Name: blastomere explants to test for cell fate commitment during embryonic development
Uploaded: 2013-01-26T13:00:00
Description: Watch this Scientific Journal Video about Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development at JoVE.com

作者要感谢GWU哈伦为支持蒙娜丽莎埃罗尔德的支持格兰特Paaqua和GWU·路德·赖斯奖学金的奖学金。这项工作是由美国国家科学基金会的资助MCB-1121711。

1。仪器仪表，文化传媒和菜肴的制备<ol><li>锐化钳使用氧化铝研磨胶片。在解剖显微镜下，监视针尖大小。作为一对钳子的背面的前端的情况下在操作过程中被损坏。高压灭菌所有四个钳子和存储在无菌容器中。 </li><li> 500毫升的0.1倍和1.0倍的培养基（马克的改良林格[MMR]或修改巴特的盐水[MBS];的食谱中15），和过滤消毒。我们经常使用MBS（1X = 88毫米氯化钠，氯化钾1毫米，0.7毫米氯?...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal-vegetal+asymmetries+influence+the+earliest+steps+in+retinal+fate+commitment+in+Xenopus.">Animal-vegetal asymmetries influence the earliest steps in retinal fate commitment in Xenopus.</a> Dev. Biol. 212, 25-41 (1999).</li><li>Yan, B., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+competence+of+Xenopus+blastomeres+to+produce+neural+and+retinal+progeny+is+repressed+by+two+endo-mesoderm+promoting+pathways.">The competence of Xenopus blastomeres to produce neural and retinal progeny is repressed by two endo-mesoderm promoting pathways.</a> Dev. Biol. 305, 103-119 (2007).</li><li>Gallagher, B. C., Hainski, A. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+differentiation+of+dorsal+axial+structures+from+an+animal+cap+cleavage+stage+blastomere+in+Xenopus.">Autonomous differentiation of dorsal axial structures from an animal cap cleavage stage blastomere in Xenopus.</a> Development. 112, 1103-1114 (1991).</li><li>Hainski, A. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+maternal+mRNAs+from+a+dorsal+animal+blastomere+induce+a+secondary+axis+in+host+embryos.">Xenopus maternal mRNAs from a dorsal animal blastomere induce a secondary axis in host embryos.</a> Development. 116, 347-3345 (1992).</li><li>Godsave, S. F., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+analysis+of+mesoderm+formation+in+the+Xenopus+embryo.">Single cell analysis of mesoderm formation in the Xenopus embryo.</a> Development. 111, 523-530 (1991).</li><li>Hainski, A. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+activin-like+signal+activates+a+dorsal-specifying+RNA+between+the+8-+and+16-cell+stages+of+Xenopus.">An activin-like signal activates a dorsal-specifying RNA between the 8- and 16-cell stages of Xenopus.</a> Dev. Genetics. 19, 210-221 (1996).</li><li>Pandur, P. D., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+maternal+influences+on+dorsal-ventral+fate+of+Xenopus+animal+blastomeres.">Multiple maternal influences on dorsal-ventral fate of Xenopus animal blastomeres.</a> Dev. Dyn. 225, 581-587 (2002).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Development+of+Xenopus+laevis.">Early Development of Xenopus laevis.</a> A Laboratory Manual. , (2000).</li><li>Sullivan, S. A., Akers, L., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=foxD5a,+a+Xenopus+winged+helix+gene,+maintains+an+immature+neural+ectoderm+via+transcriptional+repression+that+is+dependent+upon+the+C-terminal+domain.">foxD5a, a Xenopus winged helix gene, maintains an immature neural ectoderm via transcriptional repression that is dependent upon the C-terminal domain.</a> Dev. Biol. 232, 439-457 (2001).</li><li>Yan, B., Neilson, K. M., Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoxD5+plays+a+critical+upstream+role+in+regulating+neural+fate+and+onset+of+differentiation.">FoxD5 plays a critical upstream role in regulating neural fate and onset of differentiation.</a> Dev. Biol. 329, 80-95 (2009).</li><li>Vincent, J. -. P., Gerhart, 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=Subcortical+rotation+in+Xenopus+eggs:+an+early+step+in+embryonic+axis+specification.">Subcortical rotation in Xenopus eggs: an early step in embryonic axis specification.</a> Dev. Biol. 123, 526-539 (1987).</li><li>Peng, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appendix+A:+Solutions+and+Protocols.">Appendix A: Solutions and Protocols.</a> Methods in Cell Biology. 36, 657-662 (1991).</li><li>Klein, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+cleavage+furrow+demarcates+the+dorsal-ventral+axis+in+Xenopus+embryos.">The first cleavage furrow demarcates the dorsal-ventral axis in Xenopus embryos.</a> Dev. Biol. 120, 299-304 (1987).</li><li>Masho, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Close+correlation+between+the+first+cleavage+plane+and+the+body+axis+in+early+Xenopus+embryos.">Close correlation between the first cleavage plane and the body axis in early Xenopus embryos.</a> Dev. Growth Diff. 32, 57-64 (1990).</li></ol>

