这项工作由 NIH/aha R56 HL128646 和 Mindich 儿童健康和发展研究所 (MCHDI) 在 ISMMS (北达科他州) 资助。比由 NIH/NIDCR 培训 T32 HD075735 支持。显微镜和图像分析是在位于西奈山的伊坎医学院的显微镜中心进行的, 这部分由西奈山 P30 CA196521 悌癌症研究所支持。

此处描述的所有方法都已由西奈山医学院的伊坎医学院的机构动物保育和使用委员会批准. 
 1. 收集和处理心脏新月期胚胎 <ol> <li> 交配一只肥沃的雌性老鼠。
	<li> 检查是否存在阴道交配插头使用钝探针或镊子。当天中午的插头检测被认为是胚胎日 (E) 0.5 (见 图 1A 完整的时间线). 
	注: 插头应在上午检查, 因为他们是在整个一天丢失. </li>
	<li> 在 8 日 (E8.25) 的早晨, 通过 CO 2 吸入, 或根据当地和机构的规定牺牲怀孕的大坝. 
	注意: 精确的时间可以是应变依赖, 并应由形态学经验. </li>
	<li> 用70% 乙醇喷洒鼠标的腹部, 清洁区域, 减少脱落。通过腹部切开皮肤和身体壁来揭露内脏. </li>
	<li> 定位子宫并小心地从动物身上取出整个子宫角。首先从一个输卵管切开, 修剪脂肪远离子宫, 同时进行宫颈末端。通过宫颈切开并继续到另一端输卵管的上端, 切开整个子宫. </li>
	<li> 将子宫放在10厘米的盘中, 用磷酸盐缓冲盐水 (PBS, pH 值 7.4) 冲洗掉多余的血液。通过切割 mesometrium 之间的 deciduum, 其中包含了胚胎, 解剖子宫。用新鲜的 PBS 将胚胎移植到一个6厘米的盘子里. </li>
	<li> 在解剖显微镜下, 用细钳 (#5) 将子宫组织移离蜕组织. </li>
	<li> 使用镊子, 仔细切片的 deciduum 的胚胎一半的尖端, 以揭示胚胎。捏 deciduum, 把胚胎取出, 小心地把胚胎取出. </li>
	<li> 尽可能多地解剖外组织而不损害胚胎的形态学 ( 图 1B ). </li>
	<li> 使用转移吸管将胚胎放置在1.5 毫升的试管中, 并在冰上放置新鲜的 PBS 和地方。对其余的胚胎重复 1.7-1.9, 然后继续. </li>
	<li> 吸 pbs, 用 pbs 冲洗一次。吸气 PBS. 
	注: 尽量去除尽可能多的 PBS, 而不损害或干燥的胚胎。建议在所有步骤中手动删除解决方案, 以避免丢失胚胎. </li>
	<li> 在 RT 中为1小时在 PBS 中修复4% 甲醛 (粉煤灰) 的胚胎. 
	注: 胚胎可在4和 #176 一夜之间固定 (O/N); C. </li>
	<li> 用 PBS 冲洗三次。将胚胎保持在4和 #176; C 直到准备好进行免疫荧光. 
	注意: 暂停点。胚胎可以安全地储存在 PBS 4 和 #176; C 几个星期. </li>
</ol> 2。免疫荧光染色 注意: 下面的孵化条件可以调整以适应不同的时间安排。建议使用轻柔摇动或摇动所有长潜伏期的步骤. 
<ol> <li> 删除 pbs, 并在 pbs 中添加1毫升的阻断缓冲器 (0.5% 皂苷、1% 牛血清白蛋白 (BSA)). </li>
	<li> RT 时至少孵育4小时。这也可以在4和 #176 中完成; C. </li>
	<li> 删除阻塞缓冲区, 并添加在阻塞缓冲区中稀释的主抗体混合物。在4和 #176 孵育 O/N; C. 
	注: 请参阅材料部分以描述所使用的抗体和建议的稀释。抗体稀释应根据经验确定。推荐使用 Nkx2-5 作为心脏新月的参考色斑, 是下游图像分割和分析步骤的关键. </li>
	<li> 通过吸入去除主抗体. </li>
	<li> 在 PBS 中每1小时清洗3次, 每次使用0.1% 个海卫一. </li>
	<li> 去除洗涤和添加二次抗体混合物稀释在堵塞缓冲。在 RT 处孵育3小时。这也可以在4和 #176 中完成; C. </li>
	<li> 在 PBS 中用0.1% 个海卫一清洗3次, 每次1小时。这也可以在4和 #176 中完成; C. </li>
	<li> Counterstain 与4和 #39;, 6-diamidino-2-吲 (DAPI) 在 PBS 中为 10 min. 
	注: 此 counterstain 可同时进行二次抗体. </li>
	<li> 在 PBS 中用0.1% 个海卫一清洗2次, 每次5分钟. </li>
	<li> 缓慢地暂停防安装介质中的胚胎 (2% 伏无食子 nPG), 90% 甘油, 1x PBS;有关详细信息, 请参阅参考资料部分)。安装前允许平衡至少1小时。轻轻地弹管, 帮助胚胎落入防溶液. 
	注意: 暂停点。胚胎可以储存数天, 在防的解决方案, 直到准备安装和图像. </li>
</ol> 3。显微镜下安装胚胎 <ol> <li> 使用胶布磁带或硅胶垫片准备用于安装的显微镜幻灯片。如果使用胶布的磁带, 使两个平行的堆栈5-6 层约15-20 毫米分开。这将留下足够的空间来放置胚胎并确保片 ( 图 1C ). </li>
	<li> 放置15和 #181; 在胶布磁带之间的幻灯片上放置防。小心地将一个胚胎转移到滑梯上. 
