We would like to thank C. Burns, C. McRae for outlining the basic principle of this purification protocol, and F. Priller for initial implementation of this method in our lab. S.A.-S. is supported by a Heisenberg professorship of the Deutsche Forschungsgemeinschaft (DFG). This work was supported by DFG grant SE2016/7-1.

このプロトコルは、ドイツのベルリン州法の動物のケアのガイドラインに従います。ゼブラフィッシュの取り扱いは、動物保護のための地方自治体（LaGeSo、ベルリン·ブランデンブルク）によりモニターした。 1.ハート抽出のためのゼブラフィッシュ胚の取得<ol><li>そのようなTgは（myl7：EGFP）などのクロス心臓レポーターゼブラフィッシュtwu34ジェニックライン9ハート固有のGFP発現を持つ胚を得るために。 </li><li>希望胚段階10,11まで28.5℃で、卵の水で胚を維持する。収集された胚の集団が遺伝子発現の変動を制限するために、両方の遺伝的および発達段階によって均質であることを確認してください。 </li><li>手動で胚をDechorionate。 </li></ol> 2.解剖ゼブラフィッシュ胚ハーツ注：氷の上のすべてのソリューションを保管してください。可能な場合は、チームワークを行います。1人はEMBから心を解剖ryos別の人は、蛍光実体顕微鏡下で心をソートしながら。これは、数時間で胚の数百の処理を可能にする。 <ol><li> 1.5ミリリットルの遠心チューブに約100の胚を転送します。トリカイン（E3培地中0.16 mg / mlで）で胚を麻酔。胚を明確に麻酔をされると進みます（彼らは泳ぐとチューブの底に沈殿物が、その心はまだ暴行されていません）。 </li><li>トリカイン/ E3ソリューションを削除し、1ミリリットルL-15/10％FBS培地で1回の胚を洗浄し、氷上で維持する。 注：L-15/10％FBS培地の心はをRNAlaterまたはトリゾールに移されるまで、全体の解剖手順の間に生きている臓器や細胞を維持することが重要である。 </li><li>卵黄が完全に破壊されるまで、ラウンドゲルローディングチップで胚およびピペット上下に5-8回に1ミリリットルL-15/10％FBS培地を追加します。胚が滴下溶液の表面上に、ドロップが破裂するように、ピペット胚を穏やかに破壊される。 注：どのように積極的に、どのくらいの頻度胚が上下にピペッティングしなければならないことは胚の発達段階に依存し、 すなわちより多くのピペット操作（ 例えば 56 HPFで）後期段階で必要とされている。 </li><li>解剖胚の最初のバッチのために、あまりにも静かにピペットであれば、いくつかの心は胚に付着したままかもしれないので、胚の整合性と実体顕微鏡下で解剖の心の割合を評価する。それに応じて解剖の次のラウンドのためにピペット操作の仕方を調整します。 注：プラスチック製にこだわり心を最小限に抑えるために、低保持ピペットチップとマイクロチューブを使用してください。 </li><li> 50ミリリットルの遠心チューブに置か100μmのフィルター上にサンプルを適用します。 1ミリリットルL-15/10％FBS培地で1.5mlチューブをすすぎ、その後（いくつかの心臓は、チューブの壁に付着しているかもしれません）サンプルの損失を最小にするために、フィルタにこれを適用する。 1ミリリットルLで二回フィルターを洗浄-15 / 10％FBS。このステップでは、心臓は、フィルターを通過し、50 mlチューブに回収する。 </li><li> 15ミリリットルの遠心チューブの上に配置さ30μmフィルター上にフロースルーを適用し、1ミリリットルL-15/10％FBS培地で1回フィルターをすすぐ。この第2の濾過工程においては、心臓は、フィルタ内に保持され、より小さな破片を洗い流す。 </li><li>逆さまにフィルターを回して、1ミリリットルL-15/10％FBSで3つの連続した​​回の洗浄を適用することにより、1％アガロースでコーティングされたシャーレにフィルタの外心をフラッシュする。 </li><li>蛍光実体顕微鏡下にピンセットで非蛍光胚破片（ 例えばアイレンズ）から手動で独立したGFP陽性心と皿の中央でそれらを集中する。