This work was partly funded by a University of Nottingham Early Career award to DS. RM is funded by the Higher Committee for Education Development of Iraq.

倫理の声明：すべてのこれらの実験は、動物のケアとノッティンガム大学の倫理指針に従ってください。 移植のためのヘパリンビーズの作製<ol><li>使用前に徹底的にPBSにヘパリンビーズを洗ってください。注：ビーズをPBS中のスラリーとして4℃で保存することができます。 <ol><li> 1mlのPBSのドロップに20μlのピペットを用いてストックからそれらを除去することによって、移植のためのビーズを選択します。後、3センチペトリ皿に20μlのPBSのドロップにビーズを転送するために2μlに設定し、マイクロピペットを使用しています。 P2ピペットチップで選択ビーズを転送し、解剖顕微鏡ステレオを使用して、サイ​​ズに基づいてビーズを選択し;直径100μmの周りの人々は理想的です。 </li></ol></li><li>ビーズからPBSを削除します。 20μlのピペットとファイン時計職人の鉗子で毛管作用により残液と液体の大部分を削除します。注：これは、成長事実上のように、可能な限り除去することが重要ですそれがビーズに添加したときrが希釈されていません。 </li><li>ビーズ上に直接成長因子をピペット。 FGF18のために、PBS中の0.1％BSA中に0.5mg / mlで再構成された組換えFGF18の0.5μlを添加します。蒸発から液体を防ぐために、皿の縁の周りに水を数滴を置きます。 </li><li> RTで1時間ビーズをインキュベートします。パスツールピペットで皿から水滴を削除し、移植のための準備ができて氷上に置きます。 </li><li> （透明のビーズのために）必要な場合は、それらを視覚化するために移植する前に、2％のフェノールレッドに4-5のビーズを転送します。胚に転送する前に、PBSですすいでください。 </li></ol>移植のためのAG 1-X2のビーズの調製<ol><li> 0.2 Nギ酸で誘導体化ビーズを使用する前に。 <ol><li>これを行うには、1.5mlの微小管にビーズを転送するために、スパチュラを使用しています。 0.2 Nギ酸の1ミリリットルを追加し、1時間振とうプラットフォーム上で洗います。 </li><li>そして、P1000ピペットでギ酸を除去し、1と交換mlの水。 1時間洗浄し、残りのギ酸を除去するために、6回繰り返します。 </li><li> P1000ピペットを用いて残りの液体を削除し、4℃でビーズを格納します。注：この段階で、すべての残留水を除去する必要はありません。 </li></ol></li><li> 3センチメートルペトリ皿に小さなへらを使用してストックから約5〜10マイクロリットル容量のビーズの小さなペレットを削除し、1mlのDMSOを追加（または薬物を溶媒に溶解させ方）。注意：これは、正しいサイズのものを選択するために、そこから数百のビーズを提供する必要があります。 </li><li>その後、別の3センチメートルシャーレ中の溶媒の20μlの低下に約100μmの直径のビーズを転送するために2μlに設定P2ピペットを使用しています。 </li><li> 20μlのピペットと2μlのピペットを用いて任意の残留溶媒で溶媒を除去。適用される薬剤20μlを交換してください。 注：整合性（またはこれまでに溶媒が使用される）DMSO中の100μMの濃度で薬物を溶解するために、いくつかの小分子阻害剤として-80℃で20μLとストアへのアリコートは不安定であり、それを繰り返し凍結融解を回避するのが最善です。必要な場合は、ビーズを浸漬する前に、所望の濃度に薬剤を希釈します。有効濃度は、各薬物のために経験的に決定する必要があるかもしれません。 </li><li> 1時間インキュベートします。これらの分子の多くは光感受性であるため、光から保護します。 </li><li>水に溶解した2％のフェノールレッドを20μlに2μlに設定P2ピペットで転送4-5ビーズを移植する前に。ビーズを可視化するために、ステレオ解剖顕微鏡を使用してこの手順を実行します。