The authors thank Drs. Hukriede and Tsang for discussions. M.K. was supported by an NIH training grant (T32EB001026). This work was supported in part by grants from the American Liver Foundation, the March of Dimes Foundation (5-FY12-39), and the NIH (DK101426) to D.S.

ゼブラフィッシュは、標準的な手順に従って上昇して飼育しました。実験は、ピッツバーグ大学の施設内動物管理使用委員会によって承認されました。 胚/幼虫の調製<ol><li>時限交配を行うために、成人男性と女性のTg（fabp10a：CFP-NTR）を設定S931半接合またはホモ接合性魚類のO / Nを、それらの間の仕切りを配置します。相手が所望される場合は、次の朝、この分周器を取り外します。細かいプラスチックメッシュストレーナーを用いて水を負担することによって胚を収集します。 </li><li>逆さまにストレーナを回して、スクイーズボトルから分配卵の水の細い流れにメッシュ表面をすすぎます。卵、水25mlで100ミリメートルペトリ皿に100胚を移します。ペトリ皿当たりない100以上の胚を維持し、28℃でそれらを発生させます。 </li><li>色素沈着を抑制するために、10時間後に受精（HPF）の前にペトリ皿にフェニルチオ尿素（PTU）を追加し、電子メールを上げます80 HPFまでmbryos /幼虫。 PTUの最終濃度は0.2 mMです。注意：PTUは有毒です。適切な取り扱いガイドラインに従って使用してください。 </li><li>ペトリ皿の中に0.016％の最終濃度で、3-アミノ安息香酸エチルエステル（トリカイン）を加えることにより、幼虫を麻酔。そして、落射蛍光顕微鏡、ソートCFP陽性幼虫を使用。同様の肝臓サイズで幼虫を使用して、小さいまたは大きい肝臓のものを捨てます。 注：ヘミ接合性およびホモ接合性幼虫の間に肝切除の程度の差がないため、任意のCFP陽性幼虫は関係なく、強度、使用することができます。注意：トリカインは有毒です。適切な取り扱いガイドラインに従って使用してください。 </li><li>トリカインを含む卵の水を除去し、0.2 mMのPTUを補充した卵の水を加えます。 28℃での胚/幼虫を保管してください。 </li></ol> 2. MTZトリートメント<ol><li> 1にMTZ粉末0.171グラムを溶解することにより、新鮮な10mMのMTZ溶液を調製します00ミリリットルの卵の水は、0.2％DMS​​Oおよび0.2 mMのPTUを補いました。完全にMTZを溶解するために、20〜30分間急速に撹拌しながら室温で溶液を混合。 MTZを製造する場合MTZを可溶化するために役立つと幼虫にMTZの浸透を高めるために、DMSOを追加します。注意：長時間の暴露またはMTZ濃度の増加は、毒性です。適切な取り扱いガイドラインに従って使用してください。 </li><li>二つの異なるの100mmペトリ皿またはマルチウェルプレートに幼虫の所望の数を転送することにより、対照群と実験群に幼虫を区切り。実験群では、10 mMのMTZ溶液中の幼虫を保ちます。対照群のために、0.2％のDMSOを含む卵水に保管してください。 </li><li> MTZの光不活性化を防止するために、プレート又はアルミニウム箔でMTZ溶液を含むペトリ皿を覆い、そして28℃でインキュベートします。 MTZ処理の時間は、幼虫期およびトランスジェニックラインに依存しています。 </li><li>で同様の重度の肝切除を観察するために、 <eM&gt;のTg（fabp10a：CFP-NTR）ライン、（36時間の期間）3.5日後受精（DPF）のいずれかから、または（24時間の期間）5 DPFからMTZ溶液で幼虫を扱います。 </li></ol>胆道主導の肝再生の3分析<ol><li>アブレーション期間の終わりに、プレートからMTZ溶液を除去。卵の水25mlを添加することにより、MTZ処理した幼虫を洗ってください。卵の水を廃棄する前に幼虫を洗浄し、プレートを2〜3回渦巻きます。 3回繰り返し、その後、もう一度卵水に幼虫を浸します。 MTZ処理した幼虫における心臓浮腫と幼虫の死を回避するために、幼虫を固定化するために0.008％の最終濃度で、トリカインの低濃度を追加します。 </li><li>肝臓分析のために、CFPフィルターと落射蛍光顕微鏡下でMTZ処置幼虫の肝臓サイズを調べます。肝臓の相対サイズに基づいて肝切除レベルを評価：大（非アブレーション; 0〜10％の幼虫）、中（部分的に切除さを、10-20％）、及び非常に小さな（完全に切除された; 80〜90％）。 </li><li>非アブレーションされたまたは部分的に切除された肝臓と幼虫を削除します。唯一の重度の肝切除の指標である小さな肝臓で幼虫を保ちます。再生0時間の時点（R0H）で肝臓分析のために、MTZの洗い出しとCFPのソート後の幼虫を収穫。それ以外の場合は、収穫の準備ができるまで、0.2 mMのPTUを補充した卵水にソートされた幼虫を保ちます。 </li><li> 1.5ミリリットルマイクロ遠心チューブに回収した幼虫を転送し、PEM緩衝液中の3％のホルムアルデヒドの固定液で卵の水を交換してください。回転子O上/ Nチューブを4℃でインキュベートします。 </li><li> PBSDT 1mlの固定液を交換し、5分間ローテーターの幼虫を含むチューブを回転させます。 </li><li>手動で解剖顕微鏡下で卵黄と胸のフィンを取り外し、ピンセットをPBSDTを含有し、使用して100ミリメートルのペトリ皿に固定された幼虫を転送します。 1.5mlチューブにアップTP 20解剖幼虫を転送します。 </li> &lt;LI&gt;ブロッキング溶液1mlのPBSDT液を交換し、2時間室温で回転体にチューブを揺すります。 </li><li>一次抗体を含むブロッキング溶液100μlのブロッキング溶液を交換してください。ゆっくりと回転体のO / Nで4°Cのチューブを揺すります。 </li><li>一次抗体溶液を除去し、10分間PBSDTで毎回5回洗浄します。その後、二次抗体を含む溶液を阻止する100μlのを追加し、2時間室温で回転装置上でそれを揺すります。 </li><li>回転子に揺動することにより、室温で10分間PBSDTで毎回5回洗浄します。 </li><li>顕微鏡スライド上に横方向に幼虫を向けると装着メディアのドロップを追加します。慎重にカバーガラスでそれらをカバーし、ネイルポリッシャーでカバーガラスをシール。今は、共焦点顕微鏡のための準備ができています。 </li></ol>

