Name: visualization of chondrocyte intercalation and directional proliferation via zebrabow clonal cell analysis in the embryonic meckel&#8217;s cartilage
Uploaded: 2015-10-21T14:00:00
Description: Watch this Scientific Journal Video about Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic MeckelÃ¢â‚¬â„¢s Cartilage at JoVE.

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...

<ol>
	<li>Dougherty, M., 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=Distinct+requirements+for+wnt9a+and+irf6+in+extension+and+integration+mechanisms+during+zebrafish+palate+morphogenesis.">Distinct requirements for wnt9a and irf6 in extension and integration mechanisms during zebrafish palate morphogenesis.</a> Development. 140, 76-81 (2013).</li><li>Dougherty, M., 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=Embryonic+fate+map+of+first+pharyngeal+arch+structures+in+the+sox10:+kaede+zebrafish+transgenic+model.">Embryonic fate map of first pharyngeal arch structures in the sox10: kaede zebrafish transgenic model.</a> The Journal of craniofacial surgery. 23, 1333-1337 (2012).</li><li>Eberhart, J. K., Swartz, M. E., Crump, J. G., Kimmel, C. 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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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+mandibular+development+precedes+the+time+of+palate+closure+in+mice+homozygous+for+disproportionate+micromelia:+an+oral+clefting+model+supporting+the+Pierre-Robin+sequence.">Altered mandibular development precedes the time of palate closure in mice homozygous for disproportionate micromelia: an oral clefting model supporting the Pierre-Robin sequence.</a> Teratology. 65, 116-120 (2002).</li><li>Javidan, Y., 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=Development+of+cartilage+and+bone.">Development of cartilage and bone.</a> Methods in cell biology. 76, 415-436 (2004).</li><li>McCarthy, N., 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=Pdgfra+protects+against+ethanol-induced+craniofacial+defects+in+a+zebrafish+model+of+FASD.">Pdgfra protects against ethanol-induced craniofacial defects in a zebrafish model of FASD.</a> Development. 140, 3254-3265 (2013).</li></ol>

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 MeckelÃ¢â‚¬â„¢s Cartilage at JoVE.

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic MeckelÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢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. ...

Research

JoVE Journal

Developmental Biology

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Analysis of Coronary Vessels in Cleared Embryonic Hearts

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

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

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