ステップ3に従ってそれらを収集します。 注：胚が24 HPFよりも古い場合は、心は組織がまだ生理的に正常であることを示す、暴行されるべきである。 </li></ol> 3. DissecからmRNAを単離しテッドハーツ<ol><li> 0.75ミリリットルのRNAlater（氷上）を含む1.5ミリリットルチューブに可能な限り最小容量（ 例えば 、10μL）で心とピペットを収集します。何の心は、蛍光実体顕微鏡下でそれを見ることによって、ピペットチップに残っていないことを確認します。 </li><li>あるいは、化学フードは蛍光顕微鏡のすぐ近くに利用可能である場合、化学フードの下のTrizol（注意）に集め心を移す。これはピペットチップにし、また心の遠心分離中にこだわりの心の損失の可能性を回避する（3.4、3.5および3.7ステップ）。トリゾールと同じチューブに分離のいくつかのラウンドからの心をプールし、3.8のステップに直接進む。最終体積は元のトリゾール容積の10％を超えてはならない。 注：使用前にトリゾール製品安全データシート（MSDS）をお読みください。ボンネットの下にTrizol試薬を処理し、推奨される個人用保護具を着用する。皮膚、目やclothiとの接触を避けてくださいNG。発火のすべてのソースを削除します。 </li><li> RNA単離の効率が採取した組織の量を使用すると向上として、（氷上）RNAlaterで同じチューブに分離のいくつかのラウンドからの心をプール。全試料体積は、元のRNAlater体積の10％を超えると、（氷上で）保存のために0.75ミリリットルRNAlaterで追加のチューブを使用する。 </li><li>遠心機で4℃で20分間15700×gで試料は心を沈降する。 </li><li>蛍光実体顕微鏡下では、3ミリリットルL-15/10％FBS培地を含む1％アガロースでコーティングされたシャーレにできるだけ丁寧に限り多くの上清を回収したが、遠心分離管の底に沈殿し、心を乱すことがありません。心の最大15％がRNAlaterでの高粘度のために上清中に残ることができるのでステップ3.7で説明したように、それらを取得。 </li><li>化学フードの下で、沈降した心を含むチューブに0.5ミリリットルのTrizolを追加し、氷上に保つ。 </li><li>蛍光microscoの下でPEの、希釈する3ミリリットルL-15/10％FBSを有する別の1％アガロースでコーティングされた皿にRNAlater中/ L-15/10％FBS溶液を含む1％アガロースでコーティングされた皿から（ステップ3.5）からの心臓を転送RNAlaterを出。ペトリ皿の中央に心を収集し、化学フードの下のTrizol中で沈降した心のチューブに小さな音量でそれらを転送する。 </li><li>心を破壊し、室温で5分間インキュベートし、トリゾールで心を含む渦1.5mlチューブ。 </li><li>化学フードの下で、100μlのクロロホルム（注意）を追加し、完全に混合し、1.5ミリリットルに事前に紡糸しPLGチューブ（使用前に最大速度で遠心30秒）をトリゾール/クロロホルム溶液を転送する。室温で3分間インキュベートする。 注：使用前にクロロホルムのMSDSを参照してください。ボンネットの下にクロロホルム試薬を処理し、推奨される個人用保護具を着用する。皮膚、眼、そして衣服との接触を避ける。金属、アルカリなどの非互換性から遠ざけること。 </li><li> Centrifugeと4℃で15分間15700×gでのサンプルと新しい1.5mlチューブへ水相を転送する。 </li><li> 5〜10μgのグリコーゲンとイソプロパノール（-20℃で）予冷250μlのを追加します。よく混ぜ、-20℃で一晩インキュベートする。 </li><li>遠心機で4℃で30分間15700×gでサンプルし、上清を廃棄する。 </li><li> 、1ミリリットルを75％エタノールでペレットを洗浄し、4℃で15分間15700×gで遠心分離し、上清を捨てる。 </li><li>それは（10〜15分間、例えば ）の白色になるまで室温でペレットを乾燥させてエタノールを蒸発さう。 </li><li>ペレットに20μlのRNaseフリーのddH 2 Oを追加し、RNAを溶解させ、55℃で10〜15分間インキュベート。その後氷上に保つ。 </li><li>吸光度測定によってRNA濃度および純度を評価。 1％アガロースゲル上でサンプルを実行して、RNAの完全性を評価する。 </li></ol>

