We would like to thank Dr. Robert Angerer for his careful reading and editing of the manuscript. NIH R15HD088272-01 as well as the Office of Research and Development, and Department of Biological Sciences at Mississippi State University provided support for this project to RCR.

1.高スループットモルホリノデザイン戦略<ol><li>目的の遺伝子（複数可）（ 例えば 、候補遺伝子アプローチ、シス調節分析、RNAseqおよび/またはプロテオミクス差動画面）を特定します。 </li><li>頻繁に更新されるウェブサイト上で利用可能なゲノム、トランスクリプトーム、および遺伝子発現データを使用してください（ 例えば SpBase http://www.echinobase.org 20とS. purpuratusゲノム検索のhttp：///urchin.nidcr.nih.gov/blast/index .htmlの）時空間的発現プロファイルは、問題の発生機構とオーバーラップすることを決定しました。全く発現データが利用可能でない場合には、定量PCRプライマーおよび/または遺伝子の発現パターンを評価するための現場プローブアンチセンスを発生させます。 </li><li>空間的および時間的発現パターンを決定した後、ゲノムのウェブサイトからのDNA配列を得ます。 <ol><li>翻訳ブロッキングモルホリノオリゴヌクレオチドを生成するための、配列Oを得ます5 &#39;非翻訳領域（5&#39; UTR）は、f直接上流SpBaseまたはS purpuratusゲノム検索のウェブサイト上で利用可能な発現配列タグ（EST）データベースからの開始コドンから。この情報は、目的の遺伝子のために使用できない場合は、5 &#39;RACEを行います。 </li><li>スプライスブロッキングモルホリノ、SpBaseまたはS purpuratusゲノム検索ツールで足場のblastnツールを使用して、エクソンおよびイントロンを含むゲノム配列の検索を設計します。 </li></ol></li><li>目的の25塩基対のモルホリノシーケンスを設計するために、オリゴヌクレオチドの設計ウェブサイトhttp://oligodesign.gene-tools.comを使用してください。 <ol><li>翻訳ブロッキングモルホリノについては、翻訳の標的配列の供給源として、5 &#39;から3&#39;のmRNA配列を使用します。 5 &#39;UTR配列（転写開始部位の上流の約70ヌクレオチド）プラス著しいのwi開始コドンと（開始部位の下流）コード領域の25塩基を含みます番目の括弧（ATG）。 </li><li>スプライスブロッキングモルホリノについては、イントロン - エクソンまたはエクソン - イントロン境界のシーケンスを選択して、境界領域の周りの50塩基（エキソン配列の25塩基およびイントロン配列の25塩基）が挙げられます。シーケンスが一意であることを確認するために、モルホリノ配列とゲノムをスキャンします。 </li></ol></li></ol>モルホリノオリゴヌクレオチドの2マイクロインジェクション<ol><li> 300ナノモルホリノバイアルにヌクレアーゼフリー水100μLを添加することにより、モルホリノオリゴヌクレオチドの3 mMのストック溶液を準備します。 注：ピロ炭酸ジエチルがモルホリノに損傷を与えることができるので、再懸濁のためにDEPC処理水を使用しないでください。 </li><li>フルスピードで30秒のための株式オリゴヌクレオチドを含む溶液をバイアルダウン最初の再構成のスピンのための（14,000 - 16,000 XG）、簡単に渦、5〜10分間65℃で熱、簡単に渦を、ルームでモルホリノ株式を保ちます少なくとも1時間、温度。モルホリノ原液<html> sが4℃に-20°Cから格納されます。 </li><li>所望の濃度でモルホリノオリゴヌクレオチドを含む注射液を準備します。このソリューションは、通常20％グリセロールまたはキャリアとして125 mMのKCl及び15％（フルオレセインイソチオシアネートデキストラン万MWの2.5mg /μLのストック溶液）FITC-デキストランが含まれています。 FITCデキストランおよび他の蛍光デキストラン複合体は、日常的にエピ蛍光顕微鏡により注入された胚を識別するために使用されます。 -20℃で保存の注射溶液。 </li><li> 5分 - 少なくとも2つのヒートブロックまたはウォーターバス中で65℃でモルホリノ溶液を加熱します。 </li><li> （ - 16,000 XG 14000）、フルスピードで1分間遠心操作のための渦 - 少なくとも10分間（14,000 16,000×gで）簡単に言えばフルスピードで30秒間モルホリノ液をスピン。 </li><li>モルホリノ溶液で注射針をロードします。詳細なマイクロインジェクションプロトコルの場合Stepichevaや歌、2014年21、乾杯とEttensohn、2004を参照してください。&quot;&gt; 22。 </li></ol> S. purpuratus胚で24時間ポスト受精（HPF）で3固定とその場プロトコルで 注：このプロトコルは、アレナス・メナらから変更されています。 2000年23およびセティら。 2014年24。 <ol><li> 固定 <ol><li> 、胚を含有するウェルに（下記参照）固定液を数滴を追加し、ピペッティングにより穏やかに混合し、それらを解決することができます。固定溶液を外し、固定剤の二つの追加180μLの洗浄で胚を混ぜます。 注：それは徹底的に穏やかなピペッティングを数回上下することにより回の洗浄中に胚を混合することが重要です。 そうしないと、信号対雑音比を低下させることができます。 </li><li> 10mMのMOPS pHは7.0、0.1％のTween-20、および人工seawateからなる4％の電子顕微鏡グレードパラホルムアルデヒド中で室温（RT）で1時間に50分間の第二固定液の洗浄に固定するために胚を残しますR（ASW）。最良の結果を得るために毎回このソリューションは、新鮮なことを確認します。また、使いやすさと実用性のための胚を96ウェルプレートに固定されています。 <ol><li> 20 mLのための固定剤を使用5mLの16％パラホルムアルデヒド、15 mLのASW、200μLの1 M MOPS pHが7.0、および20μLのTween-20。 </li></ol></li><li> 0.1 M MOPSのpHは7.0、0.5 M NaClおよび0.1％のTween-20、少なくとも5分間、または胚は、ウェルの底に落ちるまでから成る180μLMOPSの洗浄緩衝液で室温で洗浄を5回。ここでも、徹底的に優しく上下に数回ピペッティングすることにより、洗浄緩衝液に胚を混合することが重要です。 MOPS洗浄緩衝液は4℃で保存した場合、2日間使用することができます °C。 <ol><li> 40mLのMOPS洗浄緩衝用の4 mLの1 M MOPS pHは7.0、4 mLの5 M NaClを、32 mLののdH 2 O、および40μLのTween-20。 </li><li>固定された胚は、2日間、4 O℃で保存することができます。 注：より長い固定胚を保存する場合は、細菌の増殖を防止するために、MOPS洗浄緩衝液中の0.2％アジ化ナトリウムを加えます。</li></ol></li></ol></li><li> プレハイブリダイゼーション <ol><li> MOPSは、洗浄緩衝液吸引し、2の180μLを追加します、ハイブリダイゼーションバッファにバッファ穏やかにピペッティング数倍液に胚を混合し、室温で少なくとも20分間インキュベートウォッシュMOPSの1の比率。ハイブリダイゼーション緩衝液は、70％ホルムアミド、0.1 M MOPS pH7.0の、0.5MのNaCl、1mg / mlのBSAおよび0.1％のTween-20で構成されています。 <ol><li> 40 mLのハイブリダイゼーション緩衝液用の4 mLの1 M MOPS pHは7.0、4 mLの5 M NaClを、4 mLののdH 2 O、0.04グラムのBSA、および40μLのTween-20。再びボルテックスでよく混ぜ、28 mLのホルムアミドを追加し、渦。 </li></ol></li><li> MOPS洗浄およびハイブリダイゼーション緩衝液の1の比率と1の180μL追加：2を取り外しMOPSの2比を、ハイブリダイゼーションバッファーにバッファー液に胚を混合し、室温で少なくとも20分間インキュベート洗います。 </li><li>ハイブリダイゼーション緩衝液の150μL - プローブハイブリダイゼーションの前に軽く100に胚を混ぜます。少なくとも1つの、50℃で胚をインキュベート時間。 注：一晩胚をインキュベートすることは許容可能です。インキュベーションの前に、蒸発を防ぐために、接着シートでウェルを密閉します。 </li></ol></li><li> 交配 <ol><li>その後、渦が静かにプローブ溶液を作成するには、ハイブリダイゼーション緩衝液にプローブおよび500μgの/ mLの酵母tRNAの0.3 ngの/μL - 別のチューブに0.1を追加します。酵母tRNAは、アンチセンスプローブの非特異的結合を減少させるためにプローブ溶液に添加します。この50°Cの解決策、および吸引ハイブリダイゼーション緩衝液を予熱。 </li><li> 、プローブ溶液100μLにプレハイブリダイズ胚をミックス粘着シートで密封し、プローブに応じて、2〜7日間50℃でハイブリダイズ（foxq2プローブは、2日間インキュベートすることができます）。 注：インキュベーション時間は、プローブをオフに異なります。低レベルで発現されるいくつかの遺伝子は、7日間のインキュベーションまで必要とします。 adhes場合、96ウェルプレートは、蒸発に対する保険として湿度ボックス内に配置することができアイブシートが適切に密封するために失敗しました。 </li><li>作りたてのMOPSは3時間の合計洗浄緩衝液を180μLに50℃で5回洗浄します。その後、室温で180μLMOPSの洗浄緩衝液で3回（15分間）洗浄します。静かに数回ピペッティングすることにより、各バッファ時間洗浄に胚を混合することを忘れないでください。 </li></ol></li><li> 抗体インキュベーション <ol><li>吸引しMOPSは、MOPSで10％正常ヒツジ血清および5mg / mLのBSAからなるブロッキング緩衝液180μLに胚をバッファリングし、混ぜる洗浄は、バッファー、一晩RTまたは4℃で少なくとも45分間インキュベート洗います。 </li><li>ブロッキング緩衝液外し、1含むブロッキング緩衝液に胚を混ぜる：ブロッキング緩衝液で希釈したアルカリホスファターゼ結合抗ジゴキシゲニン抗体の1500希釈。蒸発を避けるために密閉されたプレート中、室温で一晩インキュベートします。注：一晩以上の抗体を放置しないでください。 </li><li>胚を6回洗浄（5分または胚がドロップするまで）MOPSにRTでバッファを洗います。胚できます4℃で一晩保存します。 </li></ol></li><li> その場開発で <ol><li> 0.1MトリスpH9.5中、100mMのNaCl、50mMのMgCl 2を、1mMのレバミゾール、および0.1％のTween-20からなる新たに作られたpH9.5の緩衝液を用いて室温で胚を3回（10分間）洗浄します。 <ol><li> 20 mLのpHが9.5バッファ使用2 mlの1 MトリスpHは9.5、400μL5MのNaCl、1 mLの1 MのMgCl 2、20μLのTween-20、16.6ミリリットルのdH 2 O、および0.0048グラムのレバミゾール。 </li></ol></li><li>光から保護し、染色緩衝液中で37℃で胚をインキュベートします。染色バッファは、新たに作られたpH9.5の緩衝液中10％ジメチルホルムアミド、4.5μL/ mLの4-ニトロブルーテトラゾリウムクロライド（NBT）、3.5μL/ mLの5-ブロモ-4-クロロ-3-インドリル - リン酸（BCIP）からなります。 <ol><li>染色バッファー1mLのために100μLのジメチルホルムアミド、4.5μLNBT、および3.5μLのBCIPを使用しています。 </li></ol></li><li> MOPS中で3~5回洗浄することによりアルカリホスファターゼ反応を停止し、バッファを洗います。反応のwifoxq2プローブは、典型的には、1時間に30分を要し番目。いくつかのプローブは、RTまたは4℃のいずれかで一晩のインキュベーションが必要な場合があります。 </li><li> 70％のMOPS洗浄し、30％グリセロールの溶液に胚を混ぜます。グリセロールは、顕微鏡検査のために必要な屈折率を提供します。胚は、数週間のために、この溶液中で保存することができます。蒸発を防ぐためにプラスチック製のパラフィンでプレートをシール。 </li></ol></li><li> スライドの調製及び撮像 <ol><li>スライド上にストリップ間の小さなギャップを持つ三角形に両面テープの三つの小さなストリップを配置することにより、スライドを準備します。 </li><li>三角形の中央に70％のMOPS洗浄し、30％グリセロール溶液中で胚を移し、カバーガラスでカバーしています。 </li><li>カバーガラスのエッジの周りマニキュアの層を適用することにより、カバースリップをシール。 </li><li> 20Xの対物レンズと取り付けられたカメラを有する化合物の光学顕微鏡を用いて画像をキャプチャします。 </li></ol></li></ol>

