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Neuroscience

クロス種の移植による神経堤の移行および分化の解析

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

Summary

ウズラ – ニワトリキメラ胚における鳥類の神経堤細胞の遊走と最終的な運命を分析するためのアプローチが説明されています。このメソッドは、unmanipulatedニワトリ胚内で区別することはそうでなければ困難な移行および分化における神経堤細胞をトレースするためのシンプルで簡単なテクニックです。

Abstract

鳥類の胚は卵内の胚​​の簡単なアクセスのために多くの脊椎動物の発達過程を研究するためのユニークなプラットフォームを提供します。ウズラのドナー組織が​​OVOでニワトリ胚に移植されたキメラ鳥類の胚は、鳥類の胚によって提示された操作の容易さと細胞集団の消えない遺伝標識のパワーを兼ね備えています。

ウズラ-ニワトリキメラ1-3渡り鳥神経堤細胞（NCCS）をトレースするための古典的なツールです。 NCCSは、発展途上の神経管4の背側領域に由来する胚における細胞の一過性の渡り鳥集団である。彼らは、葉への移行上皮を受け、その後、彼らは軟骨5-13、メラノサイト11,14-20、ニューロンとグリア21から32までを含む様々な種類の細胞に分化する胚の他の地域に移行します。 NCCSは多能性であり、その究極の運命はインフルです。1）彼らは胚11,33-37、2 rostro -尾軸に沿って発信された神経管の領域）が38から44に移行するように隣接セルからの信号を、その究極の3）微小で積んだ胚45,46内の宛先。胚内での最終的な位置と運命に、神経管に由来それらの点から、これらの細胞を追跡し、パターニングおよび器官形成を制御する発達過程に重要な洞察を提供します。

ドナー神経管（グラフトホモトピック）またはドナー神経管（グラフト異所性）の異なる領域の相補的領域の移植はrostro-尾軸に沿って2,47 NCCSの事前の仕様の違いを明らかにすることができます。この手法は、さらに一側がドナー組織由来されるように、神経管の一方的なコンパートメントを移植するように適応し、反対側の遺跡は、ホスト胚に動じない、yiをすることができます同じサンプル2,47の内部制御を薪。前方神経管47を閉じたときにも、HH10した後、後で胚の脳セグメントの移植に適合させることができます。

ここでは、神経管の独立したセグメントから派生した渡り鳥NCCSのトレースを可能にする神経管移植を介して、ウズラ – ニワトリキメラを生成するための手法を報告します。ウズラ固有QCPN抗体を48から56のドナー由来細胞の種特異的標識は、研究者は実験終了時点で、ドナーと宿主細胞を区別することができます。この手法は、安価で、簡単で、トレース、およびrostro-尾軸45に沿って事前にパターニングイベントを識別する運命マッピング、細胞系譜を含む多くのアプリケーションを持っています。ので、鳥類の胚へのアクセスのしやすさから、ウズラ-ニワトリ移植技術は、他の操作と組み合わせて含むが、レンズのアブレーション4に限定されるものではない0の場合、胚の発達プログラムの摂動にNCCSの特定の渡り鳥ストリームの応答を識別するために、阻害分子57,58、または59から61発現プラスミドのエレクトロポレーションを介した遺伝子操作の注入。さらに、この移植技術はまた、鳥類の胚の操作のしやすさとマウス遺伝学の力を組み合わせることがNCCの顔面形態形成への貢献や、マウス、ニワトリキメラを勉強するようなウズラ、アヒルのキメラのような他の種間キメラ胚を生成するために使用することができ62

Protocol

1。希望するステージにインキュベートひよこやウズラの卵 HH9胚については、典型的なインキュベーション時間は38℃63に29から33時間の範囲ぬるま湯と卵からの破片を洗ってください。 水平トレイ上に鶏の卵を配置します。鉛筆で上を向く側をマークし、これは胚がローカライズされる領域に対応します。ウズラの卵平滑末端をインキュベートしま…

Discussion

ホストのニワトリ胚にウズラの神経管の移植はrostro-尾軸に沿って21,67-69さまざまな地域から発せられるNCCSを移行し、特定の集団を追跡するための簡単で安価な手法であるここで説明する。この手法は、実験的に調べるために、鳥類の胚へのアクセスのしやすさ（哺乳類胚に比べて）を活用し、そのような組織切除、阻害分子の注入、または発現プラスミドのエレクトロポレーション?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

著者は、原稿の批評のためにLwigale研究室のメンバーに感謝。 SLGは、国立眼研究所（F32 EY02167301）からルースL.キルシュシュタインNRSAフェローシップでサポートされています。 PYLは、国立眼研究所（EY018050）によってサポートされています。

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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