<ol>
	<li>Stainier, D. Y., 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=Mutations+affecting+the+formation+and+function+of+the+cardiovascular+system+in+the+zebrafish+embryo.">Mutations affecting the formation and function of the cardiovascular system in the zebrafish embryo.</a> Development. 123, 285-292 (1996).</li><li>Chen, J. N., 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=Mutations+affecting+the+cardiovascular+system+and+other+internal+organs+in+zebrafish.">Mutations affecting the cardiovascular system and other internal organs in zebrafish.</a> Development. 123, 293-302 (1996).</li><li>Bakkers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+to+study+cardiac+development+and+human+cardiac+disease.">Zebrafish as a model to study cardiac development and human cardiac disease.</a> Cardiovasc Res. 91, 279-288 (2011).</li><li>Jowett, T., Lettice, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+in+situ+hybridizations+on+zebrafish+embryos+using+a+mixture+of+digoxigenin-+and+fluorescein-labelled+probes.">Whole-mount in situ hybridizations on zebrafish embryos using a mixture of digoxigenin- and fluorescein-labelled probes.</a> Trends Genet. 10, 73-74 (1994).</li><li>Gibson, U. E., Heid, C. A., Williams, 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=A+novel+method+for+real+time+quantitative+RT-PCR.">A novel method for real time quantitative RT-PCR.</a> Genome Res. 6, 995-1001 (1996).</li><li>Epstein, C. B., Butow, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+technology+-+enhanced+versatility,+persistent+challenge.">Microarray technology - enhanced versatility, persistent challenge.</a> Curr Opin Biotechnol. 11, 36-41 (2000).</li><li>Qian, X., Ba, Y., Zhuang, Q., Zhong, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq+Technology+and+Its+Application+in+Fish+Transcriptomics.">RNA-Seq Technology and Its Application in Fish Transcriptomics.</a> OMICS. 18, 98-110 (2014).</li><li>Burns, C. G., MacRae, 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=Purification+of+hearts+from+zebrafish+embryos.">Purification of hearts from zebrafish embryos.</a> Biotechniques. 40, 274-278 (2006).</li><li>Huang, C. J., Tu, C. T., Hsiao, C. D., Hsieh, F. J., Tsai, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transmission+of+a+myocardium-specific+GFP+transgene+reveals+critical+regulatory+elements+in+the+cardiac+myosin+light+chain+2+promoter+of+zebrafish.">Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish.</a> Dev Dyn. 228, 30-40 (2003).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li><li>Westerfield, 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> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Veerkamp, J., 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=Unilateral+dampening+of+Bmp+activity+by+nodal+generates+cardiac+left-right+asymmetry).">Unilateral dampening of Bmp activity by nodal generates cardiac left-right asymmetry).</a> Dev Cell. 24, 660-667 (2013).</li><li>Jin, S. W., Beis, D., Mitchell, T., Chen, J. N., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+analyses+of+vascular+tube+and+lumen+formation+in+zebrafish.">Cellular and molecular analyses of vascular tube and lumen formation in zebrafish.</a> Development. 132, 5199-5209 (2005).</li><li>Brownlie, 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=Characterization+of+embryonic+globin+genes+of+the+zebrafish.">Characterization of embryonic globin genes of the zebrafish.</a> Dev Biol. 255, 48-61 (2003).</li><li>Marvin, 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=Developmental+expression+patterns+of+the+zebrafish+small+heat+shock+proteins.">Developmental expression patterns of the zebrafish small heat shock proteins.</a> Dev Dyn. 237, 454-463 (2008).</li><li>Lam, C. S., Marz, M., Strahle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gfap+and+nestin+reporter+lines+reveal+characteristics+of+neural+progenitors+in+the+adult+zebrafish+brain.">gfap and nestin reporter lines reveal characteristics of neural progenitors in the adult zebrafish brain.</a> Dev Dyn. 238, 475-486 (2009).</li><li>Wang, Y., 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=Moesin1+and+Ve-cadherin+are+required+in+endothelial+cells+during+in+vivo+tubulogenesis.">Moesin1 and Ve-cadherin are required in endothelial cells during in vivo tubulogenesis.</a> Development. 137, 3119-3128 (2010).</li><li>Heiman, 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=A+translational+profiling+approach+for+the+molecular+characterization+of+CNS+cell+types.">A translational profiling approach for the molecular characterization of CNS cell types.</a> Cell. 135, 738-748 (2008).</li><li>Tryon, R. C., Pisat, N., Johnson, S. L., Dougherty, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+translating+ribosome+affinity+purification+for+zebrafish.">Development of translating ribosome affinity purification for zebrafish.</a> Genesis. 51, 187-192 (2013).</li></ol>

