作者感谢亚历克斯Schier的亲切共享Zebrabow-M转基因株系，杰弗里·伯恩斯的pDEST载体和Renee西尔 - Daigle提供无微不至的关怀鱼设施和线路。 资金来源： 我们感谢来自NIDCR RO3DE024490和施赖纳斯医院慷慨的资金支持儿童（ECL）和博士后培训奖学金从圣地兄弟会儿童医院（LR和YK）。

马萨诸塞州总医院实验动物管理和使用委员会（IACUC）批准根据协议号＃2010N000106的所有程序。这是符合协会的评估和实验动物国际（AAALAC）的指导方针的认可。 1.试剂和材料准备<ol><li>制备1升的50X E3胚胎培养基（ 见表 1）和制备1升1X E3胚胎培养基（ 见表 2）。 </li><li>加入PTU 30毫克到1升1X E3的准备与N-苯基硫氧嘧啶（PTU）E3胚胎中期。搅拌O / N，以确保机动部队已完全溶解。存储在RT。 </li><li>为了制备链霉蛋白酶溶液，稀释150微升链霉蛋白酶（0.5毫克/毫升）的15毫升1X的E3。这一数额是足够的1培养皿。立即使用。 </li><li>准备甲基纤维素溶液。 <ol><li>制备E3-三卡因溶液通过混合75 ml的0.2％三卡因的用25ml的1X E3。煮沸3克甲基纤维素在75毫升E3-三卡因溶液。 </li><li>补足终体积到100毫升，E3-三卡因溶液。最终的甲基纤维素溶液是粘稠和不透明带黄色调。贮存：RT保护的光。 </li></ol></li><li>他莫昔芬的股票和感应解决方案<ol><li>通过将4-羟基在100％乙醇制备5mM的他莫昔芬原液。商店的股票在-80℃。 </li><li>准备新鲜的稀释他莫昔芬在1X E3的愿望，得到浓缩感应解决方案。注意！使用前请咨询MSDS。 </li></ol></li><li>制备1升0.2％三卡因的按表3中。 </li><li>获取共聚焦幻灯片。胶4玻璃罩（18×18）在一起，形成一个块，然后把2个街区，在24×60的玻璃罩相距2厘米，以便安装在中间的胚胎。 </li></ol> 2.胚胎制备<ol><li>转基因株系。 <br/&gt;注：Zebrabow-M是由亚历克斯Schier实验室的一份厚礼。 </li><li> SOX10：ERT2 Cre重组 注：使用标准的分子克隆方法ERT2 Cre重组和商业上获得重组克隆技术：生成靶向载体pDestTol2- SOX10。 <ol><li>结合5&#39;进入克隆（pENTR5&#39;- sox10p）含有7.2Kb的SOX10的启动子，一个网关中间克隆（PME-ERT2-Cre的），和一个3&#39;进入克隆（pENTR3&#39;-polyA的），其与透镜目的地LR反应矢量pDestTol2-α晶状体 ：YFP。 </li><li>净化构建pDestTol2- SOX10：ERT2 Cre重组。共注入纯化构建体（100毫微克/微升）用TOL2转座的mRNA（50纳克/微升）到WT胚胎（Fo）的一个小区的阶段。 </li><li>屏幕F0成人创始人为种系传递的基础上强劲的黄色荧光F1胚胎。 </li></ol></li><li> SOX10：ERT2 Cre重组; Zebrabow-M <ol><li>异交的Zebrabow-M行（2.1.1）与SOX10：ERT2-Cre转基因。 </li><li>选择胚胎表现出较强的无处不在RFP的表达和YFP镜头标记和成长，直到他们成年。 </li></ol></li></ol> 3.胚胎收集<ol><li>建立了几个对SOX10的：ERT2 Cre重组; Zebrabow-M转基因斑马鱼下午5点和下午6点第1天之间。 </li><li>第二天，拉早上分频器和收集中午前后鸡蛋。 </li><li>他们转移到培养皿中，并新增15毫升链霉蛋白酶的解决方案，以dechorionate胚胎。 </li><li>孵育胚胎进行1〜5分钟，直到胚胎的约60％已被dechorionated。停止反应通过滗析链霉蛋白酶溶液，并用新鲜的E3介质洗胚胎3-4倍。 </li><li>孵育在E3的dechorionated胚胎在室温O / N，让他们发展，达到10体节阶段。每孵育50-60离合器胚胎，以避免不同的发育生长。 </li></OL&gt; 4.治疗<ol><li>检查光场显微镜下胚胎的发育阶段，以确保他们在10体节阶段。 </li><li>使用荧光显微镜与红色荧光蛋白（RFP）过滤，分离出亮红色的胚胎。 </li><li>加入10的羟基他溶液到培养皿μM和孵育所述分离的胚胎在28.5℃下进行3小时进行到Cre的重组的诱导。 </li><li>滗他莫昔芬溶液和E3洗胚胎几次。 注意！丢弃在一个特殊的生物废料容器中的他莫昔芬的解决方案。 </li><li>孵育胚胎在28.5°CO / N。 </li><li>当胚胎是大约28-29体节阶段（约24小时受精后），孵化的胚胎在28.5℃，在E3-PTU解决方案以后。 注：PTU是这里用来抑制在发育中的胚胎形成黑素细胞的。这种治疗是必要的，以避免荧光的由自体荧光黑色素细胞的信号失真。 </li><li>孵育胚胎在28.5℃， </li></ol> 5.胚胎选择<ol><li>以可视化的全面发展颅面结构中，选择红色信号和表达Cre的标记阳性（黄色视网膜）的强度的胚胎在4天后施肥。 </li><li>麻醉与E3 / 0.015％三卡因溶液的胚胎。当没有活跃的走势或鳍活动时，轻轻地捅了捅胚胎观察麻醉被确认。 </li></ol> 6.安装在甲基纤维素的胚胎<ol><li>传输用于成像所选胚胎成含有一滴甲基纤维素的培养皿中，并轻轻薰胚胎与甲基纤维素。 </li><li>在干净的共焦幻灯片，将一滴新鲜的甲基纤维素和使用微加载提示下拉内安装的胚胎。位置胚背采取谨慎不要碰直接胚胎。 </li><li>覆盖安装胚用25×25盖玻片并在必要时通过操纵盖玻片调节胚胎的位置。 </li><li>胚胎是现在已经准备好形象。 </li></ol>通过荧光显微镜7.