Name: visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy
Uploaded: 2013-09-30T13:00:00
Description: Watch this Scientific Journal Video about Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy at JoVE.com

作者感谢罗伯特Kelsh的好心分享斑马鱼SOX10促进剂。

1。胚胎收集的光转化<ol><li>设置至少十对SOX10的：晚上5至下午6时枫转基因斑马鱼。 </li><li>第二天早上，拉分频器和收集中午前后鸡蛋。他们转移到培养皿中，并把它们放到28.5℃培养箱。 </li><li>在大约24小时受精后，通过去除死胚清洁的培养皿。检查是否使用了光场显微镜和任何荧光显微镜与增强型绿色荧光蛋白（EGFP）过滤胚胎13的发展阶段。枫将是可见的，通过这个过滤器。 </li><li>?...

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K., 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=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 (6), 1069-1077 (2006).</li><li>Ando, R., 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=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc. Natl. Acad. Sci. U S A. 99 (20), 12651-12656 (2002).</li><li>Kimmel, C. B., 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=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203 (3), 253-310 (1993).</li><li>Kawakami, 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=The+zebrafish-secreted+matrix+protein+you/scube2+is+implicated+in+long-range+regulation+of+hedgehog+signaling.">The zebrafish-secreted matrix protein you/scube2 is implicated in long-range regulation of hedgehog signaling.</a> Curr. Biol. 15 (5), 480-488 (2005).</li><li>Lombardo, V. 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这里会显示在斑马鱼模型颅面部发育的可视化的新方法。的SOX10：枫转基因斑马鱼线已经被成功地用于描述中集的详细的迁移模式被用来作为模式生物5。 以前的研究已经用毛地标，如眼靶细胞，并依赖于枫的mRNA注射，光转化试验或笼荧光葡聚糖光转化为10，11，14，15红袜：相比于这10枫转基因株系具有很多优点方法。神经嵴细胞已经贴有枫，符合一个非常特?...

口面裂代表了最普遍的颅面畸形，与1/700-1，000分娩的影响1。早期胚胎颅面部发育受阻可导致形成唇腭裂（CL / P）的。虽然原因综合征型唇裂已经在很大程度上表明，中颜面部的clefting的非综合征形式的遗传和表观遗传基础仍需要发现2-4。为了了解这些畸形的病因和发病机制，有必要阐明颅面结构的细胞基础上的发展。 在所有的脊椎动物物种颅神经嵴细胞（CNCC）从背神经管迁移到填充咽弓，这将有助于形成颜面部结构。早期胚胎神经嵴发育受阻可导致形成颅面畸形包括CL / P 5-7。 在广告DITION以斑马鱼和哺乳动物颅面部发育（CNCCs位于同源区域）之间的结构相似性，基因调控网络是高度保守的。还已经表明，CNCCs开发在脊椎动物物种和斑马鱼8之间以相同的方式，使斑马鱼的强大有机体为CL / P的发育和遗传基础的研究它有许多优点，包括体积小，快速，前宫内胚胎发育，以及高繁殖率。此外，胚胎是光学透明的，使得它适合于观察的显微镜9根据复杂的发育事件。这是一个理想的动物模型进行迁移和颅神经嵴细胞分化的研究。 枫转基因模型5：扩大先前发表的作品8，10，11，国家会议中心的迁徙模式是使用SOX10中详细描述。枫是一个照片兑换蛋白TU从绿色到红色RNS光活化后，使之能够追踪CNCCs精确。在此变换的肽骨架被裂解，这表明转化是稳定的，这意味着细胞可以被跟踪到其最终目的地12。标记下SOX10的转录调控枫转基因株系表明，脊椎动物味觉和斑马鱼筛片是同源融合双侧上颌骨突起（MXP）的额鼻突出（FNP），而形成的Y字型融合缝之间的相似种。 在其他应用中，SOX10：枫转基因斑马鱼模型被用来生成斑马鱼胚胎的视频在不同的发育阶段，以显示形成的正常和异常颅面结构。细胞的特定群体的光转化使得能够跟踪他们的发展。使用这种方法的方法来创建开发craniofac的实时成像IAL结构在斑马鱼被引入，因此很容易直观地展现这个复杂的发展过程。 该协议旨在共享使用筛窦板的SOX10的正常发展产生这些视频的经验：枫转基因斑马鱼作为一个例子。此协议可进一步应用于制造从颅神经嵴细胞在斑马鱼产生的任何结构的时间推移录像。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>sox10: kaede transgenic zebrafish line</td>
			<td>MGH</td>
			<td></td>
			<td>Available via the Liao lab</td>
		</tr>
		<tr>
			<td>Petri dishes 100x15 mm</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 35 mmx10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Low melting point (LMP) Agarose</td>
			<td>Invitrogen</td>
			<td>15517022</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Tek 2 Chamber SlideSystem</td>
			<td>LabTek</td>
			<td>154453</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloaders 200/pk</td>
			<td>Fisher</td>
			<td>E5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R Si Confocal Ti series</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements Software AR3.2 64-bit</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy

脊椎动物palatogenesis是一个非常精心设计和复杂的发展过程，它涉及颅神经嵴（CNC）细胞，收敛性和扩展面部日珥，以及颅面骨骼成熟的迁移。研究颅神经嵴向斑马鱼口感的特定区域一个SOX10的贡献：生成枫转基因斑马鱼线。 SOX10提供枫报告蛋白的谱系限制性的神经嵴，从而使细胞标记更精确的处理比传统的染料或报告mRNA注射。枫是光转换蛋白，由绿色变为红色光激活后，使人们有可能跟随细胞精确。该SOX10：转基因枫线被用来执行谱系分析，划定数控细胞群产生上颌下颌与元素，并说明同源性面部日珥到脊椎动物。本协议描述了一步s到产生SOX10的生存时间推移视频：枫斑马鱼胚胎。筛窦板的发展将成为一个实际的例子。这个协议可以应用于制作转基因斑马鱼任何枫或类似photoconvertible报告蛋白的时间推移共聚焦记录。此外，它可以被用来捕捉颅面结构在斑马鱼突变体不仅正常，而且还发育异常。

在SOX10：枫转基因株系，迁徙，后迁徙CNCCs被标记荧光绿色。国家会议中心的细胞标记绿色荧光枫复述内源性SOX10表达5。 在其他应用中，这种动物模型被用来更好地可视化中集依赖颅面结构的发展。具体的结构，也颅面畸形的病理发展，特别是唇腭裂的正常发育已被抓获。已经产生了一系列的斑马鱼的生存时间推移录像在不同的发育时间点。 作为...

Watch this Scientific Journal Video about Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy at JoVE.com

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

实验数据的可视化已成为展示结果给科学界的一个关键因素。越来越多的胚胎活延时录制的产生有助于复杂的发育过程中更好地介绍和了解。该协议是一步一步的通过枫蛋白在斑马鱼光转化指南细胞标记。

颅面部发育中的SOX10可视化：使用枫转基因斑马鱼线时间推移共聚焦显微镜

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

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双光子现场斑马鱼的胚胎干切断和时间推移共焦成像

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

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In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Time-lapse Imaging of Mitosis After siRNA Transfection

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

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颅面间质细胞的电

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Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

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The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...