在Vitro重建空间细胞接触模式与孤立的Caenorhabditis elegans胚胎冲击器和粘性聚苯乙烯珠
Summary
多细胞系统的组织复杂性混淆了细胞外线索与单个细胞行为之间的因果关系的识别。在这里，我们提出了一种方法，研究接触依赖线索和分裂轴之间的直接联系使用C.elegans胚胎胚芽和粘合聚苯乙烯珠子。
Abstract
在多细胞系统中，单个细胞被来自邻近细胞和环境的各种物理和化学线索所包围。这种组织的复杂性混淆了外在线索和细胞动力学之间的因果关系的识别。合成重组的多细胞系统克服了这个问题，使研究人员能够测试特定的线索，同时消除其他线索。在这里，我们提出了一种方法，以重组细胞接触模式与孤立的Caenorhabditis elegans爆炸和粘胶聚苯乙烯珠。这些程序包括蛋壳去除、通过破坏细胞粘附性进行胚分裂、制备粘胶聚苯乙烯珠子以及重组细胞-细胞或细胞珠接触。最后，本文介绍了该方法在胚胎发育中研究细胞分裂轴的定位，有助于调节发育胚胎中的空间细胞模式和细胞命运规范。这种健壮、可重复和多功能的体外方法有助于研究空间细胞接触模式与细胞反应之间的直接关系。
Introduction
在多细胞发育过程中，单个细胞的细胞行为（例如，分轴）由各种化学和物理线索指定。理解单个细胞如何解释这些信息，以及它们如何将多细胞组装作为一个紧急属性进行调节，是形态发生研究的最终目标之一。模型有机体C.elegans对细胞级形态形成调节的理解有显著贡献，如细胞极性1、细胞分裂模式1、细胞命运决定2、组织尺度调节，如神经元接线3和器官发生4、5。虽然有各种各样的遗传工具可用，组织工程方法是有限的。
在C.elegans研究中最成功的组织工程方法是经典的胚乳分离6;由于C.elegans胚胎被蛋壳和渗透屏障7包围，其去除是这种方法的主要程序之一。虽然这种爆破器隔离方法能够以简化的方式重组细胞-细胞接触，但它不允许消除不需要的线索;细胞接触仍然构成机械（例如，附着力）和化学线索，从而限制了我们完全分析线索和细胞行为之间的因果关系的能力。
本文介绍的方法使用Carboxylate改性聚苯乙烯珠子，该珠子可以共价地与包括蛋白质在内的任何胺反应分子结合为配体。特别是，我们使用一种抗胺反应形式的罗达明红-X作为配体，使珠子既可目视跟踪，又粘附在细胞上。配体分子的珠表面和原胺组的甲苯基组由水溶性甲酰胺1-乙基-3-（二甲基氨基丙基）卡博迪米（EDAC）8、9结合。获得粘合珠子允许机械提示对细胞动力学10的影响。我们已经使用这种技术来识别细胞分裂方向10所需的机械线索。
Protocol
1. 胶粘剂聚苯乙烯珠子的制备 注：此协议不需要无菌技术。 在1.5 mL微离心管中称量10mg的Carboxylate改性聚苯乙烯珠子。 要清洗珠子，在管中加入1 mL的2-（N-变形）甲酸（MES）缓冲液。由于MES缓冲液不含磷酸盐和醋酸盐，可降低卡博迪米德的活性，适合用于蛋白质耦合反应。涡旋管混合珠子。 通过台式离心机在 2，000 x g下旋转管子 60 s。 通?…
Representative Results
对于珠子制备，我们确定了表达GFP-肌苷II和mCherry-组蛋白的转基因菌株的红-X琥珀酯的最佳量（图1A-D）。我们使用 mCherry 标记组蛋白作为细胞周期进展的标记。由于罗达明红X-X和mCherry将由561nm激光照明，因此罗达明红X-X信号的最佳强度与组蛋白相当，可以同时成像细胞和珠子。例如，使用 0.005 μg/mL Rhodamine 红-X 琥珀酯处理的珠子的荧光信号太弱，无法可视化珠（…
Discussion
重建简化的细胞接触模式将使研究人员测试特定细胞接触模式在形态形成不同方面的作用。我们用这种技术来表明细胞分裂轴是由粘接珠体接触控制的。由于分轴规范对多细胞发育至关重要，有助于形成形态生成14、干细胞分裂15、16和组织平衡15、16，这种方法应该能够阐?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢詹姆斯·普里斯和布鲁斯·鲍曼的建议，并提供C.elegans菌株，唐·莫曼，小田美祖本，和生命科学研究所成像核心设施，用于共享设备和试剂，奥伊·希罗亚苏，丽莎·费尔南多，明吉金维护C.elegans和批判性阅读我们的手稿。我们的工作得到了加拿大自然科学和工程研究理事会（NSERC）（RGPIN-2019-04442）的支持。
Materials
| 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride | Alfa Aesar | AAA1080703 | For the bead preparation |
| Aspirator Tube Assembly | Drummond | 21-180-13 | For the blastomere isolation. |
| Caenorhabditis elegans strain: N2, wild-type | Caenorhabditis Genetics Center | N2 | Strain used in this study |
| Caenorhabditis elegans strain: KSG5, genotype: zuIs45; itIs37 | in house | KSG5 | Strain used in this study |
| Calibrated Mircopipets, 10 µL | Drummond | 21-180-13 | For the blastomere isolation |
| Carboxylate-modified polystyrene beads (30 µm diameter) | KISKER Biotech | PPS-30.0COOHP | For the bead preparation |
| CD Lipid Concentrate | Life Technologies | 11905031 | For the blastomere isolation. Work in the tissue culture hood. |
