成像清除胚胎和出生后心脏在单细胞分辨率
Summary
We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.
Abstract
在全心脏水平单克隆跟踪和分析可以心脏发育过程中确定心脏祖细胞的行为和分化，并允许正常和异常的心脏形态发生的细胞和分子基础的研究。回顾单克隆分析最近的新兴技术使心脏形态发生在单细胞分辨率的研究是可行的。然而，组织的不透明度和心脏的成像深度的光散射在单细胞分辨率提高阻碍全心脏成像。为了克服这些障碍，整个胚胎清算系统，可以使心脏的高度透明为照明和检测必须发展。幸运的是，在过去的几年中，已报告的全生物结算系统如透明度，爱生雅乐 ，SeeDB，ClearT，3DISCO，CUBIC，DBE，BABB和PACT许多方法。这个实验有兴趣卡的细胞和分子机制IAC形态。心脏发育过程中ROSA26-纸屑系统稀疏标签细胞;最近，我们建立了单细胞系通过ROSA26 Cre重组ERT2跟踪。我们适应几个全胚胎清算方法，包括了Sca LE和立方（清晰，通畅脑成像的鸡尾酒和计算分析）来清除组合与胚胎整装染色心脏内部的图像单一克隆。该心脏在单细胞分辨率被成功成像。我们发现的Sca 乐可以清除胚胎心脏，但不能有效地清除产后心脏，而CUBIC可以清除产后心脏，但损害胚胎心脏的组织溶解。此处所描述的方法将允许基因功能的研究在心脏形态发生，这反过来，可以揭示先天性心脏缺陷的细胞和分子基础中的单个克隆的分辨率。
Introduction
心脏形态发生是需要的四种不同类型的心脏祖细胞的时空组织进入心脏的不同扇区的连续事件，并且还需要多基因调控网络来协调这一过程以形成功能性心脏1,2。心脏规范，分化，图案，和室成熟的心源性转录因子调控3。基因突变或这些因素在心脏祖细胞转录后像差可能导致要么胚胎致死或先天性心脏缺陷（CHD）4。心脏形态发生的研究需要在三维（3D）固有的结构细节和单标记心脏祖细胞系心脏发育过程中的跟踪将促进心脏形态的理解的理解。一些高分辨率的基础部分断层扫描方法已经在日被开发Ë过去几十年来的图像器官结构5,6;然而，这些方法需要昂贵的，专门的仪器，广泛劳动，并且缺乏在单细胞分辨率的详细结构组织在最终体积重建图像7,8。
在单细胞水平的3D体积成像提供研究祖细胞分化和细胞行为体内 7的装置。然而，组织光散射仍然是主要的障碍深完整的心脏内的3D成像的细胞和结构。脂质是光散射的一个主要来源，和去除脂质和/或脂质和它们的周围区域之间的折射率差的调整的是用于提高组织透明度8潜力的方法。在过去的几年中，一些组织结算方法的发展，从而降低组织的不透明性和光散射，像BABB（苄醇和苄benzoatË混合物）和DBE（四氢呋喃和dibenzylether）;但在这些方法中，荧光猝灭仍然是一个问题8-10。溶剂为基础的亲水性的方法，如SeeDB（果糖/硫代甘油）和3DISCO（二氯甲烷/ dibenzylether），保持荧光信号，但不使整个器官透明7,8,11。相比较而言，净度组织清除方法呈现器官透明的，但它需要一个专门的电泳装置以除去脂质8,12一样，PACT（无源清晰度技术），这也需要水凝胶包埋7,13。有关所有可用的组织清除方法的详细信息，请参阅表1 Richardson和利希特曼等。7。
2011年，哈马等人偶然发现了一种亲水的混合物“的Sca 乐 '（尿素，甘油和Triton X-100混合物）呈现小鼠大脑和胚胎transpar耳鼻喉科而完全从克隆标记保持14荧光信号。这允许完整的大脑中的几个毫米和神经元群体和突起的大规模重建的深度成像以亚细胞的分辨率。须崎等人。进一步完善了Sca 乐加入氨基醇，并制定了“CUBIC”（清晰，通畅脑成像的鸡尾酒和计算分析）组织清除的方法，从而增加磷脂溶解，降低了结算时间，并允许多色荧光成像8。在本研究中，心脏发生在服用心脏内线优势在SCA 乐和立方组织清算技术和高分辨率三维光学切片，单个克隆使用Rosa26Cre ERT2 15，R26R-纸屑 16，αMHCCre重组酶17， 肌钙蛋白Cre重组酶被追踪18， </strong> NFATC1 Cre重组酶 19，和ROSA26-mTmG（mTmG）20鼠标线。整个装载染色（WMS）方法的组合预先21,22与组织结算方法还允许在标记的克隆其他蛋白质的染色和对它们的行为在3D体积上下文中研究开发的。组织清除和WMS的组合允许更好地理解不同的基因和蛋白质的心脏发育过程中的作用，并先天性心脏缺陷的病因。这个协议可以被应用开发过程中来研究其他祖细胞分化，细胞行为和器官形态发生的事件。
Protocol
Representative Results
Discussion
