Name: development of an in vitro assay to quantitate hematopoietic stem and progenitor cells (hspcs) in developing zebrafish embryos
Uploaded: 2017-11-30T15:00:00.000+00:00
Duration: 10 min 28 s
Description: Watch this Scientific Journal Video about Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos at JoVE.com

国立衛生研究所によって提供された資金 (NIH: K01-DK087814-01A1 D.L.S. に)、バイオ テクノロジーにおける教育研究のカリフォルニア州立大学プログラム (CSUPERB: D.L.S. 脊椎動物の造血ニッチの分子制御)大学院の研究室 (A.C.B.)、カリフォルニア州立大学チコ校でから。

カリフォルニア州立大学、Chico、機関動物ケアおよび使用委員会 (IACUC) 諮問委員会は、以下に述べるすべての方法を承認しました。
1. 漂白剤 48 hpf ゼブラフィッシュ胚
<ol><li>15 cm (w) x 15 cm (l) 7 cm (h) プラスチック容器 × 3 の洗い場を作成: 1000 mL 胚中 (E3;表 1参照) で 5% の漂白剤の 1 mL と 1)、2) 滅菌 E3 と 3) 滅菌 E3。胚が水没する溶液 500 mL を各コンテナーにいるを確認します。 注: テスト条件ごとに、少なくとも 10 の胚が必要でしょう。この洗浄方法は、最大 200 の胚を収容できます。</li>
	<li>転送ピペット茶漉しに胚を配置し、5 分 (内の胚) と茶漉しを削除し、洗浄駅 #2; 5 分間の手洗い所 #1 の卵が水没最後の 5 分間洗浄駅 3 番に繰り返します。</li>
	<li>最後の洗浄後茶漉しクリーン 10 cm シャーレを逆さま回すと胚を優しく洗浄からすすぎ胚は滅菌 E3 と茶漉しと転送ピペットをオフします。</li>
</ol>2. Dechorionate 胚
<ol><li>転送ピペットと削除し、シャーレからできるだけ多くの E3 を破棄、胚に dechorionation プロテアーゼ (10 mg/mL) 500 μ L を追加します。5 分は軽く絨毛を完全に削除するシャーレの側の室温で孵化させなさい。</li>
	<li>血清ピペット滅菌 E3 プロテアーゼを希釈するの 20 mL を追加します。胚を解決することを許可し、転送ピペットで E3 を削除します。このステップを 3 回繰り返します dechorionation プロテアーゼの痕跡をすべて削除します。</li>
</ol>3. 胚サンプルの準備
<ol><li>P1000 ピペットを使用して、単一の滅菌 1.5 mL 遠心チューブに 10 胚を配置します。</li>
	<li>ピペットで E3 を削除し、破棄します。 注意: ピペット ケアでの胚は容易に転置され、次の洗浄手順中に破棄されます。</li>
</ol>4. 解離過程の胚の準備
<ol><li>層流フードのサンプルを転送し、1 mL を追加することによって胚を洗って滅菌 E3。P1000 ピペットで上澄みを外し、管の底に沈殿する胚を許可します。3 洗浄の合計に対して繰り返します。</li>
	<li>最後の洗浄後、E3 を削除し、破棄します。粘液コーティング (胞子、酵母やそれに閉じ込められる可能性があります細菌) 周囲の萌芽期のゼブラフィッシュを削除する E3 で 10 mM ジチオトレイトール (DTT) の 1 つの mL を追加します。微量遠心チューブを水平に置くし、層流フードの 25 分間室温でインキュベートします。</li>
</ol>5. 胚解離
<ol><li>サンプル (Ca2 +と Mg2 +) を持つ滅菌 DPBS 1 mL で 3 回洗います。最後の洗浄後 (Ca2 +と Mg2 +) の DPBS の 500 μ L と 5 mg/mL (26 U/mL) 解離プロテアーゼの 5 μ L を追加します。 注意: この酵素は、Ca2 +と Mg2 +を必要があります、ので、DPBS にこれらのイオンが含まれていることを確認します。</li>
	<li>層流フードで 60 分位サンプルの 180 rpm 水平軌道シェーカーで 37 ° C でサンプルをインキュベートし、サンプルは完全に分離されるまで、P1000 を持つ胚をカップ刻んだ。 注意: は、彼らが解離になるときを判断する定期的に胚を確認します。胚ソリューションの組織の存在のいくつかの量が必要それは、完全に均質なしないでください。過剰のダイジェスト (補足図 1を参照) の HSPCs を破壊します。</li>
</ol>6. 解離胚の準備
<ol><li>5 mL ポリスチレンの上部の貯水池にピペット、10 解離胚はラウンド 35 μ m の細胞のストレーナー キャップと底部チューブです。</li>
	<li>ピペット、リンス (いいえ Ca2 +や Mg2 +) を持つ滅菌 PBS と転写液 5 mL ポリスチレン管を含むペトリネット チューブ フィルター ステップ 6.1 からの細胞。液 4 mL、5 mL のポリスチレン管に存在までを繰り返します。 注: PBS、DPBS、または他の等張バッファー ソリューション可能性があります使用するこの段階で彼らは、Ca2 +や Mg2 +を含まない限り、
</li>
	<li>4 ° C および均質化細胞サンプルをペレットに 5 分間、300 x g でチューブを遠心します。ピペット、削除、丸底チューブから上澄みを廃棄します。チューブの下部に小球形細胞を妨害しないように注意してください。100 μ L の PBS で細胞を再懸濁します。</li>
</ol>7. セルロースの調製
<ol><li>完全なセルロース (表 1) 原液 x 1 を生成し、16 G 針、3 mL シリンジと滅菌丸底 14 mL チューブに 2.5 mL を追加します。テスト条件ごとに 1 つの管を使用します。</li>
	<li>
サイトカイン、小さい分子、または各サンプルを調査する他のエージェントを追加します。
	<ol>
<li>骨髄性分化 1% 鯉血清および 0.3 mg/mL 組換えゼブラフィッシュ顆粒球コロニー刺激因子 (Gcsf)34メチル セルロース培地を追加します。スヴォボダらを参照してください。37鯉血清および組換えサイトカインを生成する方法について完全な説明のため。</li>