ファイン時計職人鉗子を持つ単一のビーズまたはワイヤループを削除し、胚に転送する前に、PBSですすいでください。これは活性の損失の原因となりますので、使用前に15分以上の場合は2％のフェノールレッド中のビーズを放置しないでください。 </li></ol> 3.準備タングステン針<ol><li> 3〜4cmのウェル長さに微タングステン線をカットします。の終わりに、ワイヤの一枚を挿入ガラスパスツールピペットや位置に固定するためにブンゼンバーナーで溶融。彼らが使用中に破損している場合、スペアがあることを確認するために10針まで準備します。 </li><li>ジェット機炎にワイヤの端部を配置し、（2〜3分程度）タングステンが白色に点灯し、先端が鋭利になるまでそこにそれを保持します。注：必要に応じて、使用中に針を洗浄することができ、ジェット機で再先鋭化。 注：これらの針はまた、ビーズを転送するためのループを作るために使用することができます。静かにベンチに針に触れ、それがループを形成するように曲げます。 </li></ol>ビーズ移植片のための胚の準備4 <ol><li>それらは所望の段階（ほとんどの肢芽の操作のための3〜5日）に達するまで、平滑末端を上にして卵をインキュベートします。鈍鉗子を使用すると、殻を破ると、シェルを削除し、胚を露出するために鉗子を使用するように平滑末端をタップします。卵殻膜も除去されることを確認します。 注：PBS 1-2 mlのを支援するためにパスツールピペットで追加することができますこの膜は、卵黄にこだわっている場合。静かに、卵黄の表面にPBSをピペットで卵黄を損傷しないように注意しながら、ピンセットで膜をつかみ、静かに卵から引き出します。 </li><li> 10ミリリットルの注射器と19 G針で卵から卵白を5 mlまで削除します。胚の生存率を減らすことができ、より大きなボリュームの除去などの卵を密封するために使用されるテープに接触しない胚を確保するために必要な分だけの取ります。 </li><li>胚は15 100 Uペニシリンを含むPBS mlのストレプトマイシンおよび0.1mg / mlにアーティスト墨汁の5-6滴を追加する視覚化するため。 0.5-1は直接1mlシリンジ及び25 G針で胚下mlで注入します。 </li><li>脱水しない胚を確保するために、卵子に1％ウシ胎児血清および100 Uペニシリンおよび0.1mgストレプトマイシン/ mlのPBSを2〜3mlのを追加します。先鋭化時計職人鉗子を使用すると、卵黄膜を除去し、開発肢芽の上に羊膜を開きます。胚が乾燥しないことを確認し、、必要に応じて、より多くのPBS / FCSを追加します。 注：肢芽が明らかに胚の脇腹に対になった派生物として見ることができます。これらの段階では胚は、右側が通常最上でオンになります。その結果、それが右の手足にビーズを移植片と反対側のコントロールとして残したものを使用する方が簡単です。 </li><li>ビーズが移植される肢芽の中に切開​​を作るために先鋭化タングステン線を使用してください。若い段階で、これは困難であるが可能な肢芽を介してすべての方法をカットしないでください。 注：古い胚（HHステージ22以上）においては、グラフト化が、肢自体が小さい芽、これはより困難であるより若い段階において肢芽の特定の領域を選択することが可能です。それは肢芽は、ビーズが四肢近位のままになり、特に若い段階で、そのようにグラフトされたビーズを越えて成長することを覚えておくことも重要です。 </li><li>極細WAのいずれかを使用して、成長因子または薬剤のいずれかからビーズをピックアップtchmakers鉗子やタングステン針から作られたループ。ビーズをDMSOまたはフェノールレッドのいずれかに浸漬されている場合は、胚に適用する前にPBSですすぎます。鉗子/ワイヤループで胚にビーズを転送します。その後、切開部にビードを挿入すると、鋭利なタングステン線を使用しています。 </li><li>胚が水和保たれていることを確実に卵にPBS / FCS 1-2 mlを加えたが、これは胚を損傷し、ビーズが脱落する原因となりますので、胚そのものに直接適用することは避けてください。テープで卵を密封し、インキュベーターに戻します。 </li></ol>

