斑马鱼的初始前部位置移动到成人晶状体前后轴的中心位置。高折射率透镜核位置的这种变化被认为是斑马鱼透镜光学1正常发展的要求.我们的方法允许量化镜头核集中相对于镜头半径。利用该方法, 我们证明了 Aqp0a 是透镜核集中1所必需的, 该方法可应用于其他研究的发展和生理学的镜头及其光学性质的斑马鱼模型。

<table border="0" cellpadding="0" cellspacing="0" style="width:880px;" width="881">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">1-phenyl-2-thiourea (PTU)</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:118px;">P7629</td>
			<td style="width:258px;">CAUTION &#8211; very toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:310px;">4% Paraformaldehyde aqueous solution</td>
			<td style="width:196px;">Electron Microscopy Sciences</td>
			<td style="width:118px;">RT 157-4</td>
			<td>CAUTION &#8211; health hazard, combustible</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Confocal microscope</td>
			<td style="width:196px;">Nikon</td>
			<td style="width:118px;">Eclipse Ti-E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Cryostat</td>
			<td style="width:196px;">Leica</td>
			<td style="width:118px;">CM3050S</td>
			<td style="width:258px;">Objective and chamber temperature set to -21&#730;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">DAPI</td>
			<td style="width:196px;">Invitrogen</td>
			<td style="width:118px;">D1306</td>
			<td>CAUTION &#8211; irritating to eyes and skin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Dimethyl Sulfoxide (DMSO)</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:118px;">D128</td>
			<td style="width:258px;">CAUTION &#8211; combustible, penetrates skin&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Disposable base mold</td>
			<td style="width:196px;">VWR Scientific</td>
			<td style="width:118px;">15154-631</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Disposable Pasteur glass pipets</td>
			<td style="width:196px;">Fisherbrand</td>
			<td style="width:118px;">13-678-20A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Dumont # 5 forceps</td>
			<td style="width:196px;">Dumont &#38; Fils</td>
			<td style="width:118px;"></td>
			<td>Keep forceps sharpened</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine)</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:118px;">A5040</td>
			<td>CAUTION - toxic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Glass bottom microwell dish (35mm petri dish, 14mm microwell, #1.5 coverglass)</td>
			<td style="width:196px;">MatTek Corporation</td>
			<td style="width:118px;">P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Glycerol</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:118px;">G2025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hu&#946;B1cry:mAppleCAAX DNA construct</td>
			<td style="width:196px;">Addgene</td>
			<td style="width:118px;">ID:122451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">ImageJ</td>
			<td style="width:196px;">Wayne Rasband, NIH</td>
			<td style="width:118px;">v1.51n</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Low Melt Agarose (LMA)</td>
			<td style="width:196px;">Apex</td>
			<td style="width:118px;">902-36-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">NIS-Elements</td>
			<td style="width:196px;">Nikon</td>
			<td style="width:118px;">V 4.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">NIS-Elements AR software</td>
			<td style="width:196px;">Nikon</td>
			<td style="width:118px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus&#160; with a model 2.1.1.183 controller</td>
			<td style="width:196px;">Olympus Corp</td>
			<td style="width:118px;">DP70</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus microscope&#160;</td>
			<td style="width:196px;">Olympus Corp</td>
			<td style="width:118px;">SZX12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Phalloidin-Alexa Flour 546</td>
			<td style="width:196px;">Thermo Fisher</td>
			<td style="width:118px;">A22283</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Phenol Red indicator (1% w/v)</td>
			<td style="width:196px;">Ricca Chemical Company</td>
			<td style="width:118px;">5725-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Phosphate buffered saline (PBS)</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:118px;">BP399</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Photoshop</td>
			<td style="width:196px;">Adobe</td>
			<td style="width:118px;">CS5 v12.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Photoshop software</td>
			<td style="width:196px;">Adobe</td>
			<td style="width:118px;">CS5 v12.0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plan Apo 60x/1.2 WD objective</td>
			<td style="width:196px;">Nikon</td>
			<td style="width:118px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Power source</td>
			<td style="width:196px;">Wild Heerbrugg</td>
			<td style="width:118px;">MTr 22</td>
			<td>Or equivalent power source&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Slide warmer model No. 26020FS</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:118px;">12-594</td>
			<td></td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:310px;">Sodium Hydroxide beads</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:118px;">S612-3</td>
			<td style="width:258px;">CAUTION - corrosive/irritating to eyes and skin, target organ - respiratory system, corrosive to metals</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Superfrost/Plus microscope slide</td>
			<td style="width:196px;">Fisher Scientific</td>
			<td style="width:118px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Sylgard 184 silicone Dow Corning</td>
			<td style="width:196px;">World Prevision Instruments</td>
			<td style="width:118px;">SYLG184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Tissue-Tek O.C.T. Compound</td>
			<td style="width:196px;">Sakura Finetek</td>
			<td style="width:118px;">4583</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Triton X-100 BioXtra</td>
			<td style="width:196px;">Sigma</td>
			<td style="width:118px;">T9284</td>
			<td style="width:258px;">CAUTION &#8211; Toxic, hazardous to aquatic environment, corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Vannas micro-dissection scissors</td>
			<td style="width:196px;">Ted Pella Inc</td>
			<td style="width:118px;">1346</td>
			<td>Sharp/sharp straight tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:310px;">Vectashield antifade mouting medium</td>
			<td style="width:196px;">Vector laboratories</td>
			<td style="width:118px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:310px;">Wheat Germ Agglutinin (WGA)-Alexa Flour-594</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:118px;">W11262</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of zebrafish lens nucleus localization and sutural integrity

