この作品は、GHTにニューヨーク幹細胞科学（NYSTEM C028107）グラントによってサポートされていました。

温度、栄養および明/暗サイクルのための動物の1コンディショニング<ol><li>世界中の数多くのNematostellaラボの1、または非営利業者からNematostella vectensis成人を得る（ 表1） </li><li>千あたり12部（PPT）の塩分で「1/3× &quot;人工海水（ASW）で、暗闇の中で（典型的には18と21°C）一定温度でNematostellaを維持します 。一般的に250 mlまたは1.5 L容量11、シンプルなソーダライムガラス培養皿で培養を維持します。 注：これらの単純な培養条件は、一般的にNematostellaを研究ラボの中で使用されているが、文化のケアも19を自動化することができます。 </li><li>週4倍- 2ノープリウスNematostella新しく孵化したアルテミアを養います。フル強度（36 PPT）または1/3倍の浅い長方形のガラス皿20内またはで30℃、でASWでハッチアルテミアシスト小規模、商業や自家製ブラインシュリンプ孵化場の数のいずれか。インキュベーターが使用できない場合は、エビはRTで孵化が、よりゆっくりとそうなります。 注：これは、多くの場合、完了のために24時間以上を必要とします。 </li><li>少なくとも週に一度アネモネ培養水を交換してください。最高の大人の健康のために、徹底的にクリーン（石鹸なし）文化が一度コートボウルと缶トラップ食べ残しや廃棄物の蓄積粘液分泌、の週をボウル、動物を絡ませます。 </li></ol>栄養的に馴化動物の2.選択とリラクゼーション<ol><li>ほぼ同じ長さの大きさがマッチしたポリープを選択（3〜5センチメートルを、ときに自然にリラックスした）前切断に3日間コロニーから分離ボウルに配置します。 注：切断のために選択された動物の数は、実験によって決定されますもちろん、行われているが、一般的に、我々は6反復でサンプル点当たり少なくとも5匹の動物をお勧めします。このように、典型的な実験では30匹の動物の最小値は、事前に選択されたことになります。 （下記参照）不規則な切断術の後スコアに影響を与えることができるので、一般には、より多くの最小数（30）より選択することが賢明です。 </li><li>切断する前に少なくとも1時間室内光にインキュベーターから選択した動物の食器を削除します。 注：室内光とハンドリングの振動への曝露は、おそらく動物が収縮する原因となりますので、実験台上でのインキュベーションによって適合または「リラックス」する必要があります。動物は触れるために、耐火及び露光となり、その時点での穏やかなピペッティングして移動することができます。 </li><li>オプション：（1/3×ASWに）7.5％のMgCl 2を追加することにより、動物を麻酔。静かに標準的なプラスチック5 mLのピペットでボウルへのMgCl 2溶液を追加します。 注：動物は、最終的に光へと物理的な操作に慣れになるだろうが、維持またはトンを「修正」するために動物を麻酔することが有利な場合があります彼らは細長い16,21,22となった後、彼は状態を緩和しました。 </li><li> 12 PPTのASWを含む直径100mmの滅菌ガラス切断皿の底に、切断されるように、プールから5匹の動物（1/3×ASWに）転送するために広い口径（&gt; 0.5センチメートル）プラスチックピペットを使用してください。 40X - 10の間に可変倍率の実体顕微鏡のステージに上の皿を置きます。 注：動物は、切断のために麻酔をかけ、リラックスされていない場合、彼らはまだ触れないように対応し、ステレオスコープの照明ので、再びリラックスになるために数分を必要とする場合があります。 </li></ol> 3.切断術<ol><li>滅菌メスを使用して、約限り、それは広いと全く腸間膜を含まないあるようですphysaのセクションを得ることを目標に、各ポリープから尾方physaを切断します。 注：理想的な切断部位は、腸間膜の終了にちょうど尾方です。切断面に薄いリットルに対して適切腸間膜からの移行がありますINESは、各腸間膜挿入（ 図1参照、矢印）に対応します。それは穴17,30 &#39;を差し込む」容易にすることができる粘液を生成するので、腸間膜の不在は重大です。 <ol><li>切断の所望の部位での動物と接触したメスの刃を配置します。 （フリーハンド）補助なし、または静かに＃5鉗子（デュモンスタイルまたは類似）で動物の体を把握することでこのいずれかの操作を行います。 </li><li>体全体の「ロッキング」運動にメスの湾曲した刃を活用することで、組織を通ってカット。 注：組織は、メスが揺動されるようにきれいに切断し、ドナーからphysaの所望の部分を解放する必要があります。組織の小片がまだ体とphysaを接続する場合は、メスでそれをカット。これはphysaに損傷を与える可能性として、引っ張ることにより、接続された作品を分離しようとしないでください。 </li></ol></li><li>皿から各切断「ドナー」ポリープを削除し、SEPAに戻します「プールされた切断者」の標識率ボウル。