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A., Samanta, M., Yuan, A., He, D., Davidson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SpBase:+the+sea+urchin+genome+database+and+web+site.">SpBase: the sea urchin genome database and web site.</a> Nucleic Acids Res. 37, D750-D754 (2009).</li><li>Stepicheva, N. A., Song, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+microinjections+of+sea+urchin+zygotes.">High throughput microinjections of sea urchin zygotes.</a> Journal of visualized experiments : JoVE. , e50841 (2014).</li><li>Cheers, M. S., Ettensohn, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+microinjection+of+fertilized+eggs.">Rapid microinjection of fertilized eggs.</a> Methods in cell biology. 74, 287-310 (2004).</li><li>Arenas-Mena, C., Cameron, A. R., Davidson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+expression+of+Hox+cluster+genes+in+the+ontogeny+of+a+sea+urchin.">Spatial expression of Hox cluster genes in the ontogeny of a sea urchin.</a> Development. , 4631-4643 (2000).</li><li>Sethi, A. J., Angerer, R. C., Angerer, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+labeling+in+developmental+gene+regulatory+network+analysis.">Multicolor labeling in developmental gene regulatory network analysis.</a> Methods in molecular biology. , 249-262 (2014).</li><li>Wikramanayake, A. H., Huang, L., Klein, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Catenin+is+essential+for+patterning+the+maternally+specified+animal-vegetal+axis+in+the+sea+urchin+embryo.">beta-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo.</a> Proc Natl Acad Sci U S A. 95, 9343 (1998).</li><li>Yaguchi, S., Yaguchi, J., Angerer, R. C., Angerer, L. 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+Wnt-FoxQ2-nodal+pathway+links+primary+and+secondary+axis+specification+in+sea+urchin+embryos.">A Wnt-FoxQ2-nodal pathway links primary and secondary axis specification in sea urchin embryos.</a> Dev Cell. 14, 97-107 (2008).</li><li>Molina, M. D., de Croze, N., Haillot, E., Lepage, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal:+master+and+commander+of+the+dorsal-ventral+and+left-right+axes+in+the+sea+urchin+embryo.">Nodal: master and commander of the dorsal-ventral and left-right axes in the sea urchin embryo.</a> Curr Opin Genet Dev. 23, 445-453 (2013).</li><li>Range, R. C., Glenn, T. D., Miranda, E., McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LvNumb+works+synergistically+with+Notch+signaling+to+specify+non-skeletal+mesoderm+cells+in+the+sea+urchin+embryo.">LvNumb works synergistically with Notch signaling to specify non-skeletal mesoderm cells in the sea urchin embryo.</a> Development. 135, 2445-2454 (2008).</li><li>Range, 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=Cis-regulatory+analysis+of+nodal+and+maternal+control+of+dorsal-ventral+axis+formation+by+Univin,+a+TGF-beta+related+to+Vg1.">Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by Univin, a TGF-beta related to Vg1.</a> Development. 134, 3649-3664 (2007).</li><li>Warner, J. F., Miranda, E. L., McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+hedgehog+signaling+to+the+establishment+of+left-right+asymmetry+in+the+sea+urchin.">Contribution of hedgehog signaling to the establishment of left-right asymmetry in the sea urchin.</a> Dev Biol. 411, 314-324 (2016).</li><li>Rottinger, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+signals+guide+migration+of+mesenchymal+cells,+control+skeletal+morphogenesis+[corrected]+and+regulate+gastrulation+during+sea+urchin+development.">FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis [corrected] and regulate gastrulation during sea urchin development.</a> Development. 135, 353-365 (2008).</li><li>Warner, J. F., McCarthy, A. M., Morris, R. L., McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+requires+motile+cilia+in+the+sea+urchin.">Hedgehog signaling requires motile cilia in the sea urchin.</a> Mol Biol Evol. 31, 18-22 (2014).</li><li>Technau, U., Steele, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+crossroads+in+developmental+biology.">Evolutionary crossroads in developmental biology.</a> Cnidaria. Development. 138, 1447-1458 (2011).</li><li>Yaguchi, J., Takeda, N., Inaba, K., Yaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+Wnt-Nodal+Signals+Regulate+the+Patterning+of+Anterior+Neuroectoderm.">Cooperative Wnt-Nodal Signals Regulate the Patterning of Anterior Neuroectoderm.</a> PLoS Genet. 12, e1006001 (2016).</li><li>Duboc, V., Rottinger, E., Besnardeau, L., Lepage, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+and+BMP2/4+signaling+organizes+the+oral-aboral+axis+of+the+sea+urchin+embryo.">Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo.</a> Dev Cell. 6, 397-410 (2004).</li><li>Bradham, C. A., 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=Chordin+is+required+for+neural+but+not+axial+development+in+sea+urchin+embryos.">Chordin is required for neural but not axial development in sea urchin embryos.</a> Dev Biol. 328, 221-233 (2009).</li><li>Su, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+for+ectoderm+specification+in+sea+urchin+embryos.">Gene regulatory networks for ectoderm specification in sea urchin embryos.</a> Biochimica et biophysica acta. 1789, 261-267 (2009).</li><li>Lin, C. Y., Su, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+in+sea+urchin+embryos+by+using+a+CRISPR/Cas9+system.">Genome editing in sea urchin embryos by using a CRISPR/Cas9 system.</a> Dev Biol. 409, 420-428 (2016).</li></ol>