斑马鱼特别适合于基因操作和体内成像, 使其成为逆向基因研究和体内成像转基因生成的日益流行的模型。这些独特的功能使斑马鱼成为研究眼睛发育和生理的理想平台。我们最近的发现是, Aquaporin-0, Aqp0a, 是需要的前晶状体缝合线的稳定性, 以及随着年龄的年龄将镜头核转移到镜头中心, 这导致我们开发了专门用于分析斑马鱼镜片性能的工具。在这里, 我们概述了详细的方法, 晶状体解剖, 可适用于幼虫和成人镜片, 为他们的组织学分析, 免疫组织化学和成像做准备。我们重点分析了晶状体缝合线的完整性和皮质细胞形态, 并将解剖镜片产生的数据与一种新的转基因斑马鱼系在基因上所能实现的晶状体形态体内成像数据进行了比较编码的荧光标记。通过分析垂直于其光轴的解剖镜片, 可以量化镜片核沿前后轴的相对位置。在成人斑马鱼的正常透镜光学中, 需要将晶状体核从最初的前位置移动到中心。因此, 镜头核位置的定量测量与其光学特性直接相关。

成年斑马鱼眼的解剖结构与哺乳动物的解剖结构非常相似 (图 1a)。尽管斑马鱼和哺乳动物的眼睛之间有一些差异, 如有一个睫毛区而不是一个睫毛体17, 光学特性的差异 18, 和形态发生的差异, 在胚胎发育 19,斑马鱼眼是研究眼睛发育和了解眼科疾病20,21,<su...

Watch this Scientific Journal Video about Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity at JoVE.com

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity

这些方案的开发是为了分析皮质晶状体形态, 结构完整性的斑马鱼晶状体缝合在固定和活的镜头, 并测量斑马鱼晶状体核沿前后轴的位置。

斑马鱼镜头核定位与功能完整性的评价

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for ...