<ol>
	<li>Davey, M. G., Tickle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chicken+as+a+model+for+embryonic+development.">The chicken as a model for embryonic development.</a> Cytogenetic Genome Research. 117, 231-239 (2007).</li><li>Le Douarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nogent+Institute--50+years+of+embryology.">The Nogent Institute--50 years of embryology.</a> International Journal of Developmental Biology. 49, 85-103 (2005).</li><li>Le Douarin, 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=The+avian+embryo+as+a+model+to+study+the+development+of+the+neural+crest:+a+long+and+still+ongoing+story.">The avian embryo as a model to study the development of the neural crest: a long and still ongoing story.</a> Mechanisms of Development. 121, 1089-1102 (2004).</li><li>Bell, E., Brickell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-competent+retroviral+vectors+for+expressing+genes+in+avian+cells+in+vitro+and+in+vivo.">Replication-competent retroviral vectors for expressing genes in avian cells in vitro and in vivo.</a> Molecular Biotechnology. 7, 289-298 (1997).</li><li>Abu-Elmagd, 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=Wnt/Lef1+signaling+acts+via+Pitx2+to+regulate+somite+myogenesis.">Wnt/Lef1 signaling acts via Pitx2 to regulate somite myogenesis.</a> Developmental Biology. 337, 211-219 (2010).</li><li>Munsterberg, A. E., Lassar, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+signals+from+the+neural+tube,+floor+plate+and+notochord+induce+myogenic+bHLH+gene+expression+in+the+somite.">Combinatorial signals from the neural tube, floor plate and notochord induce myogenic bHLH gene expression in the somite.</a> Development. 121, 651-660 (1995).</li><li>McGrew, M. J., 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=Efficient+production+of+germline+transgenic+chickens+using+lentiviral+vectors.">Efficient production of germline transgenic chickens using lentiviral vectors.</a> EMBO Reports. 5, 728-733 (2004).</li><li>Valasek, 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=Cellular+and+molecular+investigations+into+the+development+of+the+pectoral+girdle.">Cellular and molecular investigations into the development of the pectoral girdle.</a> Developmental Biology. 357, 108-116 (2011).</li><li>Tickle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+contribution+of+chicken+embryology+to+the+understanding+of+vertebrate+limb+development.">The contribution of chicken embryology to the understanding of vertebrate limb development.</a> Mechanisms of Development. 121, 1019-1029 (2004).</li><li>Riddle, R. D., Johnson, R. L., Laufer, E., Tabin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+mediates+the+polarizing+activity+of+the+ZPA.">Sonic hedgehog mediates the polarizing activity of the ZPA.</a> Cell. 75, 1401-1416 (1993).</li><li>Tickle, C., Alberts, B., Wolpert, L., Lee, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+application+of+retinoic+acid+to+the+limb+bond+mimics+the+action+of+the+polarizing+region.">Local application of retinoic acid to the limb bond mimics the action of the polarizing region.</a> Nature. 296, 564-566 (1982).</li><li>Rosello-Diez, A., Torres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulative+patterning+in+limb+bud+transplants+is+induced+by+distalizing+activity+of+apical+ectodermal+ridge+signals+on+host+limb+cells.">Regulative patterning in limb bud transplants is induced by distalizing activity of apical ectodermal ridge signals on host limb cells.</a> Developmental Dynamics. 240, 1203-1211 (2011).</li><li>Anakwe, 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=Wnt+signalling+regulates+myogenic+differentiation+in+the+developing+avian+wing.">Wnt signalling regulates myogenic differentiation in the developing avian wing.</a> Development. 130, 3503-3514 (2003).</li><li>Amthor, H., Christ, B., Weil, M., Patel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+importance+of+timing+differentiation+during+limb+muscle+development.">The importance of timing differentiation during limb muscle development.</a> Current Biology. 8, 642-652 (1998).</li><li>Brand-Saberi, B., Muller, T. S., Wilting, J., Christ, B., Birchmeier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scatter+factor/hepatocyte+growth+factor+(SF/HGF)+induces+emigration+of+myogenic+cells+at+interlimb+level+in+vivo.">Scatter factor/hepatocyte growth factor (SF/HGF) induces emigration of myogenic cells at interlimb level in vivo.</a> Developmental Biology. 