The authors have nothing to disclose.

このプロトコルは、遺伝子発現解析のためのゼブラフィッシュ胚の心臓組織の迅速な濃縮を可能にする。 RNA精製だけで十分で動作しますので、心の喪失は、プロトコルのすべての段階で阻止される場合は、最初の、試料の量が大幅に改善されている：心臓特異的RNAサンプルの量と質が大幅にいくつかの重要なステップに依存します出発材料。次に、実験者に完全に依存したサンプルの純度は...

ゼブラフィッシュ（ ゼブラフィッシュ ） が広くによりサイズが小さく、蛍光タンパク質の組織特異的な発現を有するトランスジェニックレポーターラインの利用可能性と合わせて、高速で透明かつ子宮外胚発生、 インビボでの器官形成を研究するために発生生物学で使用されている。初期のゼブラフィッシュ胚の酸素は、心拍、血流に依存しないので、この小さな脊椎動物は、心臓発生を研究するために特に適している。これらの機能は、心血管の変異体1,2の多数の特徴付けを可能にしたとゼブラフィッシュは現在、心疾患3を研究するための広く認識モデル生物である。 胚発生の間の遺伝子発現を研究するために、転写物は一般にホールマウントin situハイブリダイゼーション （WISH）4によって分析され、RT-qPCRを5、マイクロアレイ6、または次世代シークエンシング（RNA配列）7。同時にWISHは全体胚内の遺伝子発現の空間的·時間的な分析を可能にする、転写レベルは通常、RT-qPCRを、マイクロアレイまたはRNA-配列アプローチによって評価される。しかしながら、これらの方法は、特異的な遺伝子発現プロファイリングのための組織の濃縮を必要とする。 ゼブラフィッシュ胚の心臓は全体の胚のごく一部を表すので、心臓の開発中にトランスクリプトーム研究は心の解剖と濃縮のためのプロトコルが必要。また、生理学的に関連するデータを得るためには、RNA抽出まで、完全に機能的心臓組織を維持することが重要である。ここでは、急速に生理的に通常の単離及び効率的にさらなる分析のための高品質なRNAサンプルを得るために、ゼブラフィッシュの胚の数百人から心を打つためのプロトコルを記述します。本発明の方法は、バーンズとマクレー、2006年8によって報告されたプロトコルに基づいています。心臓組織の充実のために、両方の方法は、トランスジェニック心筋のレポーターラインを使用する他の組織に対するゼブラフィッシュ胚の心臓の差動接着特性を利用する。簡単に言えば、狭いピペットチップを通して上下に多くの胚をピペッティングすることにより、心臓を同時に胚の体から解放され、その後、2急速ろ過段階で胚の破片から分離。蛍光標識した心は、手動で残りの破片から選別し、さらなる処理のために収集されます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr >
			<td >Equipment for raising fish and collecting eggs&#160;</td>
			<td></td>
			<td></td>
			<td>see the Zebrafish Book11 for details</td>
		</tr>
		<tr >
			<td >Fluorescence stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Refrigerated Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >UV-Spectrophotometer</td>
			<td></td>
			<td></td>
			<td>eg. Thermo Scientific Nanodrop 2000</td>
		</tr>
		<tr >
			<td >Nucleic acid electrophoresis chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Petri dishes 4cm &#216;, coated with 1% agarose in E3 medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipettes and tips (P20, P100, P1000)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1,5ml centrifugation tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >15ml and 50ml centrifugation tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Pair of Dumont #5 forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >ExactaCruz&#8482; Round Gel Loading Tips in Sterile Rack, 1-200&#956;l&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-201732</td>
			<td></td>
		</tr>
		<tr >
			<td >100 &#956;m filter (BD Falcon 100 mm Cell Strainer)</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr >
			<td >30 &#956;m filter (Pre-Separation Filters-30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr >
			<td >Phase lock gel, heavy, 1,5ml tubes&#160;</td>
			<td>Prime</td>
			<td>2302810</td>
			<td></td>
		</tr>
		<tr >
			<td >Egg water medium</td>
			<td></td>
			<td></td>
			<td>60&#956;g/ml Instant Ocean Sea Salts in ddH2O, 0.00001% (w/v) Methylene Blue</td>
		</tr>
		<tr >
			<td >E3 medium&#160;</td>
			<td></td>
			<td></td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4</td>
		</tr>
		<tr >
			<td >Tricaine (3-amino benzoic acidethylester)</td>
			<td>Sigma-Aldrich</td>
			<td>A-5040</td>
			<td>4mg/ml Tricaine stock solution, pH 7</td>
		</tr>
		<tr >
			<td >1% agarose (in E3 medium)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1% agarose gel (in TBE buffer)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Leibovitz&#180;s L-15 medium&#160;</td>
			<td>Gibco</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr >
			<td >FBS (Fetal Bovine Serum)</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr >
			<td >RNAlater</td>
			<td>Ambion</td>
			<td>AM7020</td>
			<td></td>
		</tr>
		<tr >
			<td >Trizol</td>
			<td>Ambion</td>
			<td>1559606</td>
			<td></td>
		</tr>
		<tr >
			<td >Glycogen&#160;</td>
			<td>Invitrogen</td>
			<td>10814-010</td>
			<td>20 &#181;g/&#181;L in RNase-free water</td>
		</tr>
		<tr >
			<td >chloroform</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >isopropanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >75% ethanol (in DEPC-ddH2O)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Nuclease-free water or sterilized DEPC treated ddH2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Nucleic acid loading buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >TBE (Tris/Borate/EDTA) buffer for electrophoresis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

large-scale zebrafish embryonic heart dissection for transcriptional analysis

ゼブラフィッシュ胚の心臓は胚全体のごく一部を表す、わずか数百の細胞から構成されている。したがって、グローバル胚トランスクリプトによってマスクされているから心臓のトランスクリプトームを防止するためには、さらなる分析のために心臓の十分な数を収集する必要がある。ゼブラフィッシュの心臓の開発が急速に進行していくまた、心臓の収集およびRNA抽出方法は、サンプルの均一性を確保するために迅速である必要がある。ここでは、ゼブラフィッシュの胚から機能/鼓動心を収集するための迅速な手動の解剖プロトコルを提示する。これは、トランスクリプトーム解析によって、このような定量的リアルタイムポリメラーゼ連鎖反応（RT-qPCRの）として、心臓特異的な遺伝子発現レベルを決定するために、後続の心臓特異的RNA抽出のための必須の前提条件である。この方法は、他の組織と比較して、ゼブラフィッシュ胚の心臓の差動接着特性に基づいている。これは、迅速なPHYが可能になり流体せん断力の中断、段階的にろ過およびトランスジェニック、蛍光標識された心のマニュアルコレクションの組み合わせによる心外組織から心臓のsical分離。