	注意: 一个以上的胚胎可以放在一个幻灯片上。然而, 随后的方向步骤是更困难的多胚胎, 和长的成像时间可能导致光漂白. </li>
	<li> 在解剖显微镜下使用细钳, 定位胚胎, 使前侧面远离幻灯片与身体轴与幻灯片的长轴线。有关安装安装的示意图, 请参见 图 1C . </li>
	<li> 小心地将一个片放在样品上, 将一侧放在一叠胶布胶带上, 然后用镊子轻轻地降低片, 直到它与另一条带子接触. 
	注意: 步骤3.3 和3.4 是确保胚胎成像正确方向的关键。通过降低片沿后向前方的方向, 胚胎不应该滚动和心脏新月区将保持远离幻灯片, 这是理想的倒置显微镜成像. </li>
	<li> 使用吸管在幻灯片和片之间添加额外的防, 以防止样品在存储/成像过程中消失. </li>
</ol> 4。共焦成像 <ol> <li> 使用最高放大率目标获取图像, 使整个心脏新月区在一个视场中被捕获。使用奈奎斯特采样率来确定 x-y-z 像素尺寸。大多数显微镜制造商将有一个和 #34; 优化和 #34; 按钮以确保奈奎斯特取样. 
	注: Nkx2-5 染色在这里是有益的, 因为它将描绘整个心脏新月区。较小的 Z 步尺寸 (采样) 将产生更好的3D 建模结果, 但可能会导致延长采集时间和/或漂白一些样品. </li>
	<li> 使用标准 best-imaging 实践设置成像参数。即, 使用尽可能低的激光强度, 以获得良好的信噪比为每个荧光, 并选择增益和抵消水平, 产生一个广泛的动态范围不饱和的信号. 
	注: 与将直接比较的样品之间的成像参数保持一致。这对于下游3D 分析和量化尤为重要. </li>
</ol> 5。图像分析和定量3D 建模 注意:对于该协议, 使用 Imaris 软件包对图像进行了分析。类似的分析可能使用其他的软件包。下面的说明介绍了一个引用通道 ( 即 Nkx2-5) 和一个实验通道 ( 即 YFP) 的分析管道。可以通过重复这些步骤来分析其他通道。提供了一个示例数据集, 可用于复制下面的分析. 
<ol> <li> 将原始图像数据集加载到 Imaris 竞技场视图中, 并在超过视图中为所需的胚胎打开文件。数据应在3D 视图中加载 MIP (最大) 模式。打开 "显示调整" 窗口, 并只选择参考通道. </li>
	<li> 如果需要, 可调整图像的阈值, 以便更好地进行可视化。对于这个分析, 如果信噪比过低, 则调整伽玛. 
	注意: 如果执行伽玛校正, 这是非线性调整, 调整后的图像不能用于强度测量或比较。在这个阶段, 你可以进行进一步的图像预处理 (如光照校正, 高斯模糊或其他过滤), 如果你的信噪比是不满意的。如果选择进行预处理, 则必须对数据集中的所有图像执行相同的预处理步骤. </li>
	<li> 使用曲面算法对话框为参考通道创建新曲面. 
	<ol> <li> 选择和 #34; 只对感兴趣的区域和 #34 进行细分; 复选框. </li>
		<li> 调整边界框区域, 使其包含感兴趣的区域 (ROI), 即 "参考色斑" 所描述的 心脏新月区). </li>
		<li> 从源通道下拉菜单中选择参考通道。默认的平滑设置 (等于 z 像素大小) 通常是足够的, 但可能需要经验优化。对于此分析, 使用平滑等于 z voxel-size 的一半. </li>
		<li> 按绝对强度进行阈值化。要确保曲面不延伸到通道信号之外, 请使用滑块调整下限. 
		注意: 对于某些可以很容易划定细胞的图像, 可以包括 #34; 分割触摸的对象和 #34; 算法, 以便进一步将信号与背景分开. </li>
		<li> 按体素数字或体积筛选对象, 并拒绝背景 (小或在 Nkx2-5 区域之外的区域中找到) 该算法可能生成的对象。完成算法. </li>
	</ol> </li> <li> 使用新创建的曲面, 为实验通道创建一个屏蔽通道. 
	<ol> <li> 如果选定了曲面, 则从 "编辑" 窗口中选择 #34; 掩码 Sel... #863; #34; 函数. </li>
		<li> 选择感兴趣的实验通道 ( 即 YFP)。在应用掩码和 #34 之前确保 #34;D 重复通道; 选中复选框. </li>
	</ol> </li> <li> 在显示调整窗口中只选择屏蔽通道。按照步骤 5.3-5.3. 5, 为掩码通道创建一个新曲面. 
	注意: 此时调整每个掩码的外观有助于3D 模型的可视化: 选择每个曲面并选择 "颜色" 选项卡. 根据需要调整基本颜色和透明度。重叠时, 曲面颜色不合并. </li>
	<li> 若要获取所需的体积数据, 请选择 "统计" 选项卡。在和 #34;D etailed 和 #34; 子, 选择和 #34; 平均值和 #34; 从下拉菜单中。在生成的表的 Sum 列中可以找到该曲面的总体积. 
	注意: 如果生成的曲面包含错误的片段 ( 即 片段, 这些片断是在分割期间未排除的背景信号或从主表面间断的区域), 则可能需要手动计算总卷, 或按体的体积或数量过滤曲面, 并确保计算中只包含信号段。通过选择和 #34, 所有的值和 #34; 从下拉菜单中, 可以找到每个曲面部分的音量。选择每个值将使相应区域显示为黄色, 这有助于确定要排除的值. </li>
</ol>

