初期の最適化と本手法の開発を支えたアレハンドロ ・ サンチェス アルバラドに感謝したいと思います。キャロリン ・ アドラー研究所での仕事は、コーネル大学スタートアップ資金によってサポートされます。

1. 準備
<ol><li>
プラナリア水の準備17<ol>
<li>1 X Montjuïc 塩溶液でプラナリアを維持します。水をプラナリアを準備するには、個々 の在庫ソリューション 1 M CaCl2, 1 M MgSO4, 1 M MgCl2の 1 M の KCl と純水で 5 M の NaCl を確認します。0.2 μ m ボトル上部フィルターの長期的なストレージを持つフィルター滅菌します。 注:モンジュイックに塩を準備する (18.2 MΩ 25 ° C での電気抵抗) と超高純度の脱イオン水だけを使用します。</li>
		<li>5 X 塩溶液の 1 L 在庫を準備、1 M CaCl2、1 M MgSO4MgCl2、1.6 mL 900 mL 純水に食塩を 5 M の KCl の 0.5 mL の 0.5 mL の 5 mL の 5 mL を組み合わせます。このソリューションに 0.504 NaHCO3 g を加えて混ぜ合わせます。塩酸と 7.0 に pH を調整します。</li>
		<li>希釈原液を大容量カーボイなど滅菌コンテナーにおける超純水の使用濃度 X 1 X この 5 (材料の表を参照してください)。無性のプラナリアを維持するには、この 1 の X ソリューションを使用します。 注:モンジュイック食塩の代わりに、ローカル購入のばね水または市販の水槽の塩を含む超純水を使用濃度 of0.5 g/L18(材料の表を参照) をミックスします。</li>
		<li>19静置培養で細菌の感染を防ぐためには、抗生物質を含む水でプラナリアを維持します。超純水中の硫酸ゲンタマイシンの原液 50 mg/mL を準備し、フィルター殺菌します。動物の保管場所のコンテナーにゲンタマイシン硫酸塩を最終濃度 50 μ g/mL (1: 1000 希釈) に追加します。</li>
	</ol>
</li>
	<li>
レバー ペーストの準備
	<ol>
<li>プラナリアは、オーガニックの食事で繁栄する、牧草飼育牛レバー、新鮮な肝臓を購入、24 時間内のプロセス削除そっと剥がして肝臓をカプセル化膜カプセル。〜 1 cm キューブに肝臓を切るし、刃を使用してこすり、すべての肝静脈と動脈を破棄します。</li>
		<li>食品工場やフード プロセッサを使用して肝臓の部分を漬け込むし、ワイヤー メッシュの食品ストレーナを通過します。ペストリー袋に結合し、注射器や 35 mm シャーレに分注します。授乳前に空気の泡を削除に軽く遠心します。</li>
		<li>年および使用する前に解凍まで-80 ° C で肝臓の因数を格納します。解凍後、再冷凍、残った肝臓は一度、または 24 h まで 4 ° C で保存します。</li>
	</ol>
</li>
	<li>
動物のメンテナンス
	<ol>
<li>プラナリアは成長し、給餌の頻度に応じて様々 なサイズに縮小します。1 週間毎 (14 日に一度) プラナリアをフィードします。長期の大量培養 (材料表参照) 様々 なサイズのプラスチック製の容器を使用します。</li>
		<li>動物の飼料、金属のヘラまたはプラスチック ピペットを使用してボックスに肝臓の豆粒大のドロップを配置します。ボックスをクリーニングする前に食べる 1-2 削除残りの食糧のために動物を許可します。</li>
		<li>供給できない場合 (直接授乳後一度と一度 2 日後) を供給する場合週 2 回または週に一度ワームをきれい。きれいに慎重にボックスでプラナリアを維持しながら水を注ぐことによってプラスチック ビーカーに水を排出します。ボックス紙タオルで表面を拭くし、箱がきれいになるまで、すべての側面に繰り返します。</li>
		<li>(ボックスのボリュームの約 3/4) に水を交換し、50 μ G/ml の最終的な集中にゲンタマイシンを追加します。暗いキャビネットまたは 20 の ° C の定温器で動物を格納します。</li>
	</ol>
</li>
	<li>
ワーム各種
	<ol>
<li>5-7 日前に供給されたワームを選択します。 注:最近 (切断の前に 1-2 日) を供給されているワームから pharynges を切断するため困難です。動物を供給、またアジ化ナトリウムに敏感。</li>
		<li>約 6 ミリメートルの長さは、ワームを選択します。ワームのサイズを見積もるには、プラスチックの転送ピペットを使用してシャーレにそれを転送します。フラット 6 の定規またはペトリ皿の下に 5 mm × 5 mm 方眼紙を置き、それをクロール中にワームの長さを測定します。 注:長さ 6 mm より小さいワームは、切断しにくい。</li>
	</ol>
</li>
	<li>
アジ化ナトリウムの準備
	<ol>
<li>プラナリア水でナトリウム アジ化物粉末を溶解することにより 100 ミリメートルのソリューションを準備します。 注意:アジ化ナトリウムは毒性があり慎重に処理する必要があります。排水溝にアジ化ナトリウムを廃棄しないでください。次を使用、有害廃棄物としての処分のために個別に収集します。</li>
	</ol>
</li>
</ol>2. 咽頭切断
