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>

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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><tbody><tr><td>Equipment for raising fish and collecting eggs&amp;nbsp;</td><td></td><td></td><td>see the Zebrafish Book&lt;sup&gt;11&lt;/sup&gt; 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>&lt;em&gt;e.g.&lt;/em&gt; Thermo Scientific Nanodrop 2000</td></tr><tr><td>Nucleic acid electrophoresis chamber</td><td></td><td></td><td></td></tr><tr><td>Petri dishes 4 cm &amp;Oslash;, 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>1.5 ml centrifugation tubes</td><td></td><td></td><td></td></tr><tr><td>15 ml and 50 ml 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&amp;trade; Round Gel Loading Tips in Sterile Rack, 1-200&amp;mu;l&amp;nbsp;</td><td>Santa Cruz</td><td>sc-201732</td><td></td></tr><tr><td>100 &amp;mu;m filter (BD Falcon 100 mm Cell Strainer)</td><td>BD Biosciences</td><td>352360</td><td></td></tr><tr><td>30 &amp;mu;m filter (Pre-Separation Filters-30 &amp;micro;m)</td><td>Miltenyi Biotec</td><td>130-041-407</td><td></td></tr><tr><td>Phase lock gel, heavy, 1.5 ml tubes&amp;nbsp;</td><td>Prime</td><td>2302810</td><td></td></tr><tr><td>Egg water medium</td><td></td><td></td><td>60 &amp;mu;g/ml Instant Ocean Sea Salts in ddH2O, 0.00001% (w/v) Methylene Blue</td></tr><tr><td>E3 medium&amp;nbsp;</td><td></td><td></td><td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 0.33 mM MgSO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Tricaine (3-amino benzoic acidethylester)</td><td>Sigma-Aldrich</td><td>A-5040</td><td>4 mg/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&amp;acute;s L-15 medium&amp;nbsp;</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&amp;nbsp;</td><td>Invitrogen</td><td>10814-010</td><td>20 &amp;micro;g/&amp;micro;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

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

14.4K 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

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

ゼブラフィッシュ胚からの単一細胞の単離とキャラクタリゼーション

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

発生生物学

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 ...

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.

心筋機械変形(DIAMOND)の変位解析は、胚性ゼブラフィッシュにおける心機能のセグメント的不均一性を明らかにする

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

Design and Microinjection of Morpholino Antisense Oligonucleotides and mRNA into Zebrafish Embryos to Elucidate Specific Gene Function in Heart Development

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.

The present protocol describes designing, preparing, and microinjecting a translational-blocking morpholino against a representative cardiac gene; Heart And Neural Crest Derivatives Expressed2 (hand2) into the yolk of newly fertilized zebrafish embryos to knock down gene function. It also shows a transient rescue of these "morphants" by co-injection of mRNA encoding this gene product.

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression.  ...

Light-sheet Fluorescence Microscopy to Capture 4-Dimensional Images of the Effects of Modulating Shear Stress on the Developing Zebrafish Heart

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths from the myocardium. Genetic program defects in the Notch signaling cascade are involved in ventricular defects such as Left Ventricular Non-Compaction Cardiomyopathy or Hypoplastic Left Heart Syndrome. Using this protocol, it can be determined that shear stress driven trabeculation and Notch signaling are related to one another. Using Light-sheet Fluorescence Microscopy, visualization of the developing zebrafish heart was possible. In this manuscript, it was assessed whether hemodynamic forces modulate the initiation of trabeculation via Notch signaling and thus, influence contractile function occurs. For qualitative and quantitative shear stress analysis, 4-D (3-D+time) images were acquired during zebrafish cardiac morphogenesis, and integrated light-sheet fluorescence microscopy with 4-D synchronization captured the ventricular motion. Blood viscosity was reduced via gata1a-morpholino oligonucleotides (MO) micro-injection to decrease shear stress, thereby, down-regulating Notch signaling and attenuating trabeculation. Co-injection of Nrg1 mRNA with gata1a MO rescued Notch-related genes to restore trabeculation. To confirm shear stress driven Notch signaling influences trabeculation, cardiomyocyte contraction was further arrested via tnnt2a-MO to reduce hemodynamic forces, thereby, down-regulating Notch target genes to develop a non-trabeculated myocardium. Finally, corroboration of the expression patterns of shear stress-responsive Notch genes was conducted by subjecting endothelial cells to pulsatile flow. Thus, the 4-D light-sheet microscopy uncovered hemodynamic forces underlying Notch signaling and trabeculation with clinical relevance to non-compaction cardiomyopathy.

