Name: following endocardial tissue movements via cell photoconversion in the zebrafish embryo
Uploaded: 2018-02-20T14:00:00.000+00:00
Duration: 9 min 38 s
Description: Watch this Scientific Journal Video about Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo at JoVE.com

我们要感谢安妮-玛丽·韦尔坎贝里弗朗辛·杜舍芒, 丹尼斯弗朗辛·杜舍芒, 纳塔莉 Faggianelli 和巴西莱 Gurchenkov 帮助设计和制作本协议中描述的模具。这项工作得到了 FRM (DEQ20140329553)、国家情报局 (ANR-15-CE13-0015-01、ANR-12-ISV2-0001-01)、EMBO 青年调查员计划、欧洲共同体、紧急救济机构 N°682938 Evalve 和赠款 ANR-10-LABX-0030-INRT 的支助, 由一个法国国有基金管理, 由研究所的框架计划 Investissements 艾文莉标记 ANR-10-IDEX-0002-02。

1. 制备模具和安装琼脂糖
<ol><li>使用3D 打印机或传统的机械车间工具, 创建具有图 1所示尺寸的模具。</li>
	<li>吸管约1.5 毫升熔化1% 琼脂糖成35毫米塑料安装盘。将塑料模具放在液体琼脂糖中, 注意避免在霉菌和琼脂糖之间捕获空气。把盘子放在4摄氏度, 等到琼脂糖变硬 (这大约需要5分钟), 然后取出塑料模具。</li>
	<li>要储存安装盘供以后使用, 用盖子盖上盘子, 然后用 parafilm 把盘子和盖子包起来。安装盘然后可以被存放盖子边面对下来5天在4°c。</li>
	<li>提前准备1毫升整除数0.7% 低熔点琼脂糖在1.5 毫升离心管使用在安装日。 注: 安装盘的琼脂糖井会随着时间的推移而变形, 因为如果 parafilm 不紧紧缠绕在盘子里, 水就会蒸发。</li>
</ol>2. 获得 Photoconversion 的胚胎
<ol><li>Incross Tg (fli1a: gal4FF瑞银, 无人参与: 枫)线, 在28.5 摄氏度的黑暗中生长大约200胚胎。有关如何跨越斑马鱼线的详细信息, 请参阅朱庇特科学教育数据库11。</li>
	<li>5小时后, 但在24小时之前, 治疗胚胎与1苯基 2-硫脲 (PTU), 以防止色素形成 (0.003% PTU 在胚胎培养基)。</li>
	<li>在 photoconversion 开始前约1小时, 在荧光立体镜下筛选胚胎, 并选择5个健康的胚胎来表达最亮的绿色荧光。建议在胚胎可视化时使用低强度光, 以避免胚胎暴露于环境光中, 以避免非特异细胞 photoconverting 枫。</li>
</ol>3. 嵌入
<ol><li>准备10毫升的安装介质: 0.02% tricaine 和50毫米 BDM 胚培养基。</li>
	<li>添加2毫升的安装介质到安装盘, 并允许15-30 分钟的溶液扩散到琼脂糖。</li>
	<li>同时, 将管置于70°c 热块中, 熔化1毫升整除0.7% 低熔点 (LMP) 琼脂糖, 约5分钟。<ol>
<li>等待 LMP 琼脂糖稍凉, 然后添加25µL 8 毫克/毫升 tricaine 溶液和40µL 1M BDM 溶液到低熔点琼脂糖管。吹打上下混合, 然后将管子转移到38摄氏度的热块中, 以保持 LMP 琼脂糖在其液态状态。</li>
	</ol>
</li>