分析<ol><li>使用20X干透镜物镜（数值孔径0.75;针孔大小2.0微米）对焦胚胎。 </li><li>按下显微镜L100按钮，并在软件中选择的通道。 </li><li>选择激光共聚焦设置按钮，然后单击“自动”，并关闭窗口前选择以下途径：CH1：CFP，CH2：GFP，CH3：RFP和mCherry </li><li>点击“删除互锁”按钮前，打开CH2和CH3。点击“播放”开始扫描。 </li><li>专注于感兴趣的结构，这是最好的开始半帧/秒的高电压（HV）150，然后相应地调整设置完成，直到所需的图像质量是交流hieved。招标书会比其他颜色更亮，从而调节高压。改变Z位置找到这两个YFP和RFP阳性细胞。 </li><li>一旦设定的设置，捕获胚胎的形象。保存你的照片进行图像处理的每个分割通道的共焦成像软件格式和TIFF格式。 </li><li>接下来，CFP成像，关闭CH2和CH3并打开通道1。重新调整设置如在步骤5，而不改变焦点区域。 注：CFP可真难以检测由于在蓝色通道的背景信号，可以通过调节针孔大小被规避。较大的针孔大小允许更好的检测荧光的减少信号/背景噪声比。然而，一个较大的针孔会降低共焦效果，影响图像质量。因此，仔细调整的设置是必需的以获得所需的图像质量和强度。 </li><li>捕获图像，并将其保存在两种格式（共聚焦软件和TIFF）进行图像处理。图像然后可以使用通用图像处理软件来合并层和所描述的水平和同事8提高颜色设置处理。 </li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Hedgehog+signaling+from+neural+to+oral+epithelium+organizes+anterior+craniofacial+development.">Early Hedgehog signaling from neural to oral epithelium organizes anterior craniofacial development.</a> Development. 133, 1069-1077 (2006).</li><li>Dixon, M. J., Marazita, M. L., Beaty, T. H., Murray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleft+lip+and+palate:+understanding+genetic+and+environmental+influences.">Cleft lip and palate: understanding genetic and environmental influences.</a> Nature reviews Genetics. 12, 167-178 (2011).</li><li>Gfrerer, L., Dougherty, M., Liao, E. 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Birth defects research.</a> Part C, Embryo today : reviews. 93, 67-114 (2011).</li><li>Mossey, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+education+and+CPD:+are+you+being+served.">Dental education and CPD: are you being served.</a> British dental journal. Suppl, 3-4 (2002).</li><li>Pan, Y. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrabow:+multispectral+cell+labeling+for+cell+tracing+and+lineage+analysis+in+zebrafish.">Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish.</a> Development. 140, 2835-2846 (2013).</li><li>Vieira, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Journal of dental research. 87, 119-125 (2008).</li><li>Le Pabic, P., Ng, C., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat-Dachsous+Signaling+Coordinates+Cartilage+Differentiation+and+Polarity+during+Craniofacial+Development.">Fat-Dachsous Signaling Coordinates Cartilage Differentiation and Polarity during Craniofacial Development.</a> PLoS genetics. 10, e1004726 (2014).</li><li>Ricks, J. E., Ryder, V. M., Bridgewater, L. C., Schaalje, B., Seegmiller, R. 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The authors declare that they have no competing financial interests.