成功培养的单个卵裂球的最关键的步骤是：1）正确识别的卵裂球的兴趣; 2）保持无菌，健康的文化; 3）解剖细胞在细胞周期中正确的部分; 4）制定必要的手册灵巧，以防止损坏在解剖和转让过程中的文化。 操作特定的卵裂球，关键的是要能够识别的大是大非轴。在爪蟾胚胎中，被识别的背侧可以通过三种方法：（1）标志着精子入口点，与一个重要的染料18，19，（...

非洲爪蟾胚胎已被广泛利用，以确定胚胎干细胞的机制，获得其特定的命运，因为他们的鸡蛋是大到足以允许显微外科方法。此外，它们的培养基中的营养补充品，而不需要为外部发展，因为每个小区包含一个提供了内在的能量存储的蛋黄血小板丰富的细胞内供给。研究细胞命运决定的机制，一个重要的资产，是一套全面的命运地图的卵裂阶段的卵裂球（2 -通过32细胞阶段）1，2，3，4，5，6。这些地图，构建了一个检测的分子显微注射到一个单一的，可识别的卵裂球，在发展监察组织填充标记的后代。一致的地图是可能的，因为命运的大是大非轴的胚胎可以可靠地识别在许多安莉芳操作系统。首先，在所有的野生型胚胎的动物半球色素，，而植物性半球是不。其次，该条目的精子受精导致朝向未来腹侧的动物半球色素沉着的收缩，在许多胚胎色素沉着的差异，因此，可以用来区分背侧和腹侧的两侧。第三，第一卵裂沟接近大多数胚胎中期矢状面，并因此可以被用来识别的左，右两侧的胚胎。的的命运地图也依赖于一个事实，即自然受精的卵子经常切割的规律，使每个识别在一个人口众多的胚胎的卵裂球。虽然有变异本文内和之间的描述有关的色素沉着和裂解模式，使用选择的程序的鸡蛋离合器允许细胞要确定与约90％的准确度与规定的命运。 可以阻止细胞的命运开采胚胎发育过程中，由几个机制。内在因素，如差异继承细胞质的mRNA或蛋白质，有助于早期图案的几个方面。例如，特定的母亲的基因，确定哪些单元格将有助于生殖系，成为内胚层，或有助于背的身体轴（7检讨）。相邻小区或更远的胚胎信号中心提供本地的外在因素，是诱导特定的组织类型和的图案几乎每一个器官系统。信号中心的实例包括组织者/节点的原肠胚诱导的神经外胚层和图案中胚层，和偏振活性区域的模式的前后轴的肢芽。虽然命运地图的前体的不同器官和他们的后裔在正常的胚胎，揭示了发展的道路，他们无法区分内在和外在影响这些细胞的。他们也无法揭示的充分发展潜力的一个细胞，其后代分化的胚胎在复杂的信号环境。两个实验的方法可以测试是否一个细胞的命运是确定的内在因素或者是后来影响的外部因素：1）移植的细胞在胚胎中一个新的位置，或2）去除细胞从胚胎随后由文化在外源信号的情况下。 这两个实验的方法是可行的，因为细胞是在非洲爪蟾大到足以被人工分离。例如，许多研究已删除单到新的地点在宿主胚胎细胞的胚胎（改变细胞间的相互作用）或移植的细胞命运的变化（审查，7，8，9）测试。另外，第二种方法的小数量的细胞的胚胎来自不同区域的explanting我置身于文化，以澄清感性的组织在没有外源性因素的相互作用可能是因为非洲爪蟾胚胎的细胞都充满了细胞内的营养物质店，蛋黄血小板。因此，它们可以被培养在一个定义的盐介质中无营养或生长因子的培养基中补充了几天。我们用这种方法来背动物卵裂球的的自主能力产生神经和背中胚层组织，以母系遗传的基因10，11，和其他人表明，32 -细胞卵裂球中胚层规范依赖于内在和外在的信息12 。作为外植体的培养细胞的一个优点是，该介质还可以补充有定义信令因素，以便确定哪个可能影响细胞-细胞间的通信路径的取出的单元12，13，14的命运。此外，可以注入卵裂球公关的ior文化与mRNA过度表达的基因，或与反义寡核苷酸，以防止内源性的mRNA的翻译。这些收益和损失的功能分析，可以识别的分子所需要的自主表达的命运。要分析外植体的命运，识别特定的细胞类型可以通过标准的基因表达（ 例如，原位杂交，RT-PCR）和免疫组化检测。该协议提供了一个简单，但功能强大的方法来区分之间内在的和外在的机制，调节胚胎细胞发育成特定的组织。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>氧化铝研磨胶片</td><td>托马斯科学</td><td> ＃6775E-38 </td><td>场（12微米），主要维修镊子提示</td></tr><tr><td>氧化铝研磨胶片</td><td>托马斯科学</td><td> ＃6775E-46 </td><td>中等（3微米），，精细锐化的镊子提示</td></tr><tr><td>氧化铝研磨胶片</td><td>托马斯科学</td><td> ＃6775E-54 </td><td>精细（0.3微米），抛光镊子提示</td></tr><tr><td>镊子：杜蒙，Dumoxel Biologie＃5 </td><td>精细科学工具</td><td> ＃11252-30 </td><td>这些具有优良的技巧，不需要锐化在第一次购买时。耐腐蚀，使他们能够可高压灭菌。 </td></tr><tr><td>庆大霉素解决方案</td><td>西格玛</td><td> G1397 </td><td>添加到介质上使用同一天， </td></tr></tbody></table>