我们的协议为镜头发育和生理学的功能研究以及斑马鱼中镜头表型的反向基因筛选提供了一个起点。我们的协议桥接斑马鱼透镜膜形态的体外和体内分析，为斑马鱼透镜光学的定量开发提供了一种新的、直接的方法。分析斑马鱼透镜形态和光学，可以深入了解控制镜头发育、正常生理学和病理生理学的机制。这些见解反过来又会导致延迟或预防白内障的能力。除了非对称局部透镜核的斑马鱼以外的物种中，没有报告。识别镜头缝合线对于正确定位镜头以测量细胞核定位至关重要。在确认与三元鱼有足够和解后，使用微解剪刀立即将两只眼睛从成人或幼虫斑马鱼身上切除，并两只眼睛放入 35 毫米定制 Petri 盘中，其中用硅胶解剖模具填充 PBS。要从成年斑马鱼身上采集镜头，请将眼睛后部向上放置，并通过视盘以低于 45 度的角度插入钳子。使用解剖剪刀，使两个或三个径向切口通过视网膜和sclera从光学光盘到固定眼的 Ciliion 区，并剥离视网膜和sclera像花瓣。将眼睛、角膜侧向上，并使用剪刀的平端，通过操作sclera和角膜间接固定镜头，用钳子拉开视网膜和附着组织，然后小心地修剪掉镜头中多余的组织。为了收获幼虫斑马鱼镜头，将幼虫眼后侧放在PBS硅胶盘的平坦部分，并使用锐化的钨针通过视网膜和硬骨进行径向切口，同时用另一根针或钳子固定眼睛，然后用定制钨丝针的钝侧轻轻地将透镜从分离的眼睛中挖出，并小心地拉开附着的组织。为了测量前轴透镜核的定位，在 PBS 中轴向定向新切除的透镜，用盖玻璃底部与极点和缝合线平行于焦点平面。要识别镜头核，请查找折射率的差异，折射率通常发生在镜头皮层和镜头核的界面上。如果镜头缝合不明显，稍微舔一下钳子到镜头胶囊就会露出缝合线和镜头核，帮助确定镜头的测量方向。将碟子放在装有摄像头的解剖显微镜的舞台上，并在亮场照明或差分干扰对比度光学元件下对镜头核进行对焦成像。在同一放大倍数下成像千分尺以进行校准，然后单击"实时视图"。单击快照获取图像并保存图像为 TIFF 文件。要校准采集的镜头图像，请在适当的图像处理软件程序中选择直线工具，并在微米图像上绘制一条已知长度的线。单击"分析和设置比例"并输入已知距离和单位，然后选择"全局校准"，然后单击"确定"。要测量从镜头核中心到前极的距离，请确定镜头缝合的位置，并使用直线工具以轴向方向绘制穿过镜头核的图像中心。这条线的中心是镜头核的中心。从该点再绘制一条线到镜头前极，并在"分析"菜单中选择"测量"以测量距离。从前极到后极再画一条线，并测量此距离作为镜头直径，然后将这些长度从"结果"窗口复制到电子表格，并计算镜头核与镜头半径的规范化定位。到受精后三天，前极形成前缝合，通过固定胚胎中狭窄的纤维细胞膜的 phaloidin 染色，可以清楚地显示细胞的惊人收敛。在镜头膜中更强的宽纤维细胞标记局部mApple转基因允许膜亚域的实时可视化，但缺乏分离。后缝合可以在受精后三天在体外和体内可视化。在赤道平面上，phoidoidin 强烈标记外皮质纤维细胞，露出扁平的六边形。法洛丁被排除在压缩的镜头核和野生类型和突变镜头看起来无法区分。在透镜膜定位的mApple马赛克转基因中，宽纤维细胞的强标记揭示了突变透镜中细胞体积的中断。由于转基因表达不受渗透性限制，一些马赛克显示透镜核标签。在野生类动物中，镜头核在发育的早期阶段开始靠近前透镜杆，然后在成年后集中。在转基因宿主中表达绿色标记的蛋白质，其中细胞膜荧光红允许同时进行蛋白质定位以及评估体内的纤维细胞形态。斑马鱼特别适合体内研究。使用此处描述的工具，我们可以探测在体内系统中主要在体外研究的镜头机制。

Research

JoVE Journal

Biology

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

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 ...

科研

生物学

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

In vivo Electroporation of Morpholinos into the Adult Zebrafish 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 ...

Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

纳米有限元分析：蛋白质定位的光激活定位显微镜和电子显微镜

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

在单核级LINE-1逆转录转座子分析

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

无透镜视频显微术在黏附细胞培养动态定量分析中的研究

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

研究核苷酸合并: 酵母基因组 Ribonucleotides 的链特异检测及核苷酸诱变的检测

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

超快力钳谱分析合成性肌球蛋白的机械酶学特性

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...

Assessment of Gut Barrier Integrity in Mice Using Fluorescein-Isothiocyanate-Labeled Dextran

使用荧光素-异硫氰酸酯标记的葡聚糖评估小鼠肠道屏障完整性

Gut barrier integrity is a hallmark of intestinal health. While gut barrier integrity can be assessed using indirect markers such as the measurement of ...

Studying Protein Expression During Chicken Lens Development

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

Investigating Lens Development and Function Through Microinjection of RCAS(A) Retrovirus in Embryonic Chicken Lens (Video) | JoVE

重组RCAS（A）逆转录病毒显微注射到胚胎鸡晶状体中

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of ...