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著者は、彼らが競合する金融利害関係がないことを宣言します。

重度、軽度ではない、肝切除は、胆管駆動の肝臓の再生を誘発します。 CFP-NTR導入遺伝子：したがって、MTZの治療およびウォッシュアウトの後、それはfabp10aから固有のCFPの蛍光によって評価することができるMTZ処理した幼虫の肝臓サイズを調べることが重要です。 MTZ処理した幼虫の小さな割合は、非アブレーション表示（0-5％）または（10〜20％）の肝臓の一部アブレー?...

肝臓は非常に再生器官です。肝臓再生中に、肝細胞の再生、肝臓の実質細胞は、既存の肝細胞（肝細胞ドリブン肝再生）またはBECS（胆汁ドリブン肝再生）1,2から誘導されます。肝臓損傷は、通常、既存の肝細胞の増殖を誘発します。肝細胞増殖が損なわれたときただし、BECS 2-4を肝細胞に寄与することができます。肝再生のこれらの2つのモードは、臨床的に重要です。 （理由肝腫瘍やライブ肝ドナーの例えば ）ヒト肝臓の一部を外科的に除去すると、残りの肝臓における肝細胞は、失われた肝臓塊を回復するために増殖します。 BECSまたは肝臓前駆細胞（たLPC）は、肝細胞の再生5,6に寄与するように見えるように対照的に、重篤な肝疾患を有する患者において、肝細胞増殖が大きく損なわれる。げっ歯類2/3部分肝切除モデル、中肝細胞は有意に肝細胞主導の肝再生7,8の現在の理解に貢献してきました、失われた肝臓塊を回復するために増殖します。しかし、再生肝細胞は、主にBECSに由来する有効なげっ歯類モデルはありません。いくつかのげっ歯類の肝臓毒素モデルは胆道駆動型肝再生2-4の同定をもたらしたが、マウスにおける最近の系譜トレースの研究では、これらのモデル9,10に肝細胞の再生にBECSの最小の寄与を示しています。部分肝切除11-13とアセトアミノフェン誘発性肝障害14,15を含むげっ歯類の肝障害モデルのいくつかは、ゼブラフィッシュに適用され、肝臓再生に関与する新規遺伝子または経路の同定につながってきました。しかし、肝細胞主導型ではなく、BECドリブン、肝再生は、これらのゼブラフィッシュの肝障害モデルで発生します。したがって、新規な肝臓損傷モデルとは、BECSは広くregeneratに寄与します肝細胞をるがBEC主導の肝再生のよりよい理解のために必要とされます。 ここで説明肝切除モデルの全体的な目標は、（1）BECSは広範囲の肝細胞の再生に寄与し、そして（2）BECドリブン肝再生の基礎となる分子及び細胞メカニズムを解明した肝損傷モデルを生成することです。私たちは、損傷の重症度は、肝臓再生のモードを決定するという仮説を立てました。このように、我々は、胆管駆動の肝再生が厳しい肝細胞傷害の際に開始するだろうと予測しました。この仮説を検証するため、我々はトランスジェニック系統を生成することにより、ゼブラフィッシュの肝臓損傷モデルを開発し、Tgは（fabp10a：CFP-NTR）S931は 、ことは非常に肝細胞特異的fabp10a下シアン蛍光タンパク質（CFP）と融合した細菌ニトロレダクターゼ（NTR）を発現しますプロモーター。 NTRは、非毒性のプロドラッグを変換するので、メトロニダゾール（MTZ）は、細胞傷害性薬物に、それだけで意図アブレーションNTR-この場合、16-18肝細胞発現細胞。 MTZ治療の持続時間を操作することによって、肝切除の程度を制御することができます。このモデルを使用して、我々は最近、重度の肝細胞の喪失時に、BECSは広く、さらに他の二つの独立した研究20,21によって確認された肝細胞19を 、再生を生じさせることを報告しました。したがって、上記の齧歯類及びゼブラフィッシュ肝臓損傷モデルと比較して、我々の肝切除モデルはBEC駆動型肝再生を研究するため、より有利です。 このプロトコルは、ゼブラフィッシュ肝切除モデルを用いて、肝再生の実験を行うための手順を記載しています。このモデルは、胆汁駆動型肝再生のメカニズムを決定するための、および化学スクリーンが抑制または肝再生を増強することができる小分子を同定するために適切です。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>3-amino benzoic acid ethyl ester (Tricaine) powder</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td>Make 0.4% (15 mM) tricaine stock : Bring 0.4 g tricaine to 100 mL with egg water. Adjust to pH 7.&#160;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Metronidazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1547</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8532</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium sulfate dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C3771</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Phenylthiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media (Vectashield)</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>100 x 15 mm petri dish&#160;</td>