179, 303-308 (1996).</li><li>Mok, G. F., Cardenas, R., Anderton, H., Campbell, K. H. S., Sweetman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+between+FGF18+and+retinoic+acid+regulate+differentiation+of+chick+embryo+limb+myoblasts.">Interactions between FGF18 and retinoic acid regulate differentiation of chick embryo limb myoblasts.</a> Developmental Biology. 396, 214-223 (2014).</li><li>Abou-Elhamd, A., Cooper, O., M&#252;nsterberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Klhl31+is+associated+with+skeletal+myogenesis+and+its+expression+is+regulated+by+myogenic+signals+and+Myf-5.">Klhl31 is associated with skeletal myogenesis and its expression is regulated by myogenic signals and Myf-5.</a> Mechanisms of Development. , 126-852 (2009).</li><li>Schmidt, M., Tanaka, M., Munsterberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+(beta)-catenin+in+the+developing+chick+myotome+is+regulated+by+myogenic+signals.">Expression of (beta)-catenin in the developing chick myotome is regulated by myogenic signals.</a> Development. 127, 4105-4113 (2000).</li><li>Stavridis, M. P., Lunn, J. S., Collins, B. J., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+discrete+period+of+FGF-induced+Erk1/2+signalling+is+required+for+vertebrate+neural+specification.">A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification.</a> Development. 134, 2889-2894 (2007).</li><li>Martinez-Morales, P. L., 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=FGF+and+retinoic+acid+activity+gradients+control+the+timing+of+neural+crest+cell+emigration+in+the+trunk.">FGF and retinoic acid activity gradients control the timing of neural crest cell emigration in the trunk.</a> Journal of Cell Biology. 194, 489-503 (2011).</li><li>Olivera-Martinez, I., Storey, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signals+provide+a+timing+mechanism+for+the+FGF-retinoid+differentiation+switch+during+vertebrate+body+axis+extension.">Wnt signals provide a timing mechanism for the FGF-retinoid differentiation switch during vertebrate body axis extension.</a> Development. 134, 2125-2135 (2007).</li><li>Logan, M., Simon, H. G., Tabin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+T-box+and+homeobox+transcription+factors+suggests+roles+in+controlling+chick+limb-type+identity.">Differential regulation of T-box and homeobox transcription factors suggests roles in controlling chick limb-type identity.</a> Development. 125, 2825-2835 (1998).</li><li>Borycki, A. G., Mendham, L., Emerson, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+somite+patterning+by+Sonic+hedgehog+and+its+downstream+signal+response+genes.">Control of somite patterning by Sonic hedgehog and its downstream signal response genes.</a> Development. 125, 777-790 (1998).</li><li>Sweetman, 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=FGF-4+signaling+is+involved+in+mir-206+expression+in+developing+somites+of+chicken+embryos.">FGF-4 signaling is involved in mir-206 expression in developing somites of chicken embryos.</a> Developmental Dynamics. 235, 2185-2191 (2006).</li><li>Chapman, S. C., Collignon, J., Schoenwolf, G. C., Lumsden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+method+for+chick+whole-embryo+culture+using+a+filter+paper+carrier.">Improved method for chick whole-embryo culture using a filter paper carrier.</a> Developmental Dynamics. 220, 284-289 (2001).</li><li>Sweetman, D., Wagstaff, L., Cooper, O., Weijer, C., M&#252;nsterberg, 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+migration+of+paraxial+and+lateral+plate+mesoderm+cells+emerging+from+the+late+primitive+streak+is+controlled+by+different+Wnt+signals.">The migration of paraxial and lateral plate mesoderm cells emerging from the late primitive streak is controlled by different Wnt signals.</a> BMC Developmental Biology. 8, 63 (2008).</li><li>Scott, B. B., Lois, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+tissue-specific+transgenic+birds+with+lentiviral+vectors.">Generation of tissue-specific transgenic birds with lentiviral vectors.</a> Proceedings of the National Academy of Sciences of the U.S.A. 102, 16443-16447 (2005).</li><li>Seidl, A. H., 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=Transgenic+quail+as+a+model+for+research+in+the+avian+nervous+system:+a+comparative+study+of+the+auditory+brainstem.">Transgenic quail as a model for research in the avian nervous system: a comparative study of the auditory brainstem.</a> Journal of Comparative Neurology. 521, 5-23 (2013).</li></ol>