	<li>在具有胚胎培养基的培养皿中, 用镊子仔细 dechorionate 显微镜下的预选胚胎, 注意不要损伤胚胎。</li>
	<li>将胚胎移植到含有2毫升安装介质的单独盘子中。<ol>
<li>当胚胎的心脏停止 (大约5-10 分钟), 转移胚胎到安装盘和安排胚胎在井使用钳, 因此他们说谎在25度角度, 尾巴指向井的更深部分, 蛋黄面对。</li>
		<li>当胚胎大致到位时, 取出培养基, 将胚胎植入200µL 0.7% LMP 琼脂糖中, 其中含有 tricaine 和 BDM, 并调整胚胎的位置, 必要时将胚胎倾斜到左侧或右侧。</li>
	</ol>
</li>
	<li>等到 LMP 琼脂糖集 (大约5分钟), 然后添加安装培养基的菜肴。胚胎现在已经准备好 photoconverted 了。 注意: tricaine 和 BDM 是用来麻醉胚胎和停止胚胎的心脏, 分别。安装介质可以在成像前一天准备就绪, 但要确保介质不受光线的保护。 注意: 正确的胚胎定位是重要的, 让一个明确的激光路径的细胞你希望 photoconvert。把胚胎的头部靠近井的开始, 这样激光就不必远距离到达胚胎, 这样胚胎就可以很容易地在 photoconversion 后被卸载。</li>
</ol>4. Photoconversion
<ol><li>在直立共焦显微镜 (如莱卡 SP8) 配备了405毫微米, 488 毫微米和 561 nm 激光源, 找到胚胎的心脏使用透射白光 (brightfield) 和物镜 (如莱卡 HCX IRAPO L, 25 x, 符合0.95 目标)。检查红色枫荧光使用561毫微米激光 (即, DPSS 561 激光, 在5% 强度, 探测器设置在 589-728 nm)。然后, 通过使用 488 nm 激光 (即, 30 兆瓦多行氩激光器在5% 的强度, 探测器设置在 502-556 nm) 可视化心内膜。</li>
	<li>在显微镜采集软件上输入 "酶" 模式。选择1% 激光功率为488毫微米和 561 nm 激光。</li>
	<li>关注平面的兴趣, 选择一个感兴趣的区域 (ROI), 然后 photoconvert 细胞使用405纳米二极管激光器在25% 激光功率, 扫描 ROI 三倍。如果不充分枫 photoconverted 在 ROI, 扫描405毫微米激光在区域三次。对所有 ROIs 重复此步骤。</li>
	<li>返回到软件上的 "TCS SP8" 模式, 并获得一个 z 栈, 使用 561 nm 和 488 nm 激光器顺序。请确保选择 "双向" 选项以提高成像速度。 注意: photoconvert 和图像需要多长时间, 每个胚胎都有变化。心脏跳动是已知的重要的正常心脏瓣膜发展, 所以避免保持心脏停止超过30分钟, 即使它意味着你没有时间 photoconvert 所有5胚胎。 注: 在 photoconversion 过程中, 使用 PMT 探测器, 而在 photoconversion 后, 建议使用混合探测器设置为 "计数" 模式。这是因为混合探测器更敏感, 能够产生更好的质量图像, 但容易受到过度曝光的破坏。</li>
</ol>5. 卸载 photoconverted 胚胎
<ol><li>photoconversion 后, 用玻璃吸管轻轻按压胚头上方, 打破 LMP 琼脂糖, 然后轻轻吮吸胚胎。<ol>
<li>将胚胎从玻璃吸管中喷射到含有 PTU 胚培养基的35毫米培养皿中 (洗去含有 BDM 的培养基)。</li>
		<li>将胚胎移植到一个含有胚介质的6井板中, 并 PTU。在这一步, 一定要注意到哪个胚胎进入哪个好, 因为这是必要的, 以关联的位置 photoconverted 细胞在后期发育阶段。</li>
