多光子时间推移成像实时可视化发展： 迁移神经嵴细胞在斑马鱼胚胎中的可视化
Summary
激光扫描与波长多光子荧光显微镜的先进光学技术组合的实施是为了捕获高分辨率的、 三维的、 实时成像中甘油三酯 (sox10:EGFP) 和甘油三酯 (foxd3:GFP) 斑马鱼胚胎神经嵴迁移。
Abstract
先天性眼和颅颌面畸形反映神经嵴，流动人口的洄游的干细胞，导致整个身体的许多细胞类型的中断。了解神经嵴的生物学已经有限，反映出缺乏可以研究体内的转基因听话模型和实时。斑马鱼是特别重要的发展模式，为研究洄游的细胞群，如神经嵴。若要检查神经嵴迁移到眼睛发育，实施的结合激光扫描显微镜与长波长多光子荧光激发的先进光学技术是拍摄高分辨率的、 三维的、 实时的转基因斑马鱼胚胎，即甘油三酯 (sox10:EGFP) 和甘油三酯 (foxd3:GFP)，在眼睛发育如sox10和foxd3已经在众多的动物模型，以调节早期神经嵴分化，他们可能代表神经嵴细胞的标记所示。多光子延时成像用来辨别的行为和早期眼发展作出贡献的两个神经嵴细胞群的迁徙模式。本议定书提供关于作为一个例子，斑马鱼神经嵴在迁移期间，生成的视频信息，并可进一步用于可视化斑马鱼和其他模式生物的很多建筑的早期发展。
Introduction
先天性眼疾导致儿童失明，往往是由于异常的颅神经嵴。神经嵴细胞是瞬态的干细胞，起身从神经管形成许多机体全身。1,2,3,4,5前脑的两侧和中脑，神经嵴细胞，引起的骨和软骨的中部和额叶区和虹膜、 角膜、 小梁网和前段眼睛的巩膜。4,6,7,8神经嵴细胞从蛤蚧形式咽弓、 下巴和心脏流出道。1,3,4,9,10研究强调了贡献于眼部神经嵴和眼周发展，强调这些细胞在脊椎动物眼睛发育的重要性。事实上，中断的神经嵴细胞迁移和分化导致颅面部、 眼部异常如在阿克森费尔德丽格综合征和彼得斯加综合征。11,12,13,14,15,16,17因此，全面了解迁移、 增殖和分化的这些神经嵴细胞将提供洞察潜在先天性眼疾的复杂性。
斑马鱼是强大的模式生物研究眼发展，斑马鱼眼的结构类似于哺乳动物同行，以及许多基因进化保守斑马鱼和哺乳动物之间。18,19,20此外，斑马鱼胚胎是透明和卵生，促进眼发育的实时可视化。
扩大以前发表的作品，6，，720神经嵴细胞的迁移模式描述了使用多光子荧光延时成像上带有 SRY （性别决定区 Y） 的转录控制下的绿色荧光蛋白 (GFP) 的转基因斑马鱼线-框 10 (sox10) 或叉头框 D3 (foxd3) 基因调控区域。21,22,23,24.多光子荧光延时成像是一项强大的技术，结合先进的光学技术的激光扫描显微镜与长波长多光子荧光激发来捕获的标本，用荧光标记的高分辨率的三维图像。25,26,27多光子激光的使用已经超过标准的共焦显微镜，包括增加的组织的渗透和减少的荧光漂白明显的优势。
使用此方法，两个不同种群的神经嵴细胞迁移和洄游通道的时间变了可区分，即 foxd3 阳性神经嵴细胞在眼周间质和眼睛发育和颅面间质 sox10 阳性神经嵴细胞。使用此方法，介绍了可视化的斑马鱼的眼部和面部神经嵴迁移迁移方法，使其易于观察发展过程中实时调节的神经嵴迁移。
此协议提供信息以便在早期眼发育的甘油三酯 (sox10:EGFP) 和甘油三酯 (foxd3:GFP) 转基因斑马鱼作为示例生成定时视频。此协议可进一步用于任何来自神经嵴细胞在斑马鱼的眼部和面部结构的早期发展的高分辨率的、 三维的、 实时可视化。此外，这种方法进一步可以用于发展的其它组织和器官在斑马鱼和其他动物的模型可视化。
Protocol
The protocol described here was performed in accordance with the guidelines for the humane treatment of laboratory animals established by the University of Michigan Committee on the Use and Care of Animals (UCUCA). 1. Embryo Collection for Time-lapse Imaging Between 3 and 9 pm, set up male and female adult Tg(sox10:EGFP) or Tg(foxd3:GFP) transgenic zebrafish in a divided breeding tank for pairwise mating. NOTE: The Tg(sox10:EGFP) and Tg(foxd3:G…
Representative Results
多光子荧光延时成像产生一系列的视频显示斑马鱼线到眼前段中的甘油三酯 (sox10:EGFP) 和甘油三酯 (foxd3:GFP) 与颅面结构引起的颅神经嵴细胞的迁移模式。作为一个例子， sox10-12 和 30 hpf 之间的积极的神经嵴细胞迁移从神经管的边缘到颅面地区 (视频 1， 图 2)。来自前脑的两侧和中脑细胞迁移的背、 腹面的…
Discussion
多光子延时成像使体内追踪的瞬态和洄游的细胞群体。这个强大的技术可用于研究胚胎过程在真正的时间，并在本研究中，这种方法的结果加强的神经嵴细胞迁移和发展的当前知识。以前的延时成像研究通常利用共聚焦激光扫描显微镜。29,30,31,32在这里，我们目前使用的多光子技术，相比传统?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢托马斯 · 席林请送礼的甘油三酯 (sox10:eGFP) 鱼和玛丽哈洛伦的慈祥礼物甘油三酯(foxd3:GFP)鱼。
