DPは、薬物乱用の国民の協会からルースキルシュシュタイン賞1F32 DA021977 - 01A1の受信者です。この作品は、RHFへマーガレットM. Alkek財団によってサポートされていました。

<html> I.の概要図： <img src="/files/ftp_upload/old/1027_2009_1_22_2/Figure1.png" alt="図1" width="460" height="384" />このビデオでは、ニワトリ胚における神経誘導するためのアッセイの各ステップを示しています。まず、ホスト胚は、新しい文化の中で外植された[NI1]。その後、donnorの胚は、生理食塩水で外植されており、ヘンゼンのノード（ひよこの&quot;主催者&quot;）は蛍光色素DII [NI2]のラベルが付いています。ヘンゼンのノードがdonnorの胚から摘出された[NI3]とホスト胚に移植[NI4、NI5]。ホスト胚はその後donnor組織がホストの領域で異所性の軸を誘導ている期間の後に最大22時間、のために培養されopaca [NI6]。以下の文化、ホスト胚は、DABの存在下で光酸化のために固定され、処理されます：この化学反応により、光酸化する前にFITC蛍光の下に赤く写るdonnor由来の細胞は、[NI7]、DAB [NI8]と茶色の次の反応になる。胚はその後神経マーカー[NI8]とin situハイブリダイゼーションのために処理されます。宿主由来の組織はdonnorの移植片の存在下で神経組織を形成しています。この組織は、青色表示されます。 1：新しい文化のために、ホスト胚の準備<ol><li>この神経誘導アッセイのプロトコルは、ハンバーガー＆ハミルトンステージ3にインキュベートされた卵から始まります+（またはHH3 +）。これらの卵は、13時間湿ったインキュベーターで彼らの側に敷設インキュベートされています。 </li><li>新文化（ステップ3.1 5.5〜9）のホスト胚を準備します。 </li><li> 200μlの生理食塩水でホスト胚の表面を覆う。これは後で転送するとdonnorの移植片の位置決めを容易にするでしょう。 </li><li>最後に、反転ファルコン35mmの培養皿を使用してアセンブリをカバー。 </li></ol> 2：生理食塩水でdonnorの胚をExplanting <ol><li>ピンセットで殻をタップすると、すべてのシェルの周りに、シェルの部分を削除して、卵を開きます。シェルの上部を取り外して廃棄。 </li><li>鉗子と厚いアルブミンを削除して、胚が上向きに向くように粗い鉗子で卵黄嚢を傾けます。 </li><li>細かいハサミを使用して、胚の周りに卵黄嚢の正方形をカット。スプーンで卵黄から胚を取り出し、そしてPBSを含む皿の場所。 </li><li>鉗子を使用して、明域からdonnorの胚を切り離します。 </li><li> PBSを含むSylgard覆われた皿にdonnorの胚を移す。 </li></ol> 3：、ラベリング切除とdonnorグラフトを移植<ol><li>微小電極プラーを使用して、50μlのガラスマイクロキャピラリーピペットを引き出します。顕微鏡下で、微細な鉗子を用いてマイクロピペットの先端をカット。 plastelineのリボンが並んでペトリ皿の場所のマイクロピペット。 </li><li>移植前の移植片にラベルを付けるために使用される染料溶液を調製します：まず、1mlのDIIの株式を準備：色素DII（1,1&#39; - ジオクタデシル- 3、3,3&#39;、3&#39; - tetramethylindocarbocyanine過塩素酸塩）をカルボシアニン0.5％無水エタノール。この株は、暗所で- 20Cで保存することができます。 42℃に設定したウォーターバス中で、ミックスは360μl0.3Mショ糖に40μlのDIIの株式を温した。水浴の温度は、DIIと不溶性結晶の形成の析出を防止するために必要です。簡単に言えばミニspectrafuge（5〜10秒間）上でソリューションをスピン。 DIIのソリューションが使用できるようになりました。 </li><li>昆虫ピンを使用して、Sylgard覆われた皿の底にdonnorの胚を固定します。 </li><li> DIIでマイクロピペットを埋戻し。吸引管のアセンブリを使用して穏やかな圧力を適用することによって、ヘンゼンのノードにDIIの小さな塊を適用する。全体ヘンゼンのノードがラベル付けされていることを確認します。 </li><li>マイクロキャピラリーピペットまたはmicrodissectingナイフを使用して、ヘンゼンのノード（&quot;主催者&quot;、donnorの移植）をカット。 </li><li>カルミンと移植片の腹側と後端をマーク。これは、移植片は、ステップ4.3での適切な背腹方向に配置されることを保証します。 </li><li> 200μlのギルソンのピペットを使用して、ホスト胚に移植片を移す。 </li></ol> 4：移植片と宿主の培養の胚の保護<ol><li>移植片は、ホストサイトに近接して配置さ残っていることを確認し、ホスト胚から生理食塩水を削除します。 </li><li>マイクロキャピラリーピペットまたはmicrodissectingナイフを使用して、ホスト胚のヘンゼンのノードのレベルでは、明域/暗域の境界領域に小さな切開（80 -100μmの）を作る。 </li><li>その腹側があなたと後端に向かって直面しているので、マイクロキャピラリーピペットまたはmicrodissectingナイフを使用して、移植片を配置し、ホスト胚に相当する地域に平行である。 </li><li>残りの生理食塩水を削除し、81〜22時間（ステップ6.1から6.6 9）のために新しい文化のためにホストの胚を処理する。 </li><li>ホストの胚は、その後4℃（ステップ7.1から7.7 9）で一晩固定されています。 </li></ol>E&quot;&gt; 5：蛍光下で移植片をPhotooxidise <ol><li>ヒュームフードの下で、トリス緩衝液にDABワーキング溶液を調製：500μg/mlでトリス緩衝液（100mMのトリス- HCl、pH7.4）中でDAB基質（3,3&#39; - ジアミノベンジジン四塩酸塩）を溶かし、氷の上に、暗闇の中で解決策を保つ。 </li><li>トリス緩衝液でそれぞれ一回5分で5分のためにPBSで2回胚を洗浄してください。 </li><li> 1ミリリットルTris緩衝液を含むガラスの空洞のスライドに胚を移す。 </li><li> 1.5ミリリットルDAB溶液とトリス緩衝液を交換してください。 DABを除染するために10％漂白剤溶液の入ったバケツにエッペンドルフ先端処分する。空洞のスライドの上にカバースリップを置き。 </li><li> FITC（フルオレセインイソチオシアネート）蛍光下で、移植片（donnor細胞は蛍光赤を表示されています）上で顕微鏡の目的を集中し、すべての蛍光赤色細胞が茶色（これは、DIIの結果として起こるとなっているまで、FITCの蛍光に公開photoconvertedている蛍光色素DABの存在下でFITC励起波長（488nmの）への暴露によって、不溶性の褐色結晶する。このプロセスは、約20分、2時間かかります。 </li><li>光酸化反応の完了後、微細な鉗子を使用してカバーを取り外します。ガラスPBTwを（または0.1％のTween - 20を含むPBS）を含む20mlのシンチレーションバイアルをするために胚を移す。 </li><li> 10％漂白剤溶液中でインキュベートすることによってカバースリップとガラスの空洞のスライドからDABを非アクティブにします。 </li></ol> 6：in situハイブリダイゼーション、写真撮影のために胚を処理し、セクショニング<ol><li>唯一の標識プローブを掘るための胚をインキュベートし、in situハイブリダイゼーション（ステップ3.7から6.5 9）に進みます。 </li><li>撮影（ステップ8.2 9）と（ステップ8.3および8.4 9）切片を続行します。 </li></ol>代表的な結果神経誘導アッセイの次の例では、donnorの移植片は組織を派生ホストからの尾の神経マーカーの発現を誘導示されている。この特定の例では、我々は通常、&quot;主催者&quot;の能力を持っていない胚の領域の神経誘導能の獲得を評価した：donnorの移植片は、&quot;主催者&quot;領域は外科的段階でdonnorの胚から除去された胚に由来する3 +。アブレーションに続いて、donnorの胚は、穴が治癒したとヘンゼンのノードに似た構造が形成されるまで、新しい文化の発展に許可されています。この構造はその後、DIIで標識donnorの胚から切り出し、ホスト胚の明域/暗域の境界にグラフトされています。新しい文化に続いて、ミニチュアの軸は、ホスト胚に隣接して形成される：このミニチュア軸は赤血球のロッド（donnorが由来）とヘッドのような構造（ホスト派生）で構成されています：（A）において、donnor由来DIIのラベル細胞は、FITC蛍光顕微鏡下に固定前に示されています。これらdonnor由来細胞は、赤血球の棒として表示されるミニチュア脊索を形成し、増殖している。 （B）DIIの次の光変換は、これらのdonnor由来細胞は茶色に表示されます。 donnorの移植片は、ホストからの神経組織の形成を誘発している：この組織は、脳のための前駆体で構成され、のようなin situハイブリダイゼーションマーカー尾のない以下のため、その発現によって明らかにされた[6から転載]。 <img src="/files/ftp_upload/old/1027_2009_1_22_3/Figure 2.png" alt="図2" width="230" height="154" />注：この手順では、我々は&quot;神経&quot;組織（青）由来のホストからの神経誘導donnor組織（赤/茶色）を区別するために、DII標識細胞を使用している。代わりに派生ひよこを使用する代わり、DIIはdonnorグラフトのラベルが、何人かの著者はまた、代わりにニワトリからウズラ胚に由来するdonnor組織[例6]を使用してください。この代替方法はまた、宿主組織からdonnorを区別することができます。このケースでは、移植片（donnorの組織）が代わりにニワトリの卵のウズラの卵に由来する。ステップ4.5に続いて、ホストが5.7に3.4および5.1に組織（QCP1）と手順3.1をウズラに特異抗体と全体のマウント免疫組織化学のために処理されているプロトコルから省略されます。 B、​​このケースでは、我々は同じ実験手順を使用し、しかし、我々はウズラ由来donnor組織を（QCPN抗体[茶色]の式で示される）を使用：この代替手順の例を以下に示す再生ウズラ胚から派生したノードは、ホストの汎神経マーカーSox2の発現を誘導している[6から再版さ]： <img src="/files/ftp_upload/old/1027_2009_1_22_4/Figure 3.png" alt="図3" width="154" height="230" />

