作者感谢艾德琳多莉的外植体对纤维连接蛋白，埃里克Theveneau图片的有益讨论和居里研究所的动物设施。 CM是地区法兰西岛（DOMAINE D&#39;INTERET修形茎杆），巴黎大学南基（武官Temporaire德ENSEIGNEMENT等德RECHERCHE）和法新社国立德拉RECHERCHE的博士后研究员。这项工作是由巴黎大学南基（Attractivite 2011），该中心法国国家科学研究（动作Thematique等Incitative河畔计划），协会倒拉RECHERCHE CONTRE勒癌症格兰特SFI20101201882，法甲CONTRE乐癌症和法新社国立德拉RECHERCHE（法新社国立德拉RECHERCHE计划布兰克）。

实验符合的用于科学目的和更换，减少和细化的国际公认原则的动物的保护国家和欧洲的监管。 1。纤连蛋白包被的培养皿14的制备<ol><li>移液器500毫升的10毫克/毫升培养级纤连蛋白稀释在1×磷酸盐缓冲盐水（PBS）中插入标准塑料培养皿中（直径40×11毫米）。在37℃下1小时。注意：如果使用玻璃餐具或盖玻片后续成像，使用100毫克/毫升纤维连接蛋白的解决方案。 </li><li>漂洗三次，用1×PBS。 </li><li> PBS替换用500毫升1X PBS/0.1％牛血清白蛋白（BSA）的。在37°C为30分钟。 </li><li>漂洗三次，用1×PBS。 </li><li>填充用1×PBS，并保持在4℃直至使用（通常为24-48小时）。 </li></ol> 2。数控植体解剖 &lt;醇&gt; <li>准备四分之三普通两栖动物中等（NAM）（110 mM氯化钠，2mM的氯化钾，1毫米的Ca（NO 3）2，1mM的MgSO 4干燥，0.1mM的EDTA，1mM的碳酸氢钠 ，0.2×PBS，50毫克/毫升庆大霉素10,15）。不要让超过1周解剖，储存于4°C。较旧的NAM可用于制备琼脂糖包被的解剖菜（如下）。 </li><li>准备60毫米培养皿解剖，涂有四分之三NAM10毫升2％的琼脂糖。 </li><li>收集清扫工具：塑料巴斯德移液管，一个最好的昆虫针安装在销钉保持器（见材料表供参考），一“眉毛刀”（与眉毛包埋于石蜡中以玻璃巴斯德移液管16的前端制成），两个细解剖钳，P1000移液器和技巧，双目体视镜放大倍数与8-40X〜50，与光纤导一个明亮的光源。所有的解剖工具应保持清洁，每一个实验后，他们冲洗两次，用蒸馏水，一旦无线TH 100％的乙醇，并让干燥。储存远离灰尘。 </li><li>在解剖盘新鲜的不结盟运动几乎顶端填补。 </li><li>获得十根据标准程序10和老化直到它们到达神经胚阶段16（根据Nieuwkoop和费伯发育表17） 蟾胚胎。 </li><li>舞台放置16个胚胎到使用塑料巴斯德吸管解剖盘。 </li><li>开展下的双目范围内的所有后续步骤。有两对镊子取出卵黄膜。要小心，不要损坏胚胎的背侧部分。 </li><li>等到胚胎已经达到了17级，开始清扫。 17级是合适的，因为神经嵴明显设置除了神经管，而相比之下，早期阶段。 17级后，NC开始迁移，并与周围的间质交织。 </li><li>挖一个小孔，在琼脂糖用钳子做一个利基的胚胎，以保持他们在解剖。将一个胚胎背侧。