		<li>赤血球分化・ メチル セルロース培地に 1% 鯉血清と 0.1 mg/mL 組換えゼブラフィッシュ エリスロポエチン (Epo)38を追加します。</li>
		<li>血液前駆細胞を調べると、Epo と Gcsf を追加します。 注意: サイトカイン ・添加物ないの合計の総容積の 10% 以上媒体は個々 のコロニーを識別する十分な粘性できません。</li>
	</ol>
</li>
</ol>8. 解離胚セルロース添加
<ol><li>ピペットを使用して、遺伝子組換えサイトカインと鯉の血清と準備されたセルロースの表面に解離 10 胚の 100 μ L を追加します。キャップのふたとサンプルを完全に均質に優しく渦です。各チューブは今 10 胚解離の組織を含める必要があります。</li>
	<li>3 mL 注射器や 16 G 針、2 別の 35 ミリメートルのシャーレにサンプルの分注 1 mL を使用しています。サンプルはペトリ皿に完全に分散していることを確認します。サンプルごとに繰り返します。</li>
	<li>15 cm シャーレにセルロースの細胞と 35 mm ペトリ皿を配置します。それぞれの 15 cm の皿に 5 mL のサンプルを湿らせるために滅菌水 1 つの 35 mm ペトリ プレートを追加します。32 ° C および 5% CO2で 7-10 日間インキュベートします。</li>
</ol>9. HSPC 由来コロニー形成単位 (CFUs) の可視化
<ol><li>7 日間をインキュベート後 40 〜 100 倍の可視化と列挙に倒立顕微鏡にサンプルを配置します。この時点で、個々 のコロニーをピペットで分離され、染色や遺伝子解析を受けることができます。</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+kidney+stromal+cell+lines+support+multilineage+hematopoiesis.">Zebrafish kidney stromal cell lines support multilineage hematopoiesis.</a> Blood. 114, 279-289 (2009).</li><li>Svoboda, O., 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=Ex+vivo+tools+for+the+clonal+analysis+of+zebrafish+hematopoiesis.">Ex vivo tools for the clonal analysis of zebrafish hematopoiesis.</a> Nat Protoc. 11, 1007-1020 (2016).</li><li>Paffett-Lugassy, 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=Functional+conservation+of+erythropoietin+signaling+in+zebrafish.">Functional conservation of erythropoietin signaling in zebrafish.</a> Blood. 110, 2718-2726 (2007).</li><li>Stachura, D. L., Traver, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+dissection+of+zebrafish+hematopoiesis.">Cellular dissection of zebrafish hematopoiesis.</a> Methods Cell Biol. 133, 11-53 (2016).</li><li>Traver, D., 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=Transplantation+and+in+vivo+imaging+of+multilineage+engraftment+in+zebrafish+bloodless+mutants.">Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants.</a> Nat Immunol. 4, 1238-1246 (2003).</li><li>Kobayashi, I., 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+and+localization+of+side+population+(SP)+cells+in+zebrafish+kidney+hematopoietic+tissue.">Characterization and localization of side population (SP) cells in zebrafish kidney hematopoietic tissue.</a> Blood. 111, 1131-1137 (2008).</li><li>Henninger, 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=Clonal+fate+mapping+quantifies+the+number+of+haematopoietic+stem+cells+that+arise+during+development.">Clonal fate mapping quantifies the number of haematopoietic stem cells that arise during development.</a> Nat Cell Biol. 19, 17-27 (2017).</li><li>Tamplin, O. 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=Hematopoietic+stem+cell+arrival+triggers+dynamic+remodeling+of+the+perivascular+niche.">Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.</a> Cell. 160, 241-252 (2015).</li></ol>