	</ol>
</li>
	<li>现在胚胎从含有培养基的 BDM 中取出, 他们的心脏应该在5分钟内重新开始跳动. 将胚胎恢复到28.5 摄氏度的黑暗中, 让胚胎继续正常发育。</li>
</ol>6. 后期胚胎阶段的成像 photoconverted 细胞
<ol><li>在理想的阶段, 停止心脏和重新嵌入的胚胎像在这个协议的步骤 3, 具有重要的区别, 胚胎必须处理与 BDM 在单独的, 标记的菜肴, 以保持跟踪的胚胎。</li>
	<li>对于多达 62 hpf 的胚胎, 分别使用 561 nm 和488纳米的红色和绿色荧光信号获取心内膜的 z 栈。对于 62 hpf 以上阶段的胚胎, 使用多光子激光设置为 940 nm, 以图象绿色枫而不是 488 nm 激光。</li>
</ol>7. 48 hpf 下胚胎的图像分析
注: 由于其复杂的三维结构, 该组织的运动可能难以分析。为了量化在两个非 photoconverted 区域之间的 photoconverted 区域的长度, 或在两个 photoconverted 区域之间的非 photoconverted 区域的长度, 可以将三维结构映射到二维地图。在 48 hpf 之前获得的 photoconverted 胚胎的图像可以用以下方法进行分析。
<ol><li>导入 Matlab 或任何类似软件中获取的图像。</li>
	<li>界定手动执行 ROI,即心内膜, 并应用掩码来删除此区域以外的信号。</li>
	<li>在图像上应用强度阈值来识别心内膜上的点并在三维中绘制它们。通过在软件中使用 "擦除" 命令, 在心房和心室上手动抹去不感兴趣的点, 将对应于 AVC 最窄和最直部分的点隔开。</li>
	<li>使用气缸来满足代表的 AVC, 所以得到的点。</li>
	<li>定义一个参考系统来比较几个样本。例如, 作为参考系统的原点和拟合圆柱的轴线作为 z 轴, 采用了该方法的质量中心。<ol>
<li>确保阳性 z 位置始终对应于心脏的同一侧 (心室或心房)。</li>
		<li>将在 x-y 平面上的 AVC 点投射出来, 并使用一个椭圆来适应它们。</li>
		<li>旋转在 z 轴周围的心内膜点, 以便椭圆的主轴和参照系统的 y 轴是平行的, 正 y 值对应于与该胚胎有关的 AVC 的内部一侧。</li>
	</ol>
</li>
	<li>要对 AVC 进行分段, 请通过在 z 方向旋转半平面 {y=0、x > 0} 获得一些平面来剪切三维数据集。<ol>
<li>投射这些平面上相邻像素的强度, 并以一致的方式定义内皮。例如, 手动绘制一条线, 从心房到心室一侧的 AVC, 在半厚度的内皮。</li>
		<li>用三维样条来插值表示内皮的线的点, 使用样条工具箱表示与曲面的 AVC。 注: 切割平面的数量决定了分割的准确性和此步骤的时间消耗。通常, 8 架飞机足以达到良好的效果。</li>
	</ol>
</li>
	<li>用圆柱坐标系定义了 AVC 表面上每个点的方位位置。<ol>
<li>在每个方位位置上, 通过计算连续点之间的弧长来定义一个沿其表面的参数曲线。</li>
		<li>对于每个参数曲线, 在曲线上定义最接近 AVC 中心的点为零。因此, 在参数曲线上, 对其方位位置和位置进行了充分描述。</li>
	</ol>
</li>
	<li>利用点的参数位置将 AVC 展开为二维图像。</li>
	<li>找出该区域的边缘以量化为二进制图像的边缘, 以分割 photoconverted 和非 photoconverted 通道的强度比值。如有必要, 手动更正结果。</li>
	<li>计算边缘之间的弧长, 以计算组织的长度作为方位位置的函数。</li>
</ol>