注意：橡皮泥可用于保持，而不是挖一个洞到琼脂糖的胚胎。 </li><li>使前牙神经褶用眉刀和昆虫脚的外侧部切口。要小心，不要去太深入胚：目标是通过2-3层细胞（永远达不到深如原肠！）削减。在整个解剖过程中， 绝不允许解剖组织，以与自X 的气-液界面接触进入 非洲爪蟾胚胎组织是由表面张力裂解。 </li><li>拨打第二个切口平行于神经的背更加折叠部分使用相同的仪器。 </li><li>如果需要的话，转身90°的菜。使三分之一垂直切口与眉刀和昆虫针，在第一次两个切口的后侧。数控是厚厚团细胞的外胚层的色素外层下方，侧以神经管。 </li><li>使用的眉刀尖端从底层数控分离色素外胚层层，从第三个切口边缘开始。 </li><li>使用的眉刀一边拆下数控组织从第三切口开始。数控组织是相当灰色，而底层的头侧中胚层是白色的。小心地从胚层分离的NC。 </li><li>从视泡前方拆下数控组织，并削减在这个水平上，以释放外植体。至于第一个实验中，通过原位杂交技术在某些外植体检查snail2表达（一个规范的NC标记）的控制建议。 </li></ol> 3。对纤维连接蛋白电镀植<ol><li>填充在纤维连接蛋白包被的培养皿改性Danilchick中等（MDM，氯化钠53毫米，Na2CO 3 5毫米，钾葡萄糖4.5毫米，钠葡萄糖32毫米， 硫酸镁 1毫米， 氯化钙 1毫米，BSA的0.1％，调节pH值8.3用1 M BICINE <sup&gt; 18）。 </li><li>转植到使用P1000的移液管的纤维连接蛋白包被的培养皿。切勿让植触气 - 液界面。首先，吸取一些液体进入尖，然后将外植体，然后一些液体了。不要让任何空气进入尖而转植到纤维连接蛋白包被的培养皿。一旦该尖端部是纤连蛋白包被的培养皿中的液体内，让外植体慢慢地通过重力下降，或推非常平缓， 避免喷射植太快 。 </li><li>将10-30植在使用眉刀盘。避免盘的两侧，这是不足够的后续成像。 </li><li>尝试，直到植已附着在纤维连接蛋白（大约需要15-30分钟）不动的菜。如果需要，将菜非常谨慎 。 </li><li>放置在冷藏保温箱的菜（在15-20℃，温度图表见17）。当放置在15℃，将细胞ARË通常有效迁移和6小时培养后散射（此时间可能实验之间会发生变化）。 </li></ol> 4。植嫁接到主机胚胎<ol><li>切细的玻璃盖玻片到使用粗镊子非常小的碎片。件的尺寸应为大约1.5mm 2。沉浸在片放入含3/4 NAM或PBS使用镊子培养皿。 </li><li>准备主机胚胎：如3）所述特别小心，不要损坏主机胚胎同时去除卵黄膜和解剖数控解剖出主机NC 3/4 NAM。该组织张力往往会增加消融的大小：让主机胚胎愈合局部解剖，而捐赠者的胚胎。 </li><li>在另一个琼脂糖包被的培养皿或在同一盘的另一部分，如在方案3中所述解剖出NC外植体从供体胚胎。转移NC移植到含小心主机的菜，如第协议3。 </li><li>紧随数控植上放置使用镊子主机消融区。外植体应重视部分。把一块玻璃盖玻片上移植胚胎，以维持移植物到位。选择一块玻璃大于胚胎，以避免损坏。胚胎将是一个有点扁平。 </li><li>等待至少15-20分钟，然后轻轻取出盖玻片。让胚胎收回另外10分钟，小心吸取到一个干净的盘子装满3/4 NAM，使用塑料巴斯德吸管。 </li></ol>

