Name: en face endocardial cushion preparation for planar morphogenesis analysis in mouse embryos
Uploaded: 2022-07-27T14:30:00
Description: Watch this Scientific Journal Video about En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos at JoVE.com

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）。

动物研究得到了国家心血管研究中心（CNIC）动物实验伦理委员会和马德里共同体的批准（参考PROEX 155.7/20）。所有动物程序均符合欧盟指令 2010/63EU 和关于保护用于实验和其他科学目的的动物的建议 2007/526/EC，根据 Real Decreto 1201/2005 在西班牙法律中颁布。
1. 获得AVC和/或OFT（改编自Xiao等人）。25）
<ol><li>下午在雌性 mTmG 和雄?...

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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...

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。

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		<tr>
			<td>4-OH-Tamoxifen</td>
			<td>Sigma Aldrich</td>
			<td>H-6278</td>
			<td></td>
		</tr>
		<tr>
			<td>16 % Paraformaldheyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-10</td>
			<td>Dilute to 4% in water</td>
		</tr>
		<tr>
			<td>anti-GFP</td>
			<td>Aves Labs</td>
			<td>FGP-1010</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-VECadherin</td>
			<td>BD Biosciences</td>
			<td>555289</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Chicken, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11039</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse Alexa Fluor 647</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-605-174</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>AppliChem</td>
			<td>A4099,0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Slides Superfrost PLUS</td>
			<td>VWR</td>
			<td>631-0108</td>
			<td>25 mm x 75 mm x 1.0 mm</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>A4974,0500</td>
			<td>AppliChem</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000-10</td>
			<td></td>
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</table>

en face endocardial cushion preparation for planar morphogenesis analysis in mouse embryos

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。

使用该协议生成的数据显示，可以对AVC的心内膜进行面部成像。第一个目标是以细胞分辨率分析瓣膜形成过程中心内膜的细胞形状（图1）。为了突出E9.5处的单个心内膜细胞，我们使用了两种转基因小鼠品系。（1）ROSA mT/mG是一种双色荧光Cre报告等位基因（tdTomato/mT和EGFP/mG），其荧光在细胞膜处检测到。在没有Cre介导的激活的情况下，转基?...

Watch this Scientific Journal Video about En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos at JoVE.com

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

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。

En Face 用于小鼠胚胎平面形态发生分析的心内膜垫制备

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart ...