<ol><li>場所は、35 mm 径シャーレのワームします。お皿からプラスチック製のピペットを使用してすべてのプラナリア水を慎重に取り外します。 注:最大 20 の 6 mm のワームはこのサイズのディッシュ快適収容できます。</li>
	<li>顕微鏡下で皿を置き、複数のワームを同時に見ることができるように倍率を調整 (例えば10 倍)。プラナリア水を 100 mM アジ化ナトリウム溶液に置き換えます。 注:35 mm のペトリ皿のアジ化ナトリウムの約 5 mL で十分です。ソリューションは、プラナリアを水没完全にする必要があります。大きい皿のための解決の容積を必要に応じて調整します。</li>
	<li>顕微鏡と最初の 3 〜 4 分の皿を移動するからリフレインの下で動物を監視します。</li>
	<li>顕微鏡; で咽頭の押出を観察します。咽頭は咽頭嚢から突出し、完全に拡張 (長さ約 1 mm)図 1 bに示すように。 注:咽頭はさらに進む前に完全に拡張を完了するまで待機することが重要です。咽頭は、動物から出て、一度完全な拡張は約 1 分かかります。</li>
	<li>プラスチック ピペットを使用して、皿の周りに動物を噴出します。ピペットに動物を吸うし、強制的にそれらを解放、皿に幾つかの時間。咽頭が完全に場合、この積極的なピペッティングを解除を引き起こすが。</li>
	<li>また、図 1に示すように、その長さに沿って垂直方向、または水平方向に微細鉗子 (材料の表を参照してください) のペアを使用して円周に沿って咽頭を把握します。鉗子で咽頭、メニスカスに向かって上向きに動物を持ち上げます。動物が上昇するにつれて、表面張力は動物からデタッチする咽頭になります。 注:このメソッドは、強制的な旋回が不十分 (長さ 1-3 mm) な小動物の pharynges を削除する使用できます。突きまたはそれ以外の場合、動物の体を負傷を避けます。</li>
	<li>ピペットを使用して、切断された動物を新鮮なプラナリア水を含んでいる新しい皿に移動します。</li>
	<li>一度転送、リンスは完全に水を 3 回交換、プラナリア水で徹底的に動物を切断しました。50µg/ml のゲンタマイシンのプラナリア水を含んでいる新しい皿に転送します。 注:これらの 3 つの洗浄の間に完全なバッファー交換は、アジ化ナトリウムの除去を確実に不可欠です。</li>
	<li>次の日、ゲンタマイシンの 50 μ g/mL を含む新鮮なプラナリア水皿の水に置き換えます。その後にすべての他の日を繰り返します。</li>
</ol>3. 切断後咽頭除去の評価
<ol><li>図 2 aに示すように、ダーク スポットが、咽頭の成功の除去後登場しているかどうかを判断する (10-20 倍の倍率) と顕微鏡下で動物を検討します。</li>
	<li>また、修正し、ピアソンらによって記述されたプロトコルに従って動物をブリーチ20。 ソーク動物 4', 6-diamidino-2-phenylindole (DAPI) または蛍光標識ストレプトアビジンで一晩 (1 X 0.3% を含有したリン酸バッファー生食 1 Mg/ml に希釈 1: 500 トリトン X)。80% glycerol/20% 1 X リン酸バッファー生理食塩水と蛍光顕微鏡 (図 2 b) 下の画像のスライドの動物をマウントします。 注:この原稿のすべての画像は、1.0 X 目的で蛍光顕微鏡で捕えられました。</li>
</ol>4. 摂食行動を測定することにより咽頭再生の評価
<ol><li>
肝臓の準備
	<ol>
<li>遠心または円錐管にレバー ペーストの必要量を転送します。空気の泡を削除する簡単にスピンします。肝臓の容積を推定し、その量の 1/5、赤い食品着色料の合計の容積の 2% にプラナリア水を追加します。たとえば、肝臓の 1000 μ L に貼り付け、プラナリア水の 200 μ L と 24 μ L の食品着色料を追加します。</li>
		<li>食品着色料が均一でレバー ペーストになるまで徹底的にミックス プラスチック杵、ピペット チップまたは金属のヘラを使用する。もう一度簡単にスピンします。レバー ペーストは、24 h の 4 ° C で保存または因数長期保存用-80 ° C で凍結できます。</li>
		<li>最大 25 の動物をテストする場合は、一皿 (約 1 μ L あたり動物のレバー ペーストの) レバー ペーストを 25 μ l 添加を準備します。</li>
	</ol>
</li>
	<li>
摂食動物
	<ol>
<li>化学切断後 7 日間は、新しいシャーレに動物を譲渡します。10-15 動物をテストする場合を使用して、35 mm ディッシュ;以上 15 の動物は、60 mm のペトリ皿にテスト必要があります。暗闇の中、邪魔されない、供給する前に約 1 時間で動物を飼います。</li>
		<li>はさみを使用して、トリミング大体狭い端の ½ の cm で P200 ピペット チップの幅を広げます。ピペット 25 μ L の皿に赤いレバー ペースト。 注:最後をトリミング ピペッティング粘性のレバー ペーストの方が簡単になります。肝臓が表面に浮かぶことを防ぐために調剤中皿の底に先端に触れます。</li>
		<li>30 分を供給する動物を許可します。</li>
		<li>白地に動物を配置するまたは 10-20 倍の倍率での顕微鏡の下でそれらを調べることによって食べている動物の数を獲得します。</li>
		<li>授乳後お皿から肝臓を削除し、きれい。</li>
	</ol>
</li>
</ol>