胚胎隔离是一个非常关键的步骤。 E9.5胚胎的大小很脆弱的，小的，所以格外小心，应注意不要隔离时损坏胚胎/心脏结构。非胚层外包围胚胎/心脏成像应该在整个胚胎时特别小心取出。这使得抗体和清算混合穿透深胚胎组织内部，而且在成像时去除背景信号有所帮助。包括对PECAM抗体，乙酰化α微管蛋白和β1整合多种抗体进行了检查，并全部被成功检测（数据未显示）33。这证明了分子完?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank M.W. laboratory members for scientific discussion. This work is supported by AHA [13SDG16920099] to M.W., and by National Heart, Lung, and Blood Institute grants [R01HL121700] to M.W. Images were captured in the Imaging Core Facility at the Albany Medical College.
Materials
| 2,2′,2′’-nitrilotriethanol | Sigma Aldrich | 90279 | |
| 4% Paraformaldehyde in PBS | Affymetrix | 19943 | |
| BSA | Fischer Scientific | BP16000 | |
| N,N,N’,N’-tetrakis(2-hydroxypropyl) ethylenediamine | Sigma Aldrich | 122262 | |
| Phosphate Buffer Saline | Sigma Aldrich | P5368-10PAK | |
| Triton X-100 | Sigma Aldrich | T8787 | |
| Urea | Sigma Aldrich | U-1250 | |
| Sucrose | Sigma Aldrich | 84097 | |
| Glycerol | Sigma Aldrich | G8773 | |
| Tamoxifen | Sigma Aldrich | T5648 | |
| Sunflower seed oil | Sigma Aldrich | S5007 | |
| Tween 20 | Sigma Aldrich | P1379 | |
| PECAM (CD31) | BD Pharmingen | 550274 | |
| Alexa Fluor 647 | Invitrogen | A-21247 | |
| DAPI nuclear stain | Sigma Aldrich | D9542 | |
| 37oC Incubator | Thermoscientific Fischer | Heratherm, Compact Microbiological Incubators | |
| 48 well plates | Cell Treat | 229148 | |
| Analytical Balance | Metler Toledo | PB153-S/FACT | |
| Confocal microscope | Zeiss | Zeiss 510 confocal microscope | |
| Disecting Microscope | Unitron | Z850 | |
| Fluorescent microscope | Zeiss | Observer. Z1 | |
| Germinator 500 Glass Bead Sterilizer | CellPoint Scientific | GER-5287-120V | |
| Light Source | SCHOTT | ACE I | |
| Pair of Scissors | Fine Science Tools | 14084-08 | |
| Petri dish 60x15mm | TPP Techno Plastic Products AG | 93060 | |
| Rocker II Platform Rocker | Boekel Scientific | 260350 | |
| Scintillating tubes | Fischer Scientific | 03-337-26 | |
| Transfer pipette | Samco Scientific | 202 | |
| Tweezers | Fine Science Tools | 11251-20 |
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