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P., Qin, H., Pantazis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cell+tracking+using+PhOTO+zebrafish.">In vivo cell tracking using PhOTO zebrafish.</a> Methods Mol Biol. 1148, 217-228 (2014).</li></ol>

没有宣布利益冲突。

photoconversion 的定时: 尽管枫在 EdCs 中仍然保持明亮的表达, 即使在 96 hpf, 也应该注意到, 随着胚胎的生长, 激光在到达 AVC 之前扩散得更多, 使得 photoconversion 的限制枫更加困难。在胚胎阶段比 55 hpf, 心室和心房的膨胀也意味着, 用于 photoconversion 的紫激光束不能到达 AVC 细胞, 而不首先穿过心房或心室。这意味着, 在 55 hpf 以后, 为了 photoconvert EdCs, 你必须也 photoconvert 另外的 EdCs 在心房和脑室。<...

斑马鱼目前是最重要的脊椎动物模型之一, 研究细胞和发育过程的在体内。这主要是由于斑马鱼的光学透明度和顺从遗传学, 这使得它成为一个强大的模型应用光学技术涉及基因编码 photoresponsive 蛋白技术1。针对心脏发育的研究, 斑马鱼通过扩散获得足够的氧气, 即使没有心跳的突变体也能在发育的第一周存活下来, 允许分析发育基因的影响和扰动在大多数脊椎动物中, 血流在心脏形态发生是不可能的2。
斑马鱼心脏管由24小时后受精 (hpf) 形成。形成后不久, 心脏管开始积极跳动。由 36 hpf, 清晰的收缩将心房与心室隔开。这一收缩区称为房室道 (AVC), 该区域的细胞从鳞状形态学转变为立方形态学3。斑马鱼房室瓣膜形态发生开始约48小时后受精。5天后受精, 两个瓣膜小叶延伸到 AVC 和防止血液从心室的背部流到心房在心脏周期的4。当心脏的快速跳动使得通过传统的时间失效显微镜5,6时, 跟踪细胞变得困难。此协议从骏马et., 20167, 描述使用 Tg 的方法(fli1a: gal4FF瑞银, 无人参与: 枫)8斑马鱼转基因线, 其中 photoconvertible 蛋白枫表示在内皮细胞, 包括心内膜。药物 23-butanedione 2-肟 (BDM) 用于暂时停止心脏跳动, 允许在36和 55 EdCs 之间精确 photoconversion hpf, 以及在特定发育时间点的 photoconverted EdCs 的高分辨率成像。以前已显示, 使用此方法的 EdCs photoconverted 在 photoconversion7之后的五天或更远的时间内可以与未转换的邻居保持可区分性。该协议还详细介绍了一种用于在 48 hpf 的胚胎中 photoconverted EdCs 的图像分析方法, 它已成功地用于在 AVC 开发过程中的组织运动 (路易吉·博塞利et), 按下)9。我们希望读者能发现这项议定书对于研究正常胚胎中的 AVC 和瓣膜发育, 以及突变体、morphants 或药物治疗的胚胎都是有用的。有关在斑马鱼开发过程中使用 photoconvertible 蛋白进行细胞跟踪的更通用的协议, 请查看由伦巴多et, 201210的文章。

