著者は原稿、ビデオ ナレーションのパドマ Akkapeddi の批判的アントニオ Cidadão、イザベル Alcobia レオノル パレイラし感謝しているし、セルバンテス ・ デ ・ Histologia e の組織サービスからビトー プロアに Biologia を行うDesenvolvimento、Faculdade ・ デ ・ メディチーナ ・ デ ・ リスボア、パラナ ・ デ ・ リスボア、テクニカル サポートのため。Faculdade ・ デ ・ Unidade ・ デ ・ audiovisuais (視聴覚ユニット) からサンパウロ Caeiro とヒューゴ ・ シルバに特にお世話になっておりますメディチーナ ・ デ ・ リスボア、このビデオの生産への顕著なコミットメントのパラナ ・ デ ・ リスボア。我々 は親切提供するビデオ システムと Interaves を装備した堅実ライカマイクロシス テムズ株式会社を認める - Sociedade 農業 Pecuária、ウズラに貢献してくれて S.A 受精卵。この作品は、Faculdade ・ デ ・によって支えられたメディチーナ ・ デ ・ リスボア、パラナ de Lisboa (FMUL)。

これらすべての実験動物の世話やセントロ Académico ・ デ ・ メディチーナ ・ デ ・倫理的なガイドラインに従うリスボア。
1. 受精ウズラと鶏の卵の孵化
<ol><li>それぞれ、3 と 8 日間ウズラ (について) と鶏 (ギャラス) 受精卵をインキュベートしてください。<ol>
<li>空気室 (卵の鈍い端) 38 ° C で加湿のインキュベーターで上向きに卵を配置します。</li>
		<li>この実験を実行するのに約 20 のウズラの卵と 40 の鶏の卵を使用します。 注: 初めてこの手順を確立するとき、これらの番号を倍増する必要があります。</li>
	</ol>
</li>
</ol>2. 胸腺、副甲状腺の基礎の推定領域を含むウズラ胚領域の分離
メモ: 実行卵水平層流フードと滅菌器具と材料を使用して無菌状態で操作手順。
<ol><li>について大規模なホウケイ酸ガラスのボウルを準備 3/4 冷たいリン酸緩衝生理食塩水 (PBS) 液でいっぱい。</li>
	<li>シェルをタップ、曲線はさみでその鈍い端の反対側に円形の開口部を切断して 3 日間の培養後ウズラ卵を開きます。慎重に殻の部分を削除し、胚を冷 PBS で満たされたガラス ボウルに転送します。</li>
	<li>E3 でウズラ (q) 胚を保持 (qE3) (鶏の HH ステージ 21 に対応するウズラ ステージ) 細い鉗子の助けを借りて。曲線はさみを使用して卵黄を包み込む膜の膜へのカットをします。胚体外血管の周囲に外部から前後をカットし続けます。</li>
	<li>転送小さい胚ボウル 3/4 細い鉗子の助けを借りて冷 PBS でいっぱい。接続されている残りの卵黄から胚を徹底的に洗います。</li>
	<li>スキマーを使用して 100 ミリメートルのガラス シャーレと黒のベースに胚を転送する (材料の表を参照してください) 10 mL の冷 PBS を含みます。</li>
	<li>ペトリ皿を顕微鏡の下に配置します。 注: このポイントから、進歩的な倍率の顕微鏡下マイクロサージェリー手順を実行します。照明のソースとしては、限られた熱負荷を考慮した、顕微鏡や光ファイバーを組み込んだ LED ライトを使用することをお勧めします。</li>
	<li>薄い昆虫ピンとプレートの底に胚を保持します。胚体外域正方形の形成 4 本のピンを配置します。</li>
	<li>細い鉗子の助けを借りて、頭部領域の胚体外膜を外し、そこに 5 ピンを配置します。 注: 胚が正しく配置されている場合、耳胞、心臓の管と 1st、2ndと 3rd咽頭アーチ (PAs) が表示されます。</li>
	<li>時空警察ヴェッカーシグナ目はさみを使用しての関心、すなわち、3rd 4thの咽頭アーチ領域 (3/4PAR)、萌芽期の地域を分析します。<ol>
<li>長手方向と平行に体節神経・ チューブ領域と PAs の間の胚軸切断を開始します。</li>
		<li>それを切断することによって腹位置中心の管を削除します。同じ位置にはさみを維持する、カットの方向によると胚を再配置するシャーレを回転させます。</li>
