Summary

胚胎和幼体斑马鱼的高分辨率细胞移植

Published: July 05, 2024
doi:

Summary

在这里,我们提出了一种方案,可以在受精后至少 1 到 7 天之间的任何阶段将具有高空间和时间分辨率的细胞移植到斑马鱼胚胎和幼虫中。

Abstract

发育和再生是通过遗传编码的时空动态细胞相互作用过程发生的。使用动物之间的细胞移植来追踪细胞命运并诱导供体细胞和宿主细胞的遗传、空间或时间特性不匹配,是检查这些相互作用性质的有力手段。雏鸡和两栖动物等生物分别为我们理解发育和再生做出了重要贡献,这在很大程度上是因为它们适合移植。然而,这些模型的力量受到遗传易处理性低的限制。同样,主要的遗传模式生物对移植的适应性较低。

斑马鱼是发育和再生的主要遗传模型,虽然细胞移植在斑马鱼中很常见,但它通常仅限于在囊胚和原肠发育的早期阶段转移未分化的细胞。在本文中,我们提出了一种简单而强大的方法,该方法将斑马鱼移植窗口扩展到受精后至少 1 到 7 天之间的任何胚胎或幼虫阶段。这种方法的精确性允许在供体和宿主动物中以近乎完美的空间和时间分辨率移植低至一个细胞。虽然我们在这里强调胚胎和幼虫神经元的移植分别用于研究神经发育和再生,但这种方法适用于广泛的祖细胞和分化细胞类型和研究问题。

Introduction

细胞移植作为发育生物学的基础技术有着悠久而传奇的历史。在 20世纪之交,使用物理操作来扰乱发育过程的方法,包括移植,将胚胎学从观察科学转变为实验科学 1,2在一项具有里程碑意义的实验中,Hans Spemann 和 Hilde Mangold 将蝾螈胚胎的背胚孔唇异位移植到宿主胚胎的另一侧,诱导附近的组织形成次级体轴3。该实验表明,细胞可以诱导其他细胞接受某些命运,随后,移植发展成为一种强大的方法,用于询问发育生物学中有关能力和细胞命运决定、细胞谱系、诱导能力、可塑性和干细胞效力的关键问题 1,4,5

最近的科学进展扩大了移植方法的力量。1969 年,Nicole Le Douarin 发现核仁染色可以区分鹌鹑雏鸡嵌合体中的起源物种,从而可以追踪移植的细胞及其后代6。这一概念后来因转基因荧光标记物和先进成像技术的出现而得到加强5,并已被用于追踪细胞命运 6,7、识别干细胞及其效力 8,9 以及追踪大脑发育过程中的细胞运动10。此外,分子遗传学的兴起促进了不同基因型的宿主和供体之间的移植,支持对发育因子的自主和非自主功能的精确解剖11

移植还通过阐明调节再生组织生长和模式的细胞身份和相互作用,为再生研究做出了重要贡献,特别是在涡虫和蝾螈等具有强大再生能力的生物体中。移植研究揭示了效力12、空间模式的原理 13,14、特定组织的贡献15,16 以及细胞记忆12,17 在再生中的作用。

斑马鱼是研究发育和再生(包括神经系统)的主要脊椎动物模型,因为它们的遗传程序保守、遗传易处理性高、外部受精、离合器大小大和光学清晰度 18,19,20。斑马鱼在早期发育阶段也非常适合移植。最突出的方法是在囊胚或原肠胚阶段将细胞从标记的供体胚胎移植到宿主胚胎,以产生嵌合动物。在囊胚阶段移植的细胞会随着 epiboly 开始时分散和分散,在胚胎中产生标记细胞和组织的马赛克21。胃移植允许根据粗略的命运图对移植的细胞进行一些靶向,因为盾牌形成并且可以确定 A-P 和 DV 轴21。所得的嵌赛图在确定基因是否自主作用于细胞、测试细胞定型以及绘制整个发育过程中的组织运动和细胞迁移图谱方面很有价值 5,11。花叶斑马鱼可以通过多种方式产生,包括电穿孔22、重组23 以及 F0 转基因24 和诱变25,但移植在空间、时间以及细胞数量和类型方面提供了最大的可操作性和精度。斑马鱼移植的现状主要局限于早期的祖细胞,只有少数例外,包括受精后前 10-30 小时移植脊髓运动神经元26,27、视网膜神经节细胞28,29 和神经嵴细胞 (hpf)30,以及成年斑马鱼的造血细胞和肿瘤细胞 5,31.将移植方法扩展到广泛的年龄、分化阶段和细胞类型将大大增强这种方法的能力,以提供对发育和再生过程的见解。

在这里,我们展示了一种灵活而强大的高分辨率细胞移植技术,可在受精后至少 7 天内对斑马鱼胚胎和幼虫有效。在靶组织中表达荧光蛋白的转基因宿主和供体鱼可用于提取单细胞并以近乎完美的空间和时间分辨率进行移植。斑马鱼胚胎和幼虫的光学清晰度允许在宿主动物发育或再生时对移植的细胞进行实时成像。这种方法以前已被用于检查时空信号动力学如何影响胚胎中的神经元身份和轴突导向32,并检查内在和外在因素在幼鱼再生过程中促进轴突导向的逻辑 33。虽然我们在这里专注于分化神经元的移植,但我们的方法广泛适用于许多阶段和组织的未分化和分化细胞类型,以解决发育和再生中的问题。