<ol>
	<li>Waddington, 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=Experiments+on+the+development+of+chick+and+duck+embryos,+cultivated+in+vitro.">Experiments on the development of chick and duck embryos, cultivated in vitro.</a> Phil. Trans. R. Soc. Lond. B. 221, 179-230 (1932).</li><li>Waddington, 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=Induction+by+the+primitive+streak+and+its+derivatives+in+the+chick.">Induction by the primitive streak and its derivatives in the chick.</a> J. Exp. Biol. 10, 38-46 (1933).</li><li>Gallera, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excision+et+transplantation+des+diff&amp;eacute;rentes+r&amp;eacute;gions+de+la+ligne+primitive+chez+le+poulet.">Excision et transplantation des diff&amp;eacute;rentes r&amp;eacute;gions de la ligne primitive chez le poulet.</a> C. R. Ass. Anat. 49, 632-639 (1964).</li><li>Gallera, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+induction+in+birds.">Primary induction in birds.</a> Adv. Morph. 9. , 149-180 (1971).</li><li>Serbedzija, G. N., Fraser, S. E., 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=Pathways+of+neural+crest+cell+migration+in+the+mouse+embryo+as+revealed+by+vital+dye+labeling.+Development+108.">Pathways of neural crest cell migration in the mouse embryo as revealed by vital dye labeling. Development 108.</a> , 605-612 (1990).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.+Development+122.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo. Development 122.</a> , 3263-3273 (1996).</li><li>Joubin, K., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+interactions+continuously+define+the+organizer+during+the+cell+movements+of+gastrulation.">Molecular interactions continuously define the organizer during the cell movements of gastrulation.</a> Cell. 98, 559-571 (1999).</li><li>Streit, A., Berliner, A. J., Papanayotou, C., Sirulnik, A., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+neural+induction+by+FGF+signaling+before+gastrulation.">Initiation of neural induction by FGF signaling before gastrulation.</a> Nature. , 406-474 (2000).</li><li>Psychoyos, D., Finnell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+Culture+of+Early+Chick+Embryos+ex+vivo+(New+Culture.">Method for Culture of Early Chick Embryos ex vivo (New Culture.</a> JoVE. 20, 10-3791 (2008).</li></ol>