		<li>2ndと 3rd PAs の間と 4thペンシルバニアの下カットします。</li>
		<li>細い鉗子の助けを借りて 3/4PAR から残りの膜をデタッチします。</li>
	</ol>
</li>
	<li>隔離されたティッシュ (3/4PAR) と冷 PBS 2 mL 滅菌プラスチック ピペットを使用して 3/4 ガラスの皿に満ちて転送を吸い出しなさい。 注: 以下、組織 h 48 まで育てられた生体外ですることができます。 または E8 でニワトリ胚のカムに接木されるすぐに。</li>
	<li>生体外の試金の準備の間に氷の上分離 3/4PAR を含むガラスの皿を保ちます。</li>
</ol>3 切片培養の試金: 胸腺、副甲状腺の基礎の推定領域を含む萌芽期の地域の文化。
<ol><li>10 %fbs と 1% ペニシリン/ストレプトマイシン RPMI 1640 培で培養液を準備します。 注: 可溶性の薬理学的試薬は媒体 (たとえば、LY 411.575 (Ly) ・ ディ ・ benzazepine または cyclopamine とビスモデギブ、ノッチとハリネズミ (Hh) シグナル伝達経路、それぞれを阻害する追加できます。この試金のため 3 Notch シグナルを阻害する Ly の 50-200 nM または・ ディ ・ benzazepine の 5-15 μ M を使用して/4PAR. 3/4PAR5Hh シグナルを抑制する cyclopamine の 20 μ M またはビスモデギブの 10 μ M を使用します。</li>
	<li>6 ウェル プレートで 3/4 pp 外植体の in vitro文化を準備します。<ol>
<li>5 ml の培地の 6 ウェル プレートから 1 つの井戸を埋めます。24 ミリメートル ポリカーボネート膜挿入の場所 (材料表参照) 細い鉗子の助けを借りても。</li>
		<li>、実体顕微鏡下で 3/4PAR 外植体ガラス皿からに転送膜表面移植スプーン (またはヘラ) の助けを借りてをスライドさせて、と薄い鉗子。腹側と背側膜外植片を配置します。膜挿入ごとに最大 7 つの植を追加します。3.4 の手順に進みます。</li>
	</ol>
</li>
	<li>また、文化外植体膜フィルターを浮<ol>
<li>35 mm 5 ml の培地のシャーレを準備します。細い鉗子、フロート膜フィルター (材料の表を参照) と乾燥空気との接触を保つための助けを借りて。</li>
		<li>、実体顕微鏡下で 3/4PAR 植から転送ガラス皿をメンブラン フィルター移植スプーン (またはヘラ) の助けを借りて穏やかなスライドし、薄い鉗子。腹側と背側膜外植片を配置します。メンブレン フィルターあたり最大 8 植を追加します。</li>
	</ol>
</li>
	<li>植 3.2 と 3.3 5% CO2と 37 ° C で加湿のインキュベーターでの手順で準備して慎重に置きます。</li>
	<li>48 時間の潜伏期間の後に、インキュベーターから 6 ウェル プレートと 35 mm のペトリ皿を削除します。<ol>
<li>6 ウェル プレートの膜挿入から培養植を収集します。<ol>
<li>常温 (RT) 膜挿入に PBS を追加します。</li>
			<li>2 mL 滅菌プラスチック ピペットを使用して積極的なフラッシュによって膜から植をデタッチします。</li>
			<li>ヘラと細い鉗子の助けを借りて、転送した PBS でいっぱい 3/4 ガラスの皿に培養の植</li>
		</ol>
</li>
		<li>同様に、35 mm のペトリ皿でフローティングの膜フィルターから培養植を収集します。<ol>
<li>室温 PBS でいっぱい新しい 35 mm のペトリ皿に細い鉗子でメンブラン フィルターを転送します。</li>
			<li>2 mL の滅菌プラスチック ピペットを使用する積極的なピペッティングしてメンブレン フィルターから植をデタッチします。</li>
			<li>細い鉗子の助けを借りて、それを接続外植片が残存していないことを確認した後植無料メンブラン フィルターを放電します。</li>
			<li>ヘラと細い鉗子、外植片を室温 PBS でいっぱいガラス皿に転送します。</li>