日付ボウルとストック培養物に戻します。 注：切断ポリープ日以内に尾方の傷を癒してくれるし、その後正常に供給することができます。彼らは、必要に応じてphysaが再び切断することができ、その時点で2週間以内に通常見physaを再生成します。 </li><li> 12 PPT ASWで切断皿に残って切除したphysaをすすぎ、その後、すでに各ウェルに12 PPT ASWの10ミリリットルを持つマルチウェル細胞培養プレートに別々の無菌ウェルに各physaを転送します。 注：この例では、各ウェル海水の10 mL及び5切除したphysaを保持して、6ウェルプレートを使用しています。一般海水でハンドリングし、潜在的な蒸発に起因する動きに空気への暴露を避けるために十分physaをカバーする必要があります。プレートまたはウェルは、蓋を持っている必要があります。 </li><li>各実験治療のために、各ウェルの予約で少なくとも5 physaを収集するために、3.3 - 繰り返して、3.1を繰り返します。 </li><li>箴ます温度でphysaをインキュベート計画された実験的な取調べのための再生の最高の速度をIDE。再生の所望の速度によって決定される一定の温度で、温度調節インキュベーターにphysaを含むプレートを置きます。 注：physaが欠落した組織を再生し、15と27℃の間の温度でインキュベートした場合、完全なポリープを形成することになります。再生の速度は、最初の2つのステージを除いて、温度依存性です。全ての温度のための段階4に到達するための平均日は、切断後7日であり、これはまた、21℃における再生と一致します。 27℃では、ステージ4は、約3日早く到達し、15℃で、ステージ4は、21°C（参照文献10）で再生と比較して約3 dだけ遅延されます。 </li></ol> 4. Nematostella再生ステージングシステム（NRSS）で再生を評価します<ol><li>変数壮麗なステレオ-化合物顕微鏡を用いてphysaスコアエーション（10 - 80X）。ステージ0のように新鮮にカットNematostellaの physaスコアと同時に、NRSS 10を使用して、毎日ポスト切断術（DPA）をスコアリングを続けます。 注：キーのステージング基準や詳細についてはリファレンス10を参照してください。 <ol><li>オープン創傷部位で、弛緩性バルーンに似ているcup-形の質量はそう見えるようたてカットphysaが表示された場合、ステージ0（開いた傷口）としてphysaスコア。 注：傷のエッジがまた最初から一緒に固執する可能性がありますが、組織はまだ崩壊され、剛性が不足しています。開放創のエッジは、創傷治癒のような半径方向の収縮を受けて観察することができます。 </li><li>切断術創が閉じて表示された場合（閉鎖した創傷）第1段階としてphysaスコア。 注：創傷場所は、将来の経口極に対応させていただきます。将来の経口ポールの周りの外表面は、基礎となる放射対称内胚葉腸間膜の挿入に対応する明確なアーチを表示し始める可能性があります。 </li><li>スコアのpHYSAステージ2（ラジアルアーチ）などの経口極の表面が放射状に対称なパターンに配置された8つの隆起したアーチを明らかにし、膨張した溝によって分離された表示されます。アーチの頂点に小さな半球状のブレブを守ってください。彼らは広い、おそらく一過性のように背の高い、と最初は単一の外胚葉細胞層によって構成についてになります。 注：いくつかのケースでは二重層ブレブを安定化させることができます。注：この以降の段階で粘液層がphysa膜状の「鞘」で（ 図2）をカプセル化するために表示されることがあります。このカプセル化材料は、スコアリングを容易にするために削除する必要があります。 </li><li>内胚葉と外胚葉組織層を含む触手の芽を安定的に、少なくともいくつかの半径方向のアーチの経口端部に形成されている場合には、ステージ3（触手）としてphysaスコア。 注：触手は、彼らが広く、最小限の運動性よりも長くなっています。腸間膜の基礎が可視になるようにphysaは増加したが、変数インフレ率が表示されます体腔（腔腸）に腸間膜の挿入から延びるible。 </li><li> physaは、彼らが接続されているように見えたところから測定その2倍の半径方向の幅よりもある口腔尾方の長さで、体壁に挿入から腔腸中に延びる8明確な、目に見える腸間膜が含まれている場合は、ステージ4（リニア腸間膜）としてphysaスコアその尾方端（enterostome）で咽頭へ。 注：4つ以下の腸間膜には、「プリーツ」内部の自由縁を持っています。咽頭が表示されます。以上8触手は、運動性表示され、時には彼らは体内に収縮します。 </li><li> physaはプリーツとつ以上の腸間膜を有しており、プリーツは、動物はほとんど &quot;通常の&quot;大人の外観を持っていますが、目に見えるはありませんステージ4でより完全かつしなやかである場合、ステージ5（主にプリーツ腸間膜）としてphysaスコア生殖細胞。 </li></ol></li></ol>

