在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后
Summary
该协议描述了在活斑马鱼胚胎的心内膜细胞中 photoconversion 枫荧光蛋白的方法, 能够在房室管和房室心脏瓣膜发育过程中跟踪心内膜细胞。.
Abstract
在胚胎发生过程中, 细胞在细胞行为中发生动态变化, 破译这些变化背后的细胞逻辑是发育生物学领域的一个基本目标。photoconvertible 蛋白的发现和发展极大地帮助我们理解这些动态变化, 提供了一种光学突出细胞和组织的方法。然而, 虽然 photoconversion, 时间推移显微镜, 和随后的图像分析已经证明是非常成功的揭露细胞动力学的器官, 如大脑或眼睛, 这种方法一般不使用在发展中的心脏由于心脏循环过程中心的快速运动所带来的挑战。该协议由两部分组成。第一部分介绍了在斑马鱼房室管 (photoconverting) 和房室心脏瓣膜发育过程中, 对心内膜细胞 (EdCs) 进行追踪和跟踪的方法。该方法包括用药物暂时停止心脏, 以便准确 photoconversion 发生。心脏被允许恢复跳动后, 药物的清除和胚胎发育继续正常, 直到心脏再次停止的高分辨率成像 photoconverted EdCs 在以后的发展时间点。该协议的第二部分描述了一种图像分析方法, 通过将三维结构中的荧光信号映射到二维地图上来量化 photoconverted 或非 photoconverted 区域在幼胚中的长度.同时, 该协议的两个部分允许一个人检查组成斑马鱼和房室心脏瓣膜的细胞的起源和行为, 并有可能被应用于研究突变体, morphants, 或胚胎, 已治疗破坏 AVC 和/或阀门发展的试剂。
Introduction
斑马鱼目前是最重要的脊椎动物模型之一, 研究细胞和发育过程的在体内。这主要是由于斑马鱼的光学透明度和顺从遗传学, 这使得它成为一个强大的模型应用光学技术涉及基因编码 photoresponsive 蛋白技术1。针对心脏发育的研究, 斑马鱼通过扩散获得足够的氧气, 即使没有心跳的突变体也能在发育的第一周存活下来, 允许分析发育基因的影响和扰动在大多数脊椎动物中, 血流在心脏形态发生是不可能的2。
斑马鱼心脏管由24小时后受精 (hpf) 形成。形成后不久, 心脏管开始积极跳动。由 36 hpf, 清晰的收缩将心房与心室隔开。这一收缩区称为房室道 (AVC), 该区域的细胞从鳞状形态学转变为立方形态学3。斑马鱼房室瓣膜形态发生开始约48小时后受精。5天后受精, 两个瓣膜小叶延伸到 AVC 和防止血液从心室的背部流到心房在心脏周期的4。当心脏的快速跳动使得通过传统的时间失效显微镜5,6时, 跟踪细胞变得困难。此协议从骏马et., 20167, 描述使用 Tg 的方法(fli1a: gal4FF瑞银, 无人参与: 枫)8斑马鱼转基因线, 其中 photoconvertible 蛋白枫表示在内皮细胞, 包括心内膜。药物 23-butanedione 2-肟 (BDM) 用于暂时停止心脏跳动, 允许在36和 55 EdCs 之间精确 photoconversion hpf, 以及在特定发育时间点的 photoconverted EdCs 的高分辨率成像。以前已显示, 使用此方法的 EdCs photoconverted 在 photoconversion7之后的五天或更远的时间内可以与未转换的邻居保持可区分性。该协议还详细介绍了一种用于在 48 hpf 的胚胎中 photoconverted EdCs 的图像分析方法, 它已成功地用于在 AVC 开发过程中的组织运动 (路易吉·博塞利et), 按下)9。我们希望读者能发现这项议定书对于研究正常胚胎中的 AVC 和瓣膜发育, 以及突变体、morphants 或药物治疗的胚胎都是有用的。有关在斑马鱼开发过程中使用 photoconvertible 蛋白进行细胞跟踪的更通用的协议, 请查看由伦巴多et, 201210的文章。
Protocol
Representative Results
Discussion
photoconversion 的定时: 尽管枫在 EdCs 中仍然保持明亮的表达, 即使在 96 hpf, 也应该注意到, 随着胚胎的生长, 激光在到达 AVC 之前扩散得更多, 使得 photoconversion 的限制枫更加困难。在胚胎阶段比 55 hpf, 心室和心房的膨胀也意味着, 用于 photoconversion 的紫激光束不能到达 AVC 细胞, 而不首先穿过心房或心室。这意味着, 在 55 hpf 以后, 为了 photoconvert EdCs, 你必须也 photoconvert 另外的 EdCs 在心房和脑室。<…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢安妮-玛丽·韦尔坎贝里弗朗辛·杜舍芒, 丹尼斯弗朗辛·杜舍芒, 纳塔莉 Faggianelli 和巴西莱 Gurchenkov 帮助设计和制作本协议中描述的模具。这项工作得到了 FRM (DEQ20140329553)、国家情报局 (ANR-15-CE13-0015-01、ANR-12-ISV2-0001-01)、EMBO 青年调查员计划、欧洲共同体、紧急救济机构 N°682938 Evalve 和赠款 ANR-10-LABX-0030-INRT 的支助, 由一个法国国有基金管理, 由研究所的框架计划 Investissements 艾文莉标记 ANR-10-IDEX-0002-02。