The authors have nothing to disclose.

ここで紹介する方法は..多くのラボが分析する早期ウニ開発時に同様のアッセイを使用している基本的な発達のメカニズムを支配するシグナル伝達経路とGRNsを理解するために、脊椎動物未満ゲノムおよび形態学的な複雑さに胚を使用してのパワーを示している例です。他の細胞運命指定イベント（ 例えば、ノッチ、ヘッジホッグ、TGF-βの、及びFGFシグナリング）に関与する経路<sup clas...

遺伝子制御ネットワーク（GRNs）とシグナル伝達経路は、成体動物の体の計画を構築するために使用される胚発生の間の遺伝子の空間的および時間的発現を確立します。細胞から細胞へのシグナル伝達経路は、細胞が通信する手段を提供し、これらの調節ネットワークの重要な構成要素です。これらの細胞の相互作用は、胚発生1、2中の様々な地域でとの間の規制および分化遺伝子の発現を確立し、改良します。分泌された細胞外調節因子（リガンド、アンタゴニスト）、受容体および共受容体の間の相互作用は、シグナル伝達経路の活性を制御します。細胞内分子の品揃えは、遺伝子発現の変化、分割、および/または細胞の形で得られたこれらの入力を伝達します。主要な経路で細胞外および細胞内のレベルで使用する鍵分子の多くがあるが既知の、個々のシグナル伝達経路の複雑さに大部分の不完全な知識です。加えて、異なるシグナル伝達経路は、多くの場合、細胞外、細胞内での正または負のいずれかで互いに相互作用し、転写レベル3、4、5、6。重要なことには、シグナル伝達経路のコアコンポーネントは、非常にすべての後生動物種で保存され、特に密接に関連門の生物を比較する場合、著しく、主要なシグナル伝達経路の大部分は、多くの場合、多くの種において類似の発達機能を実行7、8、9、 10、11。 開発中にシグナル伝達の研究では、任意の生物で困難な作業であり、そしてそこに脊椎動物では、大きな可能性リガンドと受容体/共変調器の相互作用の数、細胞内伝達分子、ならびにがあります1）：ほとんどの新口動物モデル（脊椎動物、無脊椎動物脊索動物、半索動物、および棘皮動物）でシグナル伝達経路を研究するためのいくつかの重要な課題がありますゲノム12の複雑さに起因する異なるシグナル伝達経路の間の潜在的な相互作用、13、14; 2）脊椎動物の複雑な形態および形態形成の動きは、多くの場合、それはより困難でかつシグナル伝達経路間の機能的相互作用を解釈することを可能にします。 3）ほとんどの非棘皮動物無脊椎動物の新口動物モデル種における分析は、いくつかのホヤ種15、16を除いて妊娠の短いウィンドウによって制限されています。 ザウニ胚は、上記制限のいくつかを持っており、 生体内でのシグナル伝達経路の詳細な分析を行うために多くのユニークな資質を提供しています。これらは、以下のものが挙げられる：1）ウニゲノムの相対的なシンプルさが大幅に可能なリガンド、受容体/共受容体と細胞内伝達分子の数を減少させる17の相互対話します。 2）胚葉および主要な胚軸の仕様およびパターニングを制御GRNsはよく信号18、19セル受信/地域の規制状況の理解を助ける、ウニ胚で確立されています。胚は、その形態を分析するのが容易である単層上皮で構成されたとき3）多くのシグナル伝達経路は、早期に切断し、原腸ステージ間で研究することができます。 4）分子が関与しますウニ内シグナル伝達経路中のdは、容易に操作されています。 5）多くのウニは、10〜11ヶ月〜1年（ 例えばStrongylocentrotusのpurpuratusとLytechinusのvariegatus）のために妊娠しています。 ここでは、体系的かつ効率的に指定し、ウニ胚のパターン領土は、いくつかの無脊椎動物モデル系が複雑な分子メカニズムの研究に提供する利点を説明するためにシグナル伝達経路の構成要素を特徴づけるための方法を提示します。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Translational-blocking morpholino and/or splice-blocking morpholino</td>
			<td>Gene Tools LLC</td>
			<td>Customized</td>
			<td>More information at www.gene-tools.com</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Invitrogen</td>
			<td>15514-011</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC (dextran fluorescein isothiocyanate)</td>
			<td>Invitrogen, Life Technologies</td>
			<td>D1821</td>
			<td>Make 25mg/mL stock solution</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution EM Grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma Aldrich</td>
			<td>M1254-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>23336-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma Aldrich</td>
			<td>47671-1L-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast tRNA</td>
			<td>Invitrogen</td>
			<td>15401-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase-conjugated anti-digoxigenin antibody</td>
			<td>Roche</td>
			<td>11 093 274 910</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramisole hydrochloride (levamisole)</td>
			<td>Sigma Aldrich</td>
			<td>L9756-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base UltraPure</td>
			<td>Research Products Internationall Corp</td>
			<td>56-40-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich</td>
			<td>7786-30-3</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP (5-Bromo-4-Chloro-3-indolyl-phosphate</td>
			<td>Roche</td>
			<td>11 383 221 001</td>
			<td></td>
		</tr>
		<tr>
			<td>4 Nitro blue tetrazolium chloride (NBT)</td>
			<td>Roche</td>
			<td>11 383 213 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Formamide</td>
			<td>Sigma Aldrich</td>
			<td>D4551-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M7506-2KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>C1016-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