The authors have nothing to disclose.

卵内の組織の開発に直接適用ビーズ移植片の使用は、それらが適用されるときの発達段階と暴露時間を超える圧倒的な制御を与え、開​​発中の増殖因子シグナル伝達の役割を分析するための強力なツールです。 分子の各タイプのビーズの選択は重要です。それは、経験的にこれらのビーズのそれぞれの阻害剤の有効性をテストする必要があるが、このようなここに記?...

鳥類胚は長年1の開発のための研究のための強力なツールを提供してきました。彼らの最も有用な特徴の1つは、操作が比較的容易であるということです。外部開発は、このような細胞の運命2,3を研究するための古典的なウズラ-ニワトリキメラシステムなどの例を含む様々なmicromanipulationsを胚にアクセスするために卵を開き、実行することが可能となり、開発4,5の間に特定の組織における過剰発現のためのレトロウイルスの注入発達シグナル源6を識別し、移植片培養。さらに最近では、GFPを発現するトランスジェニックニワトリのラインからの移植片とラベルなしホストとの間で発生するキメラは、古典的な移植や遺伝子修飾の組み合わせが開発7,8に重要な洞察を提供することができることを示しています。 鳥類胚を操作することができる容易さはそれを手足を研究するための優れたモデルをしました開発9。 in vivoでの開発手足に特定の成長因子の適用は、四肢のパターニング10,11を変化させ、このプロセス12への洞察を提供し続け因子を同定してきました。このアプローチはまた、筋肉の発達を調節する因子を研究するために使用されており、そのようなのWnt 13、BMPは14およびHGF 15などの多数の信号のための役割を明らかにしました。 最近、この技術は、肢芽に筋原性遺伝子発現を制御する信号を調査するために使用されており、FGF18とレチノイン酸との間の相互作用は、MyoDの発現16のタイミングを制御することができることを示しています。ビーズ上にロードした後、定義された発達段階で特定の組織に直接移植することができる成長因子および小分子の組み合わせを使用することで、開発中のほぼすべての時間と領域に介入する機会を与えてくれます。これはinveするために使用されています体節のパターニング17,18、神経仕様19、神経堤の移行20と軸延長部21を含む多くのプロセスをstigate。 ここでは、鶏の手足の開発に増殖因子または阻害剤のいずれかに浸したビーズを移植する方法について説明します。これは、in situハイブリダイゼーションと筋特異的遺伝子発現を分析することによって筋形成におけるこれらの信号の影響を決定するために使用されてきました。我々は、レチノイン酸または特定のシグナル伝達経路の小分子阻害剤のような小さな疎水性分子のための成長因子、またはAG 1-X2ビーズのために使用されるヘパリン浸したビーズのいずれかを使用して移植片を記載しています。しかし、他のビーズは、22のFGFおよびShhの23の両方を提供するために使用されているにもご利用いただけます。

<table><tbody><tr><td>Heparin acrylic beads</td><td>Sigma</td><td>H5263</td><td>The acrylic-heparin beads used have been discontinued. However a replacement product is available, Heparin agarose beads, cat no H6508. These are transparent so harder to work with but can be stained with phenol red in the same way as AG 1-X2 beads,</td></tr><tr><td>AG 1-X2 beads</td><td>Bio-Rad</td><td>140-1231</td><td></td></tr><tr><td>Affi Gel blue beads</td><td>Bio-Rad</td><td>153-7301; 153-7302</td><td>These beads have been used with a range of growth factors including Shh and FGFs and can be used to replace heparin beads</td></tr><tr><td>FGF18</td><td>Peprotech</td><td>100-28</td><td>Resuspend in PBS with 0/1% BSA, prepare single use aliquots of 0.5-1ul and store at -80&amp;deg;C. Batches and suppliers can vary so different concentrations should be tested to determine an effective dose.</td></tr><tr><td>U0126</td><td>Cell Signalling</td><td>9903</td><td>Make to 20mM stock in DMSO and store in single use aliquots at -80&amp;deg;C. Protect from light.</td></tr><tr><td>BMS-493</td><td>Tocris Biosciences</td><td>3509</td><td>Resuspend in DMSO and store in single use aliquots at -80&amp;deg;C. Protect from light.</td></tr><tr><td>Black Indian ink</td><td>Windsor and Newton</td><td>5012572003384 (30 ml)</td><td>While alternatives to this product are available care should be taken as some inks are toxic to embryos</td></tr><tr><td>Tungsten wire, 0.1 mm dia. 99.95%</td><td>Alfa Aesar</td><td>10404</td><td></td></tr><tr><td>Penicillin / streptomycin</td><td>Sigma</td><td>P0781</td><td>Dilute 100X in PBS/ink and PBS/FCS</td></tr></tbody></table>

grafting of beads into developing chicken embryo limbs to identify signal transduction pathways affecting gene expression

