使用"彩虹"和"延时共聚焦成像"可视化活斑马鱼中发育中的脑
Summary
体内成像是一种强大的工具，可用于研究神经系统发育背后的细胞机制。在这里，我们描述了一种技术，使用延时共聚焦显微镜，以可视化大量的多色彩虹标记细胞在发展中的斑马鱼神经系统实时。
Abstract
脊椎动物神经系统的发展需要复杂的细胞行为和相互作用的精确协调。在体内使用高分辨率成像技术可以为活生物体中的这些过程提供一个清晰的窗口。例如，在神经系统形成时，分裂细胞及其后代可以实时跟踪。近年来，多色技术的进步扩大了可以调查的问题类型。多色Brainbow方法不仅可用于区分像细胞一样，还可用于对相关细胞的多个不同克隆进行颜色编码，每个克隆来自一个祖细胞。这允许在开发期间同时对许多不同的克隆及其行为进行多路复用线分析。在这里，我们描述了一种技术，使用延时共聚焦显微镜在发展中的斑马鱼神经系统中实时可视化大量多色彩虹标记细胞。这对于关注细胞之间的细胞相互作用特别有用，因为细胞很难使用传统的启动器驱动颜色进行差异标记。我们的方法可用于同时跟踪多个不同克隆之间的系系关系。使用此技术生成的大型数据集提供了丰富的信息，可以定量地比较基因或药理学操作。最终，所产生的结果有助于回答有关神经系统如何发育的系统问题。
Introduction
在发育的早期阶段，专门的祖细胞池在增殖区反复分裂，产生不同的子细胞阵列。然后，在这个发育期出生的细胞将分化和旅行，形成新生的器官。在神经系统中，前视像状胶质在心室区产生不成熟的神经元。当神经元从心室迁移并成熟时，膨胀的组织最终形成大脑高度复杂的结构1,21，2，3，4，5，6。,3,4,5,6前祖的分裂与神经元的分化和迁移之间的协调将决定大脑的最终大小、形状和功能，直接影响行为77、8、9、10。8,9,10虽然严格控制这些过程显然对于正常的大脑发育至关重要，但监管这些动力学的全球机制并不十分了解。在这里，我们描述了一个工具，以细胞分辨率研究神经系统的发展，允许研究人员可视化在发育中的斑马鱼大脑中与B彩虹的祖细胞和神经元，并通过延时共聚焦显微镜11跟踪他们的行为随着时间的推移。该方法也可以适应可视化发育中的胚胎的其他部分。
为了观察和区分正在发育中的斑马鱼大脑中的细胞，我们采用了”彩虹细胞标记技术11″。Brainbow利用随机确定的三种不同荧光蛋白（FPs）的组合表达来标记细胞群。虽然Brainbow表达式的默认表达式是红色FP dTomato，但由酶Cre重组酶的重组导致mCerulean（青色荧光蛋白，CFP）或黄色荧光蛋白（YFP）12，1312,13的表达。单元格中表达的每个 FP 的总量使其具有独特的色调，从而允许从相邻单元格中明确的视觉区别。此外，当一个祖细胞分裂时，每个子细胞将继承其母细胞的颜色，产生颜色编码的克隆，并允许研究人员追踪细胞系11，14。11,虽然最初用于分析小鼠12的神经元回路，但此后，Brainbow已在各种模型生物中表达，包括斑马鱼15。
我们的技术基于以前的多色标签和成像方法，在活斑马鱼中直接成像多个颜色编码的克隆。由于斑马鱼作为胚胎的光学透明度，非常适合成像实验16，而以前的研究已经利用斑马鱼中的B彩虹来研究各种组织，包括神经系统11、15、17、18、19、20、21、22、23、24、25。11,15,17,18,19,20,21,22,23,24,25,26，,27.直接成像生物体的能力，以及它们的快速外生发育，使斑马鱼成为脊椎动物发展的宝贵模型。与哺乳动物的大脑相比，斑马鱼后脑的整个增殖区随时可供成像，而不会破坏其内源环境6。这允许在活的有机体中进行实验，而不是在体外或固定组织制剂中进行。与固定成像实验相比，体内研究允许纵向设计，生成数小时的数据，可以分析模式，从而增加观察相对罕见事件的可能性。根据感兴趣的事件的速度和长度，研究人员可以选择进行短（1⁄2 小时）或长（高达 ±16 h）的延时成像实验。通过使用斑马鱼热冲击促进剂70（hsp70，hsp），B彩虹表达可以暂时控制28，29。28,此外，由这个启动器诱导的马赛克表达式非常适合标记和跟踪许多克隆11。
直观地识别活脑中的多个克隆的能力是这种方法的一个优点。以前研究克隆在神经系统发育中的作用的重要研究利用逆转录病毒载体使用单个FP或其他易于可视化的蛋白质标记单个祖细胞及其后代。这种标签允许研究人员观察一个克隆随着时间的推移，无论是体外或体内22，30，31，32，33，34，35，36，37，38。,30,31,32,33,34,35,36,37,38与跟踪一个克隆细胞行为的方法不同，Brainbow的不同颜色允许研究人员观察克隆之间的动态。此外，通过使用Brainbow标记大脑中的许多克隆，根据标记单个克隆11的技术，收集了有关克隆行为的其他数据。重要的是，这里描述的方法可以扩大，以产生发育比较之间的鱼经历了不同的遗传或药理操纵18。总体而言，这些优势使表达蓝彩虹的斑马鱼的体内共聚焦成像的延时技术成为探索脊椎动物神经系统发展的理想研究人员，特别是那些对克隆人作用感兴趣的人。
Protocol
涉及动物主题的程序已获得刘易斯和克拉克学院的机构动物护理和使用委员会（IACUC）的批准。 1. 斑马鱼胚胎的微注射 设置野生类型，成年斑马鱼在性别分离的交配池前，下午进行微注射39，40。39, 在微注射的早晨准备DNA溶液。稀释hsp：Zebrabow11质粒DNA浓度为±10 ng/μL，浓度为0.1 mM KCl，以及2.5%…
Representative Results
本节演示了使用本文描述的体内多色延时成像方法可以获得的结果示例。我们显示，在发育中的斑马鱼后脑14的增殖心室区，B彩虹颜色编码的细胞克隆（图1）。 通常，当B彩虹标记的细胞排列在特定的径向光纤上时，它们共享相同的颜色（图1D），可以量化为相对RGB通道权重（图1E）。这表?…
Discussion