Protocol

该程序与活体斑马鱼相关的所有方面均已获得明尼苏达大学机构动物护理和使用委员会 (IACUC) 的批准,并按照 IACUC 指南执行。 1. 移植装置的一次性初始设置(图 1) 按照制造商的说明组装移植显微镜。注意:该协议使用具有 40 倍水浸物镜的正置荧光显微镜。 组装粗略和精细的显?…

Representative Results

通过使用荧光显微镜在移植后的适当时间点观察宿主动物中荧光标记的供体细胞,可以直接观察移植实验的结果。在这里,我们以 3 dpf 移植了单个前迷走神经神经元。然后将宿主动物孵育 12 或 48 小时,麻醉,安装在玻璃盖玻片上的 LMA 中,并用共聚焦显微镜成像(图 5)。在移植后 12 小时 (hpt),我们观察到成功移植的供体神经元(<strong class="xfi…

Discussion

一个多世纪以来,发育和再生生物学一直依靠移植实验来研究细胞信号传导和细胞命运决定的原理。斑马鱼模型已经代表了遗传和移植方法的强大融合。在囊胚和原肠阶段移植以产生马赛克动物很常见,但它可以解决的问题类型有限。晚期移植很少见,尽管已经报道了分别以 16-18 hpf 和 30-33 hpf 移植胚胎脊髓运动神经元和视网膜神经节细胞的方法 26,27,28。</sup…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢 Cecilia Moens 接受斑马鱼移植培训;Marc Tye 出色的鱼类护理;以及 Emma Carlson 对手稿的反馈。这项工作得到了 NIH 对 AJI 的 NS121595 资助的支持。

Materials

10 mL "reservoir syringe" Fisher Scientific 14-955-459
150 mL disposable vacuum filter, .2 µm, PES Corning 431153
20 x 12 mm heating block Corning 480122
3-way stopcock Braun Medical Inc. 455991
3 x 1 Frosted glass slide VWR 48312-004
40x water dipping objective Nikon MRD07420
Calcium chloride dihydrate Sigma-Aldrich C3306
Coarse Manipulator Narishige MN-4
Custom microsyringe pump University of Oregon N/A Manufactured by University of Oregon machine shop (tsa.uoregon@gmail.com). A commercially available alternative is listed below.
Dumont #5 Forceps Fine Science Tools 1129500
Eclipse FN1 "Transplant Microscope" Nikon N/A
electrode handle World Precision Instruments 5444
Feather Sterile Surgical Blade, #11 VWR 21899-530
Fine micromanipulator, Three-axis Oil hydraulic  Narishige MMO-203
HEPES pH 7.2 Sigma-Aldrich H3375-100G
High Precision #3 Style Scalpel Handle Fisher Scientific 12-000-163
Kimble Disposable Borosilicate Pasteur Pipette, Wide Tip, 5.75 in DWK Life Sciences 63A53WT
KIMBLE Chromatography Adapter  DWK Life Sciences 420408-0000
Kimwipes Kimberly-Clark Professional 34120
Light Mineral Oil Sigma-Aldrich M3516-1L
LSE digital dry bath heater, 1 block, 120 V Corning 6875SB
Manual microsyringe pump World Precision Instruments MMP Commercial alternative to custom microsyringe pump
Microelectrode Holder World Precision Instruments MPH310
MicroFil Pipette Filler World Precision Instruments MF28G67-5
Nail Polish Electron MIcroscopy Sciences 72180
Nuclease-free water VWR 82007-334
P-97 Flaming/Brown Type Micropipette Puller Sutter Instruments P-97
Penicillin-streptomycin Sigma-Aldrich p4458-100ML 5,000 units penicillin and 5 mg streptomycin/mL
pipette pump 10 mL Bel-Art 37898-0000
Potassium chloride Sigma-Aldrich P3911
Professional Super Glue Loctite LOC1365882
Round-Bottom Polystyrene Test Tubes Falcon 352054
Sodium chloride Sigma-Aldrich S9888
Stage micrometer Meiji Techno America MA285
Syringes without Needle, 50 mL BD Medical 309635
Tricaine Methanosulfonate Syndel USA SYNCMGAUS03
Trilene XL smooth casting Fishing line Berkley XLFS6-15
Tubing, polyethylene No. 205 BD Medical 427445
UltraPure Low Melting Point Agarose Invitrogen 16520050
Wiretrol II calibrated micropipettes Drummond 50002010

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Cite This Article
Qian, L. S., Ibrahim, R., Isabella, A. J. High-resolution Cell Transplantation in Embryonic and Larval Zebrafish. J. Vis. Exp. (209), e67218, doi:10.3791/67218 (2024).

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