ニワトリ胚を使用して直接成長因子または遺伝子発現およびシグナル伝達経路の活性化におけるシグナル伝達経路の特異的阻害剤のいずれかの効果を試験することが可能です。この技術は、特定の時間に正確に定義された発達段階でのシグナル伝達分子の送達を可能にします。この胚を採取し、遺伝子発現は 、in situハイブリダイゼーション、または免疫染色で観察されたシグナル伝達経路の活性化により、例えば、検査することができる後。 U0126、MEK阻害剤に浸し、このFGF18に浸したビデオヘパリンビーズ、AG 1-X2ビーズでは、 卵内肢芽に移植されています。これは、FGF18はMyoDの及びERKのリン酸化の発現を誘導し、誘導された内因性の両方、およびFGF18のMyoD発現がU0126により阻害されることを示しています。レチノイン酸アンタゴニストに浸したビーズは、FGF18によって時期尚早のMyoDの誘導を増強することができます。 このアプローチは、私たちすることができます異なる増殖因子および阻害剤の広い範囲で編、容易に胚内の他の組織に適合されます。

HHステージでは21のMyoDは染色が開発体節（ 図1A）の筋節に見ることができるが、四肢の筋芽細胞の開発に表現されていない。 図1Bは、FGF18ビーズ移植以下のMyoD 6時間in situハイブリダイゼーションを示しています。対側肢では発現がないながらのMyoDは、ビーズの近くに筋芽細胞に誘導されます。 U0126、MEK、のMyoD（ 図1C）のブロックのFGF18誘導の特異...

Watch this Scientific Journal Video about Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression at JoVE.com

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

遺伝子発現に影響を​​及ぼすシグナル伝達経路を同定するために、ニワトリ胚手足の開発にビーズのグラフト

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

grafting-beads-into-developing-chicken-embryo-limbs-to-identify

Research

JoVE Journal

Developmental Biology

8.8K ビュー.  University of Nottingham. By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

ビデオ: Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

最適化された<em&gt;元OVO</em&gt;開発の進んだ段階にニワトリ胚の培養

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

発生生物学

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

鶏の胚内の神経堤細胞と血管の二重標識は、ひよこGFP 神経管移植とカルボシアニンDyI注射を用いた

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

ゼブラフィッシュ胚におけるマイクロビーズの移植

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Placing Growth Factor-Coated Beads on Early Stage Chicken Embryos

The neural tube expresses many proteins in specific spatiotemporal patterns during development. These proteins have been shown to be critical for cell fate determination, cell migration, and formation of neural circuits. Neuronal induction and patterning involve bone morphogenetic protein (BMP), sonic hedgehog (SHH), fibroblast growth factor (FGF), among others. In particular, the expression pattern of Fgf8 is in close proximity to regions expressing BMP4 and SHH. This expression pattern is consistent with developmental interactions that facilitate patterning in the telencephalon.

Here we provide a visual demonstration of a method in which an in ovo preparation can be used to test the effects of Fgfs in the formation of the forebrain. Beads are coated with protein and placed in the developing neural tube to provide sustained exposure. Because the procedure uses small, carefully placed beads, it is minimally invasive and allows several beads to be placed within a single neural tube. Moreover, the method allows for continued development so that embryos can be analyzed at a more mature stage to detect changes in anatomy and in neural patterning. This simple but useful protocol allows for real time imaging. It provides a means to make spatially and temporally limited changes to endogenous protein levels.

A variety of growth factors and proteins interact to induce cells to take on different cell fates during development. Here we demonstrate the use of an in ovo preparation to address possible interactions between different proteins in development by placing beads on E2.5 chick embryos.

アーリーステージのニワトリ胚に成長因子コートビーズの配置

The neural tube expresses many proteins in specific spatiotemporal patterns during development.   These proteins have been shown to be critical for cell ...

生物学

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

HHステージ10ニワトリ胚のOVOエレクトロポレーションで

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Application of Impermeable Barriers Combined with Candidate Factor Soaked Beads to Study Inductive Signals in the Chick

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness following surgical intervention are key experimental strengths. Mica plates were the first barriers used to prevent chick limb bud initiation1. Protocols that use aluminum foil as an impermeable barrier to wing bud or leg bud induction and or initiation are described. We combine this technique with bead placement lateral to the barrier to exogenously supply candidate endogenous factors that have been blocked by the barrier. The results are analyzed using in situ hybridization of subsequent gene expression. Our main focus is on the role of retinoic acid signaling in the induction and later initiation of the chick embryo fore and hindlimb. We use BMS 493 (an inverse agonist of retinoic acid receptors (RAR)) soaked beads implanted in the lateral plate mesoderm (LPM) to mimic the effect of a barrier placed between the somites (a source of retinoic acid (RA)) and the LPM from which limb buds grow. Modified versions of these protocols could also be used to address other questions on the origin and timing of inductive cues. Provided the region of the chick embryo is accessible at the relevant developmental stage, a barrier could be placed between the two tissues and consequent changes in development studied. Examples may be found in the developing brain, axis extension and in organ development, such as liver or kidney induction.

