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Biologia do Desenvolvimento

研究脊椎动物轴向伸长和分割的三维和四维可视化分析方法

Published: February 28, 2021
doi:
10.3791/62086
, , ,
1Instituto Gulbenkian de Ciência, 2Faculdade de Ciências da Universidade de Lisboa, 3NOVA School of Science and Technology

Summary

在这里，我们描述了计算工具和方法，这些工具和方法允许在轴向伸长和分割的背景下可视化和分析小鼠胚胎的三维和四维图像数据，通过toto光学投影断层扫描以及通过使用多光子显微镜进行实时成像和全贴装免疫荧光染色获得。

Abstract

体细胞发生是脊椎动物胚胎发育的标志。多年来，研究人员一直在使用包括离体和体外方法在内的各种技术在各种生物体中研究这一过程。然而，大多数研究仍然依赖于对二维（2D）成像数据的分析，这限制了对发育过程的正确评估，例如轴向延伸和涉及复杂3D空间中高度动态相互作用的发生。在这里，我们描述了允许小鼠实时成像采集，数据集处理，可视化和分析3D和4D的技术，以研究参与这些发育过程的细胞（例如，神经中胚层祖细胞）。我们还为小鼠胚胎中的光学投影断层扫描和全挂载免疫荧光显微镜（从样品制备到图像采集）提供分步方案，并展示了我们开发的用于处理和可视化3D图像数据的管道。我们扩展了其中一些技术的使用，并突出了不同可用软件（例如，Fiji/ImageJ，Drishti，Amira和Imaris）的特定功能，这些功能可用于提高我们当前对轴向延伸和somite形成（例如，3D重建）的理解。总而言之，这里描述的技术强调了3D数据可视化和分析在发育生物学中的重要性，并可能有助于其他研究人员在脊椎动物轴向延伸和分割的背景下更好地处理3D和4D图像数据。最后，这项工作还采用了新的工具来促进脊椎动物胚胎发育的教学。

Introduction

脊椎动物身体轴的形成是胚胎发育过程中发生的高度复杂和动态的过程。在原肠胚形成结束时[在小鼠中，在胚胎日（E）8.0左右]，一组称为神经中层祖细胞（NMPs）的上胚层祖细胞成为头到尾序列中轴向延伸的关键驱动因素，在颈部，躯干和尾巴形成过程中产生神经管和旁轴中皮组织1，2，3，4.有趣的是，这些NMP在尾部外胚层中占据的位置似乎在分化成中胚层或神经外胚层5的决定中起着关键作用。虽然我们目前缺乏NMP的精确分子指纹，但这些细胞通常被认为共表达T（Brachyury）和Sox25，6。调节NMP命运决策的确切机制（即，它们是否采用神经或中皮途径）才刚刚开始被精确定义。 Tbx6 在原始条纹区域的表达是NMP命运决定的早期标志物，因为该基因参与中胚层6，7的诱导和规范。有趣的是，早期的中胚层细胞似乎表达高水平的Epha18，Wnt / β-catenin信号传导以及 Msgn1 也被证明在轴旁中胚层分化和sumite形成中起重要作用9，10。在单细胞水平上对NMP进行完整的时空分析肯定有助于充分了解控制中胚层规范的分子机制。

体细胞（椎骨前体）的形成是脊椎动物的一个关键特征。在轴向伸长过程中，轴旁中胚层被分割成一系列称为somites的双侧重复单元。茴香的数量和形成新节段所需的时间因物种11，12而异。Somitogenesing涉及周期性信号振荡（称为“分割时钟”），可以通过预定中胚层（例如 Lfng）中Notch，Wnt和Fgf信号通路的几个基因的循环表达来观察到11，12。目前的体细胞生成模型还假设存在“成熟波前”，这是一系列复杂的信号梯度，涉及Fgf，Wnt和视黄酸信号传导，定义了每个新索米特后边界的位置。因此，“分割时钟”和“成熟波前”之间的协调相互作用对于这些椎骨前体模块的产生至关重要，因为这些关键形态发生过程中的扰动可导致胚胎致死性或先天性畸形（例如脊柱侧弯）的形成13，14，15。

尽管最近在成像技术、生物影像分析方法和软件方面取得了重大进展，但大多数轴向伸长和体分裂的研究仍然依赖于单一/分离的二维图像数据（例如切片），这不允许完整的多维组织可视化，并使胚胎发育过程中发生的病理性畸形（即由于突变） 与 正常形态变异之间的明确区分复杂化16.3D成像已经发现了新的形态发生运动，以前没有通过标准2D方法17，18，19，20识别，突出了toto成像在了解脊椎动物体发生和轴向伸展机制方面的力量。

小鼠胚胎的3D和4D显微镜，特别是实时成像，在技术上具有挑战性，需要在样品制备，图像采集和数据预处理过程中采取关键步骤，以便进行准确和有意义的时空分析。在这里，我们描述了用于小鼠胚胎的活体成像和全装载免疫荧光染色的详细方案，可用于在轴向延伸和分割期间研究NMP和中胚层细胞。此外，我们还描述了一种用于老年胚胎和胎儿的光学投影断层扫描（OPT）的方案，该协议允许3D可视化和量化可能由体细胞生成期间的问题（例如骨融合和脊柱侧弯）引起的病理异常13，21，22。最后，我们说明了3D成像重建在脊椎动物分割和轴向伸长的研究和教学中的力量。