<ol>
	<li>Sauka-Spengler, T., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+regulatory+network+orchestrates+neural+crest+formation.">A gene regulatory network orchestrates neural crest formation.</a> Nat. Rev. Mol. Cell Biol. 9 (7), 557-568 (2008).</li><li>Theveneau, E., Mayor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+delamination+and+migration:+from+epithelium-to-mesenchyme+transition+to+collective+cell+migration.">Neural crest delamination and migration: from epithelium-to-mesenchyme transition to collective cell migration.</a> Dev. Biol. 366 (1), 34-54 (2012).</li><li>Stuhlmiller, T. J., Garc&#237;a-Castro, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+perspectives+of+the+signaling+pathways+directing+neural+crest+induction.">Current perspectives of the signaling pathways directing neural crest induction.</a> Cell. Mol. Life Sci. 69 (22), 3715-3737 (2012).</li><li>Milet, C., Monsoro-Burq, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+induction+at+the+neural+plate+border+in+vertebrates.">Neural crest induction at the neural plate border in vertebrates.</a> Dev. Biol. 366 (1), 22-33 (2012).</li><li>Pegoraro, C., Monsoro-Burq, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+and+transcriptional+regulation+in+neural+crest+specification+and+migration:+lessons+from+Xenopus+embryos.">Signaling and transcriptional regulation in neural crest specification and migration: lessons from Xenopus embryos.</a> Dev. Biol. 2 (2), 247-259 (2013).</li><li>Etchevers, H. C., Amiel, J., Lyonnet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Bases+of+Human+Neurocristopathies.">Molecular Bases of Human Neurocristopathies.</a> Neural Crest Induct. Different. 589, 213-234 (2005).</li><li>Nieuwkoop, P. D., 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=Activation+and+organization+of+the+central+nervous+system+in+amphibians.+Part+II.+Differentiation+and+organization.">Activation and organization of the central nervous system in amphibians. Part II. Differentiation and organization.</a> J. Exp. Zool. 120 (1), 33-81 (1952).</li><li>Nieuwkoop, P. D., 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=Activation+and+organization+of+the+central+nervous+system+in+amphibians+Part+I.+Induction+and+activation.">Activation and organization of the central nervous system in amphibians Part I. Induction and activation.</a> J. Exp. Zool. 120 (1), 1-31 (1952).</li><li>Sharpe, C. R., Fritz, A., De Robertis, E. M., Gurdon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+homeobox-containing+marker+of+posterior+neural+differentiation+shows+the+importance+of+predetermination+in+neural+induction.">A homeobox-containing marker of posterior neural differentiation shows the importance of predetermination in neural induction.</a> Cell. 50 (5), 749-758 (1987).</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=.">.</a> Early development of Xenopus laevis: a laboratory manual. , (2000).</li><li>Monsoro-Burq, A. -. H., Fletcher, R. B., 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=Neural+crest+induction+by+paraxial+mesoderm+in+Xenopus+embryos+requires+FGF+signals.">Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals.</a> Development. 130 (14), 3111-3124 (2003).</li><li>Milet, C., Maczkowiak, F., Roche, D. D., Monsoro-Burq, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax3+and+Zic1+drive+induction+and+differentiation+of+multipotent,+migratory,+and+functional+neural+crest+in+Xenopus+embryos.">Pax3 and Zic1 drive induction and differentiation of multipotent, migratory, and functional neural crest in Xenopus embryos.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (14), 5528-5533 (2013).</li><li>Sadaghiani, B., Thi&#233;baud, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+development+in+the+Xenopus+laevis+embryo,+studied+by+interspecific+transplantation+and+scanning+electron+microscopy.">Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy.</a> Dev. Biol. 124 (1), 91-110 (1987).</li><li>Theveneau, E., 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=Collective+chemotaxis+requires+contact-dependent+cell+polarity.">Collective chemotaxis requires contact-dependent cell polarity.</a> Dev. Cell. 19 (1), 39-53 (2010).</li><li>Slack, J. M., Forman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+interaction+between+dorsal+and+ventral+regions+of+the+marginal+zone+in+early+amphibian+embryos.">An interaction between dorsal and ventral regions of the marginal zone in early amphibian embryos.</a> J. Embryol. Exp. Morphol. 56, 283-299 (1980).</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=Embryo+dissection+and+micromanipulation+tools.">Embryo dissection and micromanipulation tools.</a> CSH Protoc. , (2007).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+table+of+Xenopus+laevis+(Daudin):+a+systematical+and+chronological+survey+of+the+development+from+the+fertilized+egg+till+the+end+of+metamorphosis.">Normal table of Xenopus laevis (Daudin): a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis.</a> , (1994).</li><li>Theveneau, E., Mayor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beads+on+the+run:+beads+as+alternative+tools+for+chemotaxis+assays.">Beads on the run: beads as alternative tools for chemotaxis assays.</a> Methods Mol. Biol. 769, 449-460 (2011).</li><li>Bronner, M. E., LeDouarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evolution+of+the+neural+crest:+An+overview.">Development and evolution of the neural crest: An overview.</a> Dev. Biol. 366 (1), 2-9 (2012).