		</ol>
</li>
	</ol>
</li>
	<li>総 RNA の隔離のための試薬 1 mL にヘラで培養植を転送し、遺伝子発現の研究で使用します。 注意:この試薬への暴露は、深刻な健康被害をすることができます (材料の表を参照してください)。適切な保護眼鏡、衣類および手袋を着用します。取扱説明書に従うし、製造元から提供された安全性データシートを読みます。</li>
	<li>また、E8 で鶏胚のカムに培養組織を移植します。手順 4 に従ってください。</li>
</ol>4. カムの準備
<ol><li>インキュベーターから萌芽期の開発の 8 日間で鶏の卵を削除します。 注: 卵加湿のインキュベーターで 38 ° C で上向きに直面している空気室で培養。</li>
	<li>シェルの部分が空気室に陥るを防ぐために透明なプラスチック テープで卵の鈍端をカバーします。シェルをタップし、曲線はさみで卵円形の開口部をカットします。空気室が表示されます。</li>
	<li>細い鉗子で空気室の白い膜を慎重に削除します。カムは、表示され、異所性組織移植のアクセスです。 注: は、PBS をスライドさせ、外植片の存在を促進するため、カムを移植、前後に水和物に PBS を使用しないでください。膜が乾く場合は、卵を破棄します。</li>
</ol>5. カムに培養外植片の移植
<ol><li>ホルダーに microscalpel カムの小さい船の小血管病変/傷を作成します。 注: は、余分な出血の場合毛管現象によって血液を削除するのにパスツール ピペットの先端を使用します。</li>
	<li>ヘラと細い鉗子を使用して、カムの負傷者の領域に培養外植体を転送します。</li>
	<li>外植体よりわずかに大きいフィルター紙の一部をカットし、外植体の上に置きます。 注: フィルター ペーパー カムに開発後植場所を追跡ことができます。また、許可外植体への毎日の薬物送達ovo で開発中に必要な場合 (5.6 の手順で説明します)。</li>
	<li>透明なプラスチック テープで卵ウィンドウを密封し、木炭ペンシルを使用してそれを識別します。 注: プラスチック テープは、潜伏期間中に脱水から胚を保護します。</li>
	<li>38 ° C で加湿のインキュベーターで 10 日間の操作の卵を孵化させなさい手順 6 に従ってください。</li>
	<li>オプションのステップ: 毎日潜伏期間薬剤投与。<ol>
<li>フィルターにアクセスするため部分的にプラスチック テープを持ち上げて取り外します。一滴ずつ紙の上に薬液の 100 μ L を追加します。ウィンドウを再シールし、38 ° C で加湿のインキュベーターに卵を置き 注: この試金のため cyclopamine の 20 μ M の用量が抑制 Hh がovo で開発5時伝達します。</li>
	</ol>
</li>
</ol>6 Ovo の開発の 10 日後、カム異所性器官形成
<ol><li>培養 10 日後卵をインキュベーターから削除し、慎重にプラスチック テープを撤回します。</li>
	<li>小さなガラスにろ紙をカム派生植ボウル 3/4 冷 PBS でいっぱい曲線はさみと転送を使用して、フィルター領域の周りには、カムをカットします。</li>
	<li>細い鉗子の助けを借りて、10 mL の PBS を含むブラック ベースで 100 mm のペトリ皿にカム由来外植体を転送します。フィルター紙と時空警察ヴェッカーシグナ目はさみや細い鉗子を使用して膜の過剰を慎重に取り外します。</li>
	<li>カム外植体を固定液に転送 (3.7 %pbs で PFA) スキマーで。大きなはさみの助けを借りて、胎児の頸部の正確なカットすることによって、卵から削除せず鶏胚を安楽死させます。</li>