Materials
| Breeding Tanks with Dividers | Aquaneering | ZHCT100 | Crossing Tank Set (1.0-liter) Clear Polycarbonate with Lid and Insert |
| M205 FA Combi-Scope | Leica Microsystems CMS GmbH | Stereofluorescence Microscope – FusionOptics and TripleBeam | |
| Sodium Chloride | Millipore (EMD) | 7760-5KG | Double PE sack. CAS No. 7647-14-5, EC Number 231-598-3 |
| Potassium Chloride | Millipore (EMD) | 1049380500 | Potassium chloride 99.999 Suprapur. CAS No. 7447-40-7, EC Number 231-211-8. |
| Calcium Chloride Dihydrate | Fisher Scientific | C79-500 | Poly bottle; 500 g. CAS No. 10035-04-8 |
| Magnesium Sulfate (Anhydrous) | Millipore (EMD) | MX0075-1 | Poly bottle; 500 g. CAS No. 7487-88-9, EC Number 231-298-2 |
| Methylene Blue | Millipore (EMD) | 284-12 | Glass bottle; 25 g. Powder, Certified Biological Stain |
| Sodium Bicarbonate | Millipore (EMD) | SX0320-1 | Poly bottle; 500 g. Powder, GR ACS. CAS No. 144-55-8, EC Number 205-633-8 |
| N-Phenylthiourea | Sigma | P7629-25G | >98%. CAS Number 103-85-5, EC Number 203-151-2 |
| Dimethylsulfoxide | Sigma | D8418-500ML | Molecular Biology grade. CAS Number 67-68-5, EC Number 200-664-3 |
| Tricaine Methanesulfonate | Western Chemical Inc. | MS222 | Tricaine-S |
| Low-Melt Agarose | ISC Bioexpress | E-3112-25 | GeneMate Sieve GQA Low Melt Agarose, 25 g |
| Open Bath Chamber | Warner Instruments | RC-40HP | High Profile |
| Glass Coverslips | Fisher Scientific | 12-545-102 | Circle cover glass. 25 mm diameter |
| High Vacuum Grease | Fisher Scientific | 14-635-5C | 2.0-lb. tube. DOW CORNING CORPORATION 1658832 |
| Quick Exchange Platform | Warner Instruments | QE-1 | 35 mm |
| Stage Adapter | Warner Instruments | SA-20LZ-AL | 16.5 x 10 cm |
| TC SP5 MP multi-photon microscope | Leica Microsystems CMS GmbH | ||
| Mai Tai DeepSee Ti-Sapphire Laser | SpectraPhysics | ||
| Laser Safety Box | Leica Microsystems CMS GmbH | ||
| Leica Application Suite X (LAS X) Software | Leica Microsystems CMS GmbH | ||
| Photoshop CS 6 Version 13.0 x64 Software | Adobe | ||
| iMovie Version 10.1.4 Software | Apple |
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Cite This Article
Williams, A. L., Bohnsack, B. L. Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos. J. Vis. Exp. (126), e56214, doi:10.3791/56214 (2017).