En Face 用于小鼠胚胎平面形态发生分析的心内膜垫制备
Summary
传统上，小鼠胚胎瓣原基的心内膜已使用横向、冠状或矢状切片进行分析。我们在瓣膜生成区域对心内膜进行 面对面二维成像的新方法允许在瓣膜发育过程中对心内膜进行平面极性和细胞重排分析。
Abstract
研究哺乳动物心脏发育的细胞和分子机制对于解决人类先天性心脏病至关重要。原始心脏瓣膜的发展涉及心内膜细胞从心室管（AVC）和心脏流出道（OFT）区域的上皮到间充质转化（EMT），以响应局部诱导性心肌和心内膜信号。一旦细胞分层并侵入位于心内膜和心肌之间的细胞外基质（心果冻），就会形成原始心内膜垫（EC）。这个过程意味着心内膜必须填充分层细胞留下的间隙，并且必须重组自身以沿轴收敛（狭窄）或延伸（延长）。目前的研究已经暗示平面细胞极性（PCP）途径调节该过程所涉及的因素的亚细胞定位。传统上，心脏瓣膜发育的初始阶段已经在胚胎心脏的横截面或在胶原凝胶上培养的 离体 AVC或OFT外植体中进行了研究。这些方法允许分析顶端基底极性，但不允许分析上皮平面内的细胞行为或迁移细胞的形态变化。在这里，我们展示了一种实验方法，该方法允许将瓣膜生成区域的心内膜可视化为细胞的平面视野。这种实验方法提供了在瓣膜发育过程中研究 OFT 和 AVC 心内膜内的 PCP、平面拓扑和细胞间通讯的机会。破译涉及心脏瓣膜形态发生的新细胞机制可能有助于了解与心内膜垫缺陷相关的先天性心脏病。
Introduction
心脏是哺乳动物胚胎的第一个功能器官。在小鼠胚胎日（E）7.5左右，双侧心前中胚层细胞在腹侧形成心新月形1。心新月形包含两个心前细胞群，包括心肌和心内膜的祖细胞2。在E8.0左右，心脏前体在中线融合，形成由两个上皮组织组成的原始心脏管，即外部心肌和内部心内膜，内部心内膜是由称为心果冻的细胞外基质分离的特殊内皮。后来，在E8.5，心脏管经历右旋。环状心脏具有具有特定分子特征的不同解剖区域，例如流出道 （OFT）、心室和动脉-心室管 （AVC）3。虽然最初心脏管通过添加细胞4在其流入侧扩张，但在E9.5处，强烈的心脏增殖导致腔室气球膨胀和小梁网络建立5。瓣膜形成发生在AVC（未来的二尖瓣和三尖瓣）和OFT（未来的主动脉瓣和肺动脉瓣）中。
心内膜在瓣膜发育中起着至关重要的作用。心内膜细胞在AVC和OFT中经历上皮-间充质转化（EMT）以形成心内膜垫，这种结构出现在瓣膜发育开始时。不同的信号通路激活这一过程;在小鼠的E9.5处，响应心肌来源的BMP2在心内膜中激活的NOTCH通过激活TGFβ2和SNAIL（SNAI1）促进AVC和OFT区域中心内膜细胞的侵袭性EMT，其直接抑制血管内皮钙粘蛋白（VE-钙粘蛋白）的表达，这是粘附连接（AJ）的跨膜成分6，7，8.在OFT中，心内膜激活以启动EMT由FGF8和BMP4介导，其表达由NOTCH9，10，11，12激活。
EMT的进展涉及细胞动力学，因为细胞改变形状，与邻居断开和重建连接，分层并开始迁移13。这些变化包括AJ重塑和逐渐拆卸14，15，平面细胞极性（PCP）信号传导，顶端基底极性（ABP）丧失，顶端收缩和细胞骨架组织16，17。ABP是指蛋白质沿细胞前后轴的分布。在发育中的心脏中，心室发育需要心肌细胞中的ABP调节18。PCP是指细胞内蛋白质在组织平面上的极化分布，并调节细胞分布;具有稳定几何形状的上皮由六边形细胞组成，其中只有三个细胞在顶点19、20、21、22 处汇聚。不同的细胞过程，例如细胞分裂、邻居交换或在上皮形态发生过程中发生的分层，导致汇聚在顶点上的细胞数量增加，而给定细胞具有的相邻细胞数量增加22。这些与PCP相关的细胞行为可以通过不同的信号通路，肌动蛋白动力学或细胞内运输来调节23。
研究小鼠瓣膜发育产生的数据是从E8.5和E9.5胚胎心脏的横向，冠状或矢状面获得的，其中心内膜显示为细胞线而不是细胞场 – 心内膜覆盖心脏管的整个内表面24。胚胎切片不允许分析小鼠胚胎心内膜中的PCP。我们的新实验方法可以分析心内膜细胞分布，AJ各向异性和单细胞形状分析，如代表性结果所示。这种类型的数据是PCP分析所必需的，以及与PCP相关的其他分子的描述，本报告中未显示。全安装免疫荧光、特异性样品制备和使用转基因小鼠可在小鼠瓣膜发育开始时对心内膜进行平面极性分析。
Protocol
Representative Results
Discussion
心内膜是覆盖胚胎心脏管整个内表面的上皮单层。在瓣膜发育过程中，预期瓣膜区域的心内膜细胞经历EMT，因此心内膜细胞转换并重新排列其细胞骨架，从心内膜向心果冻分层。我们和其他人通过分析E8.5和E9.5胚胎心脏的横向切片获得了有关小鼠胚胎瓣膜发育的相关数据，其中心内膜显示为一排细胞6，8，24，32</su…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了MCIN/AEI/10.13039/501100011033到J. L. P. J.G.-B.的PID2019-104776RB-I00和CB16/11/00399（CIBER CV）的支持。由马德里市人才计划资助（2020-5ª/BMD-19729）。T.G.-C.由大学教授学院资助（FPU18/01054）。我们感谢CNIC显微镜和动态成像部门，CNIC，ICTS-ReDib，由MCIN/AEI / 10.13039 / 501100011033和FEDER“创造欧洲的方式”（#ICTS-2018-04-CNIC-16）共同资助。我们还要感谢A.加利西亚和L.门德斯的老鼠饲养。该出版物的费用部分由欧洲区域发展基金提供的资金支付。CNIC得到了ISCIII，MCIN和Pro CNIC基金会的支持，是由MCIN / AEI / 10.13039 / 501100011033资助的Severo Ochoa卓越中心（授予CEX2020-001041-S）。
Materials
| 4-OH-Tamoxifen | Sigma Aldrich | H-6278 | |
| 16 % Paraformaldheyde | Electron Microscopy Sciences | 157-10 | Dilute to 4% in water |
| anti-GFP | Aves Labs | FGP-1010 | |
| anti-VECadherin | BD Biosciences | 555289 | |
| Goat anti-Chicken, Alexa Fluor 488 | Thermo Fisher Scientific | A-11039 | |
| Goat Anti-Mouse Alexa Fluor 647 | Jackson ImmunoResearch | 115-605-174 | |
| DAPI | AppliChem | A4099,0005 | |
| Slides Superfrost PLUS | VWR | 631-0108 | 25 mm x 75 mm x 1.0 mm |
| Triton X-100 | Sigma Aldrich | X100-100ML | |
| Tween 20 | A4974,0500 | AppliChem | |
| Vectashield Mounting Medium | Vector Laboratories | H-1000-10 |
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