该协议允许在平面方向上分析原始心内膜。我们可以分析心内膜细胞的分布，其他禁令的各向异性，并描述瓣膜发育过程中单个心内膜细胞的形状。与经典组织切片相比，内瓣膜再生区域的面部成像允许分析完整胚胎瓣膜发育过程中平面细胞极性和心内膜细胞的动力学。该方法可用于加深心脏发育过程中平面细胞极性的知识。首先将从安乐死的怀孕小鼠中切除的子宫放入含有冰冷PBT的培养皿中。使用细镊子在解剖显微镜下从子宫中取出单个蜕膜。使用细镊子，在蜕膜的白色部分切开一个切口，这是胚胎所在的位置。轻轻地将其取出，避免拉扯，并使用P-1000将胚胎转移到带有新鲜PBT的新培养皿中，尖端被切断，留下足够大的直径以使胚胎通过而不会破裂。对于基因分型，取一小块约0.5平方毫米的卵黄囊或从后端取一块尾巴，向头部计数5至10个体细胞，并在100毫升适当的缓冲液中消化。在通风橱下，将胚胎转移到 4 摄氏度的两毫升微量离心管中的冰冷 4% PFA 中。将胚胎固定在章动器上，从两小时到四摄氏度过夜。在通风橱下，从微量离心管中取出4%PFA固定剂，不要接触胚胎。将 PFA 丢弃在适当的容器中。在室温下在冷PBT中洗涤胚胎5次10分钟。使用细镊子取出心脏并将其转移到带有新鲜PBT的新1.5毫升微量离心管中。用PBS Triton代替PBT，并在室温下在轨道振荡器上洗涤样品三次，每次五分钟。用含有PBS Triton和10%热灭活牛血清的封闭溶液替换PBS Triton。在轨道摇床上以四摄氏度孵育两小时至过夜。用含有所需浓度的一抗的封闭溶液替换封闭溶液。在四摄氏度的轨道摇床上孵育过夜。在 4 摄氏度下孵育过夜后，将样品在室温下在轨道摇床上孵育 1.5 小时。此步骤对于提高信噪比至关重要。取出一抗溶液并将其保持在 4 摄氏度，未来最多进行三次实验。加入叠氮化钠至终浓度为0.02%，以获得最佳保存效果。然后，用PBS Triton清洗胚胎三次，每次三分钟。用PBS Triton清洗胚胎三次30分钟，并将它们保持在4摄氏度的轨道振荡器上。最后一次洗涤后，加入一毫升含有荧光偶联二抗的封闭溶液，以对抗一抗宿主物种。然后将DAPI添加到溶液中，并将胚胎在4摄氏度的轨道振荡器上孵育过夜。在4摄氏度下孵育过夜后，将胚胎在室温下在轨道振荡器上孵育1.5小时。此步骤对于提高信噪比至关重要。重复文本手稿中提到的PBS Triton清洗步骤。将心脏放在含有PBS的35毫米培养皿上。使用钨丝和细镊子，隔离房室管或流出道并纵向切割。准备盖玻片、载玻片、镊子、带有适当吸头的 P-200 移液器、纸巾和任何基于甘油的水性封片剂，用于无 DAPI 的荧光。将两条胶带粘在相隔0.3至0.6厘米的载玻片上，以创建一个3D房间空间，使样品能够保持其原始形状而不会挤压它们。然后，使用大约 20 微升缓冲液，使用切割的 P-200 移液器吸头小心地将样品滴到胶带条之间的载玻片上。使用解剖显微镜提高精度。使用细镊子，将样品放在心内膜朝上，心肌朝下放在载玻片上。用纸巾去除多余的PBS，避免接触样品。然后，在室温下将样品干燥一到两分钟，使样品粘在载玻片上。向组织中加入40微升水性封片剂。将盖玻片放在两张胶带上，然后用针或镊子将其缓慢地放到纸巾上。避免产生气泡。在盖玻片的每个顶点上用一滴指甲油密封盖玻片。指甲油干燥后，使用吸水纸巾清洁盖玻片侧面多余的镶片介质。避免移动盖玻片。沿着盖玻片边缘添加指甲油以完全密封，防止安装介质蒸发。使用正置或倒置共聚焦显微镜，以 10 倍放大倍率拍摄图像以获得一般视图，以 63 倍放大倍率拍摄图像以获得详细图像。在瓣膜形成过程中以细胞分辨率分析心内膜的细胞形状。在心内膜中表达GFP的单个细胞或几个细胞的克隆。GFP表达和与VE-钙粘蛋白亚细胞定位的组合是研究AJ动力学与丝状伪足形成和前EMT细胞之间关系的有效方法，因为这些细胞在AJ溶解时会产生膜突起。心内膜中VE-钙粘蛋白染色强度的差异表明在瓣膜前阶段房室管的胚胎心内膜中存在各向异性收缩性。整个心脏在E8.5和E9.5处用VE-钙粘蛋白抗体染色。在E8.5时，房室管心内膜倾向于具有稳定的上皮组织，未检测到由四个以上细胞形成的顶点。在E9.5处，观察到多达六个细胞的顶点形成玫瑰花状结构，类似于先前描述的上皮细胞组织经历活性细胞重排，表明房室管心内膜在瓣膜发育过程中正在经历动态细胞重排。学习曲线是成为这种技术专家所必需的。此外，强烈建议使用主权镊子和钨丝。今天，瓣膜前心内膜的可视化仅限于横截面。我们的方法提供了从平面角度研究胚胎瓣膜心内膜行为的可能性。

Research

JoVE Journal

Developmental Biology

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

对比成像的小鼠胚胎使用高频超声

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

科研

发育生物学

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

在代谢分析准备线粒体富集级的<em&gt;果蝇</em

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

在成年小鼠V-SVZ细胞的神经分析的稳定，高效的基因修饰干细胞自主和非自主的影响

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

方法细胞或DNA的精确本地化转移到着床后早期小鼠胚胎

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

在体内果蝇蛹肌肌腱形态的影像学研究

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Wholemount In Situ Hybridization for Astyanax Embryos

Wholemount In Situ Hybridization for Astyanax Embryos

阿斯蒂亚纳克斯胚胎的全原位杂交

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...