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M., Mackler, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Transport+Systems+of+Yeast:+III.+Preparation+and+properties+of+cytochrome+oxidase.">Electron Transport Systems of Yeast: III. Preparation and properties of cytochrome oxidase.</a> J. Biol. Chem. 241, 1694-1697 (1966).</li><li>Buchan, J. R., Yoon, J. H., Parker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress-specific+composition,+assembly+and+kinetics+of+stress+granules+in+Saccharomyces+cerevisiae.">Stress-specific composition, assembly and kinetics of stress granules in Saccharomyces cerevisiae.</a> J. Cell Sci. 124, 228-239 (2011).</li><li>Cebri&#224;, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+nervous+system+in+the+model+planarian+Schmidtea+mediterranea:+An+immunocytochemical+study.">Organization of the nervous system in the model planarian Schmidtea mediterranea: An immunocytochemical study.</a> Neurosci. Res. 61, 375-384 (2008).</li><li>Shimoyama, S., Inoue, T., Kashima, M., Agata, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Neuropeptide-Coding+Genes+Involved+in+Planarian+Pharynx+Extension.">Multiple Neuropeptide-Coding Genes Involved in Planarian Pharynx Extension.</a> Zoolog. Sci. 33, 311-319 (2016).</li><li>Ito, H., Saito, Y., Watanabe, K., Orii, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epimorphic+regeneration+of+the+distal+part+of+the+planarian+pharynx.">Epimorphic regeneration of the distal part of the planarian pharynx.</a> Dev. Genes Evol. 211, 2-9 (2001).</li><li>Bueno, D., Espinosa, L., Bagu&#241;&#224;, J., Romero, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planarian+pharynx+regeneration+in++tail+fragments+monitored+with+cell-specific+monoclonal+antibodies.">Planarian pharynx regeneration in tail fragments monitored with cell-specific monoclonal antibodies.</a> Dev. Genes Evol. 206, 425-434 (1997).</li><li>Hyman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The invertebrates: Platyhelminthes and Rhynchocoela, the acoelomate Bilateria. , (1951).</li></ol>