</li><li>Alfandari, D., Cousin, H., Gaultier, A., Hoffstrom, B. G., DeSimone, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+&#945;5&#946;1+supports+the+migration+of+Xenopus+cranial+neural+crest+on+fibronectin.">Integrin &#945;5&#946;1 supports the migration of Xenopus cranial neural crest on fibronectin.</a> Dev. Biol. 260 (2), 449-464 (2003).</li><li>Fort, P., 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=Activity+of+the+RhoU/Wrch1+GTPase+is+critical+for+cranial+neural+crest+cell+migration.">Activity of the RhoU/Wrch1 GTPase is critical for cranial neural crest cell migration.</a> Dev. Biol. 350 (2), 451-463 (2011).</li><li>Matthews, H. 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=Directional+migration+of+neural+crest+cells+in+vivo+is+regulated+by+Syndecan-4/Rac1+and+non-canonical+Wnt+signaling/RhoA.">Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA.</a> Development. 135 (10), 1771-1780 (2008).</li><li>Kashef, J., K&#246;hler, A., Kuriyama, S., Alfandari, D., Mayor, R., Wedlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-11+regulates+protrusive+activity+in+Xenopus+cranial+neural+crest+cells+upstream+of+Trio+and+the+small+GTPases.">Cadherin-11 regulates protrusive activity in Xenopus cranial neural crest cells upstream of Trio and the small GTPases.</a> Genes Dev. 23 (12), 1393-1398 (2009).</li><li>Hwang, Y. -. S., Luo, T., Xu, Y., Sargent, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin-X+is+required+for+cranial+neural+crest+cell+migration+in+Xenopus+laevis.">Myosin-X is required for cranial neural crest cell migration in Xenopus laevis.</a> Dev. Dyn. 238 (10), 2522-2529 (2009).</li><li>Lee, G., 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=Modelling+pathogenesis+and+treatment+of+familial+dysautonomia+using+patient-specific+iPSCs.">Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.</a> Nature. 461 (7262), 402-406 (2009).</li><li>Mica, Y., Lee, G., Chambers, S. M., Tomishima, M. J., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Neural+Crest+Induction,+Melanocyte+Specification,+and+Disease-Related+Pigmentation+Defects+in+hESCs+and+Patient-Specific+iPSCs..">Modeling Neural Crest Induction, Melanocyte Specification, and Disease-Related Pigmentation Defects in hESCs and Patient-Specific iPSCs..</a> Cell Reports. 3 (4), 1140-1152 (2013).</li><li>Shnitsar, I., Borchers, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTK7+recruits+dsh+to+regulate+neural+crest+migration.">PTK7 recruits dsh to regulate neural crest migration.</a> Development. 135 (24), 4015-4024 (2008).</li><li>Matthews, H. 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=Directional+migration+of+neural+crest+cells+in+vivo+is+regulated+by+Syndecan-4/Rac1+and+non-canonical+Wnt+signaling/RhoA.">Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA.</a> Development. 135 (10), 1771-1780 (2008).</li><li>Matthews, H. K., Broders-Bondon, F., Thiery, J. P., Mayor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt11r+is+required+for+cranial+neural+crest+migration.">Wnt11r is required for cranial neural crest migration.</a> Dev. Dyn. 237 (11), 3404-3409 (2008).</li><li>Carmona-Fontaine, C., 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=Contact+inhibition+of+locomotion+in+vivo+controls+neural+crest+directional+migration.">Contact inhibition of locomotion in vivo controls neural crest directional migration.</a> Nature. 456 (7224), 957-961 (2008).</li><li>Guiral, E. C., Faas, L., Pownall, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+migration+requires+the+activity+of+the+extracellular+sulphatases+XtSulf1+and+XtSulf2.">Neural crest migration requires the activity of the extracellular sulphatases XtSulf1 and XtSulf2.</a> Dev. Biol. 341 (2), 375-388 (2010).</li><li>Cousin, H., Abbruzzese, G., Kerdavid, E., Gaultier, A., Alfandari, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+the+Cytoplasmic+Domain+of+ADAM13+to+the+Nucleus+Is+Essential+for+Calpain8-a+Expression+and+Cranial+Neural+Crest+Cell+Migration.">Translocation of the Cytoplasmic Domain of ADAM13 to the Nucleus Is Essential for Calpain8-a Expression and Cranial Neural Crest Cell Migration.</a> Dev. Cell. 20 (2), 256-263 (2011).</li><li>Borchers, A., David, R., Wedlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+cadherin-11+restrains+cranial+neural+crest+migration+and+influences+neural+crest+specification.">Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification.</a> Development. 128 (16), 3049-3060 (2001).</li><li>Borchers, A., Epperlein, H. -. H., Wedlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+system+to+study+migratory+behavior+of+cranial+neural+crest+cells+in+Xenopus.">An assay system to study migratory behavior of cranial neural crest cells in Xenopus.</a> Dev. Genes Evol. 210 (4), 217-222 (2000).</li><li>Kerney, R., Gross, J. B., Hanken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Runx2+is+essential+for+larval+hyobranchial+cartilage+formation+in+Xenopus+laevis.">Runx2 is essential for larval hyobranchial cartilage formation in Xenopus laevis.</a> Dev. Dyn. 236 (6), 1650-1662 (2007).</li><li>Yan, C. Y. I., Skourides, P., Chang, C., Samba Brivanlou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Xenopus+hnRNP+expressed+in+neural+and+neural+crest+tissues.">a Xenopus hnRNP expressed in neural and neural crest tissues.</a> Dev. Dyn. 238 (1), 204-209 (2009).</li><li>Acloque, H., Adams, M. S., Fishwick, K., Bronner-Fraser, M., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transitions:+the+importance+of+changing+cell+state+in+development+and+disease.">Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease.</a> J. Clin. Invest. 119 (6), 1438-1449 (2009).</li></ol>