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	<li>Lenhoff, S. G., Lenhoff, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Hydra and the Birth of Experimental Biology: Abraham Trembley's Memoirs Concerning the Natural History of a Type of Freshwater Polyp with Arms Shaped like Horns. , (1986).</li><li>Trembley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> M&#233;moires pour servir &#224; l'histoire d'un genre de polypes d'eau douce, &#224; bras en forme de cornes. , (1744).</li><li>Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+understanding+tissue+regenerative+capacity+and+mechanisms+in+animals.">Advances in understanding tissue regenerative capacity and mechanisms in animals.</a> Nat Rev Genet. 11 (10), 710-722 (2010).</li><li>Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydra,+a+fruitful+model+system+for+270+years.">Hydra, a fruitful model system for 270 years.</a> Int J Dev Biol. 56 (6-8), 411-423 (2012).</li><li>Gold, D. A., Jacobs, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+dynamics+in+Cnidaria:+are+there+unifying+principles?.">Stem cell dynamics in Cnidaria: are there unifying principles?.</a> Dev Genes Evol. 233 (1-2), 53-66 (2013).</li><li>Holstein, T. W., Hobmayer, E., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cnidarians:+an+evolutionarily+conserved+model+system+for+regeneration?.">Cnidarians: an evolutionarily conserved model system for regeneration?.</a> Dev Dyn. 226 (2), 257-267 (2003).</li><li>Amiel, A. R., 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=Characterization+of+Morphological+and+Cellular+Events+Underlying+Oral+Regeneration+in+the+Sea+Anemone,+Nematostella+vectensis.">Characterization of Morphological and Cellular Events Underlying Oral Regeneration in the Sea Anemone, Nematostella vectensis.</a> Int J Mol Sci. 16 (12), 28449-28471 (2015).</li><li>Warren, C. R., 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=Evolution+of+the+perlecan/HSPG2+gene+and+its+activation+in+regenerating+Nematostella+vectensis.">Evolution of the perlecan/HSPG2 gene and its activation in regenerating Nematostella vectensis.</a> PLoS One. 10 (4), e0124578 (2015).</li><li>Gong, Q., 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=Integrins+of+the+starlet+sea+anemone+Nematostella+vectensis.">Integrins of the starlet sea anemone Nematostella vectensis.</a> Biol Bull. 227 (3), 211-220 (2014).</li><li>Bossert, P. E., Dunn, M. P., Thomsen, G. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+staging+system+for+the+regeneration+of+a+polyp+from+the+aboral+physa+of+the+anthozoan+Cnidarian+Nematostella+vectensis.">A staging system for the regeneration of a polyp from the aboral physa of the anthozoan Cnidarian Nematostella vectensis.</a> Dev Dyn. 242 (11), 1320-1331 (2013).</li><li>Stefanik, D. J., Friedman, L. E., Finnerty, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting,+rearing,+spawning+and+inducing+regeneration+of+the+starlet+sea+anemone,+Nematostella+vectensis.">Collecting, rearing, spawning and inducing regeneration of the starlet sea anemone, Nematostella vectensis.</a> Nat Protoc. 8 (5), 916-923 (2013).</li><li>Tucker, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thrombospondin+in+the+anthozoan+Nematostella+vectensis+is+associated+with+the+nervous+system+and+upregulated+during+regeneration.">A thrombospondin in the anthozoan Nematostella vectensis is associated with the nervous system and upregulated during regeneration.</a> Biol Open. 2 (2), 217-226 (2013).</li><li>Passamaneck, Y. J., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+proliferation+is+necessary+for+the+regeneration+of+oral+structures+in+the+anthozoan+cnidarian+Nematostella+vectensis.">Cell proliferation is necessary for the regeneration of oral structures in the anthozoan cnidarian Nematostella vectensis.</a> BMC Dev Biol. 12, (2012).</li><li>Trevino, M., Stefanik, D. J., Rodriguez, R., Harmon, S., Burton, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+canonical+Wnt+signaling+by+alsterpaullone+is+sufficient+for+oral+tissue+fate+during+regeneration+and+embryogenesis+in+Nematostella+vectensis.">Induction of canonical Wnt signaling by alsterpaullone is sufficient for oral tissue fate during regeneration and embryogenesis in Nematostella vectensis.