著者が明らかに何もありません。

このプロトコルでは、アジ化ナトリウムを使用して咽頭の選択的アブレーションの方法について説明します。プラナリアの他のアブレーションの対象となる研究は、視細胞21または22ドーパミン作動性ニューロンをアブレーションするのに薬物治療を削除するのに修正手術を使用しています。既存の方法と比べて化学切断の 1 つの重要な利点は、手術が必要...

再生は、再生能力ある無脊椎動物の完全なボディ再生から脊椎動物1のより制限された能力に至るまで動物界全体が発生する現象です。機能的な組織の交換は複雑なプロセスであり、種類の細胞の同時復元をしばしば伴います。たとえば、サンショウウオの四肢、骨芽細胞、軟骨細胞、神経細胞、筋肉とする上皮細胞に必要なを再生成するには、2を置き換えられます。これらの細胞が新たに生成された型はまた新しい下肢の機能を容易にするために適切に編成する必要があります。これらの複雑なプロセスを理解すると、特定の細胞のタイプおよび器官への統合の再生に焦点を当てる技術が必要です。
再生反応に関する研究を簡素化する採用戦略は、いずれかの特定の細胞型のターゲットの切除や組織の大規模な収集があります。たとえば、ゼブラフィッシュ、特定の細胞型でニトロの式はメトロニダゾールの3、4を適用した後彼らの破壊に します。ショウジョウバエ幼虫の組織特異的発現プロモーター下プロ アポトーシス遺伝子を表現するには成虫ディスク5,6の特定の領域を切除できることは選択的に。両方これらの戦略の急速な制御された損傷の原因し、再生の分子・細胞メカニズムを解剖に使用されています。
本稿では、我々 はSchmidtea メディテラネアプラナリアの咽頭と呼ばれる全体の器官を選択的に切除する方法をについて説明します。プラナリアが再生、その多作の再生能力で知られての古典的なモデルも分の断片が全体動物7,8を再生することができます。彼らは幹細胞の多能性細胞と前駆細胞の血統制限9,,1011から成る大規模な異種の人口があります。これらの細胞は、増殖し、咽頭、神経系、消化と排泄系と筋上皮細胞9,10,12など、すべての不足している組織を置換するを区別します。これらの幹細胞が再生を開始することを私たちは知っている、すべてのこれらの異なった細胞のタイプを交換するそれらを駆動する分子メカニズムは完全に納得しません。正確な幹細胞応答を引き出す定義の人が負傷した方法は、この複雑なプロセスを明確に役立ちます。
咽頭の授乳に必要な大規模な円筒形のチューブであり、神経細胞、筋肉、上皮細胞と分泌細胞13,29を含んでいます。通常動物の腹部の側面のポーチに隠された、それは食品の存在を感じる時に開く動物の単一ボディに引かれます。咽頭を選択的に切断、化学と呼ばれるナトリウムのアジ化物、線虫14,15,16の一般的に使用される麻酔薬でプラナリアを浸します。プラナリアのアドラーらによって報告された。、201412で。アジ化ナトリウムへの露出の数分以内プラナリア押し出し、pharynges と穏やかな攪拌と咽頭を動物から切り離します。私たちは「化学切断」として咽頭の完全かつ選択的損失額を参照してください。切断後 1 週間完全に機能的な咽頭は復元された12です。咽頭は、供給する必要があるので、機能再生は摂食行動を監視することによって測定できます。化学の切断および咽頭の再生と摂食行動の回復を評価するためのプロトコルをご紹介いたします。