作者什么都没有透露。

本协议描述了一个简单的方法来植体premigratory颅NC在非洲爪蟾胚胎。用于这些实验的胚胎必须健全和愈合良好。丢弃任何批次不健康的胚胎。此外，胚胎生长在不同的温度（12〜18〜20℃），以切除神经嵴在阶段17，而不是以后。阶段17后，神经嵴可与头侧中胚层混合，并不能完全除去。 爪组织迅速愈合，然后失去其粘附到其他组织。它能够快速工作，并把神经嵴植入宿主，只要它是从...

神经嵴（NC）是一个短暂的胚胎细胞群出现从神经管在神经胚形成在脊椎动物胚胎的末端。控制NC规范的信令和遗传事件早原肠胚形成开始。 NC被指定在神经和非神经外胚层通过从周围背部组织的信号之间的边界。在神经胚形成的端部，NC细胞经历了上皮至间质转化（EMT），并广泛地在胚胎移植以下刻板航线通过响应周围指导线索。一旦他们达到他们的最终目的地，它们分化 ​​成衍生物， 如神经元，神经胶质细胞，骨，软骨和色素细胞1-5繁多。因为他们有许多类型的细胞和胚胎组织，在数控细胞发展的任何步骤中的缺陷，从感应到最终分化的贡献，可引起命名neurocristopathies 6先天性综合征。 Ë发展数控xperimental操作在不同的阶段 - 规范，EMT，迁移和分化 - 将提高我们neurocristopathies的认识，并允许潜在的治疗策略设计。 非洲爪蟾胚胎选择的模型来研究数控的发展。大量的胚胎很容易获得，并且体外受精可进入发展的第一个步骤。许多工具可用来操纵实验十蟾胚胎发育。基因增益的功能和击倒很容易通过显微注射的早期囊胚单个细胞来执行。胚胎组织可切体外复性7-11或背嫁接试验12,13。 在这个协议中，我们描述了如何解剖出premigratory颅NC在十蟾晚neurulas，迁移之前。这些外植体可以在纤连蛋白金属线进行培养D平板学习迁移和分化的控制， 在体外实验条件。数控植，也可嫁接到正常或操纵宿主胚胎，以研究它们的迁移和分化的体内 。