</a> Dev Dyn. 240 (12), 2673-2679 (2011).</li><li>Renfer, E., Amon-Hassenzahl, A., Steinmetz, P. R., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+muscle-specific+transgenic+reporter+line+of+the+sea+anemone,+Nematostella+vectensis.">A muscle-specific transgenic reporter line of the sea anemone, Nematostella vectensis.</a> Proc Natl Acad Sci U S A. 107 (1), 104-108 (2010).</li><li>Burton, P. M., Finnerty, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+and+novel+gene+expression+between+regeneration+and+asexual+fission+in+Nematostella+vectensis.">Conserved and novel gene expression between regeneration and asexual fission in Nematostella vectensis.</a> Dev Genes Evol. 219 (2), 79-87 (2009).</li><li>DuBuc, T. Q., Traylor-Knowles, N., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiating+a+regenerative+response;+cellular+and+molecular+features+of+wound+healing+in+the+cnidarian+Nematostella+vectensis.">Initiating a regenerative response; cellular and molecular features of wound healing in the cnidarian Nematostella vectensis.</a> BMC Biol. 12, (2014).</li><li>Hand, C., Uhlinger, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asexual+reproduction+by+transverse+fission+and+some+anomalies+in+the+sea+anemone+Nematostella+vectensis.">Asexual reproduction by transverse fission and some anomalies in the sea anemone Nematostella vectensis.</a> Invert Biol. 114, 9-18 (1995).</li><li>Fritzenwanker, J. H., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+gametogenesis+in+the+basal+cnidarian+Nematostella+vectensis(Anthozoa).">Induction of gametogenesis in the basal cnidarian Nematostella vectensis(Anthozoa).</a> Dev Genes Evol. 212 (2), 99-103 (2002).</li><li>Magie, C., Bossert, P., Aramli, L., Thomsen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Science's+super+star:+The+starlet+sea+anemone+is+an+ideal+tool+for+student+inquiry.">Science's super star: The starlet sea anemone is an ideal tool for student inquiry.</a> The Science Teacher. 83 (3), 33-40 (2016).</li><li>Genikhovich, G., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+of+starlet+sea+anemone+(Nematostella+vectensis)+embryos,+larvae,+and+polyps.">In situ hybridization of starlet sea anemone (Nematostella vectensis) embryos, larvae, and polyps.</a> Cold Spring Harb Protoc. (9), (2009).</li><li>Magie, C. R., Pang, K., Martindale, M. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+inventory+and+expression+of+Sox+and+Fox+genes+in+the+cnidarian+Nematostella+vectensis.">Genomic inventory and expression of Sox and Fox genes in the cnidarian Nematostella vectensis.</a> Dev Genes Evol. 215 (12), 618-630 (2005).</li><li>Chera, S., Kaloulis, K., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cAMP+response+element+binding+protein+(CREB)+as+an+integrative+HUB+selector+in+metazoans:+clues+from+the+hydra+model+system.">The cAMP response element binding protein (CREB) as an integrative HUB selector in metazoans: clues from the hydra model system.</a> Biosystems. 87 (2-3), 191-203 (2007).</li><li>Reitzel, A. M., Burton, P. M., Krone, C., Finnerty, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+developmental+trajectories+in+the+starlet+sea+anemone+Nematostella+vectensis:+embryogenesis,+regeneration,+and+two+forms+of+asexual+fission.">Comparison of developmental trajectories in the starlet sea anemone Nematostella vectensis: embryogenesis, regeneration, and two forms of asexual fission.</a> Invertebr Biol. 126, 99-112 (2007).</li><li>Ikmi, A., McKinney, S. A., Delventhal, K. M., Gibson, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TALEN+and+CRISPR/Cas9-mediated+genome+editing+in+the+early-branching+metazoan+Nematostella+vectensis.">TALEN and CRISPR/Cas9-mediated genome editing in the early-branching metazoan Nematostella vectensis.</a> Nat Commun. 5, 5486 (2014).