	<li>従来の組織および免疫組織化学によってカム派生植のパラフィン切片における器官形成を評価します。</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gcm2+and+Foxn1+mark+early+parathyroid-+and+thymus-specific+domains+in+the+developing+third+pharyngeal+pouch.">Gcm2 and Foxn1 mark early parathyroid- and thymus-specific domains in the developing third pharyngeal pouch.</a> Mech Dev. 103, 141-143 (2001).</li><li>Neves, H., Dupin, E., Parreira, L., 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=Modulation+of+Bmp4+signalling+in+the+epithelial-mesenchymal+interactions+that+take+place+in+early+thymus+and+parathyroid+development+in+avian+embryos.">Modulation of Bmp4 signalling in the epithelial-mesenchymal interactions that take place in early thymus and parathyroid development in avian embryos.</a> Dev Biol. 361 (2), 208-219 (2012).</li><li>Jerome, L. A., Papaioannou, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DiGeorge+syndrome+phenotype+in+mice+mutant+for+the+T-box+gene,+Tbx1.">DiGeorge syndrome phenotype in mice mutant for the T-box gene, Tbx1.</a> Nat Genet. 27 (3), 286-291 (2001).</li><li>Nie, X., Brown, C. B., Wang, Q., Jiao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+Bmp4+from+the+Tbx1+expression+domain+causes+abnormal+pharyngeal+arch+artery+and+cardiac+outflow+tract+remodeling.">Inactivation of Bmp4 from the Tbx1 expression domain causes abnormal pharyngeal arch artery and cardiac outflow tract remodeling.</a> Cells Tissues Organs. 193 (6), 393-403 (2011).</li><li>Zou, D., Silvius, D., Davenport, J., Grifone, R., Maire, P., Xu, P. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+of+the+third+pharyngeal+pouch+into+thymus/parathyroid+by+Six+and+Eya1.">Patterning of the third pharyngeal pouch into thymus/parathyroid by Six and Eya1.</a> Dev Biol. 293 (2), 499-512 (2006).</li><li>Uematsu, 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=Use+of+in+ovo.+chorioallantoic+membrane+engraftment+to+culture+testes+from+neonatal+mice.">Use of in ovo. chorioallantoic membrane engraftment to culture testes from neonatal mice.</a> Comp Med. 64 (4), 264-269 (2014).</li></ol>

