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Acknowledgements
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Neuroscience

跨物种移植神经嵴迁移和分化的分析

Published: February 07, 2012
doi:
10.3791/3622
,
1Department of Biochemistry and Cell Biology,Rice University

Summary

描述的分析禽流鹌鹑鸡嵌合胚胎的神经嵴细胞的迁移和最终命运的方法。此方法是一种简单明了的技术追踪神经嵴细胞迁移和分化的过程中，否则难以区分，在未添加鸡胚。

Abstract

鸡胚胎提供一个独特的平台，为研究许多脊椎动物发育过程中，由于蛋内的胚胎方便。嵌合胚胎禽流感，鹌鹑捐助组织移植到鸡胚大毛 ，结合由禽流胚胎提出的操纵方便了不可磨灭的细胞群的遗传标记的权力。

鹌鹑鸡嵌合体是一个追踪迁徙的神经嵴细胞（NCC）的1-3古典的工具。 NCC的是胚胎细胞在一个短暂的迁徙人口，这源于在区域发展中的神经管4背。他们接受的上皮间质转化，并随后迁移到其他地区的胚胎，它们分化 ​​成各种细胞类型，包括软骨5-13，11,14-20黑色素细胞，神经细胞和神经胶质21-32。 NCC的是多向的，他们的最终命运是influenced由1）该地区的神经管中，他们沿着源自胚胎11,33-37，2 rostro尾轴）从邻近的细胞信号作为他们迁移38-44，和3），其最终的微胚胎内的目标45,46。追踪从他们的起源点在神经管细胞，其最终的胚胎内的地位和命运，图案和器官的发育过程的调节提供了重要的线索。

捐助者的神经管（伦嫁接）或不同地区的捐助者的神经管（异位嫁接）互补地区移植可以揭示沿rostro尾轴2,47 NCC的预规范的差异。这种技术可以进一步适应移植的神经管单方面车厢，一边是从供体组织取得，对侧遗体在宿主胚胎，易建联泰然自若elding 2,47在同一样品的内部控制。它也可以适应在以后的胚胎移植脑段，HH10后，当前神经管已经关闭了47。

在这里，我们报告通过神经管移植，允许从离散的神经管段的候鸟NCC的跟踪产生的鹌鹑鸡嵌合体的方法。具体物种的鹌鹑，的具体QCPN抗体48-56捐助源性干细胞的标记，允许研究者区分捐助国和宿主细胞在实验终点。这种技术很简单，价格低廉，并有许多应用，包括命运的映射，细胞谱系的追踪，并确定预图案，沿rostro尾轴45。由于易于访问禽流胚胎，鹌鹑鸡移植技术可以与其他操作相结合，包括但不限于镜头消融40，注射抑制分子57,58，或通过基因表达质粒59-61电操纵，特别是在胚胎的发育程序的扰动NCC的迁徙流识别的响应。此外，这种嫁接技术也可能被用来产生其他如鹌鹑，鸭嵌合体间的嵌合胚胎研究华北贡献颅面形态，或鼠标鸡嵌合体小鼠遗传学的力量结合禽流胚胎操纵方便62

Protocol

1。孵化小鸡和鹌鹑蛋所需的阶段 HH9胚胎，典型的孵化时间范围从29-33小时，在38°C的63 鸡蛋用温水洗净任何碎片。 横向排列托盘上的鸡蛋。用铅笔标记面朝;这将对应的胚胎将局部区域。孵化鹌鹑蛋的钝端。 在38个地点°C间湿润的孵化器。打开摇摆功能。 2。准备鸡蛋的窗口和解剖从孵化器中删除的鸡蛋，并用70?…

Discussion

鹌鹑神经管移植到主机鸡胚这里描述的是一个简单和廉价的技术跟踪特定亚群的迁移沿rostro尾轴21,67-69来自不同地区产生的净捐助国。这种技术易于访问禽流胚胎（如哺乳动物胚胎相比）的优势，可与其他技术相结合，如组织消融，注射抑制分子，或通过电表达质粒的基因操纵，实验研究具体华北迁徙种群的反应不同的发展线索，在胚胎47,69。

鹌鹑，鸡嵌合体…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢批判手稿Lwigale实验室的成员。 SLG是支持国家眼科研究所（F32 EY02167301）由露丝属Kirschstein NRSA奖学金。爱国青年同盟的支持，国家眼科研究所（EY018050）。

Materials

Reagent Company Catalog number
Chick eggs Various – we use Texas A&M University’s Poultry Science Department, TX.  
Quail eggs Various – we use Ozarks Egg Company, MO.  
Egg incubator (Digital Readout 1502 Sportsman Incubator w/Humidity 110-120 Volt AC) www.poultrysupply.com 1502
Dumont AA forceps, Inox Epoxy-coated Fine Science Tools 11210-10
Scotch tape Any office supply store  
Curved Iris forceps Fine Science Tools 11065-07
India ink Any art supply store  
Pen/Strep (Penicillin, Streptomycin) Solution VWR International 101447-068
Clear Packing tape Any office supply store  
Needle pulling apparatus Narashige, Japan PE-21
Pulled glass needle, made from 1.5-1.8 x 100mm borosilicate glass capillary tube Kimble chase 34500 99
Pulled glass pipette, made from 5¾” Pasteur pipette Fisher Scientific 13-678-6A
Mouth pipette apparatus (aspirator tube assembly for calibrated microcapillary pipette) Sigma-Aldrich A5177-52A
Dumont #5 forceps Fine Science Tools 11251-30
Tungsten wire, 0.1mm diameter VWR International AA10404-H2
Needle holders (Nickel-plated pin holder) Fine Science Tools 26018-17
QCPN antiserum Developmental Studies Hybridoma Bank, University of Iowa QCPN
Alexa Fluor secondary antibody (e.g., Alexa Fluor 594 goat anti-mouse IgG1) Invitrogen A21125
Ringer’s Solution (2L):
  • 14.4g NaCl
  • 0.34g CaCl2
  • 0.74g KCl
  • 0.230g Na2HPO4
  • 0.04g KH2PO4
  • ddH2O to 2L
  • Filter and autoclave
 All reagents from Fisher Scientific
  • 7647-14-5
  • 10043-52-4
  • 7447-40-7
  • 7558-79-4
  • 7778-77-0
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Tags

Neural Crest MigrationDifferentiationCross-species TransplantationAvian EmbryosQuail-chick ChimerasMigratory Neural Crest CellsEpithelial To Mesenchymal TransitionCell TypesCartilageMelanocytesNeuronsGliaMultipotent CellsRostro-caudal AxisNeighboring CellsMicroenvironmentPatterningOrganogenesis
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Griswold, S. L., Lwigale, P. Y. Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation. J. Vis. Exp. (60), e3622, doi:10.3791/3622 (2012).
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