<table border="0" cellpadding="0" cellspacing="0" width="601">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Fibronectin from bovine plasma</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F4759-1MG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Bovine Serum Albumine. Fraction V</td>
			<td style="width:71px;">Euromedex</td>
			<td style="width:119px;">04-100-811-C</td>
			<td>Lower quality grade may&#160; be suitable for this application</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Stainless Steel Insect Pins. Size 000</td>
			<td style="width:71px;">FST</td>
			<td align="right" style="width:119px;">26001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Dumont #5 forceps 0.05 mm x 0.02 mm</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">11252-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation

神经嵴（NC）是一个短暂的背神经管细胞群，在神经胚形成年底经历了一个上皮至间质转化（EMT），广泛迁移对各个器官，并分化成多种类型的衍生工具（神经元，神经胶质细胞，软骨和骨，色素和内分泌细胞）。在这个协议中，我们描述了如何从非洲爪蟾胚胎解剖premigratory颅数控，以研究开发数控在体内和体外 。青蛙模型提供了许多优势，研究开发初期，大量批的情况下，胚胎发展迅速， 在体内增益和功能丧失的策略允许操纵基因表达前，数控夹层供体和/或主机的胚胎。数控植体可以镀在纤连蛋白和用于体外研究。它们可以在无血清的定义培养基中培养数天。我们还描述了如何嫁接数控植回到宿主胚胎研究的NC迁移和分化的体内 。

当镀上纤连蛋白，神经嵴外植体附着快速（15-30分钟）和植传播平面内2小时（ 图1A）。经过3-6小时的细胞开始分散。在24小时后在15℃下，许多细胞已开始迁移远离外植体（ 图1B和1C）。蛋黄使得细胞在相衬（ 图1B）非常明亮。细胞突起（filopodes和lamellipodes）都清晰可见鬼笔环肽与肌动蛋白细胞骨架（ 图1C）染色后。 <p class="jove_cont...

Watch this Scientific Journal Video about Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation at JoVE.com

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

本协议描述了如何从非洲爪蟾 neurulas解剖premigratory颅神经嵴（NC）。这些外植体可以镀在纤连蛋白和体外培养，或接枝回主机胚胎。这种技术可以让学习数控上皮至间质转变，迁移和分化的机制。

剖析<em&gt;非洲爪蟾</em&gt;神经嵴的<em&gt;在体外</em&gt;外植体培养或<em&gt;在体内</em&gt;移植

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

dissection-xenopus-laevis-neural-crest-for-vitro-explant-culture-or

Research

JoVE Journal

Biology

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

13.1K 次观看.  Centre Universitaire. 本协议描述了如何从非洲爪蟾 neurulas解剖premigratory颅神经嵴（NC）。这些外植体可以镀在纤连蛋白和体外培养，或接枝回主机胚胎。这种技术可以让学习数控上皮至间质转变，迁移和分化的机制。 

视频: Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

组织者和动物极植从非洲爪蟾胚胎和装配一个细胞粘附实验的剖析

科研

生物学

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

塑料嵌入和非洲爪蟾胚胎切片

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

从非洲爪蟾女性获得卵子

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

制备及分馏爪蟾卵提取

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

非洲爪蟾卵母细胞的微量注射

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

组织测定动物第移植（ACT）的含量非洲爪蟾</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

生产转基因非洲爪蟾限制性内切酶介导的整合和核移植

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

膜片钳和灌注技术研究离子通道的表达爪蟾卵母细胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

在面部移植<em&gt;非洲爪蟾</em&gt;胚胎

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.