阿尔新蓝和photoconvertible转基因株系上述相得益彰确定软骨和骨骼发育的复杂的过程。然而，器官在现场细胞迁移和组织长期以来一直推测，间接证明了，但从来没有显现。 Zebrabow-M的转基因系加上软骨特异性表达Cre允许同时实时观测参与骨和软骨形成所有这些不同的事件。这种技术允许的下颚生长缺陷在诸如罗宾序列，其中小颌是所描述的突出特征之一的条件的细胞和器官水平的研究。失败的增?...

颅面发展需要复杂的分子，细胞和组织的相互作用来驱动细胞的增殖，迁移和分化1,2,3。此严格调节和复杂的过程，受遗传和环境扰动，使得颅面畸形是其中最常见的出生畸形1-9。虽然外科手术仍然是主要的治疗方法为颅面畸形，了解发展的基础是至关重要的创新，未来的治疗方法。因此，研究的形态发生和机制中的收敛和延伸和细胞融合提供了新的见解的颅面骨1的形成。 颅神经嵴迁移和填充第一咽弓，进而形成配对下颌流程延伸到形成麦克尔软骨，这预示着下颌骨。形态Øf显示麦克尔软骨需要通过定向增殖，细胞分化和分化1,10软骨组织。然而，软骨细胞组织中的梅克尔软骨的生长和扩展的复杂仍不清楚。理解动态细胞行为是对理解影响下颌大小，如发育不全颌骨表型11先天性畸形的关键。 斑马鱼胚胎提供了对麦克尔软骨形态详细研究的许多发育和遗传的优势。其遗传易处理性，透明度， 离体快速发展的强大优势，通过实时成像6放贷很好的观察细胞的运动和组织。采用谱系追踪工具，如SOX10：枫转基因线，我们和其他人已经圈定了胚胎颅面骨1,5的神经嵴起源。 USI纳克的SOX10：ERT2 Cre重组与UBI：Zebrabow-M的转基因系，现在有可能颅面发育过程中，探索细胞运动的细节。所述Zebrabow-M，是转基因线工程与泛素启动子驱动的荧光团不同的表达，每个由lox位点8侧翼。该Zebrabow-M默认荧光红，表达RFP。诱导Cre的表达后，Zebrabow-M的构建重组和细胞表达不同的荧光团（RFP，CFP和YFP）创建在胚胎多光谱表达的组合。所有的子细胞从重组事件后的标记的细胞分裂，然后克隆标记，从而使细胞群，从不同的并列祖派生是克隆标记。由该克隆细胞标记，细胞增殖和迁移与克隆分辨率可以跟随（图1 和2）。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pronase</td>
			<td>Roche Life Sciences</td>
			<td>10165921001</td>
			<td>Prepare 500 &#956;L stock aliquots at 50mg/mL</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M0262</td>
			<td></td>
		</tr>
		<tr>
			<td>PTU (N-Phenylthiourea)</td>
			<td>Sigma-Aldrich</td>
			<td>P7619</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>4-HydoxyTamoxifen</td>
			<td>Sigma-Aldrich</td>
			<td>T176</td>
			<td></td>
		</tr>
		<tr>
			<td>24 x 60 coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-548-5P</td>
			<td></td>
		</tr>
		<tr>
			<td>18 x 18 coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-540A</td>
			<td></td>
		</tr>
		<tr>
			<td>25 x 25 coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-540C</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR5&#39;-TOPO TA Cloning Kit</td>
			<td>Life technologies&#160;</td>
			<td>K591-20</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR/D-TOPO Cloning Kit&#160;</td>
			<td>Life Technologies</td>
			<td>K2400-20</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR3&#39;-pA</td>
			<td>Tol2Kit</td>
			<td>302</td>
			<td></td>
		</tr>
		<tr>
			<td>pDEST</td>
			<td></td>
			<td></td>
			<td>Gift from Geoffrey Burns labs</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bright field microscope&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image processing software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of chondrocyte intercalation and directional proliferation via zebrabow clonal cell analysis in the embryonic meckel&#8217;s cartilage