<table border="0" cellpadding="0" cellspacing="0" width="1042">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:447px;">Calcium Chloride Dihydrate (CaCl2)</td>
			<td style="width:204px;">Fisher Chemical</td>
			<td style="width:155px;">C79-3</td>
			<td style="width:236px;">Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Sulfate Anhydrous (MgSO4)</td>
			<td>Fisher Chemical</td>
			<td>M65-3</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride Hexahydrate (MgCl2)</td>
			<td>Acros/VWR</td>
			<td>41341-5000</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride (KCl)</td>
			<td>Acros Organics/VWR</td>
			<td>196770010</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride (NaCl)</td>
			<td>Acros Organics/VWR</td>
			<td>207790050</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bicarbonate (NaHCO3)</td>
			<td>Acros Organics/VWR</td>
			<td>123360010</td>
			<td>Montju&#239;c salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalgene autoclavable polypropylene copolymer lowboy with spigot</td>
			<td>ThermoFisher Scientific/VWR</td>
			<td>2324-0015</td>
			<td>Storing planaria water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instant Ocean Sea Salt</td>
			<td>Spectrum Brands</td>
			<td>Amazon</td>
			<td>Planaria water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin Sulfate</td>
			<td>Gemini Bio-products</td>
			<td>400-100P</td>
			<td>Planaria water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>71970</td>
			<td>Mincing liver&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable pastry bags-16&#8221;, 12 pack</td>
			<td>Wilton</td>
			<td>Amazon</td>
			<td>Liver aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 mL syringes</td>
			<td>BD/VWR</td>
			<td>309647</td>
			<td>Liver aliquots</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes-35mm/60mm</td>
			<td>Greiner Bio-One/VWR</td>
			<td>82050-536/82050-544</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic containers (various sizes)</td>
			<td>Ziploc</td>
			<td>Amazon</td>
			<td>Housing planarians in static culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Azide</td>
			<td>Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipette</td>
			<td>Globe Scientific</td>
			<td>138030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps - Dumont Tweezer, Style 5</td>
			<td>Electron Microscopy Sciences&#160;</td>
			<td>72700-D (0203-5-PO)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI&#160;</td>
			<td>ThermoFisher</td>
			<td>62247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptavidin, Alexa Fluor 488 conjugate</td>
			<td>ThermoFisher</td>
			<td>S11223</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>BP229-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

chemical amputation and regeneration of the pharynx in the planarian schmidtea mediterranea

プラナリアは、扁形動物の非常に効率的な再生です。彼らは、任意のタイプの傷害に即応できる幹細胞の数が多いこの能力を借りています。これらの動物の共通の傷害モデルは、複数の臓器に損傷組織の大量を削除します。この広範な組織の損傷を克服するために我々 はSchmidtea メディテラネアプラナリアの咽頭、一つの臓器を選択的に削除する方法をここで説明します。我々 はチトクロム酸化酵素阻害剤アジ化ナトリウムを含む溶液に浸漬の動物によってこれを達成します。アジ化ナトリウムを簡単に暴露により咽頭の押出成形、「化学的切断」と呼ばれる動物から化学切断全体の咽頭を削除し、咽頭が腸へアタッチ小さな傷を生成します。広範な洗浄後は、すべての切断された動物は、約 1 週間で咽頭が完全に機能を再生成します。体の残りの部分の幹細胞は、新しい咽頭の再生を駆動します。ここで、詳細な提供化学切断のプロトコルおよび成功の切断と再生を評価する組織と行動の両方の方法について説明します。