</li><li>Jahnel, S. M., Walzl, M., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+epithelial+organisation+of+muscle+cells+in+the+sea+anemone+Nematostella+vectensis.">Development and epithelial organisation of muscle cells in the sea anemone Nematostella vectensis.</a> Front Zool. 11, 44 (2014).</li><li>Kelava, I., Rentzsch, F., Technau, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+eumetazoan+nervous+systems:+insights+from+cnidarians.">Evolution of eumetazoan nervous systems: insights from cnidarians.</a> Philos Trans R Soc Lond B Biol Sci. 370 (1684), (2015).</li><li>Nakanishi, N., Renfer, E., Technau, U., Rentzsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nervous+systems+of+the+sea+anemone+Nematostella+vectensis+are+generated+by+ectoderm+and+endoderm+and+shaped+by+distinct+mechanisms.">Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms.</a> Development. 139 (2), 347-357 (2012).</li><li>Richards, G. S., Rentzsch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+analysis+of+a+SoxB+gene+reveals+neural+progenitor+cells+in+the+cnidarian+Nematostella+vectensis.">Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis.</a> Development. 141 (24), 4681-4689 (2014).</li><li>DuBuc, T. Q., 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=In+vivo+imaging+of+Nematostella+vectensis+embryogenesis+and+late+development+using+fluorescent+probes.">In vivo imaging of Nematostella vectensis embryogenesis and late development using fluorescent probes.</a> BMC Cell Biol. 15, (2014).</li><li>Kaur, J., Debnath, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+at+the+crossroads+of+catabolism+and+anabolism.">Autophagy at the crossroads of catabolism and anabolism.</a> Nat Rev Mol Cell Biol. 16 (8), 461-472 (2015).</li><li>Carroll, B., Korolchuk, V. I., Sarkar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino+acids+and+autophagy:+cross-talk+and+co-operation+to+control+cellular+homeostasis.">Amino acids and autophagy: cross-talk and co-operation to control cellular homeostasis.</a> Amino Acids. 47 (10), 2065-2088 (2015).</li><li>Glick, D., Barth, S., Macleod, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy:+cellular+and+molecular+mechanisms.">Autophagy: cellular and molecular mechanisms.</a> J Pathol. 221 (1), 3-12 (2010).</li><li>Rodolfo, C., Di Bartolomeo, S., Cecconi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+stem+and+progenitor+cells.">Autophagy in stem and progenitor cells.</a> Cell Mol Life Sci. 73 (3), 475-496 (2016).</li><li>Guan, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+stem+cells.">Autophagy in stem cells.</a> Autophagy. 9 (6), 830-849 (2013).</li><li>Phadwal, K., Watson, A. S., Simon, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tightrope+act:+autophagy+in+stem+cell+renewal,+differentiation,+proliferation,+and+aging.">Tightrope act: autophagy in stem cell renewal, differentiation, proliferation, and aging.</a> Cell Mol Life Sci. 70 (1), 89-103 (2013).</li><li>Varga, M., Fodor, E., Vellai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+zebrafish.">Autophagy in zebrafish.</a> Methods. 75, 172-180 (2015).</li><li>Varga, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+is+required+for+zebrafish+caudal+fin+regeneration.">Autophagy is required for zebrafish caudal fin regeneration.</a> Cell Death Differ. 21 (4), 547-556 (2014).</li><li>Gonzalez-Estevez, C., Salo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+apoptosis+in+planarians.">Autophagy and apoptosis in planarians.</a> Apoptosis. 15 (3), 279-292 (2010).</li><li>Buzgariu, W., Chera, S., Galliot, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+investigate+autophagy+during+starvation+and+regeneration+in+hydra.">Methods to investigate autophagy during starvation and regeneration in hydra.</a> Methods Enzymol. 451, 409-437 (2008).</li><li>Tettamanti, G., 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=Autophagy+in+invertebrates:+insights+into+development,+regeneration+and+body+remodeling.">Autophagy in invertebrates: insights into development, regeneration and body remodeling.</a> Curr Pharm Des. 14 (2), 116-125 (2008).</li></ol>