Materials
| Materials | |||
| Necessary equipment for raising fish and collecting eggs (see the Zebrafish Book22 for details). | |||
| Stereomicroscope | |||
| Upright confocal microscope (Equipped with lasers operating at 488 nm, 561 nm, and 405 nm, a tunable multiphoton laser, and a Leica HCX IRAPO L, 25 ×, N.A. 0.95 objective) | Leica | Leica SP8 | |
| Heat block | ThermoScientific | 88870001 | |
| 35 mm x 10 mm tissue culture dish | Falcon | 353001 | |
| 6 well plate | Falcon | 353046 | |
| Petri dish | |||
| Forceps | |||
| Glass pipette | |||
| Mold | |||
| Reagents | |||
| 8 mg/mL Tricaine stock solution | |||
| 1 M BDM stock solution | |||
| UltraPure low melting point agarose | Invitrogen | 16520-050 | |
| Agarose | Lonza | 50004 | |
| PTU (1-phenyl-2-thiourea) | Sigma Aldrich | P7629 | |
| Embryo medium: 30x stock solution | |||
| Software | |||
| Matlab equipped with the Image Analysis, Curve Fitting, Bio-Formats Toolboxes | MathWorks | ||
| 8 mg/mL Tricaine stock solution | |||
| 200 mg of tricaine powder (Ethyl 3-aminobenzoate methanesulfonic acid) | Sigma-Aldrich | E10521 | |
| 25 mL Danieau | |||
| Adjust to pH 7, aliquot and store at -20 °C | |||
| 1 M BDM stock solution | |||
| 1 g BDM | Sigma-Aldrich | 112135 | |
| 10 mL ddH2O | |||
| Aliquot and store at -20 °C | |||
| Embryo medium: 30x stock solution | |||
| 50.7 g NaCl | Sigma-Aldrich | 7647-14-5 | |
| 0.78 g KCl | Sigma-Aldrich | 7447-40-7 | |
| 1.47 g Magnesium sulfate | Sigma-Aldrich | 7487-88-9 | |
| 2.1 g Calcium nitrite tetrahydrate | Sigma-Aldrich | 13477-34-4 | |
| 19.52 g HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) | Sigma-Aldrich | 7365-45-9 | |
| Adjust to pH 7.2 and store at room temperature | |||
| Mold | |||
| PlasCLEAR resin | Asiga | ||
| Plus 39 Freeform Pico 3D printer | Asiga |
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