アジ化ナトリウムへの暴露は、プラナリア、原因を伸ばし、身をよじる動物の通常の運動を混乱させます。これらの動きを強制的に咽頭、動物の腹部の側面から出てくるとアジ化ナトリウム溶液で約 6 分後、咽頭の白の先端を見ることができる(図 1 bの左側のパネル)。数分後、動物は積極的に契約し、強制的にボディからそれを押?...

Watch this Scientific Journal Video about Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea at JoVE.com

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

化学の切断とSchmidtea メディテラネアプラナリアの咽頭再生

プラナリアSchmidtea メディテラネアは、幹細胞と組織再生を研究するための優れたモデルです。この文書では、化学のアジに動物を公開することによって 1 つの器官、咽頭を選択的に削除する方法について説明します。このプロトコルはまた咽頭再生を監視する方法を説明します。

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to ...

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Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Unit 1

Regeneration and Repair

SCIENTISTS IN ACTION

10.7K ビュー.  Cornell University. プラナリアSchmidtea メディテラネアは、幹細胞と組織再生を研究するための優れたモデルです。この文書では、化学のアジに動物を公開することによって 1 つの器官、咽頭を選択的に削除する方法について説明します。このプロトコルはまた咽頭再生を監視する方法を説明します。

ビデオ: Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila melanogaster, inductive microenvironments known as larval Hematopoietic Pockets (HPs) have been identified as anatomical sites for the development and regulation of blood cells (hemocytes), in particular of the self-renewing macrophage lineage.&#160;HPs are segmentally repeated pockets between the epidermis and muscle layers of the larva, which also comprise sensory neurons of the peripheral nervous system. In the larva, resident (sessile) hemocytes are exposed to anti-apoptotic, adhesive and proliferative cues from these sensory neurons and potentially other components of the HPs, such as the lining muscle and epithelial layers. During normal development, gradual release of resident hemocytes from the HPs fuels the population of circulating hemocytes, which culminates in the release of most of the resident hemocytes at the beginning of metamorphosis. Immune assaults, physical injury or mechanical disturbance trigger the premature release of resident hemocytes into circulation. The switch of larval hemocytes between resident locations and circulation raises the need for a common standard/procedure to selectively isolate and quantify these two populations of blood cells from single Drosophila larvae. Accordingly, this protocol describes an automated method to release and quantify the resident and circulating hemocytes from single larvae. The method facilitates ex vivo approaches, and may be adapted to serve a variety of developmental stages of Drosophila and other invertebrate organisms.

Assaying Blood Cell Populations of the Drosophila melanogaster Larva

Drosophila blood cells, or hemocytes, cycle between resident sites and circulation. In the larva, resident (sessile) hemocytes localize to inductive microenvironments, the Hematopoietic Pockets, while circulating hemocytes move freely in the hemolymph. The goal of this protocol is the standardized isolation and quantification of these two, behaviorally distinct but interchanging, hemocyte populations.

の血液細胞集団のアッセイ<em&gt;キイロショウジョウバエ</em&gt;幼虫

In vertebrates, hematopoiesis is regulated by inductive microenvironments (niches). Likewise, in the invertebrate model organism&#160;Drosophila ...