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Necessary equipment for raising fish and collecting eggs (see the Zebrafish Book22 for details).</td><td></td><td></td><td></td></tr><tr><td>Stereomicroscope</td><td></td><td></td><td></td></tr><tr><td>Upright confocal microscope (Equipped with lasers operating at 488 nm, 561 nm, and 405 nm, a tunable multiphoton laser, and a Leica HCX IRAPO L, 25 &amp;times;, N.A. 0.95 objective)</td><td>Leica</td><td>Leica SP8</td><td></td></tr><tr><td>Heat block</td><td>ThermoScientific</td><td>88870001</td><td></td></tr><tr><td>35 mm x 10 mm tissue culture dish</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>6 well plate</td><td>Falcon</td><td>353046</td><td></td></tr><tr><td>Petri dish</td><td></td><td></td><td></td></tr><tr><td>Forceps</td><td></td><td></td><td></td></tr><tr><td>Glass pipette</td><td></td><td></td><td></td></tr><tr><td>Mold</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>8 mg/mL Tricaine stock solution&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>1 M BDM stock solution</td><td></td><td></td><td></td></tr><tr><td>UltraPure low melting point agarose</td><td>Invitrogen</td><td>16520-050</td><td></td></tr><tr><td>Agarose</td><td>Lonza</td><td>50004</td><td></td></tr><tr><td>PTU (1-phenyl-2-thiourea)</td><td>Sigma Aldrich</td><td>P7629</td><td></td></tr><tr><td>Embryo medium: 30x stock solution</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Matlab equipped with the Image Analysis, Curve Fitting, Bio-Formats Toolboxes</td><td>MathWorks</td><td></td><td></td></tr><tr><td>&lt;strong&gt;8 mg/mL Tricaine stock solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>200 mg of tricaine powder (Ethyl 3-aminobenzoate methanesulfonic acid)</td><td>Sigma-Aldrich</td><td>E10521</td><td></td></tr><tr><td>25 mL Danieau</td><td></td><td></td><td></td></tr><tr><td>Adjust to pH 7, aliquot and store at -20 &amp;deg;C&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;1 M BDM stock solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1 g BDM</td><td>Sigma-Aldrich</td><td>112135</td><td></td></tr><tr><td>10 mL ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>Aliquot and store at -20 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Embryo medium: 30x stock solution&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>50.7 g NaCl</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td></td></tr><tr><td>0.78 g KCl</td><td>Sigma-Aldrich</td><td>7447-40-7</td><td></td></tr><tr><td>1.47 g Magnesium sulfate</td><td>Sigma-Aldrich</td><td>&amp;nbsp;7487-88-9</td><td></td></tr><tr><td>2.1 g Calcium nitrite tetrahydrate</td><td>Sigma-Aldrich</td><td>13477-34-4</td><td></td></tr><tr><td>19.52 g HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)</td><td>Sigma-Aldrich</td><td>7365-45-9</td><td></td></tr><tr><td>Adjust to pH 7.2 and store at room temperature</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Mold&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PlasCLEAR resin</td><td>Asiga</td><td></td><td></td></tr><tr><td>Plus 39&amp;nbsp;Freeform Pico 3D printer</td><td>Asiga</td><td></td><td></td></tr></tbody></table>

following endocardial tissue movements via cell photoconversion in the zebrafish embryo

在胚胎发生过程中, 细胞在细胞行为中发生动态变化, 破译这些变化背后的细胞逻辑是发育生物学领域的一个基本目标。photoconvertible 蛋白的发现和发展极大地帮助我们理解这些动态变化, 提供了一种光学突出细胞和组织的方法。然而, 虽然 photoconversion, 时间推移显微镜, 和随后的图像分析已经证明是非常成功的揭露细胞动力学的器官, 如大脑或眼睛, 这种方法一般不使用在发展中的心脏由于心脏循环过程中心的快速运动所带来的挑战。该协议由两部分组成。第一部分介绍了在斑马鱼房室管 (photoconverting) 和房室心脏瓣膜发育过程中, 对心内膜细胞 (EdCs) 进行追踪和跟踪的方法。该方法包括用药物暂时停止心脏, 以便准确 photoconversion 发生。心脏被允许恢复跳动后, 药物的清除和胚胎发育继续正常, 直到心脏再次停止的高分辨率成像 photoconverted EdCs 在以后的发展时间点。该协议的第二部分描述了一种图像分析方法, 通过将三维结构中的荧光信号映射到二维地图上来量化 photoconverted 或非 photoconverted 区域在幼胚中的长度.同时, 该协议的两个部分允许一个人检查组成斑马鱼和房室心脏瓣膜的细胞的起源和行为, 并有可能被应用于研究突变体, morphants, 或胚胎, 已治疗破坏 AVC 和/或阀门发展的试剂。

图 2、影片 1和2中显示了一个 48 hpf 的胚胎 photoconverted 的示例, 并在 80 hpf 中再次映像。暴露枫到405毫微米光不可逆转地转换从从它的荧光绿色形式的蛋白质到荧光红色形式, 使标记以绿色或他们的红色形式的细胞的行为将跟随关于他们的差异色邻居在阀门形成期间。可以看出, 48 hpf 的几个 photoconverted 细胞后来激增, 后来主要?...