the power of simplicity: sea urchin embryos as in vivo developmental models for studying complex cell-to-cell signaling network interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

ウニ胚では、3つの異なるWntシグナル支店（のWnt /βカテニンは、Wnt / JNK、およびWnt / PKC）4、25相互作用は前後（AP）パターニングを支配するWntシグナル伝達ネットワークを形成することが示されています。これらのシグナル伝達イベントの中で最も重要な結果の一つは、初期広く発現し、前方神経外胚葉（ANE）GRNは、原腸形?...

Watch this Scientific Journal Video about The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions at JoVE.com

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

このビデオの記事では、体系的かつ効率的に多くの無脊椎動物の胚では、複雑なシグナル伝達経路および制御ネットワークの構成要素を特徴付けるために使用することができるin vivoでの方法論に簡単な詳細を示します。

シンプルの力：ウニ胚として<em&gt; in vivoで</em&gt;複雑な細胞間シグナル伝達ネットワークの相互作用を研究するための発達モデル

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the signaling pathways and the gene regulatory networks that they activate in an in vivo developmental context.Demonstrating this procedure will be Marina Martinez-Bartolom

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The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Unit 1

Principles of Cell Signaling

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7.8K ビュー.  Mississippi State University. The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the si...

ビデオ: The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

モデルマメに初期胚発生の調節を研究するための接合体と体細胞胚を得るためのプロトコル<em&gt;のMedicago truncatula</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

発生生物学

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺の特性を用いてマウス胚における前駆細胞をセトリング<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;アッセイ

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Studying Wnt Signaling During Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

誘導気道のパターニング時にWntシグナル伝達を学びます

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

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 ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

薬物治療と<em&gt;インビボ</em&gt;メダカ骨粗鬆症モデルにおける骨芽細胞破骨細胞の相互作用のイメージング

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

細胞非自律影響を研究するためのキメラゼブラフィッシュ胚における個々の網膜前駆細胞におけるトランスジェニック遺伝子発現のin vivoイメージング

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

マウス二次口蓋融合のライブイメージング

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

ノードと走査型電子顕微鏡と蛍光抗体法マウント全体を使用して Gastrulating のマウス胚における脊索板を可視化

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

紫外C(UV-C)損傷に対する主なマウス眼表面細胞/幹細胞における酸化的損傷の評価

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

ゼブラフィッシュ胚全体の延長タイムラプス共焦点顕微鏡法の層状取り付け法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...