Here, we present a protocol to visualize developing hearts in zebrafish in 4-Dimensions (4-D). 4-D imaging, via light-sheet fluorescence microscopy (LSFM), takes 3-Dimensional (3-D) images over time, to reconstruct developing hearts. We show qualitatively and quantitatively that shear stress activates endocardial Notch signaling during chamber development, which promotes cardiac trabeculation.

光シート蛍光顕微鏡のゼブラフィッシュ心臓の開発上のせん断応力を変調効果の 4次元画像をキャプチャするには

The hemodynamic forces experienced by the heart influence cardiac development, especially trabeculation, which forms a network of branching outgrowths ...

生物工学

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫の心臓の解剖、少年と大人ゼブラフィッシュ、ゼブラフィッシュ</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

生物学

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

ディテールに秘められたゼブラフィッシュ胚心臓の免疫染色

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

ハイスループットのアプリケーションのための大人のゼブラフィッシュハーツの回復

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Light Sheet Microscopy of Fast Cardiac Dynamics in Zebrafish Embryos

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid external development, tractable genetics, and translucency of the zebrafish, Danio rerio, LSFM has delivered insights into cardiac form and function at high spatial and temporal resolution without significant phototoxicity or photobleaching. Imaging of beating hearts challenges existing sample preparation and microscopy techniques. One needs to maintain a healthy sample in a constricted field of view and acquire ultrafast images to resolve the heartbeat. Here we describe optimized tools and solutions to study the zebrafish heart in vivo. We demonstrate the applications of bright transgenic lines for labeling the cardiac constituents and present novel gentle embedding and immobilization techniques that avoid developmental defects and changes in heart rate. We also propose a data acquisition and analysis pipeline adapted to cardiac imaging. The entire workflow presented here focuses on zebrafish embryonic heart imaging but can also be applied to various other samples and experiments.

We describe optimized tools to study the zebrafish heart in vivo with light sheet fluorescence microscopy. Specifically, we suggest bright cardiac transgenic lines and present new gentle embedding and immobilization techniques that avoid developmental and heart defects. A possible data acquisition and analysis pipeline adapted to cardiac imaging is also provided.

ゼブラフィッシュ胚における高速心臓ダイナミクスの光シート顕微鏡

Embryonic cardiac research has greatly benefited from advances in fast in vivo light sheet fluorescence microscopy (LSFM). Combined with the rapid ...

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological phenomena. Zebrafish have been used as an effective genetic model to study numerous questions related to heritable disease, development, and toxicology at the whole-organ and -organism level. Due to the well-annotated and mapped zebrafish genome, numerous tools for gene editing have been developed. However, the efficacy of generating and ease of detecting precise knock-in edits using CRISPR is a limiting factor. Described here is a CRISPR-Cas9-based knock-in approach with the simple detection of precise edits in a gene responsible for cardiac repolarization and associated with the electrical disorder, Long QT Syndrome (LQTS). This two-single-guide RNA (sgRNA) approach excises and replaces the target sequence and links a genetically encoded reporter gene. The utility of this approach is demonstrated by describing non-invasive phenotypic measurements of cardiac electrical function in wild-type and gene-edited zebrafish larvae. This approach enables the efficient study of disease-associated variants in a whole organism. Furthermore, this strategy offers possibilities for the insertion of exogenous sequences of choice, such as reporter genes, orthologs, or gene editors.

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene variant.

CRISPR-Cas9を介したゼブラフィッシュハートの正確なノックイン編集

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...