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H., Mohn, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+embryonic+explant+culture+system+for+analysis+of+hematopoietic+and+vascular+development.">Mouse embryonic explant culture system for analysis of hematopoietic and vascular development.</a> Methods Mol. Med. 105, 231-256 (2005).</li></ol>

作者没有什么可透露的。

上面的协议描述了从高质量的 post-implantation 小鼠胚胎免疫荧光图像中获取定量数据的策略。当正确执行时, 通过这些步骤生成的3D 体积数据可用于胚胎内多个域的比较和交叉分析。表面信号掩蔽方法的描述是特别使用时, 研究新的细胞数量与公认的参考结构比较。
我们认为, 这种方法比现有方法具有明显的优势。全贴装免疫荧光与广域成像可以提供组织级信息, 但缺乏细胞分?...

器官发生的研究一直依赖于发育中胚胎的形态发生事件的观察。这些研究往往依赖于使用荧光染料或血统追踪记者与标签的定义参考人口。1通过比较这些标签的相对位置, 可以收集有关某一群体的起源、移动或最终贡献的信息。移植和命运映射实验使用形态学地标或染料射入 non-motile 血统定义兴趣细胞的出发点, 然后被审查为贡献对被开发的胚胎。2,3,4,5遗传谱系跟踪实验使用与定义明确的报告等位基因相同的概念, 用于标记细胞数量, 而无需进行实验操作。这些方法的关键是能够确定, 具有高空间分辨率, 实验和参考标签的位置。这些方法在前发育和外植体研究方面取得了显著进展。6,7,8,9
近年来, 心脏形态发生的发展事件越来越受到人们的广泛描述。10这一研究领域的主要发现之一是描述了一些可以通过独特标记的表达来区分的祖群。11这些种群包括第一和第二个心脏领域 (FHF 和 SHF), 它们存在于胚胎前侧的心脏新月中 (E) 8.25 的小鼠发育。12这些种群经常通过广泛的显微镜的结合进行检查, 这提供了组织级的信息, 并通过免疫荧光分析进行了序列切片, 这提供了高的细胞分辨率, 但two-dimensional 空间信息。13因此, 虽然这些研究大大提高了我们对心脏发育的认识, 但现有的方法在这些阶段对形态发生的深入定量分析有限, 因此需要采取方法来检查这些人口的组织在一个整体有机体水平。
最近的进展, 共焦显微镜和3D 图像分析允许高分辨率和高通量算法重建的细胞和结构原位相对容易, 从而铺平了道路的详细研究复杂细胞结构。14随着计算能力的提高和大数据管理算法的发展, 既需要处理成像数据集大小的指数增长, 现在可以完全自动化分析。15对成像数据集的自动化分析具有无偏见的优点, 但它只与输入数据集的质量一样可靠;因此, 必须在采集和图像预处理过程中使用最佳, 以确保最高质量、无偏见的分析。16协议可以完全自动化并共享以实现重复性, 而专有软件所使用的算法也可通过库来方便地使用, 这些用户熟悉现代专有或开源开发工具。17
下面的协议解释了在心脏发育过程中对一个明确的器官形成模型进行分析的必要步骤。具体来说, 该协议描述了如何 (1) 收获和解剖心脏新月期胚胎, (2) 执行全贴装免疫荧光的参考 (Nkx2-5) 和实验 (Foxa2Cre:YFP18,19) 标记, (3)使用共聚焦显微镜制备和成像胚胎, 最后 (4) 使用先进的三维方法对结果图像进行分析和量化。在这里, 以心脏新月为例, 通过适当的修改, 本协议可用于分析肠早期节期胚胎的多血统。