独占的に心筋（図1）内twu34トランスジェニック系統9、緑色蛍光タンパク質（GFP）を発現する：ここでは、ゼブラフィッシュのTg（GFP myl7）を使用して、代表的な心臓の解剖実験について説明します。我々は、解剖心、それらが心臓サンプルの純度を評価するために誘導された胚の両方を収集した。簡単に言えば、ホモ接合のTg（myl7：GFP）は、すべて?...

Watch this Scientific Journal Video about Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis at JoVE.com

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

ゼブラフィッシュの心臓発生中の心臓遺伝子発現プロファイルを分析するために、総RNAを単離した心臓から抽出されなければならない。ここでは、心臓特異的mRNAを得るために、ゼブラフィッシュの胚からの迅速な手動の切開によって、機能/鼓動心を収集するためのプロトコルを提示する。

転写分析のための大規模ゼブラフィッシュ胚心臓解剖

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

large-scale-zebrafish-embryonic-heart-dissection-for-transcriptional

Research

JoVE Journal

Developmental Biology

14.2K ビュー.  Max Delbrück Center for Molecular Medicine. ゼブラフィッシュの心臓発生中の心臓遺伝子発現プロファイルを分析するために、総RNAを単離した心臓から抽出されなければならない。ここでは、心臓特異的mRNAを得るために、ゼブラフィッシュの胚からの迅速な手動の切開によって、機能/鼓動心を収集するためのプロトコルを提示する。 

ビデオ: Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

胚性マウスの心臓に免疫蛍光を用いて、心筋細胞の開発の分析

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

発生生物学

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain&#39;s macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution EM. Here we describe a universal method for nanotomy applied to investigate the zebrafish larval brain in health and upon non-invasive brain injury.

健康と負傷ゼブラフィッシュ脳の大規模な走査型透過電子顕微鏡（Nanotomy）

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

再現性の高い小分子ベースの分化プロトコルを使用したヒト多能性幹細胞から心筋細胞の大規模生産

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

クリア胚ハーツにおける冠状血管の分析

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

幼生ゼブラフィッシュ性腺組織の解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

マウス胎児腎臓の解剖および培養

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

ゼブラフィッシュの網膜におけるタンパク質局在を解決する相関超解像および電子顕微鏡観察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

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

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

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

鳥胚胚心筋細胞の生体内注入にマイクロインジェクション ベース システム

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

Surgical Size Reduction of Zebrafish for the Study of Embryonic Pattern Scaling

In the developmental process, embryos exhibit a remarkable ability to match their body pattern to their body size; their body proportion is maintained even in embryos that are larger or smaller, within certain limits. Although this phenomenon of scaling has attracted attention for over a century, understanding the underlying mechanisms has been limited, owing in part to a lack of quantitative description of developmental dynamics in embryos of varied sizes. To overcome this limitation, we developed a new technique to surgically reduce the size of zebrafish embryos, which have great advantages for in vivo live imaging. We demonstrate that after balanced removal of cells and yolk at the blastula stage in separate steps, embryos can quickly recover under the right conditions and develop into smaller but otherwise normal embryos. Since this technique does not require special equipment, it is easily adaptable, and can be used to study a wide range of scaling problems, including robustness of morphogen mediated patterning.&#160;

Here, we describe a method for reducing the size of zebrafish embryos without disrupting normal developmental processes. This technique enables the study of pattern scaling and developmental robustness against size change.

胚性パターンスケーリングの研究のためのゼブラフィッシュの外科的サイズ縮小

In the developmental process, embryos exhibit a remarkable ability to match their body pattern to their body size; their body proportion is maintained ...

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.