脊椎动物颅面结构的发展，需要的细胞迁移，增殖，粘附和分化精确协调。该麦克尔软骨，第一咽弓衍生物的图案涉及颅神经嵴（CNC）细胞的组织分化软骨细胞的迁移和逐步分区，增殖和。期间下颚形态几项研究描述数控迁移，但的软骨细胞如何实现组织中梅克尔软骨的生长和扩展的细节仍不清楚。的SOX10限定，化学诱导Cre重组介导的重组产生截然不同的荧光蛋白（RFP，YFP和CFP）的排列，从而产生祖细胞和它们的后代的多光谱标记，反映不同的克隆群。使用共聚焦延时摄影，也能够观察到软骨细胞behavi或斑马鱼梅克尔软骨的发育过程中。 多光谱细胞标记使得科学家能够证明延伸梅克尔软骨细胞。在麦克尔软骨，这预示了下颌骨的推广阶段，软骨细胞插层，以实现扩展，因为他们在一个有组织的单细胞层排叠加。该组织嵌入过程中未能介导细胞扩展提供了蜂窝机械的解释下颌骨发育不良，我们观察下颌骨畸形。

传统的可视化软骨通过整装阿辛兰污渍是非常宝贵的观察开发麦克尔软骨和常用的可视化最终细胞组织12（ 图1A）。为了进一步分析发展中的软骨细胞加班，使用SOX10谱系追踪：枫的转基因品系，使我们能够研究细胞的迁移，融合和延伸活胚胎2,12（ 图1B）。然而，软骨细胞在颅面骨骼结构的组织仍然知之甚少。通过实时成像，所述Zebrabow-M的?...

Watch this Scientific Journal Video about Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckelâ€™s Cartilage at JoVE.

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&amp;#8217;s Cartilage

颅面骨细胞组织长期以来一直推测但从未直接显现。多光谱细胞标记和体内活 成像可以在斑马鱼下颌动态细胞行为的可视化。在这里，我们详细的协议来操作Zebrabow转基因鱼，直接观察细胞嵌入和麦克尔软骨细胞的形态变化。

通过在胚胎梅克尔软骨Zebrabow克隆细胞分析软骨插层和定向扩散的可视化

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

visualization-chondrocyte-intercalation-directional-proliferation-via

Research

JoVE Journal

Developmental Biology

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

9.0K 次观看.  Harvard Medical School. 颅面骨细胞组织长期以来一直推测但从未直接显现。多光谱细胞标记和体内活 成像可以在斑马鱼下颌动态细胞行为的可视化。在这里，我们详细的协议来操作Zebrabow转基因鱼，直接观察细胞嵌入和麦克尔软骨细胞的形态变化。 

视频: Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

在清除胚胎心脏冠状血管分析

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

科研

发育生物学

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

5-mC点印迹测定定量软骨细胞去分化的DNA甲基化水平<em&gt;体外</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth factors, and extracellular matrix. The ability of HSCs to remain quiescent, self-renew or differentiate, and acquire mutations and become malignant depends upon the complex interactions they establish with different stromal components. To observe the crosstalk between human HSCs and the human BM niche in physiological and pathological conditions, we designed a protocol to ectopically model and image a humanized BM niche in immunodeficient mice. We show that the use of different cellular components allows for the formation of humanized structures and the opportunity to sustain long-term human hematopoietic engraftment. Using two-photon microscopy, we can live-image these structures in situ at the single-cell resolution, providing a powerful new tool for the functional characterization of the human BM microenvironment and its role in regulating normal and malignant hematopoiesis.

A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

人类骨髓微环境在小鼠中的生物工程及其通过实时成像的可视化

Human hematopoietic stem cells (HSCs) reside in the bone marrow (BM) niche, an intricate, multifactorial network of components producing cytokines, growth ...