Watch this Scientific Journal Video about Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo at JoVE.com

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

该协议描述了在活斑马鱼胚胎的心内膜细胞中 photoconversion 枫荧光蛋白的方法, 能够在房室管和房室心脏瓣膜发育过程中跟踪心内膜细胞。.

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 ...

following-endocardial-tissue-movements-via-cell-photoconversion

Research

JoVE Journal

Developmental Biology

6.4K 次观看.  Institut de Génétique et de Biologie Moléculaire et Cellulaire. 该协议描述了在活斑马鱼胚胎的心内膜细胞中 photoconversion 枫荧光蛋白的方法, 能够在房室管和房室心脏瓣膜发育过程中跟踪心内膜细胞。.

视频: Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

分离和斑马鱼胚胎单细胞的表征

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

科研

发育生物学

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, ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression.  ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

光片荧光显微术捕捉4维图像调制剪切应力对斑马鱼心脏发育的影响

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

生物工程

<meta charset="utf8"></head><body>在斑马鱼心脏中使用磁珠的体内机械转导研究

Manipulating Mechanical Forces in the Developing Zebrafish Heart Using Magnetic Beads

Mechanical forces continuously provide feedback to heart valve morphogenetic programs. In zebrafish, cardiac valve development relies on heart contraction and physical stimuli generated by the beating heart. Intracardiac hemodynamics, driven by blood flow, emerge as fundamental information shaping the development of the embryonic heart. Here, we describe an effective method to manipulate mechanical forces in vivo by grafting a 30 &#181;m to 60 &#181;m diameter magnetic bead in the cardiac lumen. The insertion of the bead is conducted through microsurgery in anesthetized larvae without perturbing heart function and enables artificial alteration of the boundary conditions, thereby modifying flow forces in the system. As a result, the presence of the bead amplifies the mechanical forces experienced by endocardial cells and can directly trigger mechanical stimulus-dependent calcium influx. This approach facilitates the investigation of mechanotransduction pathways that govern heart development and can provide insights into the role of mechanical forces in cardiac valve morphogenesis.

<meta charset="utf8"></head><body>作者聚焦:了解塑造斑马鱼心脏所涉及的机械力

This protocol outlines a method for grafting a magnetic bead into the developing zebrafish heart through microsurgery, enabling the manipulation of mechanical forces in vivo and triggering mechanical stimulus-dependent calcium influx in endocardial cells.

使用磁珠操纵发育中的斑马鱼心脏中的机械力

Mechanical forces continuously provide feedback to heart valve morphogenetic programs. In zebrafish, cardiac valve development relies on heart contraction ...

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

免疫染色的解剖斑马鱼胚胎心脏

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

在斑马鱼胚胎中通过细胞 Photoconversion 的心内膜组织运动后

摘要

探索更多视频

此视频中的章节

分离和斑马鱼胚胎单细胞的表征

成像清除胚胎和出生后心脏在单细胞分辨率

活完好斑马鱼的单细胞 Photoconversion

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

光片荧光显微术捕捉4维图像调制剪切应力对斑马鱼心脏发育的影响

使用磁珠操纵发育中的斑马鱼心脏中的机械力

使用磁珠操纵发育中的斑马鱼心脏中的机械力

使用磁珠操纵发育中的斑马鱼心脏中的机械力

免疫染色的解剖斑马鱼胚胎心脏