<table><tbody><tr><td>Blunt probe</td><td>Roboz</td><td>RS-9580</td><td></td></tr><tr><td>Forceps</td><td>Roboz</td><td>RS-8100</td><td></td></tr><tr><td>Fine forceps</td><td>Roboz</td><td>RS-5015</td><td></td></tr><tr><td>Dissection scissors</td><td>Roboz</td><td>RS-5912</td><td></td></tr><tr><td>Saponin</td><td>Sigma Aldrich</td><td>84510</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A8022</td><td></td></tr><tr><td>Triton</td><td>RPI</td><td>A4490</td><td></td></tr><tr><td>Goat anti-Nkx2-5</td><td>Santa Cruz Biotech</td><td>sc-8697</td><td>Used at 1:100-1:500</td></tr><tr><td>Chicken anti-GFP</td><td>abcam</td><td>ab13970</td><td>Used at 1:500</td></tr><tr><td>Rabbit anti-Islet1</td><td>abcam</td><td>ab109517</td><td>Used at 1:100</td></tr><tr><td>Rabbit anti-Hcn4</td><td>Millipore</td><td>AB5808</td><td>Used at 1:100</td></tr><tr><td>488 anti-chicken</td><td>Jackson Immunoresearch</td><td>703-546-155</td><td>Reconstituted in water and stored at -20 &amp;deg;C in final concentration of 50% glycerol. Used at 1:500.</td></tr><tr><td>555 anti-goat</td><td>Thermo Fisher Scientific</td><td>A21432</td><td>Used at 1:500</td></tr><tr><td>647 anti-rabbit</td><td>Jackson Immunoresearch</td><td>711-606-152</td><td>Reconstituted in water and stored at -20 &amp;deg;C in final concentration of 50% glycerol. Used at 1:500.</td></tr><tr><td>DAPI</td><td>Sigma Aldrich</td><td>D9542</td><td></td></tr><tr><td>n-Propyl gallate</td><td>Sigma Aldrich</td><td>2370</td><td>Stock solution is 20% w/v in DMSO. Working solution prepared by mixing 1 part 10x PBS with 9 parts 100% glycerol and slowly adding 0.1 part stock solution.</td></tr><tr><td>Superfrost Plus microscopy slides</td><td>VWR Scientific</td><td>48311-703</td><td></td></tr><tr><td>22x22 mm coverslips</td><td>VWR Scientific</td><td>48366-227</td><td></td></tr><tr><td>Imaris 8.4.1</td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

quantitative whole-mount immunofluorescence analysis of cardiac progenitor populations in mouse embryos

使用不断进步的成像技术, 有助于我们增加对胚胎发育的理解。植入前发育和器官移植是两个领域的研究, 得益于这些进步, 由于数据的高质量, 可以直接从成像前胚胎或活体器官。虽然前胚胎产生的数据具有特别高的空间分辨率, 但后期阶段对三维重建的顺从性较差。为已知的胚胎结构获得高质量的3D 或体积数据, 与命运图或遗传谱系追踪相结合, 可以对发生在胚胎发育过程中的形态发生事件进行更全面的分析。
本议定书描述了一个完整的免疫荧光方法, 允许标记, 可视化, 并定量的祖细胞在发展中的心脏新月, 一个关键的结构在心脏发育过程中形成。这种方法的设计方式是, 可以获得细胞和组织级的信息。利用共焦显微镜和图像处理, 该协议允许三维心脏新月的空间重建, 从而提供了分析特定祖群的定位和组织的能力心脏发育的关键阶段。重要的是, 使用参考抗体可以连续掩蔽心脏新月和随后的定量测量的区域内的新月。本议定书不仅能够对早期心脏发育进行详细的检查, 而且应适用于肠早期节期小鼠胚胎的大多数器官系统。