			<td>Denville Scientific Inc.</td>
			<td>M5300</td>
			<td></td>
		</tr>
		<tr>
			<td>clear nail polish</td>
			<td>Fisher Scientific</td>
			<td>50949071</td>
			<td></td>
		</tr>
		<tr>
			<td>fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td>Dumont #5&#160;</td>
		</tr>
		<tr>
			<td>glass slide</td>
			<td>Fisher Scientific</td>
			<td>12-544-1</td>
			<td></td>
		</tr>
		<tr>
			<td>glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-540-A and 12-542-A</td>
			<td>18 x 18 mm</td>
		</tr>
		<tr>
			<td>zn-5 (mouse anti-Alcam)</td>
			<td>ZIRC</td>
			<td>zn-5</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>goat anti-Hnf4a</td>
			<td>Santa Cruz</td>
			<td>sc-6556 (C-19)</td>
			<td>1:50 dilution</td>
		</tr>
		<tr>
			<td>rat anti-RFP</td>
			<td>Allele Biotechnology</td>
			<td>ACT-CM-MRRFP10</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>AlexaFluor 647 AffiniPure Donkey Anti-goat IgG (H+L)</td>
			<td>Jackson laboratories</td>
			<td>705-605-147</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>AlexaFluor 647 AffiniPure Goat Anti-mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A21240</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Cy3 donkey anti-rat IgG</td>
			<td>Jackson laboratories</td>
			<td>712-165-150</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>dissecting microscope</td>
			<td>Leica Microsystems</td>
			<td>S6F</td>
			<td></td>
		</tr>
		<tr>
			<td>epifluorescence microscope</td>
			<td>Leica Microsystems</td>
			<td>M205FA</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM700</td>
			<td></td>
		</tr>
		<tr>
			<td>egg water</td>
			<td></td>
			<td></td>
			<td>0.3 g/L of sea salts &#8216;Instant Ocean&#8217; and 0.5 mM CaSO4 in distilled water.</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td></td>
			<td></td>
			<td>25.6 g Na2HPO4&#183;7H2O, 80 g NaCl, 2 g KCl, 2 g KH2PO4.&#160; Bring to 1 L with distilled water.&#160; Adjust pH 7.&#160; Autoclave for 20 min at 121&#176;C.</td>
		</tr>
		<tr>
			<td>1X PBSDT</td>
			<td></td>
			<td></td>
			<td>0.2% Triton X-100 and 0.2% DMSO in 1 X PBS.</td>
		</tr>
		<tr>
			<td>PEM buffer</td>
			<td></td>
			<td></td>
			<td>30.2 g PIPES, 761 mg EGTA, 1 mM MgSO4.&#160; Bring to 1 L with distilled water.&#160; Adjust pH 7.</td>
		</tr>
		<tr>
			<td>Blocking solution</td>
			<td></td>
			<td></td>
			<td>Mix 1 ml of heat-inactivated horse serum with 9 ml of 1X PBSDT.</td>
		</tr>
	</tbody>
</table>