The authors have nothing to disclose.

創傷治癒および再生のモデルとしてNematostellaの使用は、ますます人気が高まっています。したがって、効果的な細胞および分子分析は、割り当てと比較することができる前に、特定のプロトコルの形態学的パターンを可視化できることが重要です。生活の後プラヌラの段階で、ほぼすべての場所で切断し、不足している構造を改革することができるという、回生「柔軟性」の高い学位?...

単一ヒドラにおける再生の観察は、実験科学の1,2のような生物学の出現で精液のイベントでした。再生は、生物学者に非常に広くアピールの現象のままと同様に人を置きます。人間の再生の限界を理解し、克服する発生生物学者、臨床医、生物医学科学者や組織のエンジニアの可能性はより本質的に興味深いより再生生物学になります 。 さて、このようなゲノム配列決定およびゲインと機能ツールの損失などの新たな技術を使用して、フィールドが回生機構を離れていじめるために、最終的に他の人がすることはできませんが再生成することができますどのように様々な種を理解する態勢を整えています。 、分子細胞性および形態学的応答における共通の度合いはまだ解明されていないが、今のところ再生することができる動物の間の基本的な応答がimagiであったであろうよりも類似していると思われますNEDのみ一昔前3。 特に、刺胞動物は、形態的多様性の広いスペクトルの中で、ほぼすべての身体の部分のを再生するに容易です。孤独な淡水ポリープから、ハイドラは、ポルトガル人-O-戦争のような複雑な植民地siphonophoresに、広大なサンゴ礁を構築小さな海洋ポリープと共に、再生はのための機構に加えて、多くの場合、再生モードであり、怪我や捕食に起因する破損や紛失の体の部分を修理または改。刺胞動物門の多様な種が再生のために類似または異なるメカニズムを使用するかどうかを根本的に興味深い質問4-6です。 私たち、そして他の人が再生7-17のモデルとして花虫類、Nematostella vectensisを開発してきました。我々は最近、aboraから二分組織の形態学的に均一な片から体全体の再生を記述するためのステージングシステムを開発しましたポリープ10のリットルの終わり。任意のレベルで二分したときNematostellaポリープを再生成することができますが、我々はこれが自然な無性分裂18の通常の平面に近いため一部では、ほとんどの形態学的に単純な地域、physaで尾方の位置で大人をカットすることを選んだ、ともあるため体全体が最も単純な形態学的構成要素から組み立て直されているかを観察し、分子解析を可能にします。 Nematostella再生ステージングシステム（NRSS）は、通常の培養条件下で、切断physaによって再生のいずれかの態様の進捗状況を採点するために使用することができる形態学的ベンチマークの比較的単純なセットを提供したり、実験的にそのような小分子治療、遺伝子操作などの状況を摂動しました、または環境の変化。予想通り、NRSSは再生の細胞および分子事象を参照することが可能な形態学的足場として採用されつつあります10。 最後に、切断の我々の方法は、最近の研究17で使用されるピンポイント穿刺よりも数桁大きいぽっかりと穴を生成し、まだ両方の傷は6時間の周りで癒します。創傷閉鎖の視覚的に逮捕した別個のフェーズを文書化することは傷の大きさと、それを閉じるのにかかる時間の見かけ上の独立性を説明するために実験的アプローチを示唆している必要があります。したがって、このプロトコルによって提供される尾方切断プロセスのより深い視覚的理解は、このモデル再生システムにさらなる研究を支援し、Nematostella vectensisのを使用して、この病期分類システムの適用を拡大します。

<table><tbody><tr><td>&lt;em&gt;Nematostella vectensis,&lt;/em&gt; adults</td><td>Marine Biological Lab (MBL)</td><td></td><td>non-profit supplier</td></tr><tr><td>Glass Culture Dish, 250 mL</td><td>Carolina Biological Supply</td><td>741004</td><td>250 mL</td></tr><tr><td>Glass Culture Dish, 1,500 mL</td><td>Carolina Biological Supply</td><td>741006</td><td>1,500 mL</td></tr><tr><td>Polyethylene transfer pipette, 5&amp;nbsp;mL</td><td>USA Scientific&amp;nbsp;</td><td>1022-2500</td><td>narrow bore, graduated</td></tr><tr><td>Polyethylene transfer pipet, tapered</td><td>Samco</td><td>202-205</td><td>cut off 1 inch of tip to make wide bore</td></tr><tr><td>Disposable Scalpel</td><td>Feather Safety Razor Co. Ltd</td><td>no. 10</td><td>blade should be curved</td></tr><tr><td>#5 Dumont Fine point tweezers</td><td>Roboz</td><td>RS5045</td><td>alternative suppliers available</td></tr><tr><td>Pyrex Petri dish, 100 mm diameter</td><td>Corning</td><td>3160</td><td>can substitute other glass Petri plates</td></tr><tr><td>Sterile 6-well plate</td><td>Corning Falcon&amp;nbsp;</td><td>353046</td><td>or similar from other manufacturer</td></tr><tr><td>Sterile 12-well plate</td><td>Nunc&amp;nbsp;</td><td>150628</td><td>or similar from other manufacturer</td></tr><tr><td>Sterile 24-well plate</td><td>Cellstar, Greiner bio-one</td><td>662-160</td><td>or similar from other manufacturer</td></tr><tr><td>Brine shrimp hathery kit</td><td>San Francisco Bay; drsfostersmith.com</td><td>CD-154005</td><td>option for growing brine shrimp</td></tr><tr><td>pyrex baking dish</td><td>common in grocery stores</td><td></td><td>option for growing brine shrimp</td></tr><tr><td>artificial seawater mix 50 gal or more&amp;nbsp;</td><td>Instant Ocean; drsfoster-smith.com</td><td>CD-116528</td><td>others brands may suffice</td></tr><tr><td>Plastic tub for stock ASW preparation</td><td>various</td><td></td><td>common 25 gallon plastic trash can OK</td></tr><tr><td>Polypropylene Carboy</td><td>Carolina Biological Supply</td><td>716391</td><td>For working stock of ASW @ 12 ppt</td></tr><tr><td>Beaker, Graduated, 4,000 mL</td><td>PhytoTechnology Laboratories</td><td>B199</td><td>For dilution of 36 ppt ASW to 12 ppt</td></tr><tr><td>Stereomicroscope and light source</td><td>various&amp;nbsp;</td><td></td><td>with continuous 1 - 40X magnification</td></tr></tbody></table>

inducing complete polyp regeneration from the aboral physa of the starlet sea anemone nematostella vectensis