著者が明らかに何もありません。

このメソッドの成功のための重要な側面は、ニワトリおよびウズラの両方の品質の卵。Ovoアッセイ、鶏の卵の質の良い、特に中に、長い潜伏期間の向上を考慮した生存率はプロシージャの最後で (90% 以下)。これを達成するために、別の業者から卵をテストします。長時間非操作の卵を孵化させなさい (最大 16-17 日) とその開発をチェックします。質の良いバッチを考えられる胚の 80% ?...

鳥類胚は、種子発達生物学で広く使用されています。鳥のモデルの主な利点は、卵、胚、マイクロマニピュレーションを実行する能力に比較的簡単にアクセスを開く可能性を含まれます。いくつかの例は細胞運命1カム1,3、異所性の細胞構造の成長と胚2、特定の成長因子の応用を勉強するためクラシック ウズラ鶏キメラ システムを構成します。 4。
器官形成の段階に新たな洞察を得る、萌芽期ティッシュ5体外操作と接木技術を組み合わせた方法最近開発しました。2 段階のアプローチにより、差別と探査の両方 - と後期 - の初期段階器官形成、非常にダイナミックで複雑な組織の相互作用2のために制限されます。さらに、適切な組織特異的マーカーの欠乏頻繁の使用を制限遺伝子組み換え動物モデル6。この 2 段階のアプローチ法は主としてこのような制限を克服します。
最初のステップで、器官形成の初期段階を研究するには、将来の臓器の原基を構成するウズラ胚領域は分離、48 時間培養切片システムで栽培します。この期間中に特定のシグナル伝達経路の薬理学的変調は培養培地5,7に薬を追加することによって実行できます。さらに、培養組織が体外で成長のあらゆる段階で収集され遺伝子発現プローブ ( qPCR、マイクロ アレイなどのメソッドを使用して)。
2 番目の手順では、48 h 培養組織が、接ぎ木 (cE8) (E) 8 日 (c) 鶏胚のカム (ハンバーガーとハミルトン (HH)-33-35 の段階)8。カムは栄養素の血管サプライヤーとして動作し、ガス交換1,3,4移植組織を有効にその開発ovo で時間の長い期間のためにことができます。この実験の手順は特に適しています器官形成の後期段階を研究するovo で開発5,9,10,11 の 10 日後、完全に形成された器官を得ることができるよう.形態素解析は簡単に適切な器官形成を確認する従来の組織によって実行され、(すなわち、MAb ウズラ核 (QCPN)) 特異的抗体を用いた免疫組織化学によってドナー細胞の起源を識別できます。カムの潜伏期間中に、移植できる薬理学的エージェントの存在下で成長してがも、器官形成の進行を評価するための開発のあらゆる段階で収集します。
深さで、ここで説明されている 2 段階のアプローチは、フィゲイレドらですでに採用されています。5鳥副甲状腺/胸腺共通原基開発を探検します。したがって、以下、萌芽期の地域の固有の特性と、胸腺・副甲状腺腺の器官形成に関与する開発の段階の設定が表示されます。
胸腺と副甲状腺腺上皮、咽頭の袋の内胚葉からも機能的に異なるを派生すると (PP)12。鳥、これらの器官の上皮に属します、3 番目と 4 番目 PP 内胚葉 (3/4 pp)12、哺乳類で、3PP から派生する胸腺の上皮と副甲状腺腺上皮に由来する 3PP と 3/4 pp マウスと人間、それぞれ13,14。
これらの器官の形成の初期の段階の一つは、離散胸腺・副甲状腺ドメイン共通の原基での登場です。鶏、E4.515、特定の分子マーカーの in situハイブリダイゼーションによるこれらのドメインを識別できます。開発が進むにつれて、これらの器官の基礎が個性し、神経堤由来細胞によって形成される薄い葉カプセル (E5; でそれらを囲む中咽頭から分離HH-stage 27)。後に、胸腺の上皮は造血前駆細胞 (E6.5; で植民地化します。HH-stage 30)12。
古典的なウズラ鶏研究1,12のように 2 段階のアプローチ、すなわち胸腺5リンパ ・造血器官の形成を研究に便利です。移植し、器官原基で、ウズラが外植体として鶏血液媒介性前駆細胞浸潤ウズラ胸腺上皮相手造血前駆細胞の植民地化前に鶏胚におけるキメラ胸腺が形成されて。このメソッドは、したがって、鳥の血液・ リンパ系の開発で造血細胞の貢献を探索する便利なツールです。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chicken fertilized eggs (Gallus gallus)</td>
			<td>Pintobar, Portugal</td>
			<td></td>
			<td>Poultry farm&#160;</td>
		</tr>
		<tr>
			<td>Quail fertilized eggs (Coturnix coturnix)</td>
			<td>Interaves, Portugal</td>
			<td></td>
			<td>Bird farm&#160;</td>
		</tr>
		<tr>
			<td>15 mL PP centrifuge tubes</td>
			<td>Corning</td>
			<td>430052</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL PP centrifuge tubes</td>
			<td>Corning</td>
			<td>430290</td>
			<td></td>
		</tr>
		<tr>
			<td>60 x 20 mm pyrex dishes</td>
			<td>Duran group</td>
			<td>21 755 41</td>
			<td></td>
		</tr>
		<tr>
			<td>100 x 20 mm pyrex dishes</td>
			<td>Duran group</td>
			<td>21 755 48</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate Membrane Insert&#160;</td>
			<td>Corning</td>
			<td>3412</td>
			<td>24 mm transwell with 0.4 mm Pore Polycarbonate Membrane Insert&#160;</td>
		</tr>
		<tr>
			<td>Membrane filter</td>
			<td>Millipore</td>
			<td>DTTP01300</td>
			<td>0.6 mm Isopore membrane filter</td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>Nunc, Thermo Fisher Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish, 35 x 10 mm</td>
			<td>Sigma-Aldrich</td>
			<td>P5112&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex bowls</td>