blastomere explants to test for cell fate commitment during embryonic development

命运的地图，所有的胚胎细胞谱系追踪构造，揭示下降，从每一个细胞的胚胎的组织。虽然命运地图是非常有用的，确定的前体器官移植和澄清的后裔细胞的发展路径填充该机构在正常的胚胎，他们没有说明发展潜力的前体细胞，或识别机制它的命运是确定的。为了测试细胞命运的承诺，一个细胞的正常剧目的后裔比较完整的胚胎的命运（图）实验操作后，所表达的。固定的细胞的命运（提交），无论周围的细胞环境，或者是其邻国所提供的外部因素的影响？非洲爪蟾胚胎使用全面命运的地图，我们将介绍如何识别，分离和培养单个卵裂阶段的前体，称为囊胚meres。这种方法允许1评估这些早期的细胞是否致力于获得的命运在完整的胚胎在其正常的环境中，需要与它们相邻的细胞的相互作用，或者可以影响表达如果暴露于其它类型的信号的备用命运。

在此测定法来准确地评估细胞的发育潜能的能力依赖于解剖出正确的卵裂球的基础上的命运地图1，2，3，4，5，6。因此，关键是选择正确的色素沉着在2 -细胞阶段模式中，作为示出在图1中 ，随后与符合正常卵裂模式，如在图2中示出的胚胎。如果一个人观察的排序的胚胎，因为他们达到所需的卵裂阶段和分离出来呈现出不规则的分裂的胚胎，精度大大提高。正如?...

Watch this Scientific Journal Video about Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development at JoVE.com

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

单个胚胎细胞的命运可以继承的分子和/或通过来自相邻小区的信号的影响。利用爪蟾胚胎的卵裂阶段的命运地图，单个卵裂球可确定为文化隔离的贡献进行评估的遗传分子与细胞间的相互作用。

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

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Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

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Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

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一个实用的方法遗传诱导的命运映射：一个视觉引导标记和跟踪细胞在体内</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

一个反向遗传学方法测试胚胎发育过程中的功能冗余

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Single Cell Fate Mapping in Zebrafish

单细胞在斑马鱼的命运映射

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

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A System for ex vivo Culturing of Embryonic Pancreas

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The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

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Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...