Protocols using impermeable barriers to block induction events between tissues of the main body axis and flank in the chick embryo required for limb formation are described. Beads soaked in candidate inductive signals are used to overcome the effect of barrier placement and analysis of gene expression confirms this.

チックに誘導シグナルを研究するために、候補因子浸しビーズと組み合わせることで不浸透性の障壁の応用

The chick embryo provides a superb vertebrate model that can be used to dissect developmental questions in a direct way. Its accessibility and robustness ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

鶏胚における神経誘導のためのアッセイ

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

鶏胚における単一細胞のトランスフェクション

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of developing tissues and organs. This process is actively maintained in adulthood. Cell differentiation is an ongoing process during the development and homeostasis of organs. Understanding the early steps of cell differentiation is essential to know other complex processes such as morphogenesis. Thus, recombinant chicken limbs are an experimental model that allows the study of cell differentiation and pattern generation under embryonic patterning signals. This experimental model imitates an in vivo environment; it assembles reaggregated cells into an ectodermal cover obtained from an early limb bud. Later, ectoderms are transferred and implanted in a chick embryo receptor to allow its development. This assay was mainly used to evaluate mesodermal limb bud cells; however, it can be applied to other stem or progenitor cells from other organisms.

Recombinant limbs are a powerful experimental model that allows for studying the process of cell differentiation and the generation of patterns under the influence of embryonic signals. This protocol presents a detailed method for generating recombinant limbs with chicken limb-mesodermal cells, adaptable to other cell types obtained from different organisms.

形態形成、パターニング、および細胞分化の初期段階を理解するためのニワトリ組換え四肢アッセイ

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

浸漬ビーズアッセイを用いたニワトリ胚発生時の細胞分化、形態形成、パターニングの解析

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

遺伝子発現に影響を及ぼすシグナル伝達経路を同定するために、ニワトリ胚手足の開発にビーズのグラフト

要約

さらに動画を探す

この動画の章

最適化された

鶏の胚内の神経堤細胞と血管の二重標識は、ひよこ^GFP神経管移植とカルボシアニンDyI注射を用いた

ゼブラフィッシュ胚におけるマイクロビーズの移植

アーリーステージのニワトリ胚に成長因子コートビーズの配置

HHステージ10ニワトリ胚のOVOエレクトロポレーションで

チックに誘導シグナルを研究するために、候補因子浸しビーズと組み合わせることで不浸透性の障壁の応用

鶏胚における神経誘導のためのアッセイ

鶏胚における単一細胞のトランスフェクション

形態形成、パターニング、および細胞分化の初期段階を理解するためのニワトリ組換え四肢アッセイ

浸漬ビーズアッセイを用いたニワトリ胚発生時の細胞分化、形態形成、パターニングの解析

遺伝子発現に影響を​​及ぼすシグナル伝達経路を同定するために、ニワトリ胚手足の開発にビーズのグラフト

要約

さらに動画を探す

この動画の章

最適化された

鶏の胚内の神経堤細胞と血管の二重標識は、ひよこGFP 神経管移植とカルボシアニンDyI注射を用いた

ゼブラフィッシュ胚におけるマイクロビーズの移植

アーリーステージのニワトリ胚に成長因子コートビーズの配置

HHステージ10ニワトリ胚のOVOエレクトロポレーションで

チックに誘導シグナルを研究するために、候補因子浸しビーズと組み合わせることで不浸透性の障壁の応用

鶏胚における神経誘導のためのアッセイ

鶏胚における単一細胞のトランスフェクション

形態形成、パターニング、および細胞分化の初期段階を理解するためのニワトリ組換え四肢アッセイ

浸漬ビーズアッセイを用いたニワトリ胚発生時の細胞分化、形態形成、パターニングの解析

遺伝子発現に影響を及ぼすシグナル伝達経路を同定するために、ニワトリ胚手足の開発にビーズのグラフト

鶏の胚内の神経堤細胞と血管の二重標識は、ひよこ^GFP神経管移植とカルボシアニンDyI注射を用いた