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><tbody><tr><td>Calcium Chloride Dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Fisher Chemical</td><td>C79-3</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Magnesium Sulfate Anhydrous (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Chemical</td><td>M65-3</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Magnesium Chloride Hexahydrate (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Acros/VWR</td><td>41341-5000</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Potassium Chloride (KCl)</td><td>Acros Organics/VWR</td><td>196770010</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Acros Organics/VWR</td><td>207790050</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Sodium Bicarbonate (NaHCO3)</td><td>Acros Organics/VWR</td><td>123360010</td><td>Montju&amp;iuml;c salt solution</td></tr><tr><td>Nalgene autoclavable polypropylene copolymer lowboy with spigot</td><td>ThermoFisher Scientific/VWR</td><td>2324-0015</td><td>Storing planaria water</td></tr><tr><td>Instant Ocean Sea Salt</td><td>Spectrum Brands</td><td>Amazon</td><td>Planaria water</td></tr><tr><td>Gentamicin Sulfate</td><td>Gemini Bio-products</td><td>400-100P</td><td>Planaria water</td></tr><tr><td>Razor blades</td><td>Electron Microscopy Sciences</td><td>71970</td><td>Mincing liver&amp;nbsp;</td></tr><tr><td>Disposable pastry bags-16&amp;rdquo;, 12 pack</td><td>Wilton</td><td>Amazon</td><td>Liver aliquots</td></tr><tr><td>5 mL syringes</td><td>BD/VWR</td><td>309647</td><td>Liver aliquots</td></tr><tr><td>Petri dishes-35mm/60mm</td><td>Greiner Bio-One/VWR</td><td>82050-536/82050-544</td><td></td></tr><tr><td>Plastic containers (various sizes)</td><td>Ziploc</td><td>Amazon</td><td>Housing planarians in static culture</td></tr><tr><td>Sodium Azide</td><td>Sigma</td><td>S2002</td><td></td></tr><tr><td>Transfer pipette</td><td>Globe Scientific</td><td>138030</td><td></td></tr><tr><td>Forceps - Dumont Tweezer, Style 5</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>72700-D (0203-5-PO)</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>DAPI&amp;nbsp;</td><td>ThermoFisher</td><td>62247</td><td></td></tr><tr><td>Streptavidin, Alexa Fluor 488 conjugate</td><td>ThermoFisher</td><td>S11223</td><td></td></tr><tr><td>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

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 ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

プラナリアで瞳の再生を研究するための外科切除アッセイ

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and withdrawal properties of natural products is described. Experimental assays benefitting from unique aspects of planarian physiology have been applied to studies on wound healing, regeneration, and tumorigenesis. In addition, because planarians exhibit sensitivity to a variety of environmental stimuli and are capable of learning and developing conditioned responses, they can be used in behavioral studies examining learning and memory. Planarians possess a basic bilateral symmetry and a central nervous system that uses neurotransmitter systems amenable to studies examining the effects of neuromuscular biomodulators. Consequently, experimental systems monitoring planarian movement and motility have been developed to examine substance addiction and withdrawal. Because planarian motility offers the potential for a sensitive, easily standardized motility assay system to monitor the effect of stimuli, the planarian locomotor velocity (pLmV) test was adapted to monitor both stimulation and withdrawal behaviors by planarians through the determination of the number of grid lines crossed by the animals with time. Here, the technique and its application are demonstrated and explained.

Planarian motility is used to gauge the stimulant and withdrawal properties of natural products when compared to the movement of the animals in spring water alone.

天然物の生物調節特性を測定するプラナリアン運動性アッセイ

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

生化学

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 ...

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a 'trunk' fragment is cut from an intact worm, a new 'head' will regenerate anteriorly and a 'tail' will regenerate posteriorly restoring the original 'head-to-tail' polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as 'neoblasts' which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14. 
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.