Clonal Analysis of Embryonic Hematopoietic Stem Cell Precursors Using Single Cell Index Sorting Combined with Endothelial Cell Niche Co-culture

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early embryo and the lack of assays that functionally identify the long-term multilineage engraftment potential of individual putative HSC precursors. Here, we describe methodology that enables the isolation and characterization of functionally validated HSC precursors at the single cell level. First, we utilize index sorting to catalog the precise phenotypic parameter of each individually sorted cell, using a combination of phenotypic markers to enrich for HSC precursors with additional markers for experimental analysis. Second, each index-sorted cell is co-cultured with vascular niche stroma from the aorta-gonad-mesonephros (AGM) region, which supports the maturation of non-engrafting HSC precursors to functional HSC with multilineage, long-term engraftment potential in transplantation assays. This methodology enables correlation of phenotypic properties of clonal hemogenic precursors with their functional engraftment potential or other properties such as transcriptional profile, providing a means for the detailed analysis of HSC precursor development at the single cell level.

Here we present methodology for the clonal analysis of hematopoietic stem cell precursors during murine embryonic development. We combine index sorting of single cells from the embryonic aorta-gonad-mesonephros region with endothelial cell co-culture and transplantation to characterize the phenotypic properties and engraftment potential of single hematopoietic precursors.

单细胞指数分型与内皮细胞小生境联合培养对胚胎造血干细胞前体的克隆分析

The ability to study hematopoietic stem cell (HSC) genesis during embryonic development has been limited by the rarity of HSC precursors in the early ...

Visualization of Tangential Cell Migration in the Developing Chick Optic Tectum

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture can be recorded under video microscopy. For analyzing cell migration in the developing brain, slice culture is commonly used to observe cell migration parallel to the slice section, such as radial cell migration. However, limited information can be obtained from the slice culture method to analyze cell migration perpendicular to the slice section, such as tangential cell migration. Here, we present the protocols for time-lapse imaging to visualize tangential cell migration in the developing chick optic tectum. A combination of cell labeling by electroporation in ovo and a subsequent flat-mount culture on the cell culture insert enables detection of migrating cell movement in the horizontal plane. Moreover, our method facilitates detection of both individual cell behavior and the collective action of a group of cells in the long term. This method can potentially be applied to detect the sequential change of the fluorescent-labeled micro-structure, including the axonal elongation in the neural tissue or cell displacement in the non-neural tissue.

We describe the methods for fluorescent labeling of tangentially migrating cells by electroporation, and for time-lapse imaging of the labeled cell movement in a flat-mount culture in order to visualize migrating cell behavior in the developing chick optic tectum.

发展中雏鸡光学顶盖中切向细胞迁移的可视化

Time-lapse imaging is a powerful method to analyze migrating cell behavior. After fluorescent cell labeling, the movement of the labeled cells in culture ...

In Vitro Assays to Evaluate the Migration, Invasion, and Proliferation of Immortalized Human First-trimester Trophoblast Cell Lines

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration and invasion is demonstrated by pregnancy disorders such as pre-eclampsia and intrauterine growth restriction, which are associated with inadequate trophoblast invasion of the maternal vasculature. Unfortunately, our understanding of the mechanisms by which the placenta develops from migrating trophoblasts is limited. In vitro analysis of cell migration via the scratch assay is a useful tool in identifying factors that regulate trophoblast migratory capacity. However, this assay alone does not define the cellular changes that can result in altered cell migration. This protocol describes three different in vitro assays that are used collectively to evaluate trophoblast cell movement: the scratch assay, the invasion assay, and the proliferation assay. The protocols described here may also be modified for use in other cell lines to quantify cell movement in response to stimuli. These methods allow investigators to identify individual factors that contribute to the cell movement and provide a thorough examination of potential mechanisms underlying apparent changes in cell migration.

Here, we present a highly accessible protocol for evaluating the cell movement in human trophoblast cells using three in vitro assays: the scratch assay, the transwell invasion assay, and the cell proliferation assay.

体外检测评估神仙体头三个月滋养细胞细胞系的迁移、入侵和增殖

Cell movement is a critical property of trophoblasts during placental development and early pregnancy. The significance of proper trophoblast migration ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

基于微注射的免疫胚胎心肌细胞体内植入系统

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

半高通量成像方法和数据可视化工具包，用于分析C. elegans胚胎发育

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

用于基因表达分析的小鼠胚胎软骨和骨骼的激光捕获微分

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...