このビデオでは、神経誘導のためのアッセイを行うことで、異なる手順を示し、このアッセイは、本質的にニワトリの推定神経誘導の分子の特性評価に使用されるため、発生学的micromanipulationsから1-4まで、幅広いアプリケーションのさまざまな、使用することができます。すべての脳と、残りの神経系の形成における初期段階の理解を目指す解明〜6の新しいシグナル...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Eggs</td>
<td>Animal</td>
<td>Charles River Laboratories</td>
<td>Premium Fertile</td>
<td>Fertilized, HH3+ (14 hr)</td>
</tr>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5 or similar</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Incubator</td>
<td>Equipment</td>
<td>Robbins Scientific</td>
<td>M1000</td>
<td>Use with inverted Pyrex dish and 500 ml ddH2O beaker</td>
</tr>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Equipment</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Pyrex dish </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Watchmaker's glass 50mm</td>
<td>Tool</td>
<td>VWR</td>
<td>66112-060</td>
<td>&nbsp;</td>
<tr>
<td>Glass rings</td>
<td>Tool</td>
<td>Physical Plant facility</td>
<td>&nbsp;</td>
<td>cut 4 mm thick sections of glass tubing (27 mm outer diam, 25 mm inner diam). Do not fine polish.</td>
</tr>
<tr>
<td>Curved Forceps (1)</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Forceps (2)</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11002-13</td>
<td>blunt ended using sharpening Stone and 100ul mineral oil</td>
</tr>
<tr>
<td>Fine scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td>Plastic dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Rubber Bulb</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70980</td>
<td>&nbsp;</td>
<tr>
<td>DiI</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>D-282</td>
<td>&nbsp;</td>
<tr>
<td>Aspirator tube assembly</td>
<td>Tool</td>
<td>Sigma</td>
<td>A5177-5EA</td>
<td>&nbsp;</td>
<tr>
<td>Microelectrode puller</td>
<td>Equipment</td>
<td>Sutter Instruments</td>
<td>Sutter Instruments	P-97 Flaming/Brown Micropipette</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>round edge under flame</td>
</tr>
<tr>
<td>Culture chamber</td>
<td>Tool</td>
<td>Pioneer Plastics</td>
<td>030C</td>
<td>&nbsp;</td>
<tr>
<td>Microcapillary tube</td>
<td>Tool</td>
<td>Sigma</td>
<td>P1049-1PAK</td>
<td>&nbsp;</td>
<tr>
<td>Microdissecting knife</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to puncture cavities prior to in situ hybridization</td>
</tr>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>Reagent</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>10025025</td>
<td>Add 1ml depc to 1l PBS; shake; autoclave</td>
</tr>
<tr>
<td>16% PFA</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

assay for neural induction in the chick embryo

ニワトリ胚は、初期胚発生の研究に貴重なツールです。その透明性、アクセス性と操作の容易さは、その神経系の形成と初期のパターン形成を研究するための理想的なツールとなっています。このビデオでは、ホストへの移植主催者の組織する、方法これでヘンゼンのノード（ニワトリ胚におけるオーガナイザー）がホスト有能な外胚葉にグラフトされる方法を示しています。主催者の移植は、神経誘導シグナルを介した神経運命を採用する組織をve Naの上に横たわって指示する。このメカニズムは、神経誘導と呼ばれ、羊膜の脳と脊髄の形成の初期段階を構成しています。このメソッドは本質的にニワトリの推定神経誘導の分子の特性評価に使用されます。このビデオでは、神経誘導のためのアッセイの各ステップを示し、まず、donnorの胚を外植し、皿に固定されます。その後、ホストの胚は、新しい文化のために準備されています。移植片を切除し、ホストの明域のマージンに移植される。ホストは、18〜22時間培養されています。アセンブリを固定し、さらにアプリケーション（例えば、in situハイブリダイゼーション）用に処理されます。このメソッドはもともとワディントンによって考案された<sup&gt; 1,2</sup&gt;とGallera<sup&gt; 3,4</sup&gt;。

Watch this Scientific Journal Video about Assay for Neural Induction in the Chick Embryo at JoVE.com

Assay for Neural Induction in the Chick Embryo

神経誘導、脳の形成の最初のステップです。それはヘンゼンのノード（主催者）は、神経の運命を採用する隣接する組織に指示するメカニズムである、神経系を生じさせるために、すなわち。このビデオでは、ニワトリ胚における神経誘導のためのアッセイを示しています。

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 ...

assay-for-neural-induction-in-the-chick-embryo

Research

JoVE Journal

Biology

17.1K ビュー.  Texas A&M University (TAMU). 神経誘導、脳の形成の最初のステップです。それはヘンゼンのノード（主催者）は、神経の運命を採用する隣接する組織に指示するメカニズムである、神経系を生じさせるために、すなわち。このビデオでは、ニワトリ胚における神経誘導のためのアッセイを示しています。

ビデオ: Assay for Neural Induction in the Chick Embryo

Double Whole Mount in situ Hybridization of Early Chick Embryos

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  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

初期のニワトリ胚のin situハイブリダイゼーションダブルホールマウント

生物学

Method for Whole Mount Antibody Staining in Chick

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 antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

チックの全マウントの抗体染色のための方法

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

の電気生理学的記録ショウジョウバエ胚

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

に有糸分裂研究のためのマイクロインジェクション技術キイロショウジョウバエ合胞体胚

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

チック EX卵文化と EX卵 CAMアッセイ：それが実際にどのように動く

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

活用チック神経管の神経細胞の遺伝的に定義されたグループの軸索経路の解読卵のエレクトロポレーション

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

ニワトリ胚へのヒト幹細胞の異種移植

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

クローン原性アッセイ：付着細胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

心原性誘導効率を定量化するために修飾されたマウス胚性幹細胞ベースのアッセイ

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

初期のニワトリ胚の形態形成の組織の変形を追跡

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...