			<td></td>
			<td></td>
			<td>from supermarket&#160;</td>
		</tr>
		<tr>
			<td>Transfer pipettes</td>
			<td>Samco Scientific, Thermo Fisher Scientific</td>
			<td>2041S</td>
			<td>2 mL plastic pipet</td>
		</tr>
		<tr>
			<td>Glass pasteur pipette</td>
			<td>normax</td>
			<td>5426015</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman&#160;qualitative filter paper</td>
			<td>Sigma-Aldrich</td>
			<td>WHA1001090</td>
			<td>Filter paper</td>
		</tr>
		<tr>
			<td>Clear plastic tape</td>
			<td></td>
			<td></td>
			<td>from supermarket&#160;</td>
		</tr>
		<tr>
			<td>Cytokeratin (pan; acidic and basic, type I and II cytokeratins), clone Lu-5</td>
			<td>BMA Biomedicals</td>
			<td>T-1302</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclopamine hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C4116</td>
			<td>Pharmacological inhibitor of Hh signalling</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td></td>
			<td>Standart FBS</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>GIBCO, Thermo Fisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>QCPN antibody</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>QCPN</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium, GlutaMAX Supplement&#160;</td>
			<td>GIBCO, Thermo Fisher Scientific</td>
			<td>61870010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bluesil RTV141A/B Silicone Elastomer 1.1Kg Kit</td>
			<td>ELKEM/Silmid</td>
			<td>RH141001KG</td>
			<td>To prepare the back base for petri dish</td>
		</tr>
		<tr>
			<td>Stemolecule LY411575</td>
			<td>Stemgent</td>
			<td>04-0054</td>
			<td>Pharmacological inhibitor of Notch signalling</td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen, Thermo Fisher Scientific</td>
			<td>15596026</td>
			<td>Reagent for total RNA isolation</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-30&#160;</td>
			<td>Thin forceps</td>
		</tr>
		<tr>
			<td>Extra fine Bonn scissors, curved</td>
			<td>Fine Science Tools</td>
			<td>14085-08</td>
			<td>Curved scissors</td>
		</tr>
		<tr>
			<td>Insect pins&#160;</td>
			<td>Fine Science Tools</td>
			<td>26001-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro spatula&#160;</td>
			<td>Fine Science Tools</td>
			<td>10087-12</td>
			<td>Transplantation spoon</td>
		</tr>
		<tr>
			<td>Minutien Pins</td>
			<td>Fine Science Tools</td>
			<td>26002-20</td>
			<td>Microscalpel</td>
		</tr>
		<tr>
			<td>Moria Nickel Plated Pin Holder</td>
			<td>Fine Science Tools</td>
			<td>26016-12</td>
			<td>Holder</td>
		</tr>
		<tr>
			<td>Moria Perforated Spoon</td>
			<td>Fine Science Tools</td>
			<td>10370-17</td>
			<td>Skimmer</td>
		</tr>
		<tr>
			<td>Wecker Eye Scissor</td>
			<td>Fine Science Tools</td>
			<td>15010-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Leica Microsystems&#160;</td>
			<td>MC170 HD</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscope</td>
			<td>Leica Microsystems&#160;</td>
			<td>Leica M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Leica Microsystems&#160;</td>
			<td>DM2500</td>
			<td></td>
		</tr>
	</tbody>
</table>