発生生物学

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

ゼブラフィッシュ再生の分子機構を研究するためにテトンシステムの利用

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

エバンスブルー色素でゼブラフィッシュ幼生骨格筋の整合性の分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

スターレットイソギンチャクの尾方Physaから完全ポリープ再生を誘導<em&gt; Nematostella vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. This morphological alteration is also recognized as epithelial to mesenchymal transition (EMT). Dysregulation of EMT is associated with fibrosis and cancer metastasis. There is emerging evidence that EMT is controlled by a number of molecular mechanisms. As such, many key genes that control apical constriction are also known to be important factors in the EMT observed in cancer metastasis. Like EMT during Drosophila gastrulation, epithelial cells can be induced to change their shape and be reprogrammed to redirect cell fate towards various other cell types. Here we provide a robust imaging method of Drosophila gastrulation to assay the initiation of morphogenetic cellular movements and cell fate identification during this stage of embryonic development. Using this method, we identify cell rearrangement at the time of gastrulation and demonstrate the importance of apical constriction during gastrulation using GFP labeled DE-cadherin.

Imaging of Cell Shape Alteration and Cell Movement in Drosophila Gastrulation Using DE-cadherin Reporter Transgenic Flies

Herein we describe a procedure to capture live images of Drosophila gastrulation. This has enabled us to better understand the apical constriction involved in early development and further analyze mechanisms governing cellular movements during tissue structure modification.

セル形状変化のイメージングと細胞運動で<em&gt;ショウジョウバエ</emDEカドヘリンレポータートランスジェニックハエを使用&gt;原腸陥入

Gastrulation is the first set of morphologically dynamic events that occur during the embryonic development of multicellular animals such as Drosophila. ...

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 ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

ラッパムシの再生の研究のための方法

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (&#956;CT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo &#956;CT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

成体マウスの解離と再生:哺乳類のブラステマ形成と腸内形成を調べるための簡単なモデル

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

Isolation of Rat Adipose Tissue Mesenchymal Stem Cells for Differentiation into Insulin-producing Cells

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of stem cells that does not pose any ethical concerns. However, current methods to isolate Ad-MSCs are not standardized and employ complicated protocols that require special equipment. We isolated Ad-MSCs from the epididymal fat of Sprague-Dawley rats using a simple, reproducible method. The isolated Ad-MSCs usually appear within 3 days post isolation, as adherent cells display fibroblastic morphology. Those cells reach 80% confluency within 1 week of isolation. Afterward, at passage 3-5 (P3-5), a full characterization was carried out for the isolated Ad-MSCs by immunophenotyping for characteristic MSC cluster of differentiation (CD) surface markers such as CD90, CD73, and CD105, as well as inducing differentiation of these cells down the osteogenic, adipogenic, and chondrogenic lineages. This, in turn, implies the multipotency of the isolated cells. Furthermore, we induced the differentiation of the isolated Ad-MSCs toward the insulin-producing cells (IPCs) lineage via a simple, relatively short protocol by incorporating high glucose Dulbecco&#39;s modified Eagle medium (HG-DMEM), &#946;-mercaptoethanol, nicotinamide, and exendin-4. IPCs differentiation was genetically assessed, firstly, via measuring the expression levels of specific &#946;-cell markers such as MafA, NKX6.1, Pdx-1, and Ins1, as well as dithizone staining for the generated IPCs. Secondly, the assessment was also carried out functionally by a glucose-stimulated insulin secretion (GSIS) assay. In conclusion, Ad-MSCs can be easily isolated, exhibiting all MSC characterization criteria, and they can indeed provide an abundant, renewable source of IPCs in the lab for diabetes research.

Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) can be a potential source of MSCs that differentiate into insulin-producing cells (IPCs). In this protocol, we provide detailed steps for the isolation and characterization of rat epididymal Ad-MSCs, followed by a simple, short protocol for the generation of IPCs from the same rat Ad-MSCs.

インスリン産生細胞への分化のためのラット脂肪組織間葉系幹細胞の単離

Mesenchymal stem cells (MSCs)-especially those isolated from adipose tissue (Ad-MSCs)-have gained special attention as a renewable, abundant source of ...

化学の切断とSchmidtea メディテラネアプラナリアの咽頭再生

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