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ゼブラフィッシュ モデル システムかつ決定的に原始的な脊椎動物の造血を勉強するための効率的で効果的かつ安価なモデルとなっています。高速で安価なといくつかの技術的な困難の試金の世代は、小分子をテスト、突然変異体の胚を分析、HSPC 生物学の重要な分子経路の解明に利用できます。体外メッキ HSPCs のアダルト ゼブラフィッシュからは突然変異誘発、サイトカイン、造血?...

増結は、生物の生存に必要な成熟した血液細胞の多数を作るプロセスです。成熟した血液を構成する発達制限携帯各種に造血幹細胞 (造血) の分化を含むキーの発達プロセスです。これらの造血システムが枯渇しないように、初期胚から死に至るまで維持されている必要があります更新する必要があります。ポスト mitotic の血液細胞の大半を十分に補充するために、脊椎動物の造血幹・前駆細胞 (HSPCs) の定数の分化・増殖を必要な毎日がリサイクルされていると。HSCs は制限された幹細胞のサブセットに最初の差別化によって成熟した血液細胞を生成します。一般的なリンパ球前駆細胞 (CLPs)1、最終的に T、B、NK 細胞を作り出す、および一般的な骨髄前駆細胞 (Cmp)2顆粒球、赤血球、巨核球マクロファージを生成します。これらの前駆細胞が特定の細胞系譜を生成するのにコミットしているし、骨髄赤芽球系前駆細胞 (Mep) 顆粒球や血小板、赤血球を生成するなどの詳細の発達制限前駆細胞へと分化好塩基球、好酸球、好中球、マクロファージの2を生成するマクロファージ前駆細胞 (省)。識別し、これらの前駆細胞を分離することにより重要な分子経路の同定、造血分化に関与して白血病などの多くの造血器疾患発生これらの前駆細胞が正常に失敗するとき区別します。
過去数十年間、ゼブラフィッシュ (動脈分布) モデル システム胎児および成体造血研究重要な研究ツールとなっています。ゼブラフィッシュ遺伝子解析に適しているで、系統発生的に最も低い脊椎動物のモデル種と同様血管と人間に造血システムを持つ。Zebrafish の胚が子宮、ex開発し、48 時間ポスト内受精 (hpf) は HSPCs3,4,5,6,7、8を生成します。ゼブラフィッシュは、また非常に肥沃な女性シングル クラッチで 100 個の卵を産卵大きいサンプルの大きさおよび実験的レプリケーションを可能にします。Zebrafish の胚が光学的に透明な造血系の顕微可視化を可能にします。蛍光遺伝子組換え数行ゼブラフィッシュrunx1など HSCs をマーキング: EGFP 魚9、 cd41: EGFP 魚10、およびkdrl:mCherry;cmyb: GFP3二重肯定的な動物、HSC の出現と拡大生体内で3,4,7,8、ライブ、リアルタイムの可視化を可能にします。 9。ゼブラフィッシュの速い世代時間と開発子宮 exにおける突然変異誘発の研究11,12,13,14,15と薬物使用につながっています。16,17,18,19、20人の血液疾患の治療の約束を保持する化合物をスクリーニングします。全体的にみて、造血系、存在と形質転換線、および迅速な再生時間を容易に開発の保全したゼブラフィッシュ造血の研究のための安価な迅速、柔軟、かつ理想的なモデル。
分離して造血をテストの多数の方法は、哺乳類の造血システムで開発されています。調査官は、流出色素25,26HSCs の能力を悪用すると同様、HSCs21,22,23,24をマークに細胞表面受容体の組み合わせを利用できます。彼らにラベルを付ける蛍光活性化セル (FACS) を並べ替えの物理的な分離ができます。セルが、HSC であることを証明するには、内因性 HSPCs、推定 HSCs 移植と成熟した血液細胞の種類すべての長期的な多系統の再構成を観察することを破壊するホスト動物を照射する必要があります。これらのアッセイは、造血細胞移植免疫のマッチングを容易にする近交系マウス系統と多数細胞表面の抗体があるのでマウスでよくはたらきます。ただし、いくつかのゼブラフィッシュ造血細胞表面の抗体は、生成された27, 同定と HSCs の分離を妨げることをされています。マークし、ゼブラフィッシュ HSCs を分離する最も一般的な方法は、トランスジェニック動物、セル固有のプロモーターは、蛍光タンパク質の発現を運転するという。研究は可視化して、背側大動脈の腹側の壁の HSCs の列挙にはこのテクニック3,4,8,9を利用した顕微鏡。他の研究室は、ゼブラフィッシュ28,29系統のクローンを生成しているし、MHC 一致動物30の成功移植を実行しています。ただし、これらのテクニックは、多くの実験室は膨大なコスト、技術的に困難でありは時間がかかる。これらの問題に対処するため研究所は存在、増殖率と HSPCs31,32,33、分化能力をテストするためのいくつかの in vitroアッセイを生成しています。 34,35,36。これらの試 HSPCs体外31,32,33,34,35,36, の救助の増殖・分化を示す造血障害36と発見とサイトカイン33,34,35のテストのための効率的な方法です。彼らはまた、HSPC 生物学31,32の遺伝子を識別するために利用されています。この研究では、私たちが一歩これらの試さらに、ゼブラフィッシュの胚の HSPCs の定量を可能します。これらの試また変異動物の HSPCs の数を量的に利用することができ、動物が破壊的造血薬で治療します。本質的に、これらの試金は高速、現在のいくつかの技術的な課題、HSPC 数字を量的に表わす、彼らの増殖を調べるし、分化におけるブロックを調査する安価な方法をあります。