刺胞動物、具体的にHydraは 、損傷を受けたり、切断構造を再生するために示した第一の動物であった、そして実際にこのような研究は間違いなく250年以上前にトレンブレーの仕事を通じて、現代生物学的調査を開始しました。現在再生の研究では、両方の「クラシック」の再生などヒドラ 、プラナリアやUrodelesのような生物、ならびに海綿から哺乳動物を介して、後生動物の範囲を、スパニング種の拡大スペクトルを用いて復活を見ています。生物学的現象としての本質的な関心に加えて、再生は様々な種でどのように機能するかを理解することは、再生プロセスは、共通の特徴および/または種またはコンテキスト固有の細胞および分子メカニズムを共有しているかどうかをお知らせします。女優のイソギンチャク、Nematostella vectensisは 、再生のための新たなモデル生物です。 ヒドラのように、Nematostellaは古代の門、刺胞動物のメンバーですが、トン内彼のクラス花虫綱、進化的により基礎であるヒドロ虫の姉妹クレード。したがってNematostellaでの再生の側面は、比較し、 ヒドラ及びその他の刺胞動物のものと対比するために興味深いものになります。この記事では、我々は、二等分観察し、physaと呼ばれるNematostella大人の尾方端の再生を分類する方法を提示します。 physaは自然に無性生殖の手段として、核分裂を起こし、自然核分裂またはphysaの手動切断のいずれかは、複雑な形態の再成長と改革をトリガします。ここでは、Nematostella再生ステージングシステム（NRSS）におけるこれらの単純な形態学的変化を成文化しています。私たちは、クロロキンの効果、リソソーム機能ブロックのオートファジーの阻害剤をテストするためにNRSSを使用しています。結果は、自食作用が阻害されたときにポリープ構造、特に腸間膜の再生が異常であることを示しています。

切断されたphysaで再生中の形態学的イベントの進行は、各NRSS段階でphysaの代表的な景色を備えて、図1Aに示されています。典型的なphysa切断部位は、成人（矢じり）に示されています。 図1Aの写真は完全に形成されたポリープを介して新鮮にカットphysaから口頭と身体構造のプログレッシブ再生を示しています。 図1B、Cにはそれぞ?...

Watch this Scientific Journal Video about Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis at JoVE.com

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 ...

inducing-complete-polyp-regeneration-from-aboral-physa-starlet-sea

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

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

8.5K ビュー.  Stony Brook University. 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.

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

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

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 any type of injury. Common injury models in these animals remove large amounts of tissue, which damages multiple organs. To overcome this broad tissue damage, we describe here a method to selectively remove a single organ, the pharynx, in the planarian Schmidtea mediterranea. We achieve this by soaking animals in a solution containing the cytochrome oxidase inhibitor sodium azide. Brief exposure to sodium azide causes extrusion of the pharynx from the animal, which we call &#34;chemical amputation.&#34;&#160;Chemical amputation removes the entire pharynx, and generates a small wound where the pharynx attaches to the intestine. After extensive rinsing, all amputated animals regenerate a fully functional pharynx in approximately one week. Stem cells in the rest of the body drive regeneration of the new pharynx. Here, we provide a detailed protocol for chemical amputation, and describe both histological and behavioral methods to assess successful amputation and regeneration.

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

The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

化学の切断とSchmidtea メディテラネアプラナリアの咽頭再生

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 ...

発生生物学

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 ...

Genotyping of Sea Anemone during Early Development

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the life of the animal. Following in vitro fertilization and de-jellying, zygotes are allowed to develop for 24 h at room temperature to reach the early- to mid-gastrula stage. The gastrula embryos are then placed on an agarose gel bed in a Petri dish containing seawater. Under the dissecting microscope, a tungsten needle is used to surgically separate an aboral tissue fragment from each embryo. Post-surgery embryos are then allowed to heal and continue development. Genomic DNA is extracted from the isolated tissue fragment and used as a template for locus-specific PCR. The genotype can be determined based on the size of PCR products or presence/absence of allele-specific PCR products. Post-surgery embryos are then sorted according to the genotype. The duration of the entire genotyping process depends on the number of embryos to be screened, but it minimally requires 4&#8211;5 h. This method can be used to identify knockout mutants from a genetically heterogeneous population of embryos and enables analyses of phenotypes during development.

The goal of this protocol is to genotype the sea anemone Nematostella vectensis during gastrulation without sacrificing the embryo.

