斑马鱼镜头核定位与功能完整性的评价
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 1-phenyl-2-thiourea (PTU) | Sigma | P7629 | CAUTION – very toxic |
| 4% Paraformaldehyde aqueous solution | Electron Microscopy Sciences | RT 157-4 | CAUTION – health hazard, combustible |
| Confocal microscope | Nikon | Eclipse Ti-E | |
| Cryostat | Leica | CM3050S | Objective and chamber temperature set to -21˚C |
| DAPI | Invitrogen | D1306 | CAUTION – irritating to eyes and skin |
| Dimethyl Sulfoxide (DMSO) | Fisher Scientific | D128 | CAUTION – combustible, penetrates skin |
| Disposable base mold | VWR Scientific | 15154-631 | |
| Disposable Pasteur glass pipets | Fisherbrand | 13-678-20A | |
| Dumont # 5 forceps | Dumont & Fils | Keep forceps sharpened | |
| Ethyl 3-aminobenzoate methanesulfonate salt (Tricaine) | Sigma-Aldrich | A5040 | CAUTION – toxic |
| Glass bottom microwell dish (35mm petri dish, 14mm microwell, #1.5 coverglass) | MatTek Corporation | P35G-1.5-14-C | |
| Glycerol | Sigma | G2025 | |
| huβB1cry:mAppleCAAX DNA construct | Addgene | ID:122451 | |
| ImageJ | Wayne Rasband, NIH | v1.51n | |
| Low Melt Agarose (LMA) | Apex | 902-36-6 | |
| NIS-Elements | Nikon | V 4.5 | |
| NIS-Elements AR software | Nikon | ||
| Olympus with a model 2.1.1.183 controller | Olympus Corp | DP70 | |
| Olympus microscope | Olympus Corp | SZX12 | |
| Phalloidin-Alexa Flour 546 | Thermo Fisher | A22283 | |
| Phenol Red indicator (1% w/v) | Ricca Chemical Company | 5725-16 | |
| Phosphate buffered saline (PBS) | Fisher Scientific | BP399 | |
| Photoshop | Adobe | CS5 v12.0 | |
| Photoshop software | Adobe | CS5 v12.0 | |
| Plan Apo 60x/1.2 WD objective | Nikon | ||
| Power source | Wild Heerbrugg | MTr 22 | Or equivalent power source |
| Slide warmer model No. 26020FS | Fisher Scientific | 12-594 | |
| Sodium Hydroxide beads | Fisher Scientific | S612-3 | CAUTION – corrosive/irritating to eyes and skin, target organ – respiratory system, corrosive to metals |
| Superfrost/Plus microscope slide | Fisher Scientific | 12-550-15 | |
| Sylgard 184 silicone Dow Corning | World Prevision Instruments | SYLG184 | |
| Tissue-Tek O.C.T. Compound | Sakura Finetek | 4583 | |
| Triton X-100 BioXtra | Sigma | T9284 | CAUTION – Toxic, hazardous to aquatic environment, corrosive |
| Vannas micro-dissection scissors | Ted Pella Inc | 1346 | Sharp/sharp straight tips |
| Vectashield antifade mouting medium | Vector laboratories | H-1000 | |
| Wheat Germ Agglutinin (WGA)-Alexa Flour-594 | Life Technologies | W11262 |
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