two-step approach to explore early- and late-stages of organ formation in the avian model: the thymus and parathyroid glands organogenesis paradigm

鳥類胚実験モデルとしてされている精液の発見の最も重要な発生生物学。いくつかのアプローチ、ウズラ鶏キメラ形成および漿膜 (CAM) の最後の世紀に戻る日を異所性組織の開発を維持するために使用です。今日では、最近の in vitro方法論とこれらの古典的な手法の組み合わせはさらに器官形成を探索する新しい見通しを提供しています。
ここ初期- と - の後期段階の器官形成を研究する 2 段階のアプローチを記述します。簡単に言えば、臓器の推定領域を含む萌芽期の地域は (48 h) 最大の器官システムでウズラ胚および成長の in vitroから分離されます。培養組織にその後鶏胚のカムに接木されます。Ovo の開発の 10 日後、完全に形成された臓器は移植組織から取得されます。このメソッドでは、薬理学的エージェントの通常の管理と生体外でそしてovo発達ステップ全体にわたって組織遺伝子操作によるシグナル伝達経路の調節もできます。さらに、組織の開発は遺伝子発現プロファイル (量的な PCR (qPCR)、マイクロ アレイ等を用いた)、形態 (従来の組織像と免疫評価) を分析する任意の期間で収集できます。
外に完全に形成された器官形成の初期段階と機能の器官からの鳥の胎児の器官形成に従うツールとして記述されていた実験プロシージャを使用できます。

上記のプロトコルの詳細調査の両方 - と後半-初期頻繁複雑な細胞および分子相互作用によって限られる器官形成を可能にするメソッド。
以前フィゲイレードらの手法は5ノッチと Hh 鳥副甲状腺/胸腺共通原基開発におけるシグナル伝達の役割を解明します。
<p class="jove_content" fo:keep-together.within-pa...

Watch this Scientific Journal Video about Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm at JoVE.co

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

初期と後期-段鳥モデルにおける器官形成を探索する 2 段階のアプローチ: 胸腺、副甲状腺腺器官パラダイム

この資料では、新規生体外でそしてovo実験手順を組み合わせることによって器官の形成を研究する古典的なウズラ鶏キメラ システムへの更新されたアプローチを提供します。

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

two-step-approach-to-explore-early-late-stages-organ-formation-avian

Research

JoVE Journal

Developmental Biology

7.4K ビュー.  Universidade de Lisboa. この資料では、新規生体外でそしてovo実験手順を組み合わせることによって器官の形成を研究する古典的なウズラ鶏キメラ システムへの更新されたアプローチを提供します。

ビデオ: Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

簡体字を使用した初期の器官形成の理解<em&gt;その場で</em&gt;ハイブリダイゼーションプロトコルで<em&gt;アフリカツメガエル</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

発生生物学

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

loss-が及び着床前マウス胚の初期の細胞運命決定要因を調査するための機能獲得型のアプローチ

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. The inner ear auditory sensory organ is critical for our ability to hear. It houses a highly specialized sensory epithelium that consists of mechano-transducing hair cells (HCs) and surrounding glial-like supporting cells (SCs). Despite structural differences in the auditory organs, molecular mechanisms regulating the development of the auditory organ are evolutionarily conserved between mammals and aves. In ovo electroporation is largely limited to early stages at E1 - E3. Due to the relative late development of the auditory organ at E5, manipulations of the auditory organ by in ovo electroporation past E3 are difficult due to the advanced development of the chicken embryo at later stages. The method presented here is a transient gene transfer method for targeting genes of interest at stage E4 - E4.5 in the developing chicken auditory sensory organ via in ovo micro-electroporation. This method is applicable for gain- and loss-of-functions with conventional plasmid DNA-based expression vectors and can be combined with in ovo cell proliferation assay by adding EdU (5-ethynyl-2&#180;-deoxyuridine) to the whole embryo at the time of electroporation. The use of green or red fluorescent protein (GFP or RFP) expression plasmids allows the experimenter to quickly determine whether the electroporation successfully targeted the auditory portion of the developing inner ear. In this method paper, representative examples of GFP electroporated specimens are illustrated; embryos were harvested 18 - 96 hr&#160;after electroporation and targeting of GFP to the pro-sensory area of the auditory organ was confirmed by RNA in situ hybridization. The method paper also provides an optimized protocol for the use of the thymidine analog EdU to analyze cell proliferation; an example of an EdU based cell proliferation assay that combines immuno-labeling and click EdU chemistry is provided.

Gene Transfer into the Chicken Auditory Organ by In Ovo Micro-electroporation

The auditory organ mediates hearing. Here we present a modified in ovo micro-electroporation method optimized for studying auditory progenitor cell proliferation and differentiation in the developing chicken auditory organ.