初期開発におけるアネモネのジェノタイピング

Described here is a PCR-based protocol to genotype the gastrula stage embryo of the anthozoan cnidarian Nematostella vectensis without sacrificing the ...

Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU50 (11.1 &plusmn; 0.3 &#181;g/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

簡単なバイオアッセイによる海アネモンからの粗抽出物中の溶血性およびホスホリパーゼ活性の同定

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

生化学

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

Generation and Long-term Maintenance of Nerve-free Hydra

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The interstitial cells can be eliminated through two consecutive treatments with colchicine, a plant-derived toxin that kills dividing cells, thus erasing the potential for renewal of the differentiated cells that are derived from the interstitial stem cells. This allows for the generation of Hydra that lack nerve cells. A nerve-free polyp cannot open its mouth to feed, egest, or regulate osmotic pressure. Such animals, however, can survive and be cultured indefinitely in the laboratory if regularly force-fed and burped. The lack of nerve cells allows for studies of the role of the nervous system in regulating animal behavior and regeneration. Previously published protocols for nerve-free Hydra maintenance involve outdated techniques such as mouth-pipetting with hand-pulled micropipette tips to feed and clean the Hydra. Here, an improved protocol for maintenance of nerve-free Hydra is introduced. Fine-tipped forceps are used to force open the mouth and insert freshly killed Artemia. Following force-feeding, the body cavity of the animal is flushed with fresh medium using a syringe and hypodermic needle to remove undigested material, referred to here as "burping". This new method of force-feeding and burping nerve-free Hydra through the use of forceps and syringes eliminates the need for mouth-pipetting using hand-pulled micropipette tips. It thus makes the process safer and significantly more time efficient. To ensure that the nerve cells in the hypostome have been eliminated, immunohistochemistry using anti-tyrosine-tubulin is conducted.

Generation and Long-term Maintenance of Nerve-free Hydra

Through a double treatment with colchicine, a plant-derived toxin that kills dividing cells, nerve-free Hydra vulgaris can be generated. These Hydra cannot feed or egest on their own. This paper describes an improved method for long-term maintenance of nerve free Hydra vulgaris in the laboratory.

神経フリーの生成と長期維持<em&gt;ヒドラ</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

神経科学

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

アダルトゼブラフィッシュ側線再生のためのアッセイ

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

のマイクロインジェクション法<em&gt; Patiria minata</em&gt;接合体

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Inducing Polyp Bail-out in Coral Colonies to Obtain Individualized Micropropagates for Laboratory Experimental Use

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to the rest of the colony and ultimately detach from the skeleton to continue living as separate individuals. Coral biologists have acknowledged the process of polyp bail-out for years, but only recently the micropropagates generated by this process have been recognized as a model system for coral biology studies. The use of polyp bail-out can create a high number of clonal units from a single coral fragment. Another benefit is that single polyps or patches of polyps can be easily visualized under a microscope and maintained in highly standardized low-cost environments such as Petri dishes, flasks, and microfluidic chips. The present protocol demonstrates reproducible methods capable of inducing coral micropropagation and different approaches for maintaining the single polyps alive in the long term. This methodology was capable of successfully cultivating polyps of the coral species Pocillopora verrucosa for up to 8 weeks after bail-out, exhibiting the practicality of using individual coral polyps for coral research.

Polyp bail-out is a process induced by acute stress, in which coral polyps digest the tissue connecting them to their colony and detach from it to live as individuals. The present protocol describes how to induce coral micropropagation by bail-out using hypersaline or calcium-free seawater treatments.

サンゴのコロニーでポリープ救済を誘導して、実験室での実験的使用のための個別化されたマイクロ増殖物を得る

Corals are colonial animals formed by modular units called polyps. Coral polyps are physiologically linked and connected by tissue. The phenomenon of ...

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization. Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

This paper describes a method to create wounds in the epithelium of a live Clytia hemisphaerica medusa and image wound healing at a high resolution in vivo. Additionally, a technique to introduce dyes and drugs to perturb signaling processes in the epithelial cells and extracellular matrix during wound healing is presented.

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

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化学の切断とSchmidtea メディテラネアプラナリアの咽頭再生

ラッパムシの再生の研究のための方法

初期開発におけるアネモネのジェノタイピング

簡単なバイオアッセイによる海アネモンからの粗抽出物中の溶血性およびホスホリパーゼ活性の同定

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 Dugesia

神経フリーの生成と長期維持

アダルトゼブラフィッシュ側線再生のためのアッセイ

のマイクロインジェクション法

サンゴのコロニーでポリープ救済を誘導して、実験室での実験的使用のための個別化されたマイクロ増殖物を得る

刺胞動物モデル生物Clytia hemisphaericaを用いたin vivoでの上皮創傷治癒の特徴付け