| Clorox | Clorox | N. A. | For the blastomere isolation. Open a new bottle when the hypochlorite treatment does not work well. |
| Coverslip holder | In house | N.A. | For the blastomere isolation. |
| Dissecting microscope: Zeiss Stemi 508 with M stand. Source of light is built-in LED. Magnification of eye piece is 10X. | Carl Zeiss | Stemi 508 | For the blastomere isolation. |
| Fetal Bovine Serum, Qualified One Shot, Canada origin | Gibco | A3160701 | For the blastomere isolation. Work in the tissue culture hood. |
| General Use and Precision Glide Hypodermic Needles, 25 gauge | BD | 14-826AA | For the blastomere isolation |
| Inulin | Alfa Aesar | AAA1842509 | For the blastomere isolation |
| MEM Vitamin Solution (100x) | Gibco | 11120052 | For the blastomere isolation. |
| MES (Fine White Crystals) | Fisher BioReagents | BP300-100 | For the bead preparation |
| Multitest Slide 10 Well | MP Biomedicals | ICN6041805 | For the blastomere isolation |
| PBS, Phosphate Buffered Saline, 10 x Powder | Fisher BioReagents | BP665-1 | For the bead preparation |
| Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140148 | For the blastomere isolation. |
| Polyvinylpyrrolidone | Fisher BioReagents | BP431-100 | For the blastomere isolation |
| Potassium Chloride | Bioshop | POC888 | For the blastomere isolation |
| Rhodamine Red-X, Succinimidyl Ester, 5-isomer | Molecular Probes | R6160 | For the bead preparation |
| Schneider’s Drosophila Sterile Medium | Gibco | 21720024 | For the blastomere isolation. Work in the tissue culture hood. |
| Sodium Chloride | Bioshop | SOD001 | For the blastomere isolation |
| Sodium Hydroxide Solution, 10 N | Fisher Chemical | SS255-1 | For the blastomere isolation |
| Spinning disk confocal microscope: Yokogawa CSU-X1, Zeiss Axiovert inverted scope, Quant EM 512 camera, 63X NA 1.4 Plan apochromat objective lens. System was controlled by Slidebook 6.0. | Intelligent Imaging Innovation | N.A. | For live-imaging |
| Syringe Filters, PTFE, Non-Sterile | Basix | 13100115 | For the blastomere isolation. |
| Tygon S3 Laboratory Tubing,, Formulation E-3603, Inner diameter 3.175 mm | Saint Gobain Performance Plastics | 89403-862 | For the blastomere isolation. |
| Tygon S3 Laboratory Tubing,, Formulation E-3603, Inner diameter 6.35 mm | Saint Gobain Performance Plastics | 89403-854 | For the blastomere isolation. |
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Citazione di questo articolo
Hsu, C. R., Xiong, R., Sugioka, K. In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads. J. Vis. Exp. (153), e60422, doi:10.3791/60422 (2019).