hepatocyte-specific ablation in zebrafish to study biliary-driven liver regeneration

肝臓は再生する素晴らしい能力を持っています。 hepatocyte-または胆管駆動の肝再生：肝細胞、肝臓の実質細胞は、次のいずれかの方法で再生することができます。胆管駆動の再生に、再生肝細胞は、胆管上皮細胞（BECS）から誘導され、一方、肝細胞主導の肝再生においては、再生肝細胞は、既存の肝細胞に由来します。肝細胞主導の肝臓再生のために、大幅に肝再生の現在の理解に貢献した優れたげっ歯類モデルがあります。しかし、そのようなげっ歯類モデルは胆汁駆動型肝再生のために存在しません。私たちは最近、BECSが広く重度の肝細胞の喪失時の肝細胞を生じさせているゼブラフィッシュの肝障害モデルについて報告しました。このモデルでは、肝細胞を特異的薬理遺伝学的手段によって切除されています。ここでは、肝細胞を切除するとBEC主導の肝再生過程を分析するために、詳細に方法を提示します。この肝細胞特異的切除モデルは、さらに胆汁ドリブン肝再生の基礎となる分子及び細胞メカニズムを発見するために使用することができます。さらに、これらの方法は、肝再生を増強または抑制する小分子を同定するために、化学的スクリーニングに適用することができます。

3.5へのDPF劇的に低減肝臓サイズ（ 図1B）から36時間、MTZ処理（A36h、Aは、アブレーションの略）。 MTZのウォッシュアウト後、肝再生（R）の始まりと考え、強いfabp10a：CFP-NTRとfabp10a：DsRedの発現は、30時間（ 図1B）内に再び現れました。肝内胆管ネットワークBECS 22の膜中に存在するALCAMの発現によって評価されるように、最初に崩壊したが54時間後...

Watch this Scientific Journal Video about Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration at JoVE.com

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

胆道主導の肝臓の再生を研究するためにゼブラフィッシュにおける肝細胞特異的アブレーション

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

hepatocyte-specific-ablation-zebrafish-to-study-biliary-driven-liver

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Unit 1

Regeneration and Repair

SCIENTISTS IN ACTION

8.5K ビュー.  University of Pittsburgh. To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

ビデオ: Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

ゲンタマイシン誘発性損傷によって大人ゼブラフィッシュにおける腎再生

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

発生生物学

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

ゼブラフィッシュ再生の分子機構を研究するためにテトンシステムの利用

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

前駆細胞が媒介する肝細胞の再生を研究するためにゼブラフィッシュにおけるエタノール誘発性線維症肝臓モデルの開発

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

プラナリアで瞳の再生を研究するための外科切除アッセイ

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

ゼブラフィッシュにおける急性アルコール性肝障害の組織学的解析

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

ラッパムシの再生の研究のための方法

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

ブライトフィールド顕微鏡を用いた幼虫ゼブラフィッシュの肝臓サイズの定量化

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

ゼブラフィッシュインターニューロマスト細胞における共焦点顕微鏡ベースレーザーアブレーションと再生アッセイ

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

ゼブラフィッシュモデルの筋肉再生を調べる

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

hiPSC由来内皮細胞の分化と機械的刺激

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

著者スポットライト:流体媒介性機械的刺激下で培養されたhiPSC由来内皮細胞の研究 

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.