Protocol

涉及动物的实验遵循葡萄牙（Portaria 1005/92）和欧洲（指令2010/63 / EU）关于住房，畜牧业和福利的立法。该项目由“塞内加尔古尔本基安研究所”伦理委员会和葡萄牙国家实体“食品和兽医协会”（许可证编号：014308）审查和批准。 1. 3D和4D成像的样品制备 注意：在这里，我们提供了有关如何解剖和制备小鼠E8.25至E10.5胚胎以进行活体成像（1.1），E7.5至E11.5?…

Representative Results

本文显示的活体和免疫荧光成像的代表性结果均采用双光子系统获得，其中20×1.0 NA水物镜，激发激光调谐至960nm，GaAsP光电探测器（如Dias等人（2020）43中所述。光学投影断层扫描是使用定制的OPenT扫描仪完成的（如Gualda等人（2013）28中所述）。 实时成像（4D 分析）对小鼠胚胎中轴向延伸期间LuVeLu报告活性的代表性分析，根据…

Discussion

轴向伸长和分割是脊椎动物胚胎发育过程中发生的两个最复杂和最动态的过程。一段时间以来，使用3D和4D成像与单细胞跟踪已经应用于研究斑马鱼和鸡胚胎的这些过程，其可及性和培养条件有助于复杂的成像19，44，45，46，47，48，<sup class="…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们要感谢Olivier Pourquié和Alexander Aulehla提供的LuVeLu报告菌株，SunJin实验室提供的RapiClear测试样本，Hugo Pereira帮助使用BigStitcher，Nuno Granjeiro帮助建立实时成像设备，IGC动物设施以及Mallo实验室的过去和现在的成员在这项工作过程中提供了有用的评论和支持。

我们感谢IGC高级成像设施的技术支持，该设施由葡萄牙资金参考# PPBI-POCI-01-0145-FEDER-022122和ref# PTDC/BII-BTI/32375/2017提供支持，由里斯本区域业务计划（Lisboa 2020）共同资助，根据葡萄牙2020年伙伴关系协议，通过欧洲区域发展基金（FEDER）和Fundação para a Ciência e a Tecnologia（FCT，葡萄牙）。本手稿中描述的工作得到了LISBOA-01-0145-FEDER-030254（FCT，葡萄牙）和SCML-MC-60-2014（葡萄牙Santa Casa da Misericórdia）向M.M，研究基础设施Congento，项目LISBOA-01-0145-FEDER-022170以及博士奖学金PD / BD / 128426 / 2017到A.D.的支持。

Materials

Agarose low gelling temperature Sigma A9414 Used to mounting embryos (e.g. for OPT)
Amira software Thermofisher – Commerial software tool
Anti-Brachyury (Goat polyclonal) R and D Systems AF2085 RRID:AB_2200235 For immunofluorescence
Anti-Sox2 (Rabbit monoclonal) Abcam ab92494 RRID:AB_10585428 For immunofluorescence
Anti-Tbx6 (Goat polyclonal) R and D Systems AF4744 RRID:AB_2200834 For immunofluorescence
Anti-Laminin111 (Rabbit polyclonal) Sigma L9393 RRID:AB_477163 For immunofluorescence
Anti-goat 488 (Donkey polyclonal) Molecular Probes A11055 RRID:AB_2534102 For immunofluorescence
Anti-rabbit 568 (Donkey polyclonal) ThermoFisher   Scientific A10042 RRID:AB_2534017 For immunofluorescence
Benzyl Alcohol (99+%) (any) – Used to clear embryos (component of BABB)
Benzyl Benzoate (99+%) (any) – Used to clear embryos (component of BABB)
Bovine serum albumin Biowest P6154 For immunofluorescence
Coverglass 20×20 mm #0 (any) – 100um thick
Coverglass 20×20 mm #1 (any) – 170um thick
Coverglass 20×60 mm #1.5 (any) – To use as “slides”
DAPI (4’,6-Diamidino-2- Phenylindole Dihydrochloride) Life Technologies D3571 For immunofluorescence
Drishti software (open source) – Free software tool
EDTA Sigma ED2SS For demineralization
Fiji/ImageJ software (open source) – Free software tool
Glycine NZYtech MB01401 For immunofluorescence
Huygens software Scientific Volume Imaging – Commerial software tool
HyClone defined fetal bovine serum GE Healthcare #HYCLSH30070.03 For live imaging
Hydrogen peroxide solution 30 % Milipore 1085971000 For clearing
Imaris software Bitplane / Oxford instruments – Commerial software tool
iSpacers SunJin Lab (varies) Use as spacers for preparations
L-glutamine Gibco #25030–024 For live imaging medium
Low glucose DMEM Gibco 11054020 For live imaging medium
M2 medium Sigma M7167 To dissect embryos
Methanol VWR VWRC20847.307 For dehydration and rehydration steps
Methyl salicylate Sigma M6752 Used to clear embryos
Paraformaldehyde Sigma P6148 Used in solution to fix embryos
Penicillin-streptomycin Sigma #P0781 For live imaging medium
PBS (Phosphate-buffered saline solution) Biowest L0615-500 –
RapiClear SunJin Laboratory RapiClear 1.52 Used to clear embryos
Secure-Sea hybridization chambers Sigma C5474 Use as spacers for preparations
simLab software SimLab soft – Commerial software tool
Slide, depression concave glass – 75×25 mm (any) – To mount thick embryos.
Triton X-100 Sigma T8787 For immunofluorescence
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Tags

Keywords: 3D Visualization4D VisualizationAxial ElongationSegmentationVertebrate DevelopmentMouse EmbryoLive ImagingImmunofluorescenceCongenital MalformationScoliosisIn Vitro Model
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Dias, A., Martins, G. G., Lopes, A., Mallo, M. Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation. J. Vis. Exp. (168), e62086, doi:10.3791/62086 (2021).
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