<table><tbody><tr><td>10% Bovine Serum Albumin in Iscove&#039;s MDM</td><td>StemCell Technologies&amp;nbsp;</td><td>9300</td><td></td></tr><tr><td>DPBS (10x) with Calcium&lt;sup&gt;2+&lt;/sup&gt; and Magnesium&lt;sup&gt;2+&lt;/sup&gt;</td><td>Life Technologies</td><td>14080-055</td><td></td></tr><tr><td>HyClone PBS (1x)</td><td>GE Healthcare Life Sciences</td><td>sh30256.01</td><td></td></tr><tr><td>&amp;nbsp;DMEM 500 mL</td><td>Corning CellGrow</td><td>10-017-CV</td><td></td></tr><tr><td>&amp;nbsp;Ham&#039;s F12 500 mL</td><td>Corning CellGrow</td><td>10-080-CV</td><td></td></tr><tr><td>FBS 500 mL</td><td>Gemini Bio-Products</td><td>100-108</td><td></td></tr><tr><td>HEPES 100 mL (1 M)</td><td>Gibco life technologies</td><td>15-630-080</td><td></td></tr><tr><td>Penicillin/streptomycin (5000 U/mL&lt;br/&gt; and 5000 mg/mL) with L-Glutamine (200 mM)</td><td>Corning Mediatech</td><td>30-009-CI</td><td></td></tr><tr><td>Gentamycin Sulfate 10 mL (50 mg/mL)</td><td>Corning Mediatech</td><td>30-005-CR</td><td></td></tr><tr><td>1.5 mL MCF tube</td><td>FisherBrand</td><td>05-408-129</td><td></td></tr><tr><td>3 mL 23gx1 injection needle with Luer lock</td><td>BD Safety Glide</td><td>305905</td><td></td></tr><tr><td>5 mL polystyrene round bottom tube with cell strainer cap</td><td>Corning Falcon</td><td>352235</td><td></td></tr><tr><td>Methocel MC</td><td>Sigma-Aldrich</td><td>64630</td><td></td></tr><tr><td>14 mL Polystyrene round bottom tube</td><td>Corning Falcon</td><td>352057</td><td></td></tr><tr><td>10 mm polystyrene easygrip Petri dish</td><td>Corning Falcon</td><td>351008</td><td></td></tr><tr><td>Librease TM</td><td>Roche Sigma-Aldrich</td><td>5401119001</td><td>dissociation protease</td></tr><tr><td>Pronase</td><td>Roche Sigma-Aldrich</td><td>11459643001</td><td>dechorionation protease</td></tr><tr><td>15 cm Petri dish</td><td>Corning Falcon</td><td>351058</td><td></td></tr><tr><td>AB</td><td>Zebrafish International Resource Center (ZIRC)</td><td>ZL1</td><td>zebrafish strain used</td></tr><tr><td>Dithiolthreitol (DTT)</td><td>Sigma-Aldrich</td><td>646563</td><td></td></tr></tbody></table>

development of an in vitro assay to quantitate hematopoietic stem and progenitor cells (hspcs) in developing zebrafish embryos