チキン聴覚器官への遺伝子導入によって、<em&gt;卵内</em&gt;マイクロエレクト​​ロポレーション

Chicken embryos are ideal model systems for studying embryonic development as manipulations of gene function can be conducted with relative ease in ovo. ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

郭清ときのこ体の免疫蛍光染色と大人のショウジョウバエ脳光受容器ニューロン

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain and protect cellular identities. The conversion of germ cells into specific neurons in the nematode Caenorhabditis elegans (C. elegans) is a powerful tool for performing genetic screens in order to dissect regulatory pathways that safeguard established cell identities. Reprogramming of germ cells to a specific type of neurons termed ASE requires transgenic animals that allow broad over-expression of the Zn-finger transcription factor (TF) CHE-1. Endogenous CHE-1 is expressed exclusively in two head neurons and is required to specify the glutamatergic ASE neurons fate, which can easily be visualized by the gcy-5prom::gfp reporter. A trans gene containing the heat-shock promoter-driven che-1 gene expression construct allows broad mis-expression of CHE-1 in the entire animal upon heat-shock treatment. The combination of RNAi against the chromatin-regulating factor LIN-53 and heat-shock-induced che-1 over-expression leads to reprogramming of germ cell into ASE neuron-like cells. We describe here the specific RNAi procedure and appropriate conditions for heat-shock treatment of transgenic animals in order to successfully induce germ cell to neuron conversion.

Application of RNAi and Heat-shock-induced Transcription Factor Expression to Reprogram Germ Cells to Neurons in C. elegans

This protocol describes how to study cellular processes during cell fate conversion in Caenorhabditis elegans in vivo. Using transgenic animals, allowing heat-shock promoter-driven overexpression of the neuron fate-inducing transcription factor CHE-1 and RNAi-mediated depletion of the chromatin-regulating factor LIN-53 germ cell to neuron reprogramming can be observed in vivo.

線虫のニューロンに生殖細胞を再プログラムする RNAi と熱衝撃による転写因子の発現のアプリケーション

Studying the cell biological processes during converting the identities of specific cell types provides important insights into mechanism that maintain ...

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

萌芽期ティッシュと胸腺の例を使用してウズラ鶏キメラ器官の形成の分離

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

鳥胚胚心筋細胞の生体内注入にマイクロインジェクション ベース システム

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

iPSC-ICにおけるゼブラフィッシュ胚の全山ハイブリダイゼーションとチューブ形成アッセイは血管開発におけるエンドリンの役割を研究する

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

歯科幹細胞の制御を研究するためのトロウェル型臓器培養の使用

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, and stress response. More than a decade ago, stem cells were identified in the pituitary gland. However, despite the application of transgenic in vivo approaches, their phenotype, biology, and role remain unclear. To tackle this enigma, a new and innovative organoid in vitro model is developed to deeply unravel pituitary stem cell biology. Organoids represent 3D cell structures that, under defined culture conditions, self-develop from a tissue's (epithelial) stem cells and recapitulate multiple hallmarks of those stem cells and their tissue. It is shown here that mouse pituitary-derived organoids develop from the gland's stem cells and faithfully recapitulate their in vivo phenotypic and functional characteristics. Among others, they reproduce the activation state of the stem cells as in vivo occurring in response to transgenically inflicted local damage. The organoids are long-term expandable while robustly retaining their stemness phenotype. The new research model is highly valuable to decipher the stem cells' phenotype and behavior during key conditions of pituitary remodeling, ranging from neonatal maturation to aging-associated fading, and from healthy to diseased glands. Here, a detailed protocol is presented to establish mouse pituitary-derived organoids, which provide a powerful tool to dive into the yet enigmatic world of pituitary stem cells.

Development of Organoids from Mouse Pituitary as In Vitro Model to Explore Pituitary Stem Cell Biology

The pituitary gland is the key regulator of the body's endocrine system. This article describes the development of organoids from the mouse pituitary as a novel 3D in vitro model to study the gland's stem cell population of which the biology and function remain poorly understood.

下垂体幹細胞生物学を探るための In Vitro モデルとしてのマウス下垂体からのオルガノイドの開発

The pituitary is the master endocrine gland regulating key physiological processes, including body growth, metabolism, sexual maturation, reproduction, ...