最终数据和分析的质量很大程度上取决于 (1) 解剖胚胎的完整性和形态学, (2) 使用高特异性抗体, (3) 正确设置成像参数。受损的胚胎会混淆表面生成过程, 阻碍定量分析。正确解剖和分期胚胎的例子如图 2B所示。胚胎可以通过去除卵黄囊进行进一步的解剖, 抗体会经常非。然而, 去除卵黄囊会使胚胎变得不那么健壮, 因此在下游处理过程中应注意。
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Watch this Scientific Journal Video about Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos at JoVE.com

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

在这里, 我们描述了一个协议的全贴装免疫荧光和图像定量容积分析早期小鼠胚胎。我们提出这项技术是一个强有力的方法, 定性和定量评估心脏结构在发展过程中, 并提出它可能会广泛适用于其他器官系统。

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 ...

quantitative-whole-mount-immunofluorescence-analysis-cardiac

Research

JoVE Journal

Developmental Biology

9.5K 次观看.  Icahn School of Medicine at Mount Sinai. 在这里, 我们描述了一个协议的全贴装免疫荧光和图像定量容积分析早期小鼠胚胎。我们提出这项技术是一个强有力的方法, 定性和定量评估心脏结构在发展过程中, 并提出它可能会广泛适用于其他器官系统。

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

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

在胚胎小鼠心脏采用免疫荧光心肌发展分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

科研

发育生物学

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

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

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 ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

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.

成像清除胚胎和出生后心脏在单细胞分辨率

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

评估心肌亚型以下鼠标胚胎成纤维细胞的转录因子介导的重编程

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

低血清条件下心脏祖细胞内皮分化的诱导作用

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

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

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.

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

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

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 ...

Live Imaging of Early Cardiac Progenitors in the Mouse Embryo

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail specific developmental stages in a still snapshot, live imaging captures dynamic morphogenetic events, such as cell migration, shape changes, and differentiation, by imaging the embryo as it develops. This complements fixed analysis and expands the understanding of how organs develop during embryogenesis. Despite its advantages, live imaging is rarely used in mouse models because of its technical challenges. Early mouse embryos are sensitive when cultured ex vivo and require efficient handling. To facilitate a broader use of live imaging in mouse developmental research, this paper presents a detailed protocol for two-photon live microscopy that allows long-term acquisition in mouse embryos. In addition to the protocol, tips are provided on embryo handling and culture optimization. This will help understand key events in early mouse organogenesis, enhancing the understanding of cardiovascular progenitor biology.

We present a detailed protocol for mouse embryo culture and imaging that enables 3D + time imaging of cardiac progenitor cells. This video-toolkit addresses the key skills required for successful live imaging otherwise hard to acquire from text-only publications.

小鼠胚胎中早期心脏祖细胞的实时成像

The first steps of heart development imply drastic changes in cell behavior and differentiation. While analysis of fixed embryos allows studying in detail ...

Cell Labeling and Injection in Developing Embryonic Mouse Hearts

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect their in vivo potential. Preferably, these cells should be placed in a proper embryonic environment in order to acquire their definite phenotype. Furthermore, cell lineage tracing studies in the mouse after labeling cells with dyes or retroviral vectors has remained mostly limited to early stage mouse embryos with still poorly developed organs. To overcome these limitations, we designed standard and ultrasound-mediated microinjection protocols to inject various agents in targeted regions of the heart in mouse embryos at E9.5 and later stages of development. &#160;Embryonic explant or embryos are then cultured or left to further develop in utero. These agents include fluorescent dyes, virus, shRNAs, or stem cell-derived progenitor cells. Our approaches allow for preservation of the function of the organ while monitoring migration and fate of labeled and/or injected cells. These technologies can be extended to other organs and will be very helpful to address key biological questions in biology of development.

We describe a series of methods to inject dyes, DNA vectors, virus, and cells in order to monitor both cell fate and phenotype of endogenous and grafted cells derived from embryonic or pluripotent cells within mouse embryos at embryonic day (E)9.5 and later stages of development.

细胞标记和注射胚胎发展小鼠心脏

Testing the fate of embryonic or pluripotent stem cell-derivatives in in vitro protocols has led to controversial outcomes that do not necessarily reflect ...

生物学

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

从发展胚胎小鼠心脏瓣膜区蛋白分离

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...