Pharmacological and Functional Genetic Assays to Manipulate Regeneration of the Planarian Dugesia japonica

An attractive model for studying stem cell differentiation within a live animal is the planarian flatworm. Regeneration is studied by simple amputation experiments that are easily performed in a basic laboratory and are amenable to pharmacological and genetic (in vivo RNAi) manipulation as detailed by protocols in this article.

プラナリアの再生を操作するために薬理学的および機能的遺伝学的アッセイ<emジャポニカ> Dugesia</em

Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from ...

生物学

Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a na&iuml;ve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of na&iuml;ve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

プラナリアの固定化、部分照射し、組織移植

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian ...

Planarian Scrunching as a Quantitative Behavioral Readout for Noxious Stimuli Sensing

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce musculature-driven forms of locomotion: peristalsis or scrunching. While peristalsis results from a ciliary defect, scrunching is independent of cilia function and is a specific response to certain stimuli, including amputation, noxious temperature, extreme pH, and ethanol. Thus, these two musculature-driven gaits are mechanistically distinct. However, they can be difficult to distinguish qualitatively. Here, we provide a protocol for inducing scrunching using various physical and chemical stimuli. We detail the quantitative characterization of scrunching, which can be used to distinguish it from peristalsis and gliding, using freely available software. Since scrunching is a universal planarian gait, albeit with characteristic species-specific differences, this protocol can be broadly applied to all species of planarians, when using appropriate considerations. To demonstrate this, we compare the response of the two most popular planarian species used in behavioral research, Dugesia japonica and Schmidtea mediterranea, to the same set of physical and chemical stimuli. Furthermore, the specificity of scrunching allows this protocol to be used in conjunction with RNA interference and/or pharmacological exposure to dissect the molecular targets and neuronal circuits involved, potentially providing mechanistic insight into important aspects of nociception and neuromuscular communication.

Freshwater planarians exhibit three gaits (gliding, peristalsis, and scrunching) that are distinguishable by quantitative behavioral analysis. We describe a method to induce scrunching using various noxious stimuli, quantification thereof, and distinction from peristalsis and gliding. Using gene knockdown, we demonstrate the specificity of scrunching as a quantitative phenotypic readout.

有害刺激センシングのための定量的行動読み出しとしてのプラナリアンスクランチング

Freshwater planarians normally glide smoothly through ciliary propulsion on their ventral side. Certain environmental conditions, however, can induce ...

Planarian Ovary Dissection for Ultrastructural Analysis and Antibody Staining

Accessibility to germ cells allows the study of germ cell development, meiosis, and recombination. The sexual biotype of the freshwater planarian, Schmidtea mediterranea, is a powerful invertebrate model to study the epigenetic specification of germ cells. Unlike the large number of testis and male germ cells, planarian oocytes are relatively difficult to locate and examine, as there are only two ovaries, each with 5-20 oocytes. Deeper localization within the planarian body and lack of protective epithelial tissues also make it challenging to dissect planarian ovaries directly. 
This protocol uses a brief fixation step to facilitate the localization and dissection of planarian ovaries for downstream analysis to overcome these difficulties. The dissected ovary is compatible for ultrastructural examination by transmission electron microscopy (TEM) and antibody immunostaining. The dissection technique outlined in this protocol also allows for gene perturbation experiments, in which the ovaries are examined under different RNA interference (RNAi) conditions. Direct access to the intact germ cells in the ovary achieved by this protocol will greatly improve the imaging depth and quality and allow cellular and subcellular interrogation of oocyte biology.

<meta charset="utf8"></head><body>超微細構造解析および抗体染色のためのプラナリア卵巣解剖

This protocol presents steps taken to dissect ovaries in the freshwater planarians, Schmidtea mediterranea. The dissected ovaries are compatible for antibody immunostaining and ultrastructural analysis with transmission electron microscopy to study the cell biology of the oocytes and somatic cells, providing an imaging depth and quality that were previously inaccessible.

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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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