该协议描述了一种方法，可视化在发育中的斑马鱼后脑中，前祖细胞和神经元的克隆，并跟随它们在体内使用B彩虹和延时共聚焦显微镜11。与体外或活体研究相比，该协议的主要优点是能够长期直接观察脊椎动物大脑的自然环境中的增殖区。这项技术建立在以前使用逆转录病毒载体标记单个克隆的研究的基础上。相反，使用hsp：Zebrabow 同时构造了许多克隆的颜色代码，允?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢潘文杰、J.Livet和Z.托比亚斯在技术和智力方面的贡献。这项工作得到了国家科学基金会（1553764奖）和M.J.默多克慈善信托基金的支持。
Materials
| 1.5mL transfer pipet | Globe Scientific, Inc. | 134020 | |
| 1-phenyl-2-thiourea (PTU) | Alfa Aesar | L06690 | Diluted to 0.2 mM in E3 to prevent embryo pigmentation |
| 50ml conical tubes | Corning | 352070 | For heat shocking embryos |
| 6 lb nylon fishing line | SecureLine | NMT250 | For making embryo manipulators |
| 7.5mL transfer pipet | Globe Scientific, Inc. | 135010 | |
| CaCl2 | Sigma | C3881 | For E3 |
| Cotton swabs | Puritan | 867-WC NO GLUE | For making embryo manipulators |
| Cre recombinase | New England Biolabs | M0298M | |
| Digital dry bath | Genemate | 490016-616 | Used to store LMA at 40°C |
| Epifluorescence dissection scope | |||
| Glass capillary tubes | World Precision Instruments | TW100F-4 | |
| Incubator | Forma Scientific | 3158 | To maintain embryos at 28°C |
| Injection plate molds | Adaptive Science Tools | TU-1 | |
| Isotemp water bath | Fisher Scientific | 2320 | For heat shocking embryos |
| KCl | AMRESCO | 0395 | For E3 and for DNA solution for injections |
| Laser-scanning confocal microscope | Zeiss | LSM710 | |
| LE agarose | Genemate | E3120 | To create agarose injection plates |
| Low-melt agarose (LMA) | AMRESCO | J234 | |
| Mating tanks | Aquaneering, Inc. | ZHCT100 | |
| Methylene blue | Sigma | M9140 | For E3 |
| MgSO4 | Sigma | 9397 | For E3 |
| Micromanipulator | World Precision Instruments | M3301 | |
| Micropipette Puller | Sutter Instrument Co. | P-97 | |
| MS-222 Tricaine-S | Western Chemical, Inc. | Stock made at 4 mg/mL in reverse osmosis (RO) water, then added dropwise to E3 to final concentration of 0.2 mM to anesthetize embryos | |
| NaCl | J.T. Baker | 4058-01 | For E3 |
| Petri dishes (90 mm, 60 mm) | Genesee Scientific | 32-107G | To house embryos and create imaging chamber (60 mm) |
| Phenol red | Sigma | P0290 | |
| Soft stitch ring markers | Clover Needlecraft, Inc. | 354 | For creating imaging chamber with Petri dish |
| Super glue (Ultra gel control) | Loctite | 1363589 | For making embryo manipulators |
| Syringe needles | Beckton Dickinson | BD329412 | For dechorionating embryos |
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Cite This Article
Cook, Z. T., Brockway, N. L., Weissman, T. A. Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging. J. Vis. Exp. (157), e60593, doi:10.3791/60593 (2020).