造血は造血幹・前駆細胞 (HSPCs) が分化成熟した血液を構成する異なる細胞系譜の多数、重要な細胞プロセスです。これらの HSPCs の分離・同定は難しいので定義された事後彼らは、特定のセルにその分化後だけ定義できます。過去数十年間、ゼブラフィッシュ (動脈分布) 造血を調査するモデル有機体となっています。ゼブラフィッシュ胚の子宮 ex、開発し、48 時間後受精 (hpf) によって HSPC 分化を評価するために決定的な HSPCs アッセイを生成していると移植を利用した増殖機能が開発されているとそれ以降。共焦点顕微鏡を用いた特殊な形質転換線の可視化に加えて造血システムの再構成。ただし、これらの試金はコストが法外な技術的に困難で、多くの実験室では時間がかかるです。HSPCs を評価するための in vitroモデルの開発はより速く、費用対効果になるし、以前に比べて現在の少ない困難に HSPC 生物学に影響を与える突然変異や薬物の画面をすばやく評価する所を可能にするメソッドが記述されています。HSPCs を評価するためにこの小説の in vitroアッセイは、めっき解離全体ゼブラフィッシュ胚とだけ HSPC の分化と増殖を促進する追加の外因性の要因によって実行されます。胚が単一のセルに分離される、単一の前駆細胞から生じるコロニー形成単位 (CFUs) を生成させる HSPC 支持のコロニー刺激因子とメッキします。これらの試金からの分子経路のより注意深い検査ように HSPC 増殖、分化および脊椎動物の造血の基盤とその調節不全を理解する研究者を可能にする規制を担当中に病。

HSPC ゼブラフィッシュ胚の数を評価するために 48 の hpf 胚消化、造血支持の外因性の成長因子とセルロースでメッキされ (図 1A) の 7 日間の培養します。7 日後、コロニー形成単位 (CFUs) が列挙 (図 1B) とイメージ (図 1C)。追加別のサイトカインを制御することによ?...

Watch this Scientific Journal Video about Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos at JoVE.com

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

造血幹・前駆細胞 (HSPCs) ゼブラフィッシュ胚を量的に簡単な方法を紹介します。成熟した血液へと分化支持の要因とセルロースでめっき解離ゼブラフィッシュから HSPCs います。これにより血液の検出欠陥や薬剤のスクリーニングを簡単に行なうことができます。

ゼブラフィッシュ初期胚細胞 (HSPCs) を造血幹・前駆細胞の量的にIn Vitroアッセイの開発

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

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Research

JoVE Journal

Developmental Biology

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

7.9K ビュー.  California State University, Chico. 造血幹・前駆細胞 (HSPCs) ゼブラフィッシュ胚を量的に簡単な方法を紹介します。成熟した血液へと分化支持の要因とセルロースでめっき解離ゼブラフィッシュから HSPCs います。これにより血液の検出欠陥や薬剤のスクリーニングを簡単に行なうことができます。

ビデオ: Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

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 cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

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 ...

発生生物学

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

大人のゼブラフィッシュ脳由来神経球の単離および培養

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

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 ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

胞胚の開発の外科的融合による並体結合ゼブラフィッシュ胚の生成

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

Here, we present an effective, easy, and fast 3D culture protocol for the formation of spheroids of two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte).

ゼブラフィッシュ胚および肝臓細胞株のスフェロイドの3D培養法

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known ...

環境学

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

iPSC-ICにおけるゼブラフィッシュ胚の全山ハイブリダイゼーションとチューブ形成アッセイは血管開発におけるエンドリンの役割を研究する

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

ゼブラフィッシュの化学スクリーンのための自動化された画像と解析の開発。

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

生物学

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

発達中のゼブラフィッシュにおける変化した血液形成を検出し定量するためのフローサイトメトリーの使用

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

ゼブラフィッシュのMyc誘導性T細胞急性リンパ芽球性白血病における側方集団の単離

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

免疫・感染学

<meta charset="utf8"></head><body>再生・開発研究のための堅牢なゼブラフィッシュ細胞移植

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>著者スポットライト:発生および再生中のゼブラフィッシュ神経系における細胞接続性のマッピング